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Acknowledgements
My supervisors:
Dr. Charlie Corke. Thankyou for your never ending enthusiasm, support and
fantastic opportunities over the past five years.
Associate Professor Tom Watson, thanks for all of your support and encouragement.
Thanks for sparking my interest in biochemistry all those years ago. Thanks also for
playing the devil’s advocate in research meetings!
Laboratory assistance:
Dr. Kieran Scott, Megan Taberner, Dr. Katherine Bryant, Garvan Institute of Medical
Research. Thankyou, particularly to Kieran for your guidance, and help over many
years.
Australian Proteome Analysis Facility, in particular David Basseal and Dr. Stuart
Cordwell, thankyou for the opportunity to learn Proteomic techniques in such a
professional environment.
Dr. Wojtek Michalski, Brian Shiell, Dr. Mark Lanigan, Gary Beddome, Megan
Retallick, Australian Animal Health Laboratories, CSIRO. Thankyou for your
support over an extended period of time, and for making me feel like another member
of the lab.
Dr. Mark Raftery, Dr. Valerie Wassinger, BMSF, UNSW. Thankyou for the mass
spectrometry work.
Dr. Peter Hoffman, Professor Bruce Kemp, Sid Murthy, St. Vincent’s Institute of
Medical Research. Thankyou for the opportunity to use your mass spectrometer.
Rosemay Condron, La Trobe University. Thankyou for early sequencing attempts.
Carolina Lopez, Matt Constable, Cathy Aitken, Jason Hodge, Department of Clinical
and Biomedical Sciences, thankyou for the loan of various bits of equipment!
Personal Communications:
Professor Gregory Bulkley, for personal communications relating to
ischaemia/reperfusion injury.
IV
Acknowledgements
Dr. Ben Herbert, for personal communications relating to low abundance proteins and
Proteomics.
Dr. Margaret Henry, for personal communications relating to multivariate analysis.
Dr. Bruce Kemp, for personal communication relating to mass spectrometry of silver
stained proteins.
Phospholipase A2 practical assistance:
Dr. Tanweer Ahmad, Leeds University, thanks for the thesis, and ideas!
Dr. Anthony Lawrence, University of Glasgow, thankyou for ideas and hints.
Dr. David Wilton, University of Southampton, thanks for the problem solving session.
Laboratory members:
Fiona Collier, Caryll Waugh, Courtney Talbot, Dr. Liem Vo, Dr. Claudia Gregorio
King, Associate Professor Mark Kirkland, Karyn Bolton, Gavin van Der Meer, Dr.
Janet McLeod, Jane Hosking, Paul Fell, Sarah Roberts, Tamara Gough, Douglas
Hocking Research Institute. Thanks for everything!
Mark Rigby, Joanne Spence, Emilio Baldonado, Garth Stephenson, Andrew Krich,
Michael Lovelace, thanks for the laughs.
Other assistance:
Helen, Rosemary and Ruth, Intensive Care Unit, Geelong Hospital, Barwon Health.
Surgical Staff, Geelong Hospital, Barwon Health.
Dr. Harry Armstrong, Pathcare.
Peter Gumley, Pathcare.
Financial support:
Deakin University Postgraduate Association.
Geelong and Region Medical Research Foundation.
Moral support and Motivation:
Dr. Neil Barnett and Jane Pappin, for moral support over a number of years.
V
Acknowledgements
Debbie and Geoff Gill, the teachers and the kids at Geelong Aquatic Centre, thankyou
for a different perspective on life, and your support over the past five years.
Fellow PhD. Students, in particular Rachel, Pete, Benge and Emma, thanks for your
discussions and help. Thanks Caz for the emails, coffee and late night microscope
assistance.
My housemates (and mates) Weasel, Meaghan, Clairey, Rachelle, Tania, Michael and
Ellie, thanks for putting up with me!
My family: Mum, Dad, Jacqui, Dave, Grandpa G., Grandma and Grandpa W, where
do I start? Thanks for your support, I couldn’t have done it without you!
My friends: Lisa, Peter, Andrew, Jane P. & Adam, Jane W., Benge, Emma, Kit, Matt,
Rachel, Caz, Rachelle, Claire, Tania, Raelene, Jenny, Megan R., Heleen, Megan
McG., Meaghan & Ben, Kim, thanks for everything.
VI
Table of contents and figures.
Table of Contents: SECTION: Pages: Acknowledgements IV-VI Table of Contents VII List of Figures VIII-IX List of Tables X Abbreviations XI-XIII List of Publications arising from this Research XIV-XV Abstract XVI-XVIII Thesis summary XIX Aims of study XX CHAPTER ONE Introduction and Literature Review 1-18 CHAPTER TWO Methods section 19-41 CHAPTER THREE Plasma Proteomics
Introduction 42-60 Results 61-74 Discussion 75-82
CHAPTER FOUR Phospholipase activity in Mesenteric Ischaemia/Infarction
Introduction 83-95 Results 96-102 Discussion 103-106
CHAPTER FIVE Protein Purification
Introduction 107-114 Results 115-146 Discussion 147-151
CHAPTER SIX Cyclophilin B
Introduction 152-157 Results 158-169 Discussion 170-176
CHAPTER SEVEN Alkaline Urea PAGE
Introduction 177-178 Results 179-197 Discussion 198-201
Conclusions and Future Directions 202-212 Bibliography 213-236
VII
Table of contents and figures.
Table of Figures: Figure number: Description: Page: 1.1 The axis of ischaemic damage 4 1.2 The development of ischaemia/reperfusion injury. 5 3.1 The Plasma Proteome. 50 3.2 2D gels of control patients’ plasma proteins. 63 3.3 2D gels of bowel infarction patients. 64 3.4 A typical 2D gel with annotated protein identities. 65 3.5 Relative quantities of proteins of interest. 66 3.6 Serum amyloid A identification. 68 3.7 Sequence alignment of Serum Amyloid A subtypes. 69 3.8 Serum Amyloid A variant quantities. 70 3.9 Serum Amyloid A peptide functions. 71 3.10 PLA2 activity in plasma. 72 3.11 Five plasma variables. 73 3.12 ROC plot of five plasma variables. 74 3.13 The Acute Phase Proteins. 81 4.1 Sites of phospholipase activity on
a generalised phospholipid. 83 4.2 Products of phospholipase A2 hydrolysis. 86 4.3 Immunoprecipitation of PLA2-IIA from recombinant PLA2
standard. 97 4.4 Immunoprecipitation of PLA2 from human bowel. 98 4.5 Western blotting of PLA2 from human bowel. 99 4.6 PLA2 activity in infarcted bowel tissue and lumen content. 100 4.7 PLA2 activity in infarcted and ischaemic bowel tissue. 101 4.8 PLA2 activity versus tissue damage score. 102 5.1 Protein purification process 1. 118 5.2 Protein purification process 2. 119 5.3 Protein purification process 3. 120 5.4 Absence of PLA2 activity in haemoglobin. 121 5.5 Absence of PLA2 activity in haemoglobin & buffers. 122 5.6 Absence of PLA2 enhancing effects of haemoglobin. 123 5.7 Heparin binding of PLA2 isoforms. 124 5.8 Sequence alignment of sPLA2 isoforms. 125 5.9 Typical elution profile of total protein from a heparin affinity
column. 127 5.10 Elution of total protein and PLA2 activity from heparin affinity
column. 128 5.11 Effect of column volume on protein eluted. 129 5.12 Effect of dialysis. 130 5.13 Non-interference of Rotofor buffer in PLA2 assay. 131 5.14 Typical running conditions during a Rotofor run. 132 5.15 The pH and PLA2 profiles of a typical Rotofor experiment. 133 5.16 SDS PAGE gel of active fractions from a typical Rotofor
experiment. 134 5.17 Rotofor system reproducibility. 135 5.18 Rotofor refractionation. 136 5.19 Determination of isoelectric point by refractionation. 137
VIII
Table of contents and figures.
5.20 Electroelution of the protein of interest. 138 5.21 Optimal pH of the protein of interest. 139 5.22 Purification overview. 140 5.23 Silver stained SDS gel of protein of interest. 141 5.24 Protein purification system 4, the final purification process. 142 5.25 Mass spectrometry of the unknown protein, BisTris gel. 143 5.26 Mass spectrometry of the unknown protein, TrisGly gel. 144 5.27 Concentration of the protein of interest. 145 6.1 SDS gel of Jurkat cell lysate and cell culture medium. 159 6.2 Western blot of Cyclophilin B from infarcted human bowel
tissue. 160 6.3 Western blot of Cyclophilin B from infarcted human bowel
tissue. 161 6.4 Western blot of Cyclophilin B from normal human bowel
tissue. 162 6.5 Western blot of Cyclophilin B from infarcted human bowel
lumen samples. 163 6.6 Phospholipase activity before and after immunoprecipitation of cyclophilin B. 164 6.7 Western blot of Cyclophilin B from plasma. 165 6.8 Amino acid sequence of human Cyclophilin B. 166 6.9 Immunohistochemistry of cyclophilin B in human bowel. 167 6.10 Cyclosporin A does not alter phospholipase activity. 169 6.11 Cyclophilins and pathological pore opening. 175 7.1 Electrotransfer of proteins, appearance of membranes. 181 7.2 Electrotransfer of proteins, appearance of gels. 182 7.3 Transfer efficiency of CAPS and Towbin transfer buffers. 183 7.4 Amino acid sequencing of electrotransferred protein. 184 7.5 Running temperature of modified alkaline urea gels. 185 7.6 Protein mobility in modified alkaline urea gel. 186 7.7 Extraction of crude venom for SDS PAGE. 187 7.8 Protein size estimation. 188 7.9 Carbamylation of bee venom PLA2. 189 7.10 Alkaline urea gel of haemoglobin. 190 7.11 Extraction of PLA2 activity from alkaline urea gels. 191 7.12 PLA2 activity of tiger snake venom proteins. 192 7.13 PLA2 isoform mobility in alkaline urea gels. 193 7.14 Infarcted and normal bowel PLA2 protein mobility in alkaline
urea gels. 194 7.15 Alkaline urea gels of partially purified and crude bowel
phospholipase. 195 7.16 Alkaline urea gels of normal bowel and synovial fluid
phospholipase. 196 7.17 Effect of sodium chloride concentration on protein mobility.
197
IX
Table of contents and figures.
List of Tables: Table number: Description: Page(s): 1.1 Clinically assessed markers of intestinal ischaemia
and infarction. 9-11 1.2 Animal models of mesenteric ischaemia
and infarction. 12-13 2.1 Gross tissue rating system of Sun et al, 1997. 28 3.1 Advantages and disadvantages of nucleic acid
based global techniques. 42 3.2 Advantages and disadvantages of protein
based global techniques. 43 3.3 Acute phase Serum Amyloid Variants. 53 3.4 Protein spots present in 2D gels of plasma. 67 4.1 Phospholipase A2 in Intestinal Disease. 83 5.1 Purification of PLA2 using heparin
affinity chromatography. 111 5.2 Heparin affinity of PLA2 isoforms. 112 6.1 The Major Human Cyclophilins. 154
X
Abbreviations
2DE; two dimensional electrophoresis
ALP/AP: alkaline phosphatase
APACHE: acute physiology and chronic health evaluation
APS: Ammonium persulfate
ASB14: tetra decanol amidopropyl dimethylammonio propane sulfonate
ALT: alanine amino transferase
ARDS: adult respiratory distress syndrome
AST: aspartate amino transferase, formerly known as SGOT
BCA: Bicinochoninic Acid
BIS: N,N’-Methylene-bis-acrylamide
BMSF: Biomedical Mass Spectrometry Facility, University of New South Wales
BSA: Bovine serum albumin
CAPS: 3-[cyclohexylamino]-1-propanesulphonic acid
CBB: Coomassie brilliant blue
cDNA: complementary deoxyribonucleic acid
CHAPS: (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate)
CHO: Chinese hamster ovary
CK: creatine kinase
CNBr: Cyanogen bromide
CRP: C-reactive protein
CsA: cyclosporin A
CT: computed tomography
CyPB: cyclophilin B
CyPB-CsA: cyclophilin B-cyclosporin A complex
DC: detergent compatible
DD-PCR: differential display polymerase chain reaction
DTE: dithioerythritol
DTT: dithiothreitol
ECL: enhanced chemiluminescence
EDTA: Ethylene diamine tetra-acetic acid
ELISA: Enzyme linked immunosorbent assay
ESI: electrospray ionization
EST: expressed sequence tag
GGT: γ glutamyl transferase
XI
Abbreviations
HCl: hydrochloric acid
H&E: Hematoxylin and eosin
HEK: human embryonic kidney
HPLC: High performance liquid chromatography
HRP: horseradish peroxidase
HUPO: human proteome organisation
IAA: iodoacetamide
ICU: intensive care unit
IEF: iso-electric focusing
I-FABP: intestinal fatty acid binding protein
IL 1, 6, 8: interlukin 1, 6, 8
IP: immunoprecipitation
IPG: immobilised pH gradient
I/R: ischaemia reperfusion
kDa: kilo Dalton
KLH: keyhole limpet hemocyanin
LC/MS/MS: liquid chromatography- tandem mass spectrometry
LDH: lactate dehydrogenase
LDS: lithium dodecylsulphate
MALDI-TOF: matrix assisted laser desorption ionization-time of flight
MDA: malondialdehyde
MES: 2-(N-morpholino)ethane sulphonic acid
MOF: multiple organ failure
MODS: multiple organ dysfunction syndrome
MPO: myeloperoxidase
MRI: magnetic resonance imaging
mRNA: messenger ribonucleic acid
MS: mass spectrometry
MS/MS: tandem mass spectrometry
MWCO: molecular weight cut off
NOGS: N-Octyl β-D-glucopyranoside or n-Octyl glucoside
NOMI: non-occlusive mesenteric ischaemia
PAF: platelet activating factor
PAGE: polyacrylamide gel electrophoresis
XII
Abbreviations
PBS: Phosphate buffered saline
PC: Phosphatidylcholine
pCO2: partial pressure carbon dioxide
PE: Phosphatidylethanolamine
PES: polyethersulphone
pI: isoelectric point
PLA2: Phospholipase A2
PMN: polymorphonuclear neutrophil
PMSF: Phenyl methyl sulfonyl fluoride
PNPP: p-nitrophenyl phosphate
PPO: Diphenyl oxazole
P&T: phloxine and tartrazine
PVDF: polyvinyl difluoride
ROC: receiver operating characteristic curve
RP-HPLC: reversed phase high performance liquid chromatography
RT-PCR: reverse trancriptase polymerase chain reaction
SAA: serum amyloid A
SDS: Sodium dodecyl sulfate or lauryl sulfate
SDS PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SGOT: serum glutamic oxaloacetic transaminase
sPLA2: secretory phospholipase A2
TBS: Tris buffered saline
TBST: Tris buffered saline, Tween-20
TCA: trichloroacetic acid
TEMED: N,N,N’,N’ tetramethyl ethylene diamine
TFA: trifluroacetic acid
TLC: Thin layer chromatography
TNFα: tumour necrosis factor α
TRICINE: N-tris[hydroxymethyl] methyl glycine
TRIS: Tris (hydroxy methyl) methylamine
XIII
Publications
Papers:
1. Corke C. and Glenister K., Monitoring intestinal ischaemia. Crit. Care Resuscitation,
2001. 3(3): p. 176-180.
2. Corke C., Glenister K., and Watson T., Circulating secretory phospholipase A2 in
critical illness- the importance of the intestine. Crit. Care Resuscitation, 2001. 3(4): p.
244-249.
3. Kristen M. Glenister, Charlie F. Corke. The Infarcted Intestine: a Diagnostic Void.
ANZ J. Surg. , 2004. 74 (4): p. 260-265.
4. Kristen M. Glenister, Charlie F. Corke (2003). Extensions of alkaline urea gels for
venom proteins. (in preparation).
Abstracts:
Australian Society of Medical Research, 2000:
Phospholipase A2 activity as a marker of bowel ischaemia.
Kristen Glenister, Charlie Corke, and Tom Watson, (2000).
Lorne Protein Structure and Function, 2001:
Phospholipase A2 isoenzymes as diagnostic markers of Bowel Ischaemia/Infarction.
K. Glenister, C.Corke and T. Watson (2001).
Australian and New Zealand Intensive Care Society, 2002:
Kristen Glenister, Charlie Corke & Tom Watson, (2002).
PLA2 in intestinal Infarction- a key to intestinal involvement in critical illness?
Melbourne protein group, Melbourne, 2002:
Glenister, K.M., Corke, C.F. and Watson, T.G. (2002).
Phospholipase A2 in Infarcted Human Bowel.
XIV
Publications
ComBio, Sydney, 2002:
Glenister, K.M., Corke, C.F. and Watson, T.G. (2002).
Phospholipase A2 in Infarcted Human Bowel.
Melbourne Protein group, Melbourne, 2003:
Kristen Glenister and Charlie Corke (2003).
Improved method for separation of basic venom proteins: alkaline urea PAGE.
ComBio, Melbourne, 2003:
Kristen Glenister and Charlie Corke (2003).
Improved method for separation of basic venom proteins: alkaline urea PAGE.
XV
Abstract
Currently, diagnostic tests for mesenteric ischaemia and infarction are inadequate due
to poor sensitivity and specificity. In addition, many potential markers appear too
late to be clinically useful. At present, definitive diagnosis can only be made at the
time of surgery, which is not ideal as surgery is often to be avoided in critically ill and
elderly patients. A clinically useful, minimally invasive test is likely to decrease the
currently very high mortality rate and allow monitoring of ‘at risk’ patients during
their hospital stay.
A two-dimensional electrophoresis based proteomic approach was undertaken to
assess plasma protein differences between patients with surgically confirmed bowel
infarction and control Intensive Care patients. The major protein differences were
found to be members or variants of acute phase proteins. Serum amyloid A showed
the largest difference between the two patient groups, and this protein was
investigated in greater depth. An analysis was performed to compare the diagnostic
ability of several commonly used indicators of critical illness and bowel infarction
with serum amyloid A and phospholipase A2. Although none of the variables were
ideal for clinical use, plasma phospholipase A2 activity showed the best
discriminatory power, as determined by Receiver Operating Characteristic curves.
From a review of the literature, phospholipase A2 (PLA2) appeared to be increased in
the bowel as a result of ischaemia and infarction. In one patient, matched tissues
were obtained, and PLA2 activity was found to be significantly higher in infarcted
bowel tissue compared to ischaemic bowel tissue. PLA2 activity was significantly
greater in bowel lumen than tissue, suggesting that the protein was being released, and
may enter the circulation. PLA2 activity was increased in the plasma of bowel
infarction patients compared with control patients, though the difference was not
significant. The phospholipase activity exhibited a number of similarities to typical
phospholipase A2 proteins, but also showed a number of inconsistent characteristics.
For this reason, we wished to identify the protein responsible for the increased
phospholipase activity in infarcted human bowel.
The PLA2 activity in human bowel could not be abolished by immunoprecipitation of
the PLA2 isoforms IIA (well described in bowel) and V (a closely related isoform).
To investigate these proteins, a native urea protein gel devised for snake venom
XVI
Abstract
phospholipase A2 was modified for use with mammalian phospholipase A2. The
modified gel was used to show that the protein with phospholipase activity from
infarcted gut was different from normal gut PLA2 and type IIA PLA2. A number of
extensions were devised for these native gels and were found to be useful both in this
investigation and for venom investigations.
Protein purification was undertaken to identify the protein responsible for the
increased phospholipase activity in infarcted bowel. Protein was purified from
infarcted human bowel using a number of techniques that exploited unusual
characteristics of the protein. The purification techniques each retained the native
activity of the protein and the purification could therefore be monitored with a
phospholipid hydrolysis assay at each stage.
The protein identified by mass spectrometry was an excellent match for cyclophilin B,
an inflammatory protein that had previously been identified in rat bowel at the mRNA
level (Hasel et al, 1991, Kainer & Doris, 2000). As the purification progress had
been monitored throughout with a phospholipid hydrolysis assay, cyclophilin B was
an unexpected identification, as it is not known to have phospholipase activity.
Cyclophilin B was removed from the highly purified samples via
immunoprecipitation and this process abolished all phospholipase activity. The
addition of cyclosporin A, (the pharmaceutical ligand of cyclophilin B), did not effect
the phospholipase activity. Cyclophilin B protein was found in normal and infarcted
human bowel using Western blotting. Cyclophilin B protein also appeared to be
present in the bowel lumen and plasma of several patients with bowel infarction, but
not in control patients. Immunohistochemistry confirmed the ubiquitous nature of
cyclophilin B that had been reported by other groups.
This project has investigated the use of two dimensional gel electrophoresis based
proteomics to identify proteins present in the plasma of patients with confirmed bowel
infarction and control intensive care patients. The major protein classes observed
were members of the acute phase proteins, which highlights the need for pre-
fractionation of plasma to identify lower abundance, disease associated proteins. A
series of potential plasma markers were compared using Receiver Operating
Characteristic Curves. Although no ideal marker was clear from this analysis,
XVII
Abstract
phospholipase activity appeared to warrant further investigation. Phospholipase
activity was investigated in human infarcted bowel. Protein purification identified
cyclophilin B as a bowel protein that showed unusual phospholipid hydrolysing
activity. Cyclophilin B is a ubiquitous protein in intestinal cell types in both normal
and infarcted tissue. There appears to be release of cyclophilin B into bowel lumen
and plasma under conditions of mesenteric ischaemia and infarction.
XVIII
Thesis Summary
DEAKIN UNIVERSITY.
Summary of Thesis Submitted for the Degree of:
DOCTOR OF PHILOSOPHY
Title of Thesis:
Phospholipase Activity in Human Mesenteric Ischaemia and Infarction. Diagnostic tests for mesenteric ischaemia and infarction are currently inadequate due
to low sensitivity and specificity. Many potential markers of mesenteric ischaemia
and infarction have been proposed but are not clinically useful as they appear too late
in the disease development. Proteomic techniques have great potential in medicine to
identify diagnostic markers and potential drug targets. Proteomic investigations have
previously identified proteins differentially expressed between benign and cancerous
tissues.
This thesis examines the proteins in the plasma of patients with surgically confirmed
bowel infarction and control Intensive Care patients by two dimensional
electrophoresis proteomics. A number of disease associated proteins are investigated
in greater depth using appropriate assays. This thesis examines the protein
purification of one of the proteins of interest, and the examination of this protein in
human mesenteric ischaemia and infarction. The investigations suggest that the
levels of a number of these disease associated proteins are increased during
mesenteric ischaemia and infarction in the bowel and in plasma.
Name: Kristen Glenister Signed: Date: Supervisors: Dr. Charlie Corke and Associate Professor Tom Watson.
XIX
Aims of this Study
The aims of this study are as follows:
1. To use Proteomics to assess whether an abundant and specific disease
associated protein was evident in the plasma of patients with confirmed bowel
infarction.
2. To assess the potential of a variety of disease associated proteins as clinically
useful diagnostic markers of mesenteric ischaemia and infarction.
3. To study potential disease associated proteins in greater depth in relation to
mesenteric ischaemia and infarction using appropriate biochemical and
antibody assays and protein purification techniques where necessary.
4. To develop or refine techniques that are useful to the study of disease
associated proteins where necessary.
XX
Chapter 1 Introduction
Chapter 1: Clinical introduction:
In 1926 A.J. Cokkinis stated starkly:
“occlusion of the mesenteric vessels is apt to be regarded as one of those conditions of which
diagnosis is impossible, the prognosis hopeless and the treatment almost useless”.
Despite the significant medical advances that have been made since this statement, the
problems associated with the diagnosis and prognosis of mesenteric ischaemia remain
substantial. Ischaemia results from the insufficient blood supply to an organ or tissue, in the
case of mesenteric ischaemia the organ affected is the intestine. If the ischaemia persists,
tissue necrosis, or infarction will invariably follow. Infarction of the intestine often leads to
gangrene and perforation of the gut wall.
As the period of ischaemia increases, the prognosis becomes more grave, and by the time
infarction has occurred, the condition has a 50-90% mortality rate during hospital treatment
(Aydin et al, 1998, Flinn & Bergan, 1997, Howard et al, 1996, Klempnauer et al, 1997).
Interestingly, not long after Cokkinis made his grave synopsis the mortality rate due to
mesenteric infarction was 70% (Hibbard, Swenson & Levin, 1931), which has not changed
significantly in the past 70 years. Early diagnosis is critical for effective treatment, and to
keep the mortality rate as low as possible but this remains difficult due to vague symptoms,
lack of clinically useful diagnostic tests (Bradbury et al, 1995) and broad groups of ‘at risk’
patients.
The occurrence of mesenteric ischaemia/infarction is quite low in comparison to other
circulatory disorders (Flinn & Bergan, 1997), accounting for approximately 0.1-0.2% of
hospital admissions (Aydin et al, 1998, Howard et al, 1996, Wadman, Syk & Elmstahl, 2000).
Mesenteric infarction can occur as a complication of hospitalization. In a study spanning six
years, Newman and colleagues (1998) found that a quarter of mesenteric infarction cases
occured as secondary events after hospitalisation, and, in the majority of cases, was the cause
of death. Approximately one half of these cases were due to non-occlusive mesenteric
ischaemia or NOMI (Newman et al, 1998). NOMI is a particularly severe condition due to a
high mortality rate and lack of any treatment other than resection of necrotic tissue. NOMI is
associated with a high mortality rate due to the critical nature of the illness and typically
advanced age of the patients involved (Boley et al, 1977). The use of drugs to reverse
1
Chapter 1 Introduction
hypotension in shock patients in ICU is common, but is likely to compromise blood supply to
the small bowel (Boley, Brandt & Sammartano, 1997, Newman et al, 1998).
Elderly people, particularly those with cardiac conditions, are most at risk of mesenteric
ischaemia. When considering the incidence of this condition, other factors must be
considered:
- the number of recognised cases has increased over the past few decades due to increased
awareness of the condition and an increased number of intensive care units (Bradbury et al,
1995, Rogers & David, 1995),
- the reported incidence is likely to be a substantial underestimation, as up to half of cases are
only detected at autopsy (Wilson et al, 1987), and the rates of autopsy in Victorian hospitals
have decreased by approximately 50% since the introduction of the Human Tissue Act of
1982 (Vic.), (McKelvie & Rode, 1992),
- the incidence is likely to continue to increase with the ageing population,
- patients with ‘low grade sepsis’ may have sub-clinical mesenteric ischaemia, particularly
those that later develop Multiple Organ Dysfunction Syndrome (MODS) (Montgomery,
Venbrux & Bulkley, 1997). The prevalence of sub-clinical mesenteric ischaemia cannot be
assessed without accurate diagnostic tests.
Mesenteric ischaemia can be divided into chronic or acute conditions. Chronic mesenteric
ischaemia is rare (Flinn & Bergan, 1997, Grendell & Ockner, 1989). This condition is
associated with marked weight loss due to avoidance of food as eating aggravates the
condition (Grendell & Ockner, 1989). Acute mesenteric ischaemia can be categorised as
being due to arterial occlusion (due to an embolus or thrombosis) or venous occlusion
(thrombosis or strangulation), or alternatively may be non occlusive in nature as a result of
inadequate blood flow (NOMI).
The high mortality rate is the result of not only the severity of the disease and its tendency to
cause remote organ injury (Montgomery, Venbrux & Bulkley, 1997) but also due to:
- the failure to make a diagnosis until infarction has already occurred
- the tendency of the gut to develop signs of ischaemic damage or infarction even after the
vascular problems have been corrected (Rogers & David, 1995)
2
Chapter 1 Introduction
- the proportion of NOMI cases, where the condition reflects poor general circulatory
perfusion and cannot be treated until infarction has occurred (Boley, Brandt & Sammartano,
1997).
The question of whether the gut is particularly susceptible to ischaemic episodes is
controversial. Villi tips have been observed to be hypoxic even under normal perfusion
states, due to short circuiting of oxygenated blood at the base of the villi (Kong et al, 1998).
The mucosa is much more dependent on constant blood supply than the underlying
connective tissue layers (Marshak & Lindner, 1967). In a normal resting state the mesenteric
vascular bed holds a third of the body’s total blood volume and receives a quarter of the
cardiac output (Schoenberg & Berger, 1993). Under conditions of low blood volume or
pressure this reserve is called upon, and for this reason many clinicians consider the bowel
susceptible to ischaemia during shock states (Deitch, 1992, Koike et al, 1992, Mansour, 1999,
Montgomery, Venbrux & Bulkley, 1997). The gut is thought to show poor tolerance to
hypoxic insult (Mansour, 1999).
Other groups consider the gut to be resistant to hypoxic damage. The bowel wall is capable
of extracting extra oxygen under conditions of decreased blood flow (Patel, Kaleya &
Sammartano, 1992, Rogers & David, 1995) and the splanchnic region in general has a high
capacity to increase oxygen extraction as required (Rowell et al, 1984). In an animal model
of septic shock, the microcirculation to the jejunal mucosa has been shown to remain constant
despite systemic blood flow reduction of approximately 50% (Hiltebrand et al, 2000).
Signs and symptoms of mesenteric ischaemia:
The signs and symptoms associated with mesenteric ischaemia are characteristically vague,
non specific and can vary greatly between patients. The first sign may be severe abdominal
pain which is perceived by the clinician as being ‘out of proportion to physical findings’ on
abdominal examination (Gredell & Ockner, 1989). Other signs that suggest the diagnoses
are an elevated white blood cell count, vomiting, diminished or hyperactive bowel sounds,
tachycardia and hypotension, however, all of these features are non-specific.
Intestinal pathophysiology in response to ischaemia:
The ischaemic bowel initially turns a paler colour (Rogers & David, 1995), but darkens as the
ischaemic period progresses (Carter & Camilleri, 1997). Affected regions of the bowel wall
3
Chapter 1 Introduction
then haemorrhage and appear purple to blackish red. The mucosa ulcerates and appears white
to green (Carter & Camilleri, 1997). The mucosa sloughs off into the lumen, which can
cause gastrointestinal bleeding (Flinn & Bergan, 1997). After 24 to 48 hours the bowel
becomes thin and friable (Carter & Camilleri, 1997). The lumen fills with blood and mucous
and ultimately perforates.
At a microscopic level, damage due to ischaemia begins at the villi tips and this superficial
damage is evident within minutes of occlusion (Carter & Camilleri, 1997). Histological signs
such as mucosal cell necrosis and submucosal venous congestion are evident within an hour
(Rogers & David, 1995). As the ischaemic insult continues damage progresses deeper into
the mucosa (Montgomery, Venbrux & Bulkley, 1997), see Figure 1.1 below.
Figure 1.1 The axis of ischaemic injury.
Mucosal damage during ischaemia occurs from the villi tips in toward the mucosa.
Acknowledgement to Jacqui Glenister for the design and drawing of this figure.
4
Chapter 1 Introduction
Biochemical level changes during ischaemia:
The insufficient blood supply to the gut during mesenteric ischaemia causes significant
cellular damage, but the resumption of blood supply may cause further damage (reperfusion
injury), see Figure 1.2. This became more evident when increasing numbers of patients who
had successful revascularisation of their gut developed intestinal and cardiopulmonary
problems post surgery (Schoenberg & Beger, 1993).
Figure 1.2. The development of Ischaemia Reperfusion injury.
Modified from Bulkley G.B. Free radical-mediated reperfusion injury: A selective
review. Br. J. Cancer 1987; 55: 66-73, with permission from author and Nature
Publishing Group.
If the period of ischaemia is relatively short, the damage caused by reperfusion is greater than
that caused by the ischaemia (Bradbury et al, 1995). As the period of ischaemia increases the
ischaemic component becomes more detrimental than reperfusion, and when infarction
occurs, there is no effect due to reperfusion (Bradbury et al, 1995).
5
Chapter 1 Introduction
During ischaemia xanthine dehydrogenase is converted to xanthine oxidase (Montgomery,
Venbrux & Bulkley 1997). When oxygen supply is restored the purines which have
accumulated during ischaemia are metabolised by xanthine oxidase (which requires oxygen,
unlike its precursor) (Montgomery, Venbrux & Bulkley 1997) forming excess amount of
tissue damaging superoxide O2•-. Reperfusion injury is thought to be primarily due to
reactive oxygen species and increased activity of phospholipase A2 (Schoenberg & Beger,
1993). Free radicals can cause cell membrane damage, mucosal edema and ulceration
(Schoenberg & Beger, 1993). Phospholipase A2 hydrolyses phospholipids to produce fatty
acids and the corresponding lysophospholipids. Lysophospholipids can cause cellular
damage, for example, lysophosphatidylcholine (LysoPC) is known to be cytotoxic
(Schoenberg & Beger, 1993) and increases intestinal permeability (Otamiri et al, 1987).
Inhibitors of phospholipase A2 have been shown to be protective against reperfusion damage
to gut tissue (Otamiri et al, 1987, Otamiri & Tagesson, 1989).
Efforts to limit tissue damage from reperfusion have been unsuccessful, which is likely to be
due to the lengthy and severe periods of hypoxia seen in clinical situations (Montgomery,
Venbrux & Bulkley, 1997).
Current diagnosis of mesenteric ischaemia:
Radiographic investigations are often of little use in the diagnosis of mesenteric ischaemia
(Grendell & Ockner, 1989) particularly in the early stages (Carter & Camilleri, 1997). Plain
films often only display definite signs when necrosis has already developed (Sutton, 1987).
Reviews of the value of computerised tomography (CT) in the diagnosis of mesenteric
ischaemia and infarction have been mixed. Rogers and David (1995) concluded that CT was
able to suggest intestinal ischaemia but was unable to provide a definite diagnosis.
Conversely, Klein and colleagues (1995) found CT to be highly sensitive (82%), able to
correctly diagnose 18 of 22 cases of intestinal ischaemia. This study also concluded that CT
was able to rule out other causes of surgical acute abdomen (Klein et al, 1995). For the
diagnosis of arterial occlusion, angiography is preferred due to the accuracy of diagnosis and
the ability to infuse vasodilatory drugs or even fibrinolytic agents in rare cases (Klein et al,
1995). Angiography is associated with a number of disadvantages including potential
nephrotoxicity, exposure to radiation and its invasive nature (Sreenarasimhaiah, 2003).
Angiography is of less value after the condition has proceeded to infarction (Corder & Taylor,
6
Chapter 1 Introduction
1993). Improvement in ultrasound technology and expertise mean that the absence of flow
can be reliably demonstrated in major abdominal arteries and veins (unless there is significant
gaseous distension). Where expertise exists this can be useful. In one small German study,
few cases (2 of 11 patients) were correctly diagnosed pre-operatively by imaging (plain X-
ray, sonography, CT or angiography) (Klempnauer et al, 1997).
In 1995 a paper stated that ‘little clinical work has been done to evaluate the use of MRI’
(Magnetic Resonance Imaging), (Rogers & David, 1995), but more recently, MRI has been
reported to be a ‘highly accurate and non invasive method of diagnosing mesenteric
ischaemia’ (Sreenarasimhaiah, 2003).
Intraluminal pCO2, measured by gastric tonometry, has been proposed as a way of monitoring
ischaemia and infarction. This technique has not been widely adopted because of the
relatively high cost of the catheters required, the inability of the probes to monitor large areas
of bowel and poor correlation between the disease state and measurement results (Corke &
Glenister, 2001).
As mesenteric infarction is a disease of older patients, often with significant co-morbidities
and a frequently unclear clinical picture, there may be a useful role for laparoscopy. Where
laparoscopy demonstrates profound, extensive infarction the patient is spared full laparotomy
(since resection in massive infarction is often impractical). When laparoscopy reveals
normal bowel laparotomy may also be avoided, though unfortunately bowel ischaemia cannot
be completely excluded by laparoscopy as the serosal surface can appear normal despite
mucosal necrosis (Kurland, Brandt & Delaney, 1992). Observation of limited bowel
infarction on laparoscopy can be followed by laparotomy and resection. Where the bowel
viability is unclear the laparoscope may be left in situ (with a purse ring suture to secure it)
and a second look bedside laparoscopy performed 12-24 hours later (Dr. C. Corke, personal
communication).
Diagnosis of mesenteric ischaemia via blood tests:
Many plasma based pathology tests have been proposed to diagnose mesenteric ischaemia but
none have been useful in a clinical setting because of low specificity, low sensitivity, but most
importantly markers appear only after infarction has already occurred. Lange and Jackel
(1994) concluded that elevated serum lactate was ‘the best marker of mesenteric ischemia to
7
Chapter 1 Introduction
date’. Raised serum lactate is not a specific finding but suggests that a life threatening
condition exists. The usefulness of lactate increases as a diagnostic marker when the possible
diagnoses of shock, diabetic ketoacidosis, renal and hepatic failure can be excluded (Lange &
Jackel, 1994).
Amylase was shown to be significantly elevated in a canine model of mesenteric ischaemia
(Aydin et al, 1998) but in a clinical setting has been shown to be only moderately elevated
(Corder & Taylor, 1993). In addition, approximately one half of patients with confirmed
infarction show normal levels of amylase (Wilson & Imrie, 1985). Total creatine kinase
(CK) is of little value in differentiating patients with gut infarction from other patients with
abdominal symptoms, or from healthy controls (Fried et al, 1991). The isoform of creatine
kinase CK-BB has been suggested to have some value in predicting gut infarction, with 63%
sensitivity at 100% specificity (Fried et al, 1991), but this protein is labile and prompt
analysis is critical, which limits its practicality for clinical use (Smirniotis, Labrou & Tsiftses,
1989). Serum phosphate has been shown to be a strong indicator of mesenteric ischaemia
(Jamieson et al, 1982) but normal concentrations cannot rule out ischaemia or infarction
(Carter & Camilleri, 1997). Hexosaminidase levels only become elevated after necrosis
(Polson, Mowat & Himal, 1981). Diamine oxidase (Bounous et al, 1984) and intestinal fatty
acid binding protein (I-FABP) (Kanda et al, 1995, Kanda et al, 1996) have shown some
promise but have only been investigated in very small groups of patients. Lactate
dehydrogenase has been found to be elevated during intestinal infarction compared with
‘surgical abdomens’, with a sensitivity of approximately 73% (Calman et al, 1958). In a
recent review, discussion of any blood based diagnostic tests of intestinal ischaemia was
notably lacking, highlighting the void in this area (Sreenarasimhaiah, 2003). The comparison
of the various markers that have been proposed is reviewed and summarised in the following
tables.
8
Chapter 1 Introduction
Potential marker Reference Control group(s) How accurate are these markers? Lactate dehydrogenase.
Calman et al, 1958.
Peritonitis, obstructive jaundice, distended abdomen, fractures & normal controls.
73% sensitive in patients with infarction. Elevated LDH could indicate necrotic intestine or another source of damaged tissue. A single normal LDH reading could not exclude intestinal infarction.
Hexosaminindase Polson,Mowat & Himal, 1981.
Abdominal pain or tenderness.
Indicator of necrosis.
Phosphate Jamieson et al, 1982.
Only patients with acute abdomen & gut ischaemia.
80% had elevated phosphate. The 20% that did not show elevated phosphate had blood taken late after onset of symptoms, so phosphate may have been cleared via urine.
Diamine oxidase Bounous et al, 1984.
Normal, healthy controls & cardiac patients with no abdominal symptoms & one NOMI patient.
One bowel infarction patient showed 7 times normal value, but two showed below normal levels. The elevated level seen in the bowel infarction patient fell to an approximately normal level even though necrotic tissue was not resected. High diamine oxidase levels could indicate pregnancy or lung carcinoma. Low levels are seen in celiac disease & Crohn’s disease.
Amylase, lactate dehydrogenase, phosphate.
Wilson et al, 1987.
Only acute mesenteric ischaemia patients.
Amylase elevated in 27 of 52 patients tested, lactate dehydrogenase elevated in 5 of 7 patients tested, phosphate elevated in 2 of 3 patients tested.
Creatine kinase (total CK) and isoforms MB and BB.
Fried et al, 1991.
Group I: normal healthy controls. Group II: acute abdominal signs.
Total CK and CK-MB levels were not significantly raised in patients with infarction. CK-BB showed 100% specificity, 63% sensitivity.
Table 1.1. Clinically assessed markers of intestinal ischaemia/infarction:
9
Chapter 1 Introduction
Lactate Lange &
Jackel, 1994.
Acute abdominal signs.
100% sensitive, 42% specific, but elevated levels were not indicative of bowel ischaemia/infarction.
Intestinal fatty acid binding protein
Kanda et al, 1995.
Healthy volunteers.
Only two patients in study. I-FABP was undetectable in healthy controls and after bowel resection.
Intestinal fatty acid binding protein, AST, LDH, ALP & CK.
Kanda et al, 1996.
Normal healthy controls and acute abdominal pain.
Small study group, (only 5 cases of mesenteric infarction). Patients with strangulated bowel and patients with mesenteric ischaemia showed high levels of I-FABP, significantly different from healthy controls and patients with other sources of abdominal pain. AST: 60% sensitivity, LDH: 60% sensitivity, ALP: 0% sensitivity, CK: 40% sensitivity.
Lactate Klein et al, 1995.
None. 17/22 bowel infarction patients showed elevated levels.
Lactate, amylase, alkaline phosphatase
Newman et al, 1998.
Mesenteric infarction patients only, divided into primary or secondary development.
Lactate was the only predictor of mortality.
α glutathione S-transferase, AST, ALT, amylase
Delaney et al, 1999.
Acute abdominal patients.
Sensitivity, specificity results: α glutathione S-transferase (100%, 86%, though there may be significant levels seen in liver insult, see Gearhart et al, 2003, below), AST (70%, 50%), ALT (73%, 60%), amylase (25%, 63%).
Lactate Klotz et al, 2001.
Open heart surgery patients +/- NOMI.
No significant difference found.
Table 1.1 continued. Clinically assessed markers of intestinal ischaemia/infarction:
10
Chapter 1 Introduction
Table 1.1 continued. Clinically assessed markers of intestinal ischaemia/infarction: α glutathione S-transferase, amylase, lactate, AST, ALT.
Gearhart et al, 2003.
Patients with suspected acute mesenteric ischaemia. 60% of patients showed some form of mesenteric ischaemia (small bowel ischaemia, colonic ischaemia or both).
α glutathione S-transferase was the most accurate predictor of acute mesenteric ischaemia (74% accuracy) compared with conventional tests (47-69% accuracy). α glutathione S-transferase could not distinguish between ischaemia and necrosis. Elevated levels of α glutathione S-transferase may reflect hepatic ischaemia. The negative predictive value of combined α glutathione S-transferase, white blood cell count and lactate was high.
11
Chapter 1 Introduction
Table 1.2. Animal models of mesenteric ischaemia/infarction:
Potential marker Reference Animal model & Controls used.
How accurate are these markers?
Alkaline phosphatase.
Barnett, Davidson & Bradley, 1976.
Dog. Control laparotomy.
50% of animals with necrotic bowel tissue had no detectable alkaline phosphatase. 2/6 dogs with induced pancreatitis also showed detectable de novo levels of intestinal alkaline phosphatase but also showed duodenal necrosis.
Diamine oxidase Wollin, Navert & Bounous, 1981.
Rat. Sham laparotomy.
Occlusion of superior mesenteric artery for either 30, 60 or 90 minutes resulted in increased levels of diamine oxidase in serum. Diamine oxidase was thought to be released from intestinal epithelium into interstitial fluid. A portion of this enzyme thought to then be released into blood via lymphatics.
Phosphate Jamieson et al, 1982.
Dog. Controls not described, SMA or SMV occluded.
Phosphate levels were elevated at 2 hours and continued to rise until 6 hours. Statistics were not reported.
ALP, CK, LDH & AST, GOT, amylase.
De Toma et al, 1983.
Dog. 6 laparotomy controls, 6 ligated superior mesenteric artery.
Non specific. There was no significant difference between control dogs and those dogs with bowel infarction.
ALP, creatine kinase, lactate dehydrogenase. SGOT, DAO.
Thompson, Bragg & West, 1990.
Dog. SMA ligation, or occluded then released & laparotomy controls.
LDH, SGOT and ALP had low sensitivity. Creatine kinase had greater sensitivity and overall accuracy than other enzymes tested.
ALP, LDH, AST & CK.
Kurland, Brandt & Delaney, 1992.
Dog. Controls not described.
No markers were found to be sensitive or specific to intestinal ischaemia.
12
Chapter 1 Introduction
Table 1.2 continued. Animal models of mesenteric ischaemia/infarction: MDA, ALP, LDH, amylase, AST, GGT & CK.
Aydin et al, 1998.
Dog. Sham laparotomy.
MDA levels significantly higher after ligation. Other proteins were significantly elevated compared with sham operated controls.
Von Willebrand’s factor, myeloperoxidase, protein carbonyl.
Abu-Zidan et al, 1999.
Rat. Sham laparotomy & anaesthesia controls.
Protein carbonyl significantly higher in I/R rats than sham controls. Protein carbonyl correlated with Von Willebrand’s factor. Myeloperoxidase levels were not significantly different between groups.
Cytosolic β-glucosidase.
Morris et al, 1999.
Guinea Pig. Sham laparotomy & anaesthesia controls.
Low specificity. Closed loop obstruction resulted in increased levels.
13
Chapter 1 Introduction
In conclusion successful early diagnosis of mesenteric ischaemia and infarction is
currently dependent on a high degree of suspicion by the physician. Recently, the
mortality rate associated with ischaemic bowel was 35%, but when the condition had
progressed to infarction the mortality rate increased to 68% (Rogers & David, 1995).
At present the only method of definitive diagnosis is surgery (Fried et al, 1991) but
this is often avoided in critically ill or elderly patients. However, an accurate
diagnostic marker would allow surgeons to make risk assessments prior to surgery.
A diagnostic marker would also allow post operative monitoring of patients that have
undergone bowel resection, to ensure that the condition has been rectified. At present
post operative monitoring is not practical and definitive post operative monitoring for
ongoing ischaemia depends upon clinical examination or ‘second look surgery’ 12 to
24 hours after resection (Rogers & David, 1995).
Were early diagnosis to become more attainable and reliable, treatment could be
administered earlier and mortality would decrease. Therefore the ability to make
accurate diagnosis at an early stage would decrease the mortality rate.
Treatment of mesenteric ischaemia:
When mesenteric ischaemia is suspected general treatments such as intravenous
fluids, thrombolytic agents (Flinn & Bergan, 1997), anticoagulants and broad
spectrum antibiotics (Mansour, 1999, Rogers & David, 1995) can be administered to
try to improve outcomes. Surgical treatment primarily aims to revascularize the
bowel when possible (in early ischemia), but irreversible ischaemia and/or gangrene
requires intestinal resection (Klempnauer et al, 1997).
Outcomes after mesenteric ischaemia:
Relatively few studies have focused on the long term prognosis of the patients
discharged from hospital after mesenteric ischaemia. One study conducted follow up
investigations of 31 patients for up to five years after discharge (Klempnauer et al,
1997). It was found that only one patient suffered recurring mesenteric ischaemia,
and died as a result. This patient, unlike the others was not receiving ongoing anti-
coagulant therapy. The major causes of mortality during this study were
cardiovascular events or malignancies. One fifth of the group suffered from short
14
Chapter 1 Introduction
bowel syndrome. The severity of this syndrome is inversely correlated to the length
of small bowel remaining (in this study there was an average of 170cm of small bowel
remaining). An earlier study of patients with infarction due to venous occlusion
found recurrence of thrombosis in up to 29% of cases, most commonly due to residual
thrombi (Clavien, 1990). Of the 31 patients studied, there was a 70% two year
survival rate and 50% 5 year survival rate. The average age of the patients was not
reported.
When large sections of the small intestine require resection, patients must receive
long term parenteral nutrition for survival. Parenteral nutrition allows the delivery of
nutrients independent of the alimentary tract, typically intravenously. Usually
parenteral nutrition is only administered on a short term basis (during a hospital stay
for instance) but longer term nutrition is possible. Prolonged parenteral nutrition is
associated with significant complication and expense (Pennington, 1997). It can be
difficult to estimate the nutritional requirements of patients, and prolonged nutrition
independent of the alimentary tract can cause mucosal atrophy (Braga & Gianotti,
2002), infectious complications, imbalance of gut flora (Deitch, 1993), and decreased
host immune function (Deitch, 1992).
Importance of gut ischaemia/reperfusion injury to other conditions:
Gut ischaemia/reperfusion injury (I/R) is an important condition in its own right, but
is also involved in the development of several other serious conditions. Under
conditions of mucosal damage and hypoxia the gut releases factors into circulation
that may trigger remote organ injury (for example, adult respiratory distress syndrome
(ARDS), or multiple organ dysfunction syndrome (MODS)) and sepsis. The identity
of these gut derived factors and the mechanism of their release are not yet understood.
Research has focussed on gut derived:
- bacteria,
- bacterial endotoxin,
- proteases,
- enzymes (for example, phospholipase A2),
- nitric oxide,
- free radicals,
- cytokines.
15
Chapter 1 Introduction
Some of the gut derived mediators (eg cytokines, free radicals) produced under
ischaemic conditions cause mucosal damage leading to a loss of gut barrier integrity
(Kong et al, 1998), and leakage into the peritoneal cavity. Other factors are
postulated to be transported via the portal circulation or mesenteric lymph. A
complex interplay of a variety of factors is likely. Conditions such as sepsis may
play a role in the development of gut dysfunction (Bradbury et al, 1995) or may
develop because of gut dysfunction (Kong et al, 1998).
Phospholipase A2 is thought to be involved in ischaemia induced remote organ injury,
as PLA2 inhibitors have been shown to reduce bacterial translocation and mucosal
injury in a canine model of shock (Xu, Lu & Deitch, 1995). Interestingly, PLA2
inhibition and calcium channel blocking failed to be fully protective, suggesting that
other factors, such as oxidising agents, are involved (Xu, Lu & Deitch, 1995).
Inhibitors of cyclooxygenase, phospholipase A2 or xanthine oxidase have been shown
to protect against the lung injury caused by laparotomy/intestinal handling in a rat
model (Thomas, Karnik & Balasubramanian, 2002). Animal studies suggest that the
gut serves as a priming bed for circulating polymorphonuclear neutrophils (PMN)
which then promote remote organ injury, a process that is likely to involve
phospholipase A2 (Koike et al, 1992, Koike et al, 1995).
Multiple Organ Dysfunction Syndrome:
Multiple organ failure syndrome (MOFS) was first recognised in 1973 (Tilney, Bailey
& Morgan, 1973) when it was observed that patients with ruptured aortic aneurysms
suffered injury to initially uninvolved organs after surgery. Later it was proposed
that the term MOFS be updated to Multiple Organ Dysfunction Syndrome (MODS) to
reflect the continuum of the syndrome (Fry, 2002). MODS is not an uncommon
event [incidence of 5-20% in ICU patients (Poole, 2002)] and accounts for up to 90%
of the deaths of surgical ICU patients (Montgomery, Venbrux & Bulkley, 1997), a
rate that has not changed significantly since the condition was first described.
MODS is a condition defined as:
“a generalized autoaggressive inflammatory process” (Goris, 1985, Nyman et al,
1996). MODS involves the ‘sequential failure of vital organs caused by generalized
cellular damage’ (Nyman et al, 1996). The most common predisposing factors are
16
Chapter 1 Introduction
shock and infection (Deitch, 1992). The exact cause of MODS is still unclear but
may be due to exogenous factors (bacteria and toxins), but more importantly,
mediators produced by the patient (Deitch, 1992), such as cytokines (Magnotti et al,
1998) or inflammatory mediators. MODS is diagnosed by the identification of the
level of function of several organs by general tests (elevated bilirubin, creatinine,
hematological changes) and clinical observations (requirement for ventilation,
intolerance to enteral feeding, progressive coma) (Deitch, 1992). In reference to the
diagnosis of MODS, Fry (2002) stated that: “Current diagnosis sophistication is
inadequate”.
The condition is characterized by three stages. The first stage involves sepsis, failure
of the pulmonary system and decreasingly utilisation of oxygen in the periphery
(McMenamy et al, 1981). The second stage involves the failure of the hepatic,
intestinal and renal systems (Deitch, 1992), and the third stage involves cardiac failure
(McMenamy et al, 1981). As the number of organs involved increases, so too does
the mortality rate (Deitch, 1992). When a single organ is involved, the mortality rate
is between 15 and 20%, when two organs are involved the mortality rate rises to 40%,
three organs shows 60-80% mortality, and four or more organs involved is associated
with close to 100% mortality (Poole, 2002). The realisation that the organs fail either
simultaneously or in similar sequences has prompted investigations into a common
cause of MODS (Goris et al, 1985). The contribution of gut dysfunction to remote
organ injury and MODS is yet unknown.
Gut derived factors; bacteria and endotoxin:
The suspected involvement of bacteria and bacterial endotoxin in the development of
MODS, have been studied in numerous animal models and in clinical settings. The
gut is thought to be the source of bacteria and toxins. Although healthy duodenum
and jejunum are relatively sterile (Welsh & Reynolds, 2002), bacteria are present in
the distal small bowel and colon at levels of 1010 anaerobic and 105 to 108 aerobic
organisms per gram of bowel tissue (Deitch & Sambal, 2002). It has been clearly
demonstrated that the integrity of the gut epithelial barrier is compromised after sepsis
(Ziegler et al, 1988). This theory may explain why no identifiable source of sepsis is
found in 30% of bacteremic patients with MODS (Goris, 1985). Research has also
focussed on the route that bacteria and endotoxin pass from the gut into the
17
Chapter 1 Introduction
circulation. Initially research focused on the portal circulation, but no bacteremia or
endotoxemia was detectable (Magnotti et al, 1998, Moore et al, 1991). It was also
found that endotoxin concentrations were not significantly different in gut I/R or
laparotomy controls in rats (Koike et al, 1994). This latter study also demonstrated
that endotoxin elimination failed to prevent lung injury due to gut I/R (Koike et al,
1994). A similar clinical study failed to find bacteria or endotoxin in the portal blood
of trauma patients, even those that went on to develop MODS (Moore et al, 1991).
Later studies in animals focussed on the mesenteric lymph as the route of release.
Entry into the circulation via the mesenteric lymph is an interesting possibility as it
reaches the lung before any other vascular bed (Deitch & Sambal, 2002, Magnotti et
al, 1998) (possibly explaining why the lung is the first organ to fail in MODS). It
was found that gut derived factors that increase cell permeability and contribute to
shock induced lung injury were more prevalent in mesenteric lymph than portal
plasma (Magnotti et al, 1998). Deitch and colleagues (2001) also concluded that gut
derived factors carried in mesenteric lymph appeared to be responsible for lung injury
after shock but bacteria were not responsible. It has been proposed that although
bacteria and/or endotoxin are rarely found in either the portal circulation or lymph
they may still play a role in the development of MODS. A change in the bacterial
balance or bacterial overgrowth may trigger a more potent cytokine response in the
gut after ischaemia/reperfusion injury or shock (Deitch et al, 2001). It has been
demonstrated that IL-6 (Biffl, Moore & Moore, 1995, Grotz et al, 1999) and TNF-α
are released from ischaemic gut and that there is a relationship between the magnitude
of the ischaemic injury and the cytokine response (Grotz et al, 1999). Cytokines
(IL-1, IL-6, TNF) and neutrophils can decrease oxygen supply to the gut by altering
microcirculation, possibly leading to a self sustaining cycle (Deitch, 1992).
18
Methods
POLYACRYLAMIDE GEL STAINING METHODS:
SILVER STAINING:
Silver Staining (BioRad method):
Immediately after electrophoresis, gels were placed in fixative (40% methanol v/v, 10% acetic
acid, 30 minutes). The gels were then placed in oxidising solution (1:10 dilution of BioRad
Silver Staining Kit stock solution oxidiser, 5 minutes, BioRad, Hercules, CA). The gels were
then washed with water (2L with several changes over 15 minutes). The gels were then
stained with silver reagent (1:10 dilution of BioRad Silver Staining Kit stock solution, 20
minutes) and then quickly rinsed with water. The gels were then developed (8g BioRad
Silver Staining developer/250mL water, with multiple changes of solution). The
development was stopped with an acetic acid solution (5% v/v).
Silver staining: (SilverQuest™, Invitrogen method):
Silver staining of NuPAGE gels was performed using SilverQuest™ mass spectrometry
compatible silver staining kit (Invitrogen, Carlsbad, CA). Immediately after the completion
of the electrophoretic run the gels were rinsed quickly (ultrapure water). The gels were then
fixed (40% ethanol, 10% acetic acid, 20 minutes). The gels were then washed (30% ethanol,
10 minutes) before the sensitizing step (10% sensitizing solution, 30% ethanol, 10 minutes).
The gels were then washed in ethanol (30%, 10 minutes) and then ultrapure water (10
minutes). The gels were then stained (100mL, 1% staining solution, 15 minutes) and quickly
rinsed (ultrapure water, 1 minute). The developing solution was then added (10% developer,
1 drop enhancer, 6 minutes) until the desired level of staining was achieved after which the
stop solution was added directly.
Silver staining (PlusOne Pharmacia Biotech method):
All solutions were prepared in ultrapure water. Immediately after electrophoretic run was
complete the gels were placed in fixative (40% ethanol, 10% acetic acid v/v, 60 minutes).
The gels were then placed in sensitizing solution (30% ethanol, 0.125% glutardialdehyde
solution, 0.2% sodium thiosulphate solution, 6.8% sodium acetate w/v, 30 minutes). The
gels were then washed with ethanol solution (20% v/v) followed by two washed with water (5
minutes per wash). The gels were then stained with silver reagent (0.25% solution of silver
nitrate, 0.04% formaldehyde, 20 minutes) and then washed with water (2x1 minute). The
19
Methods
gels were then developed (2.5% sodium carbonate, 0.02% formaldehyde, 10 minutes). The
development was stopped with an EDTA solution (1.5% w/v). Gels were then placed in
water before scanning.
MS Compatible Modification of the PlusOne method:
This method was modified by Yan et al, 2000 for use with ESI and MALDI.
The PlusOne method was followed with the following modifications:
Two fixing steps were employed (15 minutes each). The sensitiser did not contain
glutardialdehyde. The silver solution did not contain formaldehyde. Formaldehyde (100µL)
was added to the developer solution. Acrylamide (1%) was added to the samples after
heating, before loading onto the gel (Dr. Bruce Kemp, personal communication), to prevent
re-formation of disulfide bonds.
Protein bands and spots of interest stained by this method were excised from the gels and
rinsed briefly (ultrapure water). The gel pieces were destained (50µL, fresh 1:1 solution of
30mM potassium ferricyanide, 100mM sodium thiosulphate, 8 minutes, then repeated). The
destaining solution was discarded. The pieces were then washed (ultrapure water, two
washes). The gel pieces were then incubated in ammonium hydrogencarbonate (100µL,
50mM, 20 minutes room temperature). The gel pieces were then cut into small pieces,
washed in ultrapure water and dried (four changes of acetonitrile). The gel pieces were then
dried under vacuum, trypsin digested and submitted for nano-MS.
COOMASSIE STAINING:
Protein stains for gels of samples containing ampholytes:
Coomassie G250 (CBB-G250) containing stains (Neuhoff et al, 1988) were used in
preference to Coomassie R-250 (CBB-R250) stains when running gels of samples that
contained ampholytes. CBB-G250 did not show the high background and ampholyte staining
that CBB-R250 did. For quantitative studies gels were stained with Colloidal Coomassie for
three days with minimal destaining (Herbert, personal communication).
20
Methods
Copper protein stain:
Copper containing protein stain could also be used for staining gels that contained
ampholytes. Copper sulphate (0.5%) was dissolved in water. Ethanol (final concentration
27%) and acetic acid (final concentration 10%) and Coomassie R-250 (0.04%) were added
and solution made up to volume with water. Gels were destained with ethanol/acetic
acid/copper sulphate (12%v/v, 7%v/v, 0.5%w/v respectively). The ethanol could be replaced
with isopropanol.
ANTIBODY TECHNIQUES:
Preparation of immobilised antibodies, with CNBr:
Dialysis tubing (6-8MWCO) was boiled in buffer (100mM bicarbonate, 10mM EDTA, 3
minutes). Antibody (1mL) of 10B2 (~0.93mg/mL, Cayman Chemicals, Ann Arbor, MI) or
4A1 (1mg/mL, Boehringer Mannheim) in coupling buffer were dialysed against coupling
buffer (4L, 4oC, 18 hours). CNBr Sepharose 4B beads (Pharmacia Biotech, 0.06g) were
swelled in dilute HCl (1mM HCl, 5 changes of acidic solution). Beads and dialysed antibody
were mixed and rotated (4oC, 47 hours). The beads were then washed with coupling buffer
(5 volumes, 0.1M NaHCO3 pH 8.3, 0.5M NaCl) and active sites remaining were blocked
(0.1M Tris-HCl, pH 8.0, 2 hours).
Immunodepletion of PLA2:
CNBr sepharose-antibody beads (50µL) and samples suspended in PBS were mixed with
protease inhibitors (1mM PMSF, 50µg/mL aprotinin, 200µM leupeptin) and incubated with
rotation (4oC, 3.25hours). The mixture was then centrifuged (750g, 1 minute) and
supernatent removed. The beads were washed with PBS (4 times) and SDS loading buffer
was added (20µL). The beads were boiled (100oC, 7 minutes) and loaded onto a 4-20%
gradient Tris-Glycine gel (Invitrogen pre-cast mini-gel), and run according to manufacturer’s
instructions. Gels were then silver stained (BioRad silver staining kit).
Immunodepletion of Cyclophilin B:
Protein G agarose (50µL of 50% solution per sample) was mixed with ice cold PBS (500µL)
and centrifuged (12 000g, 4oC, 20 seconds). Supernatent was discarded and the washing
process was repeated twice.
21
Methods
Pre-clearing samples:
Samples (500µg total protein, cold PBS/protease inhibitor cocktail (Sigma, St. Louis, MO) to
a volume of 1mL) were added to the protein G agarose beads. The samples were incubated
with rotation (4oC, overnight).
Immunodepletion:
Pre-cleared samples were centrifuged (12 000g, 4oC, 30 seconds) and supernatent removed to
a fresh tube. Aliquots were removed for other assays. Antibody (1µg of polyclonal
cyclophilin B antibody, Abcam Ltd, Cambridge, UK.) was added to each sample and rotated
for one hour (4oC). Washed protein G-agarose (50µg) was then added and the samples were
again incubated with rotation (4oC, 3 hours). The supernatent was then collected (12 000g,
4oC, 30 seconds) for other assays. The beads were then washed (three washes in ice cold
PBS) before addition of SDS-PAGE sample buffer (50µL).
Cyclophilin B Immunohistochemistry:
Cyclophilin B was investigated in sections of human gut (control and infarcted) using the
Dako immunoperoxidase method, according to manufacturer’s instructions (Dako, Glostrup,
Denmark). The primary antibody dilution was optimised to 1:1200, by performing a
preliminary serial antibody dilution experiment. The sections were counterstained with
Gill’s haematoxylin. Acknowledgement must go to the Pathcare histology laboratory staff
for the preparation of tissue sections and slides for this work.
Phospholipase A2 sandwich ELISA:
ELISA plates (Nunc, Weisbaden, Germany) were coated with the monoclonal antibody 9C1
(100µL per well, 2mg/mL diluted 1:1000 in PBS, Cayman Chemicals, 4oC, 8 hours). The
plates were then blocked (1% skimmed milk powder, 0.1% BSA in PBS, 37oC, 16 hours).
The wells were then washed twice with wash buffer (200µL per well). A series of sPLA2
standards were prepared in the range 0-200ng/mL in PBS/0.1% BSA. The samples were also
prepared in a series of dilutions from 1/5 to 1/50 in PBS. The standards and samples were
added (100µL per well) and were incubated at 37oC for 2 hours. The wells were then washed
twice with wash buffer (200µL per well). Conjugate binding was performed by adding 4A1-
AP (100µL of 0.1% conjugate antibody in 0.1% BSA/PBS). The plates were incubated
22
Methods
(37oC, 60 minutes). The plates were washed three times with wash buffer and three times
with carbonate buffer (200µL per well). p-Nitrophenyl Phosphate (pNPP) (100µL of 15mg
pNPP in 15mL carbonate buffer) was added and incubated at room temperature for 10
minutes. The absorbance of the plates were then read at 405 nm.
PLA2 Western Blotting:
16% polyacrylamide gels (Invitrogen) were used in this section, and were run according to
manufacturer’s instructions under non-reducing conditions. Lanes contained 30µg of total
protein. Multimark Multi Coloured Standard (Invitrogen), (5µL) were used for size
estimation.
The transfer apparatus used was the X-cell-II Blot Module, (Invitrogen). The transfer was
completed at 30volts for 1½ hours (12mM Tris, 96mM glycine, 15% methanol). The
membrane was then washed with Tris buffered saline (TBS) and carefully dried. The
membrane was then blocked (50mL 5% skim milk powder in TBS) overnight at 4oC. The gel
was stained with Coomassie R-250.
The blocked membrane was washed in Tris buffered saline-Tween-20 (TBST) (50mL), and
then incubated in the primary antibody (10mL of 9C1 antibody (5µL) in 1%BSA, 1% skim
milk powder in TBS) for 1 hour. The antibody solution was then removed and the membrane
washed three times with TBST (50mL). The membrane was incubated in the secondary
antibody (3.5µL anti-mouse IgG-HRP in 1%BSA, 1% skim milk powder in TBS, 10 mL final
volume). The antibody solution was removed and the membrane washed in TBST (3x50mL,
3x15 minutes) and TBS (50mL, 15 minutes). Enhanced Chemiluminescence Reagents
(Renaissance, NEN, Irvine, CA), luminol (700µL) and oxidiser (700µL) were mixed and
placed on the membrane for 1 minute. The membrane was blotted dry and covered with
plastic wrap. The membrane was exposed to film (Hyperfilm-ECL, Amersham, Little
Chalfont, Buckinghamshire, UK) for varying time periods (1-60 minutes).
Serum Amyloid A ELISA Assay (TriDelta Phase™ range SAA kit):
The SAA ELISA (TriDelta, Greystokes, Ireland) assay was performed according to
manufacturer’s instructions. Samples of plasma were diluted 1:500, 1:1000 or 1:5000 in
diluent. Biotinylated anti-SAA (1:100 in diluent) was applied to wells containing SAA
23
Methods
antibody (50µL per well). Samples and standards then added (50µL per well in duplicate)
and incubated (60 minutes, 37oC). The wells were then washed four times with wash buffer.
Plates were read at 450nm.
Determination of plasma lactate, C-reactive protein and the APACHE-II score.
Acknowledgement must go to the laboratory staff at Pathcare Pathology, Geelong for the
analysis of plasma C-reactive protein and lactate. Acknowledgement must also go to the
staff of the Intensive Care Unit, Geelong Hospital for the APACHE-II scoring of patients.
Cyclophilin B Western blotting:
SDS PAGE gels (8-16% iGEL, TrisGlycine, Gradipore) were run according to manufacturers
instructions, under reducing conditions. Sample lanes contained 30µg of total protein.
Prestained protein molecular weight markers (MBI Fermentas, St. Leon Rot, Germany) were
included for size comparison. The positive and internal control was a whole cell lysate
(15µL) from Jurkat cells. Acknowledgement must go to Caryll Waugh, Douglas Hocking
Research Institute, for the culture of Jurkat cells. The same cell lysate was used in all
Western analyses. The Jurkat cell pellet (1× 106 cells) was collected (10 minutes, 4oC,
9000g). The supernatent (cell culture medium) was removed, and stored (-80oC) for the
positive and internal control in the Western analyses of plasma samples. SDS-PAGE sample
buffer (100µL, containing 0.05M DTT) was added to cell pellet. The solution was
thoroughly mixed and incubated (room temperature, one hour). The solution was then boiled
(100oC, 5 minutes). If the Jurkat cell lysate sample was stored, fresh DTT (10% v/v, 0.5M)
was added and the samples re-boiled before applying to a gel.
Proteins were transferred from SDS-PAGE to PVDF (Amersham) using the CAPS buffer
system (15% methanol, 0.01% SDS, 10mM CAPS) for 45 minutes at 180 mA. Membranes
were blocked (5% skim milk powder in TBS-T) overnight at 4oC. The primary antibody
(rabbit polyclonal anti-cyclophilin B, Abcam Ltd.) was applied (0.025µg/mL in TBS-T, 5%
skim milk powder) for one hour, room temperature with gentle agitation. The membrane was
washed extensively (TBS-T). The secondary antibody (goat anti-rabbit IgG conjugated to
HRP, Abcam Ltd.) was applied (diluted 1:50 000 in TBS-T, 5% skim milk powder) for one
hour at room temperature. The membrane was washed extensively (TBS-T). The signal was
detected with chemiluminescence (ECL plus kit, Amersham), according to manufacturer’s
24
Methods
instructions. Images were recorded and image analysis was performed using Kodak Digital
Science 1D, version 3.0.0.
Membranes were stripped (50oC, 30 minutes) with stripping buffer (2% SDS, 62.5mM Tris-
HCl, pH 6.8, 100mM β-mercaptoethanol), according to the method of Kaufman, Ewing &
Shaper (1987). Following stripping membranes were washed extensively (TBS-T), dried and
stored at 4oC until re-probing.
ALKALINE UREA GELS:
Gels for mammalian phospholipases A2:
The method used with these gels is a modification of Ahmad, Lawrence and Moores (1994).
The resolving gel contained acrylamide (12.5%), bis-acrylamide (0.5%), urea (8M) and
ethanolamine (2%) in ultrapure water. The gels were polymerised by the addition of
ammonium persulphate (0.005%) and TEMED (0.1%) under an iso-butanol overlay. The
gels were cast in mini gel format. The surface of the resolving gel was rinsed thoroughly
with water and then stacking gel solution. The stacking gel contained acrylamide (7%), bis-
acrylamide (0.3%), Tris (0.01M, pH 6.8) and urea (8M) in ultrapure water. The gels were
polymerised by the addition of ammonium persulphate (0.05%) and TEMED (0.2%). The
running buffer was dilute ethanolamine (2% in ultrapure water). The gels were pre-run at
200V for two hours to remove free radicals and prevent artifactual protein bands, after which
the buffer was changed. Wells were rinsed with running buffer before the application of
samples to remove the products of secondary polymerisation. Samples were applied to the
wells in sucrose (50%) or glycerol (50%) containing bromophenol blue as a tracking dye.
Typical lanes contained 4µg (purified proteins) to 20µg (crude venom) of total protein. Gels
were run at 200V in an ice bath until the tracking dye had migrated at least 7cm. The gels
were stained with the Neuhoff stain/Colloidal Coomassie (Neuhoff et al, 1988). Crude
venom samples were obtained from Sigma.
Blotting/ electrotransfer of proteins from alkaline urea gels to PVDF membrane:
The PVDF membrane (PALL BioTrace ™ PVDF (Pall, Ann Arbor, MI) or BioRad
SequiBLOT) was thoroughly wet with methanol before use but was not equilibrated in
transfer buffer. Proteins were transferred to PVDF membrane from alkaline urea gels with
25
Methods
the CAPS transfer buffer (10mM CAPS, pH 11.0 with 20M NaOH, 0.01% SDS, 10-15%
methanol) or the Towbin buffer (25mM Tris, 192mM glycine, 20% methanol, Towbin,
Staehelin & Gordon, 1970). CAPS transfer buffer is recommended for basic proteins
(Szewczyk & Kozloff, 1985). The transfer was performed at 100-180mA for 45 minutes
(CAPS) or 150mA for 60 minutes (Towbin) in a stirred blotting apparatus (Mini Trans Blot,
BioRad). After the transfer was complete the gel was stained in the Neuhoff stain (to check
transfer efficiency) and the membrane was washed in ultrapure water (5 minutes). The
membrane was then stained quickly (10 seconds) in Coomassie R-250 stain (0.1% w/v
Coomassie R-250, 40 % v/v methanol in deionised water) before rinsing the membrane with
methanol. If required the staining/methanol rinse was repeated until an appropriate level of
differential staining was achieved. Both the gel and the membrane were destained in
methanol solution (30% v/v methanol in ultrapure water).
Transfer of proteins of interest to SDS-PAGE:
Protein bands of interest from alkaline urea gels were excised with a clean scalpel. Bands
were more easily handled if frozen before transfer to SDS-PAGE. Gel pieces were placed in
wells of SDS-PAGE or Tricine gels and embedded in molten agarose (0.5% agarose, trace
bromophenol blue in appropriate running buffer), or reduced and then applied to the gels.
Gels were run according to the method of Laemmli (1970) for SDS-PAGE or Schagger & von
Jagow (1987) for Tricine gels.
Reducing method “A”: Gel pieces were placed in fresh microcentrifuge tubes
containing molten agarose (100µL, 0.5% agarose, trace bromophenol blue in SDS-PAGE
running buffer) containing DTT (1 part 0.5M DTT to 9 parts agarose). Tubes were then
heated (100oC, 10 minutes). Gel pieces and the agarose were then loaded into wells of the
SDS gels.
Reducing method “B”: According to the method of Davie, 1982. Briefly, gel pieces
were incubated (room temperature, 30 minutes) in equilibration buffer (10% glycerol, 5% β
mercaptoethanol, 2.3% SDS, 62.5mM Tris-HCL pH 6.8). The gel pieces were then placed in
the wells of the SDS gel and embedded in agarose (0.5%, agarose (Davie, 1982 reported the
use of 1% agarose) trace bromophenol blue in SDS running buffer).
26
Methods
Phospholipase A2 enzyme activity assay of gel pieces:
The gel was sliced into 1mm slices with a clean scalpel. Each piece was quickly transferred
to a microcentrifuge tube containing ethanolamine solution (100µL of 10mM ethanolamine in
ultrapure water), or Tris buffer (100µL, 50mM pH 6.8) or PBS (100µL) or ultrapure water
(100µL). Each piece was macerated with clean forceps. Aliquots (25µL) were removed
from each tube to analyse phospholipase A2 activity.
PHOSPHOLIPASE A2 ASSAYS:
Phospholipase A2 enzyme activity assay:
This method follows that published by Green and colleagues (1991).
Phosphatidylethanolamine L-1-palmitoyl, 2-arachidonyl, [arachidonyl-1-14C] (NEN), L-3-
Phosphatidyethanolamine 1-acyl-2-[1-14C arachidonyl] (specific activity 54 mCi/mmol)
(Amersham) or Phosphatidylcholine 1-stearoyl-2-[1-14C arachidonyl] (specific activity
55mCi/mmol) (Amersham) (5.5nmol) were dried under a stream of cold air or nitrogen. The
phospholipid was taken up in deoxycholate (2% w/v in ultrapure water). Assay buffer (25µL
per sample, 100mM Tris, pH 8.0, 300mM NaCl, 10mM CaCl2) was then added and this
mixture pre-warmed (37oC, 10 minutes). The sample solutions were also pre-warmed (37oC,
5 minutes). The substrate was then added to the samples (25µL per sample) and incubated
(37oC, 30 minutes). The reaction was terminated with EDTA (10µL per sample, 100mM).
The reaction solutions (each 30µL) were spotted onto TLC plates and dried with warm air.
The TLC plates were run in a chloroform/methanol/glacial acetic acid system (90:10:1 v/v/v).
Autoradiography was used to establish the position of the product (fatty acid) and then un-
reacted phospholipid. These regions were scraped from the plates and analysed by
scintillation counting (United technologies, Packard 2000CA Tricarb Liquid Scintillation
Analyzer).
When the pH profile was being examined the buffers used were: Tris (100mM) for pH 7-9,
Glycine (100mM) for pH 9.5-10 and sodium acetate (100mM) for pH 6-6.5 to ensure
adequate buffering capacity.
27
Methods
Samples were diluted as required to maintain the hydrolysis to less than 30%, which
represents the upper limit of linearity for this assay (Seilhamer et al, 1989). Where the
phospholipase activity of samples with very different quantities of total protein were
compared, results were expressed as the specific activity.
The addition of cyclosporin A to the assay:
Cyclosporin A (Sigma) was dissolved in ethanol (1mg/mL) to form the stock solution. The
stock solution was diluted in phospholipase A2 assay buffer (containing the phospholipid) to
the working concentration (2µg/mL). The cyclosporin A/assay buffer solution (25µL) was
then added to the enzyme source (25µL).
Phospholipase A2 and purified protein sources:
Pancreatic PLA2: Porcine, Sigma.
Bee venom PLA2: Sapphire Biosciences (Crow’s Nest, Sydney, Australia).
Haemoglobin: Human, Sigma.
Cytochrome C: Bovine heart, >99% pure, Sigma.
Bovine serum albumin: Fraction V, >96% pure, Sigma.
BOWEL TISSUE ANALYSIS:
Ethics approval for use of human bowel and plasma samples:
All experimental procedures were approved by the Deakin University (Ethics approval
number EC 116-2001) and Geelong Hospital (Ethics approval numbers 01/31 and 01/44)
ethics committees. Tissue was obtained from patients undergoing surgical removal of
infarcted bowel tissue or removal of normal bowel tissue from patients undergoing right
hemicolectomy. Acknowledgement must go to Geelong Hospital theatre staff for their help
in this regard.
Gross tissue damage rating system:
Damage to bowel tissue was assessed according to the scale proposed by Sun and colleagues
(1997).
28
Methods
Table 2.1: Gross tissue rating system of Sun and colleagues (1997):
Rating Features
1 Mild damage. Slight reddish discoloration.
2 Moderate damage. Red discoloration often with haemorrhage.
3 Severe damage. Grossly necrotic, blackish red, friable and lustreless.
ONE AND TWO DIMENSIONAL GEL ELECTROPHORESIS:
NuPAGE:
Bis-Tris gels (10%) were run with the MES buffer system according to manufacturer’s
instructions. SeeBlue®Plus2 (Invitrogen) prestained molecular weight markers and
Mark12™ wide range protein standards (Invitrogen) were used in these gels for protein size
estimation. Blotting from these gels was performed using the Mini Trans-Blot® apparatus
(BioRad) with a CAPS transfer buffer (10mM CAPS, pH 11, 15% methanol) at 180mA for 45
minutes. The PVDF membrane (ProBlot™, Applied Biosystems) was wet in methanol and
equilibrated in transfer buffer. The gel was rinsed briefly (Milli-Q) and also equilibrated in
transfer buffer. The gel was stained (0.25% Coomassie brilliant blue R-250 in 45%
methanol, 10% acetic acid). The membrane was stained quickly (60 seconds, 0.1%
Coomassie brilliant blue R-250 in 40% methanol, 1% aldehyde free acetic acid) before
quickly rinsing with methanol and soaking in ultrapure water.
Two-Dimensional Electrophoresis of heparin fraction samples:
Samples were desalted and concentrated by methanol or TCA precipitation. Samples
(100µL) were mixed with cold methanol (-20oC, 900µL) and stored overnight (-20oC).
Alternatively samples (100µL) were mixed with TCA (10mg) and left on ice (30 minutes).
The samples were then centrifuged (10 minutes, 4oC, 18000g). The pellets were rinsed with
cold methanol or acetone (TCA precipitation) before re-centrifugation and were then air dried
(room temperature, 30 minutes). 1st dimension rehydration buffer containing DTT (2.9mg
per mL) and biolytes (0.5%) was added (130µL per sample) and samples were vortexed
repeatedly and quickly centrifuged before placing in contact with IPG strips (pH 3-10, 7cm
strips). The proteins were focussed using a ramping current:
29
Methods
Stage Voltage (V) Time (hours)
1 50 12
2 500 1
3 1000 1
4 8000 3.5-8
TOTAL 18 500-26 000 volts
The IPG strips were then equilibrated firstly in DTT buffer (15 minutes) and then in IAA
buffer (15 minutes). The strips were then loaded onto NuPAGE® gels (4-12% BisTris,
1.0mm, MES running buffer) or Tris Glycine gels (4-20% Zoom™, SDS running buffer,
Invitrogen) along with Mark 12 ™Standards (strip loaded) and embedded in agarose (0.5%
agarose, trace bromophenol blue, in MES buffer). The gels were run according to
manufacturer’s instructions. The gels were then stained with either Coomassie R-250 (0.25%
CBB-R250 in 45% methanol, 10% acetic acid) or silver stained (PlusOne, Amersham
protocol).
Proteomics:
Samples of human plasma were thawed and added to chilled methanol (-80oC, 1.0mL).
Chilled methanol was added to a final volume of 2mL for each sample. Samples were
thoroughly vortexed and placed in a freezer overnight (-80oC). Samples were centrifuged in
a pre-cooled centrifuge (12 000g, -4oC, 60 minutes). The methanol supernatent was removed
and discarded. The pellet was resuspended in solubilisation solution containing 1% carrier
ampholytes (pH 3-10), (230µL solubilisation solution per 100µL original volume of plasma).
The mixture was repeatedly vortexed and sonicated until the pellet had been dissolved. If
required the samples were also subjected to bead beating (4 X 15 seconds, Bio 101 Fast
Protein™ blue tubes). Immobilised pH gradient strips (IPG), (pH 7-10, 7cm, BioRad) were
re-hydrated overnight in solubilisation solution containing 1% carrier ampholytes (pH 3-10,
200µL per strip). Samples were centrifuged quickly before cup loading onto the IPG strips.
The IPG strips were subjected to isoelectric focussing with a ramping current:
30
Methods
Stage Votage (kV) Time (hours)
1 0.1 5
2 0.3 3-7
3 0.6 2-3
4 1 1-3
5 2 1-3
6 3-3.5 9-12
At the completion of isoelectric focussing the IPG strips were equilibrated in equilibration
solution (20 minutes with agitation). The IPG strips were then embedded in 0.5% agarose in
cathode buffer containing bromophenol blue (0.001%) on the top of either pre-cast Criterion
gels (4-20%, Tris-HCl) or freshly prepared large format gradient gels (18x20cmx1.5mm, 8-
18% acrylamide). The second dimension was performed using the following programme for
Criterion gels:
Stage Current (mA) Time (hours)
1 3 per gel 0.5
2 30 per gel 2
The second dimension was performed using the following programme for large format gels:
Stage Current (mA) Time (hours)
1 3 per gel 2
2 50 overnight
The programme was continued until the dye front had begun to run off the gel. Gels were
then fixed for 30 minutes (10% methanol, 7% glacial acetic acid in ultrapure water). Gels
were then stained with Sypro Ruby Protein gel Stain (BioRad) for the required length of time.
Gels were destained (10% methanol, 7% glacial acetic acid in ultrapure water) for 30 minutes.
Gel images were captured with BioRad Molecular Imager® FX and BioRad Quantity One
version 4.1.1 software. Image analysis was performed using Melanie version 3. Gels
previously stained with Sypro Ruby were re-stained with colloidal Coomassie G-250
overnight. Coomassie stained gels were scanned with the Molecular Dynamics personal
31
Methods
densitometer SI and Corel Photo Paint version 8. Protein differences were assessed using
Melanie version 3.
PROTEIN IDENTIFICATION:
Montage™ In-Gel Digest96 Kit (Millipore):
Gel spots were cut and placed in the MultiScreen plate (Millipore, Bedford, MA). Destaining
solution (100µL) was added to each well and incubated (room temperature, 20 minutes). The
solution was removed under vacuum. This process was repeated twice. Acetonitrile
(100µL) was then added to each well and incubated (room temperature, 10 minutes). The
solvent was then removed under vacuum. The samples were reduced with DTT (100µL,
10mM in 0.2M ammonium bicarbonate) and incubated (room temperature for 60 minutes or
37oC for 30 minutes). The solution was removed under vacuum. The gel pieces were
washed using three changes of ammonium bicarbonate (100µL, 0.2M) followed by
acetonitrile (100µL) with removal of solutions under vacuum. The proteins were then
alkylated (100µL of 50mM IAA in 0.2M ammonium bicarbonate) for 20 minutes at room
temperature. The solution was then removed under vacuum. The gel pieces were washed
using three changes of ammonium bicarbonate (100µL of 0.2M) followed by acetonitrile
(100µL) with removal of solutions under vacuum. Trypsin solution (50µL diluted to
approximately 1/10 the mass of protein in the spot in 0.1M ammonium bicarbonate) was then
added to each well and the plate was incubated overnight (37oC). The peptides were then
extracted by adding extraction solution (50µL per well) and incubating (room temperature, 30
minutes) before collecting the solution in a microtitre plate. Extra extraction solution (50µL)
was added to each well, incubated (room temperature, 30 minutes) and then collected. This
process was repeated. The peptide solutions were then concentrated (SpeedVac, Savant).
Nano MS/MS:
Samples for nano MS/MS were run in 4-12% TrisGly gels (Invitrogen), according to
manufacturer’s instructions. Proteins were thoroughly reduced with DTT containing sample
buffer and heating (100oC, 7 minutes). After heating the samples were allowed to cool
before the addition of acrylamide (final concentration of 1%) to form cysteine adducts to
prevent reformation of disulphide bonds. Potassium ferricyanide (30mM) and sodium
thiosulphate (100mM) were mixed in equal proportions. Excised bands from Tris-Glycine
32
Methods
gels were incubated in the ferricyanide/thiosulphate solution (50µL per tube) until the brown
colour of the silver stain had been removed. The solution was removed and the gel pieces
were washed with two changes of water. The gel pieces were then incubated in ammonium
hydrogen carbonate (100µL, 50mM, room temperature, 20 minutes). The gel bands were cut
into small pieces, washed with water and transferred to new tubes. Acetonitrile was used to
rinse and shrink the gel pieces (3x100µL, 1x500µL). The gel pieces were dried (SpeedVac,
Savant). Trypsin (20µL, 0.1µg/mL in 1mM HCl, Promega, Madison, WI) was added,
followed by digestion buffer (100µL, 50mM NH4CO3, 1mM CaCl2, pH 7.8), and incubated
overnight at 37oC.
Extraction of peptides :
Ammonium bicarbonate (100-150µL, 25mM) was added to each tube, centrifuged and then
incubated, (37oC, with sonication). Acetonitrile (150µL) was added to each tube, vortexed
and incubated. Formic acid (50µL, 5%) was added, vortexed and incubated. Acetonitrile
(150µL) was added to each tube, vortexed and incubated. The pooled extracts were then
dried to approximately 10µL (SpeedVac, Savant).
The peptide solutions were desalted using miniature C18 columns constructed in gel loading
pipette tips (Eppendorf). The columns were prepared using C18 resin (Vydac) in
acetonitrile/water. The columns were rinsed extensively with methanol, then formic acid
(5%). The peptides were bound to the column, and then washed with formic acid (5%). The
peptides were eluted into the sample applicator with methanol.
The mass spectrometer used was the API Q-STAR Pulsar i, LC/MS/MS (Applied
Biosystems). MS/MS spectra were submitted to Mascot and SwissProt. Acknowledgment
must go to Dr. Peter Hoffman (St. Vincent’s Medical Research Institute) for assistance with
sample preparation and mass spectrometry analysis.
MALDI-TOF MS:
Protein spots of interest were excised from the gels and placed in microtitre plates with care
taken to avoid contamination between other spots and outside sources. Wash buffer (120µL
per gel piece, 50% acetonitrile, 25mM NH4CO3, pH 7.8) was added to each well. The plate
was incubated with shaking (37oC, 10 minutes). The wash solution was removed and
33
Methods
discarded. This washing process was repeated three times, after which there was no colour
remaining in the gel pieces. The gel pieces were then dried under vacuum for 15 minutes.
Sequencing grade trypsin (8µL, 15ng/mL porcine trypsin, (Promega) in NH4CO3, pH 7.8) was
added to each gel piece. The plate was then sealed and incubated overnight with agitation
(37oC). The plate was spun (SpeedVac Plus, Savant, 5 minutes). Extract solution (8µL,
50% acetonitrile, 1% TFA) was added to each well and sonicated for 20 minutes.
An aliquot of each sample (1.5µL) was spotted onto a MALDI plate. Matrix (1.0µL, α-
cyano-4-hydroxycinnamic acid, 8mg/mL in 50 % acetonitrile, 1% TFA) was added to each
sample spot on the MALDI plate. Samples were air dried and analysed. Acknowledgment
must go to Dr. Stewart Cordwell (Australian Proteome Analysis Facility) for analysis of
samples by MALDI-TOF.
Bioinformatics:
The protein sequences of the ‘theoretical proteins’ identified by MALDI were analysed using
software from the National Centre for Biotechnology Information (BLASTN, BLASTP) and
the EXPASy Molecular Biology Server of the Swiss Institute of Bioinformatics.
Nano LC-MS/MS:
Protein bands of interest were excised from BisTris (Invitrogen) and TrisGly (Gradipore) gels
with appropriate positive (protein markers) and negative (gel pieces from no protein regions)
controls. The protein bands were destained and digested with trypsin. The proteins were
analysed by nano LC-MS/MS. Acknowledgement must go to Dr. Mark Raftery, Biomedical
Mass Spectrometry Facility, University of New South Wales for the protein digestion and
mass spectrometry analysis.
Amino terminal amino acid sequencing;
Rotofor fractions were applied to a ProSorb sample preparation cartridge (Applied
Biosystems, Foster City). The cartridge membrane was washed several times with TFA
solution (0.1%), changing the filtration absorbance filter after each 750µL load.
Alternatively the samples were applied via PVDF membrane:
34
Methods
Protein bands of interest were excised from PVDF membranes and subjected to automated
amino acid sequencing (Edman degradation) using the Procise automated sequentor (Applied
Biosystems, Foster City, California). The phenylthiodantoin (PTH) derivatised amino acids
were analysed to determine the amino acid sequence. The Procise PTH system is comprised
of the Micro-gradient delivery system (model 140C), a UV detector (model 785) and data
analysis software (model 610A). Acknowledgement must go to Gary Beddome and Brian
Shiell (Australian Animal Health Laboratories, CSIRO) for amino acid sequencing.
PROTEIN PURIFICATION:
Ammonium sulphate precipitation:
The appropriate amount of ammonium sulphate to reach saturations of 15 to 80% (ACS
reagent grade) was added to samples and agitated by rotating (5 minutes). The samples were
then centrifuged (7500g, 4oC, 10 minutes) and supernatent removed. The precipitate was
resuspended in buffer or water to the original sample volume. The supernatent was diluted as
required to attain original sample volume. The supernatent and precipitate at each saturation
level were analysed for total protein (Bradford assay) and phospholipase A2 activity
(phosphatidylcholine hydrolysis).
Methanol precipitation:
Methanol (-80oC) was added to samples to equivalent or greater volume and mixed
thoroughly. Samples were then stored in freezer overnight (-80oC). Samples were then
centrifuged (12000g, 0oC, 60 minutes). The extended storage followed by extended
centrifugation at low temperature was essential for pellet formation. The supernatent was
removed and discarded. The pellet was then air dried for a maximum of 5 minutes. The
pellet was resuspended in appropriate buffer or electrophoresis sample buffer.
Homogenising bowel tissue:
The mesentery was removed from bowel tissue. Bowel tissue was roughly chopped with a
scalpel. Tissue was weighed and placed in 5vol/g buffer (10mM Tris, pH 7.4). Homogenate
was clarified by centrifugation (2000g, 4oC, 10 minutes ).
35
Methods
Heparin Sepharose Affinity Chromatography;
Heparin Sepharose columns (Amersham Pharmacia Biotech, HiTrap columns) were
equilibrated with binding buffer (10 volumes, 10mM Tris-HCl, pH 7.4, flow rate of
approximately 1mL/minute). Samples were centrifuged (7500g, 5 minutes) before being
loaded onto pre-equilibrated column. Non-bound proteins were eluted with buffer (10 to 25
column volumes, 10mM Tris, pH 7.4) until eluent was clear and contained no protein (by
detection of absorbance at 280nm). The bound fractions were then eluted with increasing
concentrations of NaCl (0-5M NaCl, 10mM Tris, pH 7.4) in stepwise fashion either using a
single concentrated salt solution or a series of increasingly concentrated salt solutions.
Fractions were collected at regular time intervals (typically 30 seconds). Fractions were
analysed for total protein, phospholipase A2 activity and protein sizes (Tricine PAGE or SDS-
PAGE).
Preparative Isoelectric focusing using the BioRad Rotofor®:
Heparin column fractions with PLA2 activity eluted under conditions of 0.5M NaCl were
pooled. Glycerol and n-octyl glucoside (NOGS) were added to the sample to final
concentrations of 1% and 0.5% respectively. Biolytes (pH 3-10, BioRad, 1.5mL) were added
to the sample (total volume 40 mL). The Rotofor was pre-run with ultrapure water (15W
constant power, 5 minutes) to clear any contaminants from the apparatus. The Rotofor was
set to run at constant power (15watts, 4oC). The current was monitored throughout the run
and the experiment was stopped when the current had reached a plateau (approximately 3.5
hours). The fractions were then harvested and analysed for PLA2 activity. The pH of each
fraction was determined. The fractions were also run on a gel (NuPAGE Bis-Tris 4-12% gel
with Mark 12™ protein standards). After completion of the gel run the gel was fixed (30%
methanol, 10% TCA) with sulfosalicylic acid (3.5%) for 1 hour to remove ampholytes. The
gel was fixed for a further 2 hours (30% methanol, 12% TCA) to remove excess sulfosalicylic
acid. These gels were then silver stained using the PlusOne protocol.
Active Rotofor fractions of pH 8.98 to 9.8 were pooled and NaCl was added to a
concentration of 1M to electrostatically remove ampholytes. This solution was mixed and
incubated (45 minutes, 4oC). This solution was dialysed against 0.5X PBS (24 hours, 4oC)
using Slide-A-Lyzer® cassettes (Pierce) with a 3,500MWCO.
36
Methods
Electroelution:
The Mini Whole Gel Eluter apparatus (BioRad) was used to electroelute protein from SDS
gels. The gels were run under non-reducing conditions. The gel was equilibrated in elution
buffer (20mM CAPS pH 11, 15 minutes with three changes of buffer). The elution was
performed (20 minutes, 100mA), and at completion the electrodes were briefly reversed. The
fractions were collected and analysed for PLA2 activity. Pre-stained molecular weight
standards (See Blue®, Invitrogen) were used to construct a standard curve of mobility and
protein size.
Vivaspin concentration and desalting:
Vivaspin centrifugal filter devices (PES membrane, 5 000 MWCO, 500µL capacity,
Sartorius) were used to concentrate protein samples and exchange buffers. Samples were
applied to the upper chamber and centrifuged (10 000g, 10 minutes, 4oC). PBS (500µL) was
added and centrifuged (10 000g, 10 minutes, 4oC). The washing process was then repeated.
If ampholytes needed to be removed NaCl was added to the sample to a final concentration of
1M to electrostatically strip protein bound ampholytes (BioRad Rotofor application guide).
TOTAL PROTEIN ASSAYS:
BioRad DC Protein High Range Assay:
Protein assays were performed according to manufacturer’s instructions. Briefly, a standard
curve was constructed using BSA diluted in PBS in the range 0-2mg/mL. Samples were
diluted 1:50, 1:100, 1:200 or 1:300 in PBS. Samples and standards (5µL per well) were
assayed in triplicate. The absorbance was read at 750nm.
Bradford Total Protein Assay:
The protein assay of Bradford (1976) was used routinely. Standard curve composed of BSA
in PBS in the range 1-10 µg per 100µL. Samples diluted in PBS until the absorbance of the
solution is less than the absorbance of the highest standard. Protein reagent was added to
samples and standard in the ratio of 10 parts reagent to 1 part sample or standard. The
solutions were mixed thoroughly and the absorbance read at 595nm within 60 minutes of
addition of protein reagent.
37
Methods
Pierce BCA Protein Assay:
The BCA Protein Assay was used for samples containing carrier ampholytes. The Pierce
BCA Protein Assay Kit standard protocol was followed using a sample to working reagent
ratio of 1:20. A standard curve was constructed using BSA in the range 0-2000µg/mL.
Samples were prepared using a method to remove carrier ampholytes (Brown, Jarvis and
Hyland, 1989). Briefly, samples containing carrier ampholytes (100µL) were diluted in
ultrapure water (900µL). Deoxycholate solution (100µL, 0.15%) was added and solutions
were incubated (room temperature, 10 minutes). TCA solution was then added (100µL of
72% solution) and samples vortexed, then centrifuged (2000g, room temperature, 15
minutes). Supernatent was discarded and pellet immediately resuspended in detergent
solution (5% SDS in 0.1M NaOH). The BCA working reagent was immediately added
(2mL). Absorbance was read at 562nm.
38
Methods
RECIPES:
Bradford Protein reagent:
Method according to Bradford, 1976.
Coomassie Brilliant Blue G-250 final concentration 0.01%
Ethanol final concentration 4.7%
Phosphoric acid final concentration 8.5%
In ultrapure water.
Colloidal Coomassie/Neuhoff reagent:
Ammonium sulphate (17% w/v)
Phosphoric acid (4.2% v/v)
Methanol (34% v/v)
Coomassie G-250 (0.1% w/v)
in ultrapure water.
Solubilisation solution:
5M urea
2M thiourea
2mM tributylphosphine
2% CHAPS
2% sulfobetaine 3-10
0.2% carrier ampholyes
40mM Tris
0.002% bromophenol blue
1% ASB14 (BioRad)
20mM DTT
0.2% carrier ampholytes pH 3-10
Proteomics: Equilibration solution:
2.5% acrylamide
5mM tributylphosphine
20% glycerol
6M urea
39
Methods
2% SDS
0.375M Tris-HCl pH 8.8
Tris Glycine SDS running buffer:
29g Tris base
144g Glycine
10g SDS
to 1L ultrapure water.
Tris Glycine sample buffer:
2.5mL 0.5M Tris HCl pH 6.8
2mL glycerol
4mL 10% SDS
0.5mL 0.1% bromophenol blue
ultrapure water to 10mL
Two-dimensional electrophoresis equilibration buffer:
50mM Tris pH 8.8
6M urea
30% glycerol
2% SDS
bromophenol blue
2mL per strip, containing either 10mg/mL DTT or 25mg/mL IAA.
ELISA wash buffer:
Amounts for 1Litre of buffer:
NaCl (8g)
KCl (0.2g)
NaH2PO4.H2O (0.2g)
Na2HPO4 (1.96g)
Tween 20 (0.5mL)
BSA (10g) added directly before use
40
Methods
Carbonate buffer:
Amount for 500mL, pH 9.8.
Na2CO3 (1.1g)
NaHCO3 (1.5g)
MgCl2 (0.2g)
Phosphate buffered saline:
Amounts for 1L, pH 7.4.
Na2HPO4 (1.44g)
NaH2PO4 (0.24g)
NaCl (8g)
KCl (0.2g)
NuPAGE 4X LDS sample buffer:
Glycerol 40%
Tris base 6.82% w/v
Tris HCl 6.66% w/v
LDS 8% w/v
EDTA 0.06% w/v
Serva Blue G-250 0.075% solution
Phenol red 0.025% solution
In ultrapure water
NuPAGE 20X MES running buffer:
MES 1M
Tris Base 1M
SDS 69.3mM
EDTA 20.5mM
In ultrapure water.
41
Chapter 3 Introduction
Chapter 3: Plasma and Proteomics.
Global techniques:
Global techniques aim to detect all of the expression products (RNA or protein) in a
particular sample in a non-targeted, single experiment. Global analysis may be
undertaken to identify drug targets, diagnostic markers, markers of infection, markers
of developmental stages, or organism specific markers.
Nucleic acid based examples of global analysis include differential display RT-PCR,
serial analysis of gene expression (SAGE) and cDNA micro-arrays. Protein based
examples of global analysis include surface enhanced laser desorption ionisation
(SELDI) protein chips, multi-dimensional HPLC, antibody arrays and two-
dimensional electrophoresis. There are several advantages and disadvantages
associated with either nucleic acid and protein based global techniques, as
summarised in the table below.
NUCLEIC ACID BASED GLOBAL TECHNIQUES:
Table 3.1: The advantages and disadvantages of nucleic acid based global
techniques:
Advantages: Disadvantages:
Less material required (~5X103cells)
(Seliger & Kellner, 2002).
No detection of isoforms is possible
(Seliger & Kellner, 2002).
Amplification steps are possible (Seliger
& Kellner, 2002).
Alternate gene splicing adds complexity
(Banks et al, 2000, Wilkins et al, 1995).
Only potential functional state is
indicated (Griffin & Aebersold, 2001).
42
Chapter 3 Introduction
PROTEIN BASED GLOBAL TECHNIQUES:
Table 3.2: The advantages and disadvantages of protein based global techniques:
Advantages: Disadvantages:
Drug targets generally work on proteins
(Banks et al, 2000).
Diversity in protein size, pI and
solubility.
Post translational modifications can be
investigated, these are not apparent from
nucleic acid information (Krishna &
Wold, 1993).
Dynamic range varies over 6 orders of
magnitude (Corthals & Nelson, 2001)
May provide information about function
(Griffin & Aebersold, 2001, Seliger &
Kellner, 2002).
More material is required (~5×106 cells)
Abundance and sub-cellular location are
identified (Griffin & Aebersold, 2001).
No amplification step is available.
Proteomics:
Proteomics describes the study of the ‘entire protein complement of the genome’.
Unlike the genome, the proteome is not a static entity but changes according to:
- tissue or cellular source or cell culture conditions,
- stage of development of the organism or cell,
- pathophysiological state.
Proteomics is a relatively new field, but is becoming increasingly important to
biologists with the completion of the Human Genome project and similar milestones
involving other organisms. It is believed that approximately one third of the gene
sequences in genome databases encode for proteins of yet unknown function (Banks
et al, 2000).
It is proposed that the number of genes in the human genome is in the order of 20 000
to 25 000 (Stein, 2004) and that at any one time, up to five thousand of these genes
are being transcribed. The number of genes provides an underestimate of the number
of proteins due to:
43
Chapter 3 Introduction
- post translational modifications (Herbert & Righetti, 2000), of which 200
have been described (Krishna & Wold, 1993),
- alternate gene splicing (Wilkins et al, 1995).
Preliminary studies suggest that the number of protein forms per gene ranges from
one to two in bacteria to three to six per gene in humans (Banks et al, 2000), resulting
in up to one million proteins (Herbert & Righetti, 2000). Another group has
proposed that up to 10 protein variants exist per locus [Traini, Herbert, Wilkins &
Williams, unpublished as cited by (Gabor Miklos & Maleszka, 2001)].
Proteomics involves:
- preparation of the sample to extract the maximum number of proteins in the
maximum yield,
- separation of the proteins, or their peptides,
- visualisation and differential analysis,
- identification of the proteins.
Proteomics has traditionally been associated with two dimensional electrophoresis as
the technique for separation.
Two dimensional electrophoresis involves the separation of the proteins firstly on the
basis of the isoelectric point (pI) and then on the basis of molecular weight. The first
dimension of two dimensional electrophoresis involves isoelectric focusing (IEF)
which separates proteins according to their isoelectric point. In the past this step has
been achieved using carrier ampholytes embedded in polyacrylamide filled tubes to
create the pH gradient. Immobilised pH gradient (IPG) strips have largely replaced
carrier ampholyte based IEF, as they have several advantageous features. IPG strips
are composed of a thin layer of polyacrylamide on a supporting plastic film. The pH
gradient is covalently coupled to the acrylamide. IPG strips can tolerate higher
protein loads, have stable and precisely determined pH gradients and are not
susceptible to distortion (which could occur with ampholyte based IEF gels). IPG
strips are also very accurate and commercially available which limits interlaboratory
reproducibility problems.
The second dimension involves polyacrylamide gel electrophoresis in the presence of
sodium dodecyl sulphate (SDS), which separates the isoelectrically focused proteins
44
Chapter 3 Introduction
by molecular weight. The gels used for the second dimension can be gradient gels or
gels of a single percentage of acrylamide, depending on the application. It has been
reported that two dimensional gels are able to resolve 3000 proteins on a single gel
(Wilkins et al, 1996). Although there are several disadvantages of 2DE based
proteomics it has remained the leading technique because of its ability to:
a) ‘visualise a very large number of proteins simultaneously’ (Haynes &
Yates, 2000) and
b) be used in a ‘differential display format’ (Haynes & Yates, 2000).
The deficiencies of two dimensional electrophoresis based Proteomics:
It is accepted that two dimensional gels are deficient in the resolution of several
classes of proteins including those with:
- extreme pI (<4 and >10) (Gygi & Aebersold, 2000, Gygi, Rist & Aebersold,
2000, Washburn, Wolters & Yates, 2000).
- extreme molecular weight (Gygi & Aebersold, 2000, Gygi, Rist &
Aebersold, 2000, Washburn, Wolters & Yates, 2000), for example proteins
>100kDa are under-represented and those >150kDa are usually not separated
in the 2nd dimension (Corthals et al, 1999), those <10kDa are unable to be
detected (Seliger & Kellner, 2002).
- low codon bias*/low abundance and therefore unable to be detected (Gygi &
Aebersold, 2000, Simpson et al, 2000, Washburn, Wolters & Yates, 2000),
- membrane associations (Gygi, Rist & Aebersold, 2000, Han et al, 2001,
Simpson et al, 2000)
- a high degree of hydrophobicity (Gygi, Rist & Aebersold, 2000, Han et al,
2001, Simpson et al, 2000)
* Codon bias is the tendency for a given gene to preferentially use one of several potential codons to incorporate a specific amino acid into a protein (Liebler, 2002). Highly expressed proteins have high codon bias value, conversely low abundance proteins have low codon bias values. Low abundance is defined by a codon bias of <0.1.
45
Chapter 3 Introduction
Various modifications have been proposed to address the limitations of two
dimensional gel based proteomics including:
- pre-fractionation of sample by size, solubility or isoelectric point,
- using a series of gels of restricted pH gradients (subproteomics),
- improved solubilization and extraction procedures.
Other approaches have sought alternatives for two dimensional electrophoretic gels in
the separation step. Technologies that have emerged involve the use of one or more
of the following techniques:
- multidimensional chromatography, or MudPit ,*
- ion exchange chromatography,
- size exclusion chromatography,
- capillary electrophoresis,
- fragmentation of proteins into peptides before separation,
- the isotope coded affinity tag peptide labelling (ICAT) approach.
Work has been completed to assess how much of a proteome is likely to be seen in
2DE based proteomics, using yeast as a model. Yeast is a relatively simple organism
in terms of its genome and proteome given that it contains only 6 000 open reading
frames and simplified post translational modifications (Liebler, 2002). Yeast
proteins vary over 5 orders of magnitude (Gygi et al, 2000), and an estimated 80% of
the proteome is composed of low abundance proteins (Dr. Ben Herbert, personal
communication). Almost 90% of the yeast proteome can be solubilised with existing
technology (Pedersen et al, 2003) but half of the proteins are too low in abundance to
be detected (Griffin, Goodlet & Aebersold, 2001). The analysis of low abundance
yeast proteins can be improved with pre-fractionation (Gygi et al, 2000).
* The multidimensional protein identification technology or MudPIT approach involves the digestion of all of the proteins in a sample, and then separation of the resulting peptides by strong cation exchange chromatography and reverse phase chromatography. The peptides are then submitted to tandem mass spectrometry and database searching (Link et al, 1999, Washburn, Wolters & Yates, 2001). The advantage of this approach is that the peptides generated have more uniform characteristics than the proteins from which they were derived, in terms of size, pI and hydrophobicity (Washburn, Wolters & Yates, 2001).
46
Chapter 3 Introduction
When the maximum amount of whole cell lysate was run in a 2D gel the average spot
detected represented 51 000 copies per cell (Gygi et al, 2000). When the sample was
pre-fractionated, proteins present at 1 000 copies per cell could be detected (Gygi et
al, 2000).
To allow the detection of low abundance proteins the relative concentrations must
obviously be increased (Simpson et al, 2000). Theoretically two approaches can be
used:
- increase the sample load (which can be impractical due to sample load
limitations of 2DE gels),
- increase the concentration of the low abundance proteins (which alters the
composition of the sample),
- fractionate the sample and analyse each fraction separately (pre-fractionation
or sub-proteomics).
If the sample composition is altered to allow the detection of low abundance proteins
the final sample will not
1. reflect the proteome
2. remain quantitative
Sample alterations may involve:
- the removal of high abundance proteins such as albumin,
- pre fractionation.
Proteomics in medicine:
There is significant potential for proteomic research in the biomedical field.
Proteomics can identify disease specific proteins which could then be used as
diagnostic markers, markers for the monitoring of disease progression, therapy
effectiveness, toxicity or drug targeting. Proteomics has several advantages over
genomic based research in the biomedical field. For example:
- examination of the genome cannot provide information on multi-gene
processes such as aging, stress or many diseases,
- most drugs target proteins rather than genes,
47
Chapter 3 Introduction
- post translational modifications may be critically important in disease and
these modifications cannot be predicted from the genetic sequence (Banks et
al, 2000).
The vast potential of proteomics in biomedical research is yet to be realised (Banks et
al, 2000). This is thought to be due to a lack of awareness of the advances that have
been made in the field (Banks et al, 2000) and the concept that the technique is
unrefined. Proteomic investigations of pathophysiological conditions have generally
focussed on a variety of cancers. Cancer proteome databases have been constructed
for a hepatocellular carcinoma cell line (Liang et al, 2002) and a colon cancer cell line
(Simpson et al, 2000). Human proteome maps also exist for plasma, urine,
cerebrospinal fluid, breast tissue and heart tissue (Banks et al, 2000). Proteomics has
allowed the identification of:
- several protein variants associated with the development of liver cancer
(Zeindl-Eberhart et al, 1994),
- proteins differentially expressed between non tumorigenic and metastatic
prostate epithelial cells (Griffin, Goodlet & Aebersold, 2001),
-several proteins that are differentially expressed between benign prostate
tissue and prostate carcinoma (Alaiya et al, 2001),
-a number of protein alterations in a neuropsychiatric disease (Merril &
Goldman, 1982),
- a number of novel serum markers in patients with lung cancer (Hanash,
Brichory & Beer, 2001),
- microheterogeneity of proteins (perhaps glycoproteins) in pancreatic juice of
patients with pancreatic cancer and pancreatitis (Scheele, 1982),
- a putative urinary marker of bladder squamous cell carcinoma (Celis &
Gromov, 1999).
The importance of low abundance proteins in medicine:
A large section of the proteome is composed of low abundance proteins (Gygi et al,
2000). The use of proteomics to study disease raises the issue of low abundance
proteins for a number of reasons:
- many important classes of proteins, for example transcriptional control
proteins are present in low abundance (Hoffman et al, 2001),
48
Chapter 3 Introduction
- many disease associated proteins are expected to be present in low copy
number (Griffin, Goodlet & Aebersold, 2001),
- the relative changes in the amount of a particular protein between a normal
and disease state are expected to be quite low (Gabor Miklos & Maleszka,
2001),
- micro-dissection of tissue and tumours is often used to obtain pure cell
populations, resulting in prohibitively small samples, in which low abundance
proteins cannot be identified (Adam et al, 2001).
Plasma Proteomics:
In this review, the focus will be on the plasma proteome rather than the serum
proteome, for simplicity and to include the largest range of proteins of the soluble
component of blood (Anderson & Anderson, 2002). An assessment of the
advantages and disadvantages of investigating proteins either in serum or plasma is
currently a priority of HUPO (the worldwide Human Proteome Organisation).
Plasma is the primary clinical specimen because it is one of the most easily obtained
and reflects many physiological and pathophysiological processes. The plasma
proteome can be regarded as the ‘largest and deepest’ version of the human proteome,
because plasma contains not only classical plasma proteins, but leakage proteins from
all tissues in addition to immunoglobulins (Anderson & Anderson, 2002). Plasma is
also one of the most difficult proteomes to work with, because of the number of
protein present, the number of protein variants (degradation products and post
translationally modified proteins) and the large dynamic range. It is common for
small number of proteins (albumin, α1-antitrypsin, α2 macroglobulin, transferrin, γ
globulins) to represent over 80% of the total protein in plasma (Georgiou, Rice &
Baker, 2001). The abundance of plasma proteins varies over ten orders of magnitude
(Anderson & Anderson, 2002). It is difficult to analyse a large proportion of the
plasma proteome using any single method. For example: two dimensional gel
electrophoresis based proteomics is likely to detect only the high abundance and/or
long lived proteins of a sample (Haynes & Yates, 2000), at the expense of the short
lived or low abundance proteins.
49
Chapter 3 Introduction
Detectable in 2D gels Likely to be not detectable in 2D gels
↓C-reactive protein
↑Haptoglobin
↑Serum amyloid A
↓PLA2
↑Haemoglobin
Figure 3.1:
The Plasma Proteome. Modified from Anderson N.L. & Anderson N.G. The
Human Plasma Proteome: History, Character and Diagnostic Prospects.
Molecular and Cellular Proteomics 2002; 1 (11): 845-867, with permission from
the author.
Plasma proteins encompass proteins with function within the plasma (haemoglobin,
albumin), proteins that have been released from tissues, immunoglobulins and
cytokines. Tissue leakage proteins are those proteins that are generally retained
within a particular tissue but are released into plasma during tissue damage because of
cell death or injury (Anderson & Anderson, 2002). In this way, cellular factors
including proteins can be used as markers of tissue damage in plasma (Noe, 2001).
An ideal marker of tissue damage would be present only in the tissue or organ of
interest (Noe, 2001). A number of highly tissue specific proteins have been
identified, such as pancreatic α amylase and cardiac muscle creatine kinase MB (Noe,
2001). The concentration of tissue leakage proteins in plasma varies greatly
according to the amount present in the tissue, the distribution of the protein within the
tissue, the mode of release into circulation and the clearance or catabolism of the
protein from circulation. A highly abundant protein in a particular tissue can become
a very attractive marker, as even moderately elevated levels in plasma can reflect
small amounts of tissue damage (Anderson & Anderson, 2002). For example,
cardiac myoglobin is released during myocardial infarction. A myocardial infarction
50
Chapter 3 Introduction
affecting less than 3g of cardiac tissue (a typical moderate infarct affects 45g) would
release cardiac myoglobin into plasma to a substantial concentration of 3µg/mL
(Anderson & Anderson, 2002).
Tissue damage proteins may be released from tissues during injury due to:
- leakage of cytoplasmic proteins,
- heat shock response,
- inflammatory response,
- signalling,
- protective mechanisms.
Plasma proteomics offers a method of evaluating protein differences between patients
with a disease of interest and control patients. If a suitable tissue damage marker can
be identified by a proteomic investigation, this becomes a significant result because
the test sample (plasma) is the same in a clinical situation. Obtaining plasma is a
minimally invasive procedure and samples would be routinely taken from patients for
a multitude of other tests, during diagnosis and treatment.
Serum Amyloid A, an important and abundant disease associated protein.
Serum amyloid A (SAA) is a protein family comprised of several isoforms, that have
high homology (50-95%) and similar size (see Figure 3.11, for sequence alignment of
human serum amyloid A variants). The SAA family can be divided into two groups-
the acute phase SAA isoforms (A-SAA) and the constituent isoforms (C-SAA). The
acute phase SAA are highly conserved and have been found in all vertebrates studied
(Uhlar & Whitehead, 1999). As the name suggests, the acute phase SAA are
involved in the acute phase response, where the concentration in blood can increase
up to 1000 fold (Uhlar & Whitehead, 1999). Acute phase SAA isoforms are among
the most responsive acute phase proteins (Ensenauer et al, 1994), with mRNA
detectable within 2 hours of inflammation (Pruzanski et al, 1995). Plasma levels of
acute phase SAA can increase from baseline levels (2-5µg/mL (Malle et al, 1997)) to
1mg/mL within 20 hours (de Beer et al, 1994). SAA is also one of the most rapidly
cleared acute phase proteins, with a half life of only 90 minutes under both normal
and inflammatory states (Husby et al, 1994). The acute phase SAA are the
51
Chapter 3 Introduction
precursors of the amyloid fibril protein A found in secondary or reactive amyloidosis
(Husby et al, 1994). Amyloidosis refers to a group of diseases characterised by
deposition of amyloid in a variety of tissues and organs (Husby et al, 1994), most
commonly the spleen, liver and kidney (Buxbaum & Tagoe, 2000). Constitutive
SAA (C-SAA) is maintained at a relatively constant concentration in blood at
approximately 50µg/mL (Yamada et al, 2001). C-SAA is only minimally inducible
during the acute phase response, for example, during renal allograft transplantation,
C-SAA only rises approximately 3 fold (Yamada et al, 2001). Constitutive SAA has
only been found in humans and mice (Uhlar & Whitehead, 1999). The relevance of
C-SAA is unknown (Husby et al, 1994). Unlike the acute phase SAA, the C-SAA
gene lacks IL-1 and IL-6 responsive elements (Watson, Coade & Woo, 1992).
In humans one isoform of SAA is predominant (SAA1), and this isoform comprises
95% of the total SAA (Malle et al, 1997). In humans four SAA genes have been
detected, SAA1 (five alleles), SAA2 (the acute phase isoforms, two alleles), SAA3 (a
pseudogene) and SAA4 (the constitutive isoform, one allele). SAA3 is a pseudogene
due to a single base insertion which causes an early stop signal. No mRNA or
protein have been found for this gene (Uhlar & Whitehead, 1999). These genes are
all located on chromosome 11, and are thought to be the result of gene duplication and
conversion (Buxbaum & Tagoe, 2000). The serum amyloid A proteins are produced
primarily in the liver, and circulate in blood complexed to lipids (Buxbaum & Tagoe,
2000). SAA synthesis has also been detected in epithelial and endothelial cells,
smooth muscle, macrophages and lymphocytes (Sipe, 2000). The precise function of
SAA is unknown, but the massive increase of SAA during the acute phase and high
degree of conservation across species suggests an important role in defence (Uhlar &
Whitehead, 1999). Possible SAA functions include lipid metabolism or transport,
regulation of extracellular matrix degrading enzymes, inflammatory cell recruitment
(Uhlar & Whitehead, 1999), bacteria clearance (Alsemgeest et al, 1995), tissue repair
and cholesterol metabolism during inflammation (Ensenauer et al, 1994).
There are six electrophoretic variants of human acute phase SAA which occur in three
possible phenotypes (Betts et al, 1991). Two of these isoforms occur in all
52
Chapter 3 Introduction
phenotypes, and in addition two or four other isotypes distinguish the phenotypes.
The acute phase SAA isoforms are summarised in Table 3.1:
Table 3.3. Acute Phase Serum Amyloid A Variants. Isoelectric point
N-terminal amino acids
Identification Reference
6.0 SFFSFL SAA-1α des arg Dwulet, Wallace & Benson, 1988.
6.0 FFSFL SAA-1α des arg minor variant
Strachan et al, 1989.
6.4/6.6 RSFFSFL SAA-1α Foyn Bruun et al, 1995, Strachan et al, 1989.
7.0 SFFSFLG SAA-2α des arg Beach et al, 1992.
7.0 FFSFL SAA-2α des arg minor variant
Strachan et al, 1989.
7.4 SFFSFLG SAA-2β des arg Beach et al, 1992.
7.5 RSFFSFL SAA-2α Beach et al, 1992.
8.0 RSFFSFL SAA-2β Beach et al, 1992.
All humans express the pI 6.0/6.4 pair, and individuals can be divided into three
groups based on the patterns of the additional acute phase SAA expressed:
Phenotype 1: isotypes with pI values of 7.0, 7.4, 7.5 & 8.0, exists in 33% of
individuals.
Phenotype 2: isotypes with pI values 7.0 & 7.5, exists in 61% of individuals.
Phenotype 3: isotypes with pI values 7.4 & 8.0, exists in 6% of individuals, (Strachan
et al, 1989).
Most Europeans and Americans show the SAA1 +/- N terminal arginine (pI 6) and
SAA 2α +/- N terminal arginine (pI 7 & 7.5) while the SAA 2β pair is rare (Husby et
al, 1994). Constitutive serum amyloid A (SAA4) also occurs as a number of
electrophoretic variants. The smaller variant (14kDa) is the non-glycosylated form
and has three alternate isoelectric points (7.3, 7.9 and 8.1) (Whitehead et al, 1992).
53
Chapter 3 Introduction
The larger variant (19kDa) is glycosylated and has two alterative isoelectric points
(7.3 and 7.9) (Whitehead et al, 1992).
Serum Amyloid A as a disease marker:
The elevation of SAA concentration in the acute phase response is a generalised
reaction to inflammation or infection, but several groups have sought to investigate
SAA as a disease marker.
In cattle with experimental respiratory infection SAA responded more quickly than
other acute phase proteins (Heegard et al, 2000). In a murine model of colitis SAA
levels in plasma preceded inflammatory signs in intestinal tissue and levels reflected
the severity of the disease (de Villiers et al, 2000). In the synovium of patients
suffering from rheumatoid arthritis, SAA appears to induce degradation of the
extracellular matrix (Migita et al, 1998). Patients with cerebral infarction showed
elevated SAA levels and the levels appeared to correlate with the clinical severity
(Ilzecka & Stelmasiak, 2000).
The previous examples examined the net level of SAA in disease. Several groups
have investigated the SAA subtype patterns in disease. To quote Alsemgeest and
colleagues (1995):
“if isoforms of SAA are differentially expressed during different disease
processes this might be of potential value in diagnostics”.
In a murine model different expression patterns of SAA1, SAA2 and SAA3 were
found and tissues could be categorised into those that expressed all three isoforms,
those expressing SAA1 and SAA2 and those with predominant SAA3 expression
(Meek & Benditt, 1986). In cows with a variety of diseases a heterogenous pattern of
isoforms was reported (Alsemgeest et al, 1995). In contrast, human patients with
sepsis, arthritis, renal or liver allograft rejection or administration of inteferon,
showed that SAA subtype response was similar irrespective of the ‘initiating
stimulus’ (Maury, Enholm & Lukka, 1985).
Phospholipase A2, a low abundance disease associated protein.
Phospholipase A2 is a protein implicated in several facets of disease. Its involvement
in inflammatory conditions has been widely studied because of its role in the
54
Chapter 3 Introduction
generation of eicosanoids by the liberation of arachidonic acid from phospholipid
molecules. Hydrolysis of phospholipids by phospholipase A2 also releases
lysophospholipid which can be metabolised to platelet activating factor, both of which
can elicit cell responses (Bomalaski & Clarke, 1993), see Figure 4.2. The
inactivation of phospholipase A2 by alkylation or heat prevents the inflammatory
response, which suggests that the inflammatory response is due to the enzyme’s
action rather than a non specific response to an inflammatory initiator such as
endotoxin (Bomalaski & Clarke, 1993).
Phospholipase A2 in bowel injury:
Phospholipase A2 levels have been shown to be increased in the plasma (Minami et
al, 1992) and the intestinal tissue (Lilja et al, 1995) of patients with Crohn’s disease.
Phospholipase A2 may be involved in the pathogenesis of bowel injury. The isoform
of phospholipase A2, type II is thought to be involved in the development of intestinal
inflammation in both Crohn’s disease and ulcerative colitis (Minami & Tojo, 1997).
Serum phospholipase A2 correlates well with the activity of ulcerative colitis and
Crohn’s disease (Nevalainen, Gronroos & Kortesuo, 1993). Inhibition of
phospholipase A2 has been shown to be partially protective against intestinal mucosal
injury in a model of shock (Xu, Lu & Deitch, 1995) and against lung injury caused by
intestinal handling (Thomas, Karnik & Balasubramanian, 2002).
Phospholipase A2 in gut ischaemia/reperfusion:
In rat models of intestinal ischaemia/reperfusion injury, increased levels of
phospholipase hydrolysis products have been observed in the mucosa (Otamiri et al,
1987, Otamiri & Tagesson, 1989). Phospholipase activity in the portal circulation of
rats with experimental ischaemia/reperfusion was ten fold higher than in the systemic
circulation suggesting that the enzyme was being released from the injured gut (Koike
et al, 2000). Inhibition of phospholipase A2 reduced mucosal injury and permeability
in a rat model of ischaemia/reperfusion (Otamiri, Lindahl & Tagesson, 1988). The
involvement of phospholipase A2 in disease including bowel injury is discussed in
greater detail in the introduction to Chapter 5.
55
Chapter 3 Introduction
Pre-fractionation to identify low abundance proteins:
The problem of analysing low abundance proteins in complex mixtures can be
overcome by pre-fractionation of the sample into a number of discrete sub-samples.
The sub-samples can then be further separated using two dimensional gel
electrophoresis based proteomics or another technique such as a series of
chromatographic columns. The pre-fractionation approach can be applied to a global
analysis scheme, which allows greater resolution, particularly in regions which are
usually distorted by high abundance proteins. Pre-fractionation can also be applied in
a targeted approach to purify a particular protein of interest. A number of pre-
fractionation techniques have been designed to overcome the limitations of current
proteomic, and include subproteomics or preparative isoelectric focussing prior to the
global protein separation.
Subproteomics:
Subproteomics involves the use of sequential protein extraction procedures followed
by several 2DE gels of each fraction. By using a series of protein extraction
solutions a biological sample can be fractionated according to relative solubility,
which also reflects the protein’s cellular compartment (secreted, cytosolic or
membrane). Numerous 2DE gels (down to a single pH unit IPG strips) can then be
run from each fraction and a composite map constructed (Cordwell et al, 2000). By
using this approach low abundance proteins can be identified because of increased
sample loading and resolving power (Cordwell et al, 2000). An additional advantage
if that the sequential protein extraction provides some information about the cellular
location of proteins identified (Cordwell et al, 2000).
Preparative IEF techniques:
The use of preparative iso-electric focussing as the preliminary pre-fractionation step
is a powerful approach that is particularly suited to low abundance and/or membrane
proteins. Isoelectric focussing (IEF) separates proteins on the basis of their
isoelectric point or pI, the pH at which there is no net charge on the protein.
Isoelectric focussing can be conducted using small amounts of sample on an IEF gel,
or an IPG strip. Preparative IEF uses large amount of sample and can be performed
using large gel tubes or in the liquid phase. The multi-compartment electrolyzer, free
flow electrophoresis and the Rotofor® apparatus are examples of preparative
56
Chapter 3 Introduction
isoelectric focussing systems. For further discussion of the Rotofor® apparatus, see
Chapter 4.
Multi-compartment electrolyzer:
The multi-compartment electrolyzer is a liquid based preparative IEF system
composed of a variable number of compartments (typically six) separated by
isoelectric membranes (Herbert & Righetti, 2000). Each compartment is capable of
producing a sub-sample with a discrete range of isoelectric points. The fractions can
then be analysed (without alteration of the chemical composition) using 2DE gels.
Disadvantages of this approach include possible loss of protein on the membrane
surface or precipitation of the proteins onto membranes at their pI (Herbert &
Righetti, 2000). This approach led to the identification of several acidic proteins
from human plasma that were unable to be seen in un-fractionated plasma (Herbert &
Righetti, 2000).
Free flow electrophoresis:
Free flow electrophoresis is another liquid based IEF system that can be used to
fractionate samples prior to 2DE gels. Sample recovery is optimal due to the absence
of solid membrane supports and sample load is less of a concern than with other
techniques due to continuous sample application (Hoffman et al, 2001). Free flow
electrophoresis has allowed the identification of a number of intact protein complexes
and membrane associated proteins (Hoffman et al, 2001).
Determining the Accuracy of Diagnostic Markers:
The effectiveness of a new diagnostic marker can be assessed in a number of different
ways. The accuracy of a test is assessed by the sensitivity and specificity.
Sensitivity is the ability of a test to correctly identify patients that have the condition
in question. Specificity is the ability of the test to correctly discount the patients who
do not have the condition in question. The positive predictive value of a test is the
probability that a subject is positive if a positive result is obtained. Conversely, the
negative predictive value is the probability that a negative subject results in a negative
result. Positive/negative predictive values are dependent on the prevalence of the
disease. Diagnostic likelihood ratios are independent on the prevalence of the
disease, and represent the odds ratio of obtaining a positive result among a negative
57
Chapter 3 Introduction
population, compared with the likelihood of obtaining a positive result among a
positive population. Other techniques to measure the effectiveness of a diagnostic
marker include univariate/multivariate analysis and receiver operating characteristic
(ROC) curves. The Receiver Operating Characteristic Plot:
The accuracy of a particular test in the diagnosis of a particular disease is often
evaluated using the concepts of sensitivity and specificity. The specificity and
sensitivity of a test depend on the decision threshold or cut-off point for the test.
ROC plots were first introduced to evaluate radar in the 1950s and have since been
employed in radiography, experimental psychology and pathology testing fields
(reviewed by Zweig & Campbell, 1993). ROC plots evaluate the test’s
‘ability to discriminate between alternating states of health over the complete
spectrum of operating conditions’ (Zweig & Campbell, 1993).
ROC graphs plot sensitivity against 1-specificity. Each point represents the
specificity/sensitivity pair for a particular cut-off point for a particular test. ROC
plots are useful in situations where
- there is no ‘gold standard’ with which to compare a new test (Zweig &
Campbell, 1993),
- if the accepted test has an associated bias (Zweig & Campbell, 1993),
- if the decision threshold has not been decided.
ROC plots are advantageous because
- the entire range of threshold values is assessed,
- a common scale is used, unlike a frequency diagram
which usually has different scales (Zweig & Campbell,
1993).
The ROC plot has a number of disadvantages:
- the decision threshold is not visually obvious from the graph (Zweig &
Campbell, 1993),
- the number of subjects is not shown by the graph (Zweig & Campbell, 1993).
58
Chapter 3 Introduction
When a plot is created the area under the curve is a measure of the discriminatory
power of that test. The closer the area under the curve is to 1, the better
discriminating power the test has. Conversely, an area of 0.5 has no more
discriminating ability than random chance.
Plasma Variables assessed in this Study:
APACHE II:
The APACHE system is a scoring system that was devised by a group at Washington
University and was first published in 1981 (Knaus et al, 1981). APACHE II
represented an improved version of the original APACHE system designed to predict
patients’ outcome in ICU based on twelve physiological variables, the Glasgow Coma
Score, chronic health status and age (Knaus et al, 1985, Siegel & Rixen, 2002).
APACHE II has been used as an indicator of patient status and to predict mortality in
patients with multiple trauma (Knaus et al, 1985, Rhee et al, 1990).
SAA:
SAA is one of the most reactive and most quickly cleared acute phase proteins in
humans. SAA can increase in concentration in the circulation from baseline levels to
200 to 500 times that level within 20 hours (de Beer et al, 1994). The half life of
SAA in normal and inflammatory states is only 90 minutes (Husby et al, 1994), and
therefore the clearance of this protein from circulation can be very rapid.
CRP:
CRP is the most widely used marker of inflammation in clinical practice at present
(Bellamy, Lansbury & Murdoch, 2002). CRP can reach levels of 500mg/L within 6
hours of the induction of the acute phase response (Bellamy, Lansbury & Murdoch,
2002). Ongoing infection or inflammation usually results in continually elevated
levels of CRP whereas declining levels usually indicate resolution of the condition
(Bellamy, Lansbury & Murdoch, 2002).
PLA2:
PLA2 is a rate limiting enzyme in the generation of a variety of inflammatory
mediators including the eicosanoids, lysophospholipid and platelet activating factor
(Dennis, 1997). Phospholipase A2 production and release into plasma/extracellular
59
Chapter 3 Introduction
fluid is increased during inflammation (Kallajoki & Nevalainen, 1997, Kudo et al,
1993). Extracellular phospholipase A2 levels can reach concentrations of up to 1000
times baseline during inflammation (Weinrauch et al, 1998).
LACTATE:
Elevated lactate concentration has been described as
“the best marker of mesenteric ischaemia to date” (Lange & Jackel, 1994).
A high lactate concentration is not a finding that is unique to mesenteric ischaemia,
but indicates the need for immediate surgery to avert life threatening deterioration.
Shock, diabetic ketoacidosis, convulsion and hepatic or renal failure can also lead to
elevated lactate levels, but if these conditions can be discounted then the likelihood of
mesenteric ischaemia increases (Lange & Jackel, 1994).
60
Results
Chapter 3: Proteomics and plasma investigations.
A proteomic investigation of potential differences between the plasma proteins of
patients with surgically confirmed bowel infarction and control patients was
undertaken. The control patients in this study were intensive care patients that were
being successfully fed, and therefore had functioning bowels. An initial investigation
of the neutral to acidic (pI of 4 to 7) proteins was undertaken as a starting point. The
major protein differences were found to be variants of haptoglobin (results not
shown). The basic plasma proteins (pI of 7 to 10) were investigated in greater depth
as it was thought that small basic proteins may be the first to transverse the gut barrier
into circulation. It is known that gut permeability increases as a result of ischaemia
(Kong et al, 1998). Well resolved plasma protein gels were achieved using cup
loading rather than in-gel rehydration, and a methanol precipitation cleanup step
(Figures 3.2 and 3.3). A number of protein differences were evident between the
samples from patients with bowel infarction and control patients (Figure 3.4).
Proteins of interest were identified using MALDI-TOF mass spectrometry and
database searching. If MALDI-TOF did not provide an adequate database match, the
protein was identified using Q-TOF tandem mass spectrometry and database
searching. The relative quantities of each of the proteins were assessed using image
analysis software (Figure 3.5). The presence or absence of each spot in each gel was
also noted (Figure 3.6). From the analysis of protein quantity, serum amyloid A
appeared to warrant further investigation. The mass spectrometry analysis of serum
amyloid A gave good sequence coverage (Figure 3.7), and Q-TOF was also able to
distinguish between SAA2 and SAA1 (Figure 3.8), two proteins that share very high
homology (96%). Three serum amyloid protein variants were identified in this study,
and the relative proportions of each is shown in Figure 3.9, though there is no clear
pattern that distinguishes the bowel infarction patients from controls.
An analysis was conducted to compare the ability of the following variables in
distinguishing patients with bowel infarction from control patients:
- a commonly used clinical marker (C-reactive protein)
- a commonly used clinical scoring system (APACHE II)
- an indicator of bowel infarction (lactate)
- a protein suspected to be released from infarcted bowel (phospholipase A2)
- serum amyloid A
61
Results
The analysis of serum amyloid A was conducted using an ELISA system, due to a
superior limit of detection than image analysis of gel spots. SAA levels were not
significantly different between the plasma of control and bowel infarction patients
(Figure 3.13).
Previous research had shown that phospholipase A2 activity was increased in the
intestinal mucosa in rat models of ischaemia/reperfusion injury (Koike et al, 1992,
Koike et al, 1995, Otamiri & Tagesson, 1989). In this investigation we wished to
investigate the possible release of phospholipase activity into the plasma of patients
with bowel infarction. Phospholipase A2 activity was measured in the plasma of
patients involved in this study (Figure 3.10). Each variable was assessed in groups of
patients with bowel infarction and control patients (Figure 3.11). C-reactive protein
showed a trend (p<0.025) toward increased levels in the patients with bowel
infarction, but the other variables showed poor discrimination between the two groups
of patients, as assessed by t-tests of the mean data. Receiver operating characteristic
curves of the five variables were constructed to allow comparison on a common scale
(Figure 3.12). The variable that showed the best discrimination power was
phospholipase A2, though none of the variables assessed were ideal for use in a
clinical setting. The next investigations focused on the phospholipase A2 activity in
normal and infarcted human bowel (Chapter 4) and the purification of the protein
responsible for the increased phospholipase A2 activity (Chapter 5).
62
63
A B C
D E F
Figure 3.2: Two Dimensional gels of plasma proteins from control patients A to F. Large
format gels (8-18% gradient) and basic IPG strips (pH 7-10) were employed. These gels
were stained with the colloidal Coomassie stain. These gels have been cropped to show
proteins smaller than 50kDa.
pH7 10
kDa
~50
~25
~15
64
1 2 3
6 8 9
kDa~50
~25
~15
pH7 10
Figure 3.3: Two Dimensional gels of plasma from patients with confirmed bowel infarction,
labelled here as patients G to L. Large format gels (8-18% gradient) and basic region IPG strips
(pH 7-10) were employed. These gels were stained with the colloidal Coomassie stain. These
gels have been cropped to show proteins smaller than 50kDa.
65
Figu
re 3
.4: T
ypic
al tw
o-di
men
sion
al g
el sh
owin
g pl
asm
a pr
otei
ns fr
om a
pat
ient
with
bow
el in
farc
tion
(left
hand
side
) and
pla
sma
from
a c
ontro
l pat
ient
(rig
ht h
and
side
).
The
gel h
as b
een
crop
ped
to sh
ow o
nly
the
smal
l pro
tein
s. I
mm
obili
sed
pH g
radi
ents
(pH
7-1
0) a
nd la
rge
form
at g
radi
ent g
els (
8-18
%) w
ere
empl
oyed
. M
ass s
pect
rom
etry
was
per
form
ed o
n sp
ots l
abel
led
in re
d (A
1-7)
. M
ass S
pect
rom
etry
ana
lysi
s em
ploy
ed
MA
LDI a
nd Q
-TO
F.
66
00.020.040.060.08
0.10.120.140.160.18
0.2
Haemoglobinbeta
fragment a
Haemoglobinbeta
fragment b
Haemoglobinalpha
Serumamyloid
Serumamyloid A2
A
Serumamyloid A2
Q9NV34
Qua
ntity
.
Infarcted bowel Normal bowel
* ** ***
Figure 3.5: Relative quantities of spots selected for Mass Spectrometry analysis. Analysis
was performed using BioRad Quantity One Software, version 4.1.1. Proteins that were
significantly different by the student’s t-test are highlighted with asterisks (* for p<0.05,
** for p<0.02, *** for p<0.01 levels of significance).
67
Protein: Control Plasma: Bowel Infarction plasma: Haemoglobin α 6/6 6/6 Haemoglobin β fragment a 6/6 6/6 Haemoglobin β fragment b 5/6 6/6 Q9NV34 theoretical protein 6/6 6/6 Q9P1I9 theoretical protein 1/6 0/6 SAA (insufficient peptide information to distinguish the specific variant)
5/6 6/6
SAA 2 3/6 6/6 SAA A2 5/6 6/6
Table 3.4: Protein spots present in 2DE gels of plasma.
68
Spot Type of Mass Spec.
Identity Number of peptides obtained
Sequence coverage
A2 MALDI SAA, (unknown variant)
7 58.70%
A5 MALDI SAA 2-α 7 61.51% A7 MALDI - 0 - A7 Q-TOF SAA2 3+sequence data 25.96% by amino
acids
Figure 3.6: Serum amyloid identification by Mass Spectrometry.
69
SAA1 MKLLTGLVFCSLVLGVSSRSFFSFLGEAFDGARDMWRAYSDMREANYI SAA2 MKLLTGLVFCSLVLGVSSRSFFSFLGEAFDGARDMWRAYSDMREANYI SAA3 MKLSTGIIFCSLVLGVSSQGWLTFLKAAGQGAKDMWRAYSDMKEANYK SAA4 MRLFTGIVFCSLVMGVTSESWRSFFKEALQGVGDMGRAYWDIMISNHQ SAA1 GSDKYFHARGNYDAAKRGPGGAWAAEVISDARENIQRFFGHGAEDSLA SAA2 GSDKYFHARGNYDAAKRGPGGAWAAEVISNARENIQRLTGHGAEDSLA SAA3 KSDKYFHARGNYDAVQRGPGGVWATEVISDARENVQRLTGDHAEDSLA SAA4 NSNRYLYARGNYDAAQRGPGGVWAAKLISRSRVYLQGLIDYYLFGNSS SAA1 DQAANEWGRSGKDPNHFRPAGLPEKY SAA2 DQAANKWGRSGRDPNHFRPAGLPEKY SAA3 GQATNKWGQSGKDPNHFRPAGLPEKY SAA4 TVLEDSKSNEKAEEWGRSGKDPDRFRPDGLPKKY PEPTIDE A SFFSFLGEAFDGAR PEPTIDE B SFLGEAFD PEPTIDE C GPGGAWAAEVISNAR
Figure 3.7: Sequence alignment of serum amyloid isoforms SAA1-4. Q-TOF Mass
Spectrometry allowed the spot ‘A7’ to be identified as SAA2 from peptide denoted ‘C’.
SAA1 and SAA2 differ by only 5 from 104 amino acids, and share the same Swiss-Prot entry
because of this homology. The high degree of homology between the isoforms
is likely to be the reason that it was not possible to determine the specific isoform
corresponding to spot A2.
70
SAA gel spots- control patients.
00.050.1
0.150.2
0.25pl
asm
a11
plas
ma1
2
plas
ma1
3
plas
ma1
4
plas
ma1
7
plas
ma2
0
Qua
ntity
.
spot A2spot A5spot A7
SAA gel spots- Bowel infarction patients.
00.050.1
0.150.2
0.25
plas
ma1
plas
ma2
plas
ma3
plas
ma6
plas
ma8
plas
ma9
Qua
ntity
.
spot A2spot A5spot A7
Figure 3.8: Serum amyloid proteins present in plasma of control patients and
patients with confirmed bowel infarction. Analysis was performed using
BioRad Quantity One software version 4.1.1. Spot A2= SAA, spot A5=
SAA 2α, spot A7= SAA 2.
71
SAA1 MKLLTGLVFCSLVLGVSSRSFFSFLGEAFDGARDMWRAYSDMREANYISAA2 MKLLTGLVFCSLVLGVSSRSFFSFLGEAFDGARDMWRAYSDMREANYISAA3 MKLSTGIIFCSLVLGVSSQGWLTFLKAAGQGAKDMWRAYSDMKEANYKSAA4 MRLFTGIVFCSLVMGVTSESWRSFFKEALQGVGDMGRAYWDIMISNHQ
SAA1 GSDKYFHARGNYDAAKRGPGGAWAAEVISDARENIQRFFGHGAEDSLASAA2 GSDKYFHARGNYDAAKRGPGGAWAAEVISNARENIQRLTGHGAEDSLASAA3 KSDKYFHARGNYDAVQRGPGGVWATEVISDARENVQRLTGDHAEDSLASAA4 NSNRYLYARGNYDAAQRGPGGVWAAKLISRSRVYLQGLIDYYLFGNSS
SAA1 DQAANEWGRSGKDPNHFRPAGLPEKYSAA2 DQAANKWGRSGRDPNHFRPAGLPEKYSAA3 GQATNKWGQSGKDPNHFRPAGLPEKYSAA4 TVLEDSKSNEKAEEWGRSGKDPDRFRPDGLPKKY
Leader peptide: residues 1-18Heparin binding sites: residues 78-104SAA4 octapeptide extension: residues 70-77
Figure 3.9: Serum amyloid A isoform sequence alignment. The leader peptide (18
amino acids in length) is cleaved to form the mature protein.
The serum amyloid 4 octapeptide extension is not present in the acute phase isoforms.
This octapeptide is the only potential N-linked glycosylation site in the molecule.
Studies have shown that 50% of the proteins are glycosylated giving rise to two size
classes differing by 5kDa (Whitehead et al, 1992).
72
01234567
BOWEL INFARCTION CONTROL
PLA
2 ac
tivity
(%
hyd
roly
sis)
Figure 3.10: PLA2 activity in the plasma of bowel infarction patients and
control patients. Results are expressed as the mean +/- standard error of
the PLA2 activity of bowel infarction (n=10) and control plasma (n=9).
The PLA2 activity in the plasma of bowel infarction patients and control
patients is not significantly different.
73
LACTATE
01234567
Bowel Infarction Control
LACT
ATE
(mm
ol/L
)
PLA2 ACTIVITY
0
2
4
6
8
Bowel Infarction ControlPLA
2 H
YDRO
LYSI
S (%
)
CRP
0
100
200
300
400
Bowel Infarction Control
CRP
(ug/
mL)
APACHE
05
1015202530
Bowel Infarction Control
APAC
HE
SCO
RE *
SAA
0
200
400
600
800
Bowel Infarction Control
SAA
(ug/
mL)
Figure 3.11: A visual comparison of the five variables measured in the plasma of
control patients and patients with confirmed bowel infarction.
The differences for each of these variables was not significant except for CRP
levels, (* p<0.025, student’s t test). Data are represented as the mean +/- standard
error. Number of samples in bowel infarction group and control group
respectively: CRP n=7 in each group, Lactate n=7 & n=10, APACHE n=10 in each
group, PLA2 n=10 & n=9, and SAA n=10 & n=9. The numbers of samples in each
group varies because of differences in the availability of clinical testing.
74
Figure 3.12: The ROC plot and the area under the curve obtained for each variable
measured in the plasma of control patients and patients with surgically confirmed
bowel infarction. The separator variable was the presence or absence of bowel
infarction. As the area under the ROC curve approaches 1, the test approaches
perfect discrimination power.
Discussion
Chapter 3 Discussion:
Our proteomic investigation examined possible plasma protein differences between
patients with surgically confirmed bowel infarction and control patients. This
investigation was undertaken as a starting point for this project and to assess whether
a highly abundant marker of tissue damage or a diagnostic protein was evident in the
plasma of bowel infarction patients but not in controls. The control patients were
Intensive Care patients that were being successfully fed, and thus had functioning
bowels. The control patients had been admitted to intensive care for a wide variety
of conditions. Our investigation did not recruit normal healthy volunteers as
controls, to try to limit protein differences that were simply indicative of health status.
The majority of proteomic investigations of disease associated proteins have focussed
on a variety of cancers, often at the tissue level. The control patients in proteomic
investigations of plasma proteins tend to be healthy volunteers. We chose to use
plasma (rather than bowel tissue, for example) as our sample of choice, as a clinically
useful diagnostic test would be minimally invasive, and be able to be used to
continually monitor ‘at risk’ patients. Our pilot investigation focussed on the neutral
to acidic (pH 4-7) proteins. The specific proteins identified from this region in
patients with confirmed bowel infarction were variants of haptoglobin.
Haptoglobin variants have been observed in two dimensional electrophoretic gels of
human serum during and after the acute phase, and were predictably more abundant
during the acute phase (Bini et al, 1996). Haptoglobin is thought to aid in tissue
repair by stimulating angiogenesis, which may compensate for the decreased oxygen
supply during ischaemia (Cid et al, 1993). Haptoglobin also has antioxidant
properties and protects against damage by reactive oxygen species (Gabay &
Kushner, 1999). The increased levels of haptoglobin observed during infection and
inflammation are a non-specific response triggered by cytokines (Cid et al, 1993) and
is therefore not of specific diagnostic value for bowel infarction. Although the role
of haptoglobin in ischaemic injury to bowel may be an interesting area for further
research, it was not investigated in greater detail in this project due to its non specific
increase during the acute phase.
75
Discussion
Cup loading and methanol precipitation:
Our investigation then focused on the small basic proteins present in the plasma of
patients with and without surgically confirmed bowel infarction. The small, charged
proteins were thought to be the first to be released from injured bowel (into the lumen
and subsequently into general circulation). Bowel permeability is known to increase
as a result of ischaemia (Kong et al, 1998). Basic proteins are known to be difficult
to resolve by in gel rehydration, which is the usual method of uptake of proteins into
IPG strips. Cup loading is commonly recommended for uptake of proteins into
alkaline IPG strips (Gorg et al, 1999, Gorg et al, 2000). Our work confirmed that
cup loading of basic proteins was more successful than in gel rehydration (results not
shown). It is also thought that plasma proteins in general are more successfully
resolved with cup loading than in gel rehydration (Australian Proteome Analysis
Facility, personal communication). Methanol precipitation was also found to give
superior results, in terms of superior resolution of protein spots and less streaking.
Methanol precipitation has been reported to improve resolution by removing non-
protein contaminants such as lipids, polysaccharides, salts and nucleic acids
(Amersham Pharmacia applications manual). The improved resolution provided by a
methanol precipitation step was even more evident with gels of bowel content
samples, which are likely to contain more non-protein contaminants than plasma.
The gels of bowel content samples are not shown in this report, as the protein patterns
were too heterogenous to draw any conclusions or locate particular proteins for
identification by mass spectrometry.
PROTEIN DIFFERENCES FOUND BY 2D-PAGE:
A number of protein differences were evident from a composite synthetic gel
constructed from the gels of plasma proteins from the two groups of patients. A
number of haemoglobin variants were found, and, not surprisingly, the levels of these
proteins were similar between the two groups of patients. Two proteins that were
identified by MALDI-TOF mass spectrometry had been entered into the protein
databases as ‘theoretical proteins’. These two proteins had little potential as
diagnostic proteins as one was present in all samples at similar levels (Q9NV34), and
the other was present in only one sample (Q9P1I9), but were interesting nevertheless.
76
Discussion
Q9NV34 was present in all controls and all patients with confirmed bowel infarction.
Q9NV34 was an excellent match for v-maf musculoaponeurotic fibrosarcoma
oncogene family proteins (BLASTN, BLASTP). Maf genes are highly conserved in
vertebrates (Iwata et al, 1998) and have been implicated in a variety of physiological
processes including the stress response (Moran, Dahl & Mulcahy, 2002). The maf
proteins in disease would be an interesting area for further investigation. It is
interesting to note that the level of Q9NV34 was similar in the plasma of each control
patient, despite the heterogenous disease pattern of this group. Q9P1I9 was present
in only 1/6 control patients and no patients with bowel infarction. The amino acid
sequence of Q9P1I9 showed no matches to any entered sequence in either the
nucleotide or protein databases. Q9P1I9 showed limited similarity to a
metalloproteinase inhibitor (Propsearch), but matched no recognised motifs (Prosite).
Propsearch uses amino acid composition instead of sequence to find similar proteins.
Propsearch can provide information about molecular weight, hydrophobicity, charged
residues and distribution of residue sizes. Motif scan (Prosite) identifies similarities
to recognised protein motifs (Falquet et al, 2002).
SERUM AMYLOID A:
Three variants of serum amyloid A were identified in the plasma of patients with
surgically confirmed bowel infarction by mass spectrometry in this study. Serum
amyloid A exists as a complex family of proteins, and includes six electrophoretic
variants of the acute phase types (Betts et al, 1991) in addition to five electrophoretic
variants of the constitutive type (Whitehead et al, 1992). Serum amyloid A variants
exist in three recognised phenotypes, with either four and six acute phase variants, in
addition to up to five constitutive variants. All of the constitutive variants and either
two or four acute phase variants (depending on phenotype) will theoretically resolve
in the pH region 7-10 of a two dimensional gel, though some are likely to be below
the detection limit of the gel stains. In this study, the quantity of the SAA2 and
SAA2α variants appeared to be elevated in the plasma of patients with bowel
infarction, compared with control patients. This is an interesting finding as the level
of SAA and other acute phase proteins would be expected to be similar among
intensive care patients with similar levels of critical illness.
77
Discussion
If the expression profile of the SAA variants was different in various tissues and/or
different diseases then this protein family may be of diagnostic significance. This
idea has been proposed by groups studying a variety of diseases in both cattle
(Alsemgeest et al, 1995) and humans (Maury, Enholm & Lukka, 1985). Human
patients with a variety of diseases showed a similar SAA subtype pattern regardless of
the disease (Maury, Enholm & Lukka, 1985). Serum amyloid A variants have been
observed in two dimensional gels of human serum during the acute phase, but never
after recovery (Bini et al, 1996). In cattle, a heterogenous pattern of SAA isoforms
was observed (Alsemgeest et al, 1995). In our study the SAA variants were
generally higher in the patients with confirmed bowel infarction than in controls, but
no clear pattern of variants was evident between the two patient groups.
The function of serum amyloid A during the acute phase is not yet known, but
functions such as lipid metabolism, recruitment of inflammatory cells (Uhlar &
Whitehead, 1999), cell adhesion and chemotaxis of phagocytes and lymphocytes
(Berliner et al, 1995), tissue repair (Ensenauer et al, 1994) or bacterial clearance
(Alsemgeest et al, 1995) have been proposed.
Mass spectrometry analysis of serum amyloid A:
One variant of SAA (SAA2) was unable to be identified by MALDI-TOF mass
spectrometry, but was successfully identified by Q-TOF tandem mass spectrometry.
The variant submitted to Q-TOF analysis was identified as SAA2, which was a
fortuitous result, as SAA1 and SAA2 differ by only 4 out of 105 amino acids (Betts et
al, 1991), and share the same entry in protein databases because of their very high
homology. SAA1 is thought to be the major SAA variant in humans, comprising
over 95% of the total SAA in plasma (Malle et al, 1997), so it was interesting to
identify variants other than SAA1 in this study. The SAA1 variants have the
isoelectric points 6.0, 6.4 and 6.6, and therefore would not have resolved within the
IPG strip range employed in this study.
Serum Amyloid A ELISA:
The plasma levels of serum amyloid A in bowel infarction and control patients were
assessed in greater depth using an ELISA. It was expected that the levels of SAA
would be greater in the plasma of patients with bowel infarction compared with
78
Discussion
controls, from the proteomic results. The ELISA was employed because of its
superior limit of detection compared to the quantification of gel spots (ELISA limit of
detection 5ng/mL, compared to a gel spot which represents 0.8-1µg/mL). There was
a trend toward increased serum amyloid A levels in the plasma of bowel infarction
patients compared with controls, but the difference was not significant The antibody
used in the ELISA cross reacts with both SAA1 and SAA2 (TriDelta laboratories,
personal communication). Any subtle differences in the subtype patterns of SAA
between the two patient groups is therefore likely to have been overlooked with this
approach. To our knowledge, no antibodies have been raised that can distinguish the
SAA subtypes, therefore subtype specific ELISA are not yet possible. As SAA1 is
the predominant SAA isoform, this may have masked subtle differences in other SAA
variants. To test this hypothesis, western blotting could be undertaken using an
acute phase SAA antibody and proteins arrayed by pH 3-10 two dimensional gels.
Although the use of an acute phase protein as a possible diagnostic marker may seem
inappropriate, a number of examples of this approach have been reported:
- CRP has been recently recommended as a marker of coronary disease risk
(Pearson et al, 2003).
- All of the patients with bacterial infections in one study showed elevated
SAA, while only 6/16 patients with viral infection had elevated SAA levels
(Bini et al, 1996).
Until subtype specific SAA antibodies can be generated and corresponding ELISAs
developed, the increased levels of SAA and possible involvement of SAA variants in
bowel ischaemia/infarction are difficult areas to study in greater depth.
PHOSPHOLIPASE A2:
Another protein liberated in response to tissue injury, phospholipase A2, was
suggested in the literature to be of possible importance in bowel ischaemia and
infarction. For example, a rat model of ischaemia/reperfusion injury showed that
phospholipase A2 was increased in the portal circulation to a tenfold higher level than
in the systemic circulation, suggesting that circulating phospholipase A2 in ischaemia
was released by the injured gut (Koike et al, 2000). Phospholipase A2 could not be
studied using a two dimensional proteomic approach because of its low abundance,
and was studied using catalytic activity assay as an alternative. The patients with
79
Discussion
confirmed bowel infarction in this study showed higher plasma PLA2 activity than
control patients, but the difference was not significant. The phospholipase
complement of bowel is complex, and a subtle difference between bowel infarction
and control patients could not be examined using a catalytic activity assay. The
phospholipase A2 assay employed in this project, and many other publications
quantifies the net in vitro catalytic activity and is not able to determine the presence or
contribution of the various isoenzymes (Farrugia et al, 1993). The aim of these
experiments was to assay the catalytic activity of the enzyme (rather than the amount
of protein which may not be active or bound to inhibitors). It is possible that the
hydrolysis products of this enzyme may be of greater importance than the protein
isoform itself. Protein purification was undertaken to identify the protein responsible
for the increased phospholipase activity (Chapter 5).
APPLICATION OF PROTEOMICS TO BIOMARKER DISCOVERY:
A number of issues were presented by the proteomic investigations. Although the
number of patients in each group was large in terms of a proteomic investigation (10
samples from each group), this number is small in terms of a clinical investigation.
Biological variability results in large differences in protein expression between
patients. There are also significant fluctuations in protein levels within a patient.
For example; one study investigated C-reactive protein in a group of healthy
individuals and found that protein levels fluctuated over a six month period (Macy,
Hayes & Tracy, 1997). These fluctuations equated to coefficients of variation of
42% within an individual and 93% between individuals (Macy, Hayes & Tracy,
1997). There are also fluctuations in disease associated proteins, such as the acute
phase proteins (see Figure 3.14), over the time course of the development of the
disease. These fluctuations add even greater complexity to the development of
reference ranges and subsequent diagnostic tests.
80
Discussion
Figure 3.14 The Acute Phase proteins.
Modified from Gitlin J.D. & Colten H.R., Molecular Biology of the acute phase
plasma proteins. Lymphokines 1987; 14: 123-153 with permission from Elsevier.
Low abundance proteins:
Low abundance plasma proteins are more likely to be more clinically important than
highly abundant or long lived proteins (Griffin, Goodlet & Aebersold, 2001).
However, the majority of the proteins identified by proteomics in this study were high
abundance and/or acute phase proteins. There was a clear need for pre-fractionation
of a clinical sample prior to two-dimensional gels in order to identify low abundance
proteins, and this would become the basis for future proteomic investigations for this
project. Calculation based on our sample volumes and protein stain capabilities show
that a protein would need to present in a concentration of greater than 0.8µg/mL to be
identified in our two dimensional gel electrophoresis system, which is a relatively
81
Discussion
high level. This level of abundance is likely to represent very few tissue
damage/leakage proteins (as shown by Figure 3.1).
Multiple biomarker approach:
The process of identifying a diagnostic test for bowel infarction may benefit from the
simultaneous investigation of several potential markers. A diagnostic test for bowel
ischaemia/infarction need not be based on the detection of a single protein, but may
involve a combination of protein(s), radiographic features and/or clinical signs. The
use of a panel of diagnostic markers has been proposed by a number of groups.
Anderson and Anderson recently stated that there is a:
“significant theoretical problem with the notion that there should be a single
protein in plasma whose levels change in response to one specific disease”.
Adam and colleagues (2002) state that ‘no single marker is likely to prove sufficiently
predictive’, while Seliger and Kellner (2002) suggest that ‘preferably small clusters of
proteins represent the ideal diagnostic markers”. Conversely, the number of
diagnostic variable must be kept as low as possible to keep costs to a minimum.
ROC PLOTS:
Receiver Operating Characteristic curves are very useful in assessing a number of
diagnostic tests on a common scale. ROC plots are also very useful when the cut-off
point has not been decided, or even if a cut-off point is known it can be omitted from
the analysis and thus does not bias the evaluation. In this project, the numbers of
patients were too low to obtain an accurate assessment of the value of the variables by
univariate or multivariate analysis. It has been recommended that multivariate
analysis only be undertaken when the study involves more than thirty subjects in each
group (Dr. M. Henry, personal communication). If the numbers of patients recruited
in the future were increased, then multivariate analysis is likely to be valuable in
identifying the most useful diagnostic marker, or combination of diagnostic markers.
Higher numbers of patients in each group (bowel infarction and control) would also
begin to overcome problems associated with biological variation and protein
fluctuation.
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Chapter 4 Introduction
Chapter 4: Phospholipase A2 in Mesenteric Ischaemia and Infarction: Phospholipase A2: The phospholipases are a diverse range of enzymes that have been grouped by their ability to
hydrolyse phospholipids. The phospholipase family is divided into the subgroups A1, A2, B,
C and D according to the site of action within the generalised phospholipid molecule (see
Figure 4.1). The subgroup A2 hydrolyses phospholipids at the middle or sn-2 fatty acid to
produce a free fatty acid and the corresponding lysophospholipid molecule. Phospholipase
A2 has been studied extensively because of the physiological importance of the hydrolysis
products and the role of these products in disease (see Figure 4.2). For example: the most
common fatty acid occurring in the sn-2 position is arachidonic acid, the fatty acid central to
the eicosanoid mediator pathway in inflammation. The lysophospholipids liberated by the
action of phospholipase A2 have also been implicated in various pathophysiological
processes.
CH
CH2
CH2
O C
O
R'
O C R'
O
OP
O
O-
XO
A1
B
A2
CD
Figure 4.1: Site of action of the phospholipase enzymes on a generalised phospholipid
molecule. X can represent choline, ethanolamine, inositol, serine, glycerol,
diacylglycerol etc (Mansbach, 1990).
The physiological roles that phospholipase A2 plays are diverse, ranging from digestion of
dietary phospholipids, cell membrane remodelling, host defence (Hanasaki & Arita, 1999),
cell signalling, biosynthesis of eicosanoids (Lambeau & Lazdunski, 1999) to envenomation
83
Chapter 4 Introduction
(Gelb et al, 2000). Phospholipase A2 exists as a family of isoforms, the recognised number
of which has increased rapidly over the last five to ten years. The isoforms can be grouped
into secretory isoforms, cytosolic isoforms, calcium independent isoforms and platelet
activating factor (PAF) acetylhydrolases (Hanasaki & Arita, 2002).
Phospholipase A2 proteins utilising a catalytic Histidine:
Typical phospholipase A2 isoforms (Group I, II, III, V, IX, X and XI) have phospholipid
hydrolysing capability due to a catalytic histidine which is conserved along with an aspartate
residue in the active site. All secretory phospholipase A2 (groups I, II, II, V, X, XII) isoforms
show a catalytic histidine and share a number of common features including:
- a conserved calcium binding loop,
- low molecular weight (13-18kDa1),
- low milli-molar calcium dependency (Hanasaki & Arita, 2002) and
- high sensitivity to reduction by chemicals such as DTT (Clark, Milona & Knopf,
1990).
The number of secretory phospholipase A2 isoforms recognised in mammals has increased
from two isoforms in 1990 (IB and IIA) to ten (Hanasaki & Arita, 1999, Scott, Graham &
Bryant, 2003). The mammalian secretory phospholipase A2 isoforms that have identified
(IB, IIA, IIC, IID, IIE, IIF, III, V, X and XII) all occur in humans except for IIC which
appears to be a pseudogene. The secretory phospholipases A2 have low homology and
Singer et al (2002) state ‘different sPLA2 paralogs are not closely related isoforms since
amino acid identity between any two is in the range <15% to 50%’.
Phospholipase A2 proteins utilising a catalytic Serine:
As discussed in the previous section, the catalytic activity of secretory phospholipase A2
isoforms is dependent on a conserved histidine that occurs in a Histidine-Aspartate dyad.
Several phospholipase A2 variants exist that do not exhibit a catalytic histidine, and the
phospholipase A2 activity of these ‘orphans’ is dependent on a catalytic serine (Six & Dennis,
2000). Platelet activating factor (PAF) acetylhydrolase is one of the serine phospholipase A2
variants. PAF acetylhydrolase (group VIIA PLA2) shows the lipase consensus motif Gly-X-
Ser-X-Gly and the classic hydrolase triad of Ser273Asp296His351 (Six & Dennis, 2000). Group
1 Human type III is an exception, the active protein forms a section of a larger protein of 55kDa (Scott, Graham & Bryant, 2003).
84
Chapter 4 Introduction
VIIIA and B are subunits of the PAF acetylhydrolase Ib protein and both exhibit the ‘pseudo
lipase’ consensus motif of Gly-X-Ser-X-Val (Six & Dennis, 2000). Group VIA
phospholipase A2 (iPLA2) also contains the hydrolase (Gly-X-Ser-X-Gly) motif (Six &
Dennis, 2000). Groups IVA (cPLA2), IVB and IVC phospholipases A2 also exhibit a serine
aspartate active site (Six & Dennis, 2000). 1-cysteine peroxiredoxin, (or bifunctional lung
enzyme) was another unusual protein reported to exhibit phospholipase A2 activity (Chen et
al, 2000, Kim et al, 1997). Six and Dennis (2000) argue that this protein should not be
classified as a phospholipase A2 because mutation of the serine (and cysteine) had no effect
on phospholipase A2 activity (Kang, Baines & Rhee, 1998). Six and Dennis (2000) proposed
that the classification of a phospholipase A2 may only occur when a protein can be shown to
catalyse the hydrolysis of the sn-2 bond of a phospholipid substrate, and when the complete
amino acid sequence is known.
The complexity of Phospholipase A2 Research:
The enzyme characteristics and tissue distribution of phospholipase A2 isoforms show
considerable overlap, making the study of specific isoforms at the protein level difficult. The
functions of many of the recently identified isoforms are yet to be elucidated and detailed
investigations of tissue and cellular distribution and enzyme characteristics are yet to be
performed. The source of circulating secretory phospholipase A2 activity is unknown
(Nevalainen, Gronroos & Kallajoki, 1995, Nevalainen, Haapamaki & Gronroos, 2000).
The precise roles of phospholipase A2 has not been clearly defined in physiology, membrane
maintenance or cellular signalling. Even less is understood about the roles and interactions
of the multitude of secretory PLA2 isoforms. The complexity of the functions of the
secretory PLA2 isoforms is likely to increase further during pathophysiological situations.
Organs (Scott, Graham & Bryant, 2003), cells and tissues (Yang et al, 1999) are all known to
contain multiple forms of phospholipase A2 proteins.
Phospholipase A2 in disease:
Since the physiological roles of recently described PLA2 isoforms is unclear, the role in
disease states is essentially unknown.
The study of phospholipase A2 in relation to disease has focussed on several key areas:
- the role of phospholipase A2 at the site of inflammation or tissue damage,
- the use of phospholipase A2 as a diagnostic or prognostic marker,
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Chapter 4 Introduction
- the role of phospholipase A2 in the generation of eicosanoids,
- the possibility of inhibition of phospholipase A2 to treat inflammatory conditions.
Figure 4.2: The multitude of products of phospholipase A2 Hydrolysis and their
Cellular Response (Bomalaski & Clark, 1993).
PLA2 involved in the pathogenesis of disease:
Synovial fluid from patients with rheumatoid arthritis shows elevated levels of PLA2 (Hara et
al, 1989, Lai & Wada, 1988). Type II PLA2 is proposed to be involved in the pathogenesis
of intestinal inflammation in Crohn’s disease and ulcerative colitis (Minami & Tojo, 1997).
PLA2 levels correlate to mortality rates in patients with established MODS (Uhl et al, 1990,
Uhl et al, 1995). Recently it has been proposed that PLA2-II may “contribute to the
induction of organ dysfunction but less of a role in sustaining organ system failure” (Abraham
et al, 2003). Elevated levels of sPLA2 have been shown to be a significant risk factor for
presence of coronary artery disease (Kugiyama et al, 1999). PLA2 is thought to be a
circulating mediator of typhoid fever (Keuter et al, 1995).
Elevated levels of PLA2 in serum of patients:
Elevated levels of phospholipase A2 type II have been reported in the plasma of patients with
septic shock (Green et al, 1991, Nevalainen et al, 1993), Crohn’s disease (Minami et al,
1992), systemic lupus erythrematosus (Pruzanski et al, 1994) and ARDS (Arbibe, Vial &
86
Chapter 4 Introduction
Touqui, 1997, Romashin et al, 1992). Serum concentrations of PLA2-II are considerably
elevated in patients with surgical intensive care patients (Nyman et al, 1996).
PLA2 as a marker:
PLA2 is regarded as a sensitive marker for inflammatory activity in rheumatoid arthritis
(Nevalainen & Gronroos, 1997). Serum PLA2 correlates with the activity of Crohn’s disease
and ulcerative colitis (Minami et al, 1992). PLA2 type I in serum is useful in the diagnosis of
acute pancreatitis but does not aid in predicting the severity of the condition (Nevalainen,
Gronroos & Kortesuo, 1993). Enhanced PLA2-IIA expression has been correlated to poor 5
year survival in patients with prostate cancer (Graff et al, 2001, Jiang et al, 2002). Elevated
levels of secretory phospholipase A2 have been found to predict coronary events in patients
with coronary artery disease, independent of other risk factors (Kugiyama et al, 1999).
PLA2 as a eicosanoid producing enzyme:
Synovial fluid from patients with arthritis shows elevated levels of arachidonic acid and
downstream factors (PAF, leukotrienes and prostaglandins) derived from the hydrolysis by
PLA2 (Bomalaski & Clark, 1993).
PLA2 as a therapeutic target:
The inhibition of PLA2 as a therapeutic strategy has been proposed for many years. PLA2
could be directly inhibited, inactivated by dephosphorylation or indirectly regulated by
inhibiting mRNA translation of agents such as TNF or interlukins that activate PLA2
expression (Waage & Bakke, 1988). There are many considerations for the use of
phospholipase A2 inhibition as a therapeutic strategy including:
- difficulty in targeting single isoforms,
- potential disruption to essential phospholipase A2 functions (membrane remodelling,
digestion),
- the presence of multiple PLA2 receptors may mean that catalytically inactive
enzymes may still be able to act via signalling pathways (Scott, Graham & Bryant,
2003).
Recent results of the clinical trial of a type IIA selective inhibitor have shown no survival
benefit but a statistically significant dose dependent improvement in patients with sepsis
induced organ failure in the first 18 hours (Abraham et al, 2003).
Assay of Phospholipase A2.
Phospholipase A2 can be studied using biochemical enzyme activity assays based on the
hydrolysis of phospholipids. Biochemical assays are generally of limited value in the study
87
Chapter 4 Introduction
of phospholipase A2 isoenzymes in crude samples because of the difficulty in differentiating
isoforms and the low abundance of PLA2 in mammalian tissue. Conversely, biochemical
assays for PLA2 can be very sensitive and robust. PLA2 assays can be used for monitoring
protein purification progress and examining enzyme characteristics. Results obtained from
different types of PLA2 assays (vesicle assays, mixed micelle assays, synthetic or natural
membrane assays) can be difficult to compare on absolute terms, but one study showed that,
in general, different assays showed similar relative results (Ghomashchi et al, 1999).
Species differences:
Although the use of an animal model would seem advantageous to study such a complex
enzyme, there are significant limitations associated with phospholipase A2. The species
differences that exist encompass distinct enzyme characteristics, function and tissue
distribution (Dr. K. Scott, personal communication). Some examples of the species
differences in phospholipase A2 are noted below:
- In rats and humans the predominant secretory isoform is type IIA (Murakami et al,
1998), whereas in mice the major secretory isoform is type V (Chen et al, 1994 (a),
Sawada et al, 1999),
- In some strains of mice, type IIA is found only in the intestine (Sawada et al, 1999)
- Humans have a soluble form of PLA2 receptor which is not found in other animals
(Tischfield, 1997),
- Patterns of PLA2 receptors are distinctly different in rabbits and humans (Tischfield,
1997),
- Expression profiles show that genes are regulated differently in humans and rodents
(Scott, Graham & Bryant, 2003).
General PLA2 Assay difficulties:
The complexity of the kinetics of phospholipase A2 hydrolysis has ensured that this enzyme is
a popular kinetics model. Part of the complexity of the PLA2 kinetics arises from the ability
of PLA2 to act at the interface of the hydrophobic membrane and the hydrophilic extracellular
fluid. Phospholipase A2 hydrolysis is dependent on traditional enzyme activity variables (for
example, temperature, pH, cofactors, ionic strength, aqueous environment). In addition PLA2
activity is dependent on :
- substrate presentation (vesicles, micelles, bilayers),
- size of substrate aggregates (small or large vesicles),
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Chapter 4 Introduction
- presence of detergents or other enhancing agents,
- whether substrate contains one or more phospholipid species.
These variables can make interpretation of results and the comparison of results from different
assays very difficult. For example:
- type V shows a preference for choline phospholipids over ethanolamine
phospholipids when deoxycholate is present and the opposite preference when the
detergent is omitted (Chen et al, 1994),
- the production of lysophospholipid can inhibit the hydrolysis reaction (Lawrence &
Moores, 1975).
Many groups have tried to differentiate PLA2 isoforms using hydrolysis assays with different
substrates, but it is not possible to draw conclusions from the results obtained due to
significant overlap of results. Studies of PLA2 inhibitors have shown that in general, PLA2
assays show similar patterns, even though absolute results cannot be compared (Ghomaschi et
al, 1999).
Phospholipase A2 in the gastrointestinal tract:
The study of phospholipases in the human gastrointestinal tract is complicated by difficulty
obtaining tissue in suitably good condition (as the gut undergoes spontaneous autolysis after
death). In animal models it is difficult to obtain gut tissue without sacrificing the animal.
Access to human tissue is very difficult but is essential in investigations of human gut
pathologies due to significant species differences.
Biochemical PLA2 assays;
Early investigations found clear patterns in the distribution of phospholipases throughout the
gastrointestinal tract, through cross sections of small intestinal mucosa and were able to
discount a number of possible sources of the phospholipase activity. Mansbach and
colleagues (1982) showed that the phospholipase A2 in rat gut did not originate from
pancreatic, salivary or gastric secretions by diverting relevant ducts away from the small
intestine. This group also showed that the phospholipase A2 activity was not bacterial by
repeating their experiments in germ free rat lines. They also found that the phospholipase A2
activity in the small intestinal mucosa was ten fold greater in the crypts than at the villi tips.
Secretory PLA2 was also found to increase with increasing distance from the ligament of
Treitz to the ileo-cecal valve in rats (Sonnino & Pigatt, 1996).
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Chapter 4 Introduction
Type IIA Phospholipase A2 in Human Gut:
Human Paneth cells (particularly the secretory apparatus) have been shown to have strong
immunoreactivity to type IIA PLA2 (Kiyohara et al, 1992). Morita and colleagues (1999)
found that human ileal mucosa reacted with an antisynovial PLA2 antibody (and was therefore
likely to be type IIA). The Paneth cells were found to be the only cell type in the gut found
to secrete type II PLA2 mRNA (Nevalainen, Gronroos & Kallajoki, 1995). Differential
display polymerase chain reaction (DD-PCR) was used to examine genes that were
differentially expressed between the small and large intestine in mice (Keshav et al, 1997).
A secretory phospholipase A2 isoform was found using this technique (Keshav et al, 1997)
and was found to have significant homology to mouse enhancing factor and to rat and human
secretory PLA2 type II proteins.
Other phospholipase A2 isoforms in Human Gut:
In the past five years attempts have been made to study the phospholipase A2 isoenzymes in a
clear and systematic manner. With the identification of each new secretory isoform came the
probing of panels of human tissue for mRNA transcripts. In normal human gut the
transcripts corresponding to four of the isoforms have been detected (reviewed by Scott,
Graham & Bryant, 2003): namely IIA (Cupillard et al, 1997), IID (Ishizaki et al, 1999), V
(Cupillard et al, 1997) and XII-1 (Gelb et al, 2000). These results were obtained using either
RT-PCR or Northern blotting. The expression of type X in normal human gut has been
shown by one group (Suzuki et al, 2000) but not by another group (Cupillard et al, 1997).
These results are an indication of the complexity of phospholipase A2 in human gut but even
then may be an oversimplification because:
- northern hybridisation panels can be composed of a single sample (Scott, Graham & Bryant,
2003) which may not be representative of the typical pattern,
- they are an indication of the normal situation, likely to be complicated further in disease
states,
- as yet unidentified isoforms may exist (Scott, Graham and Bryant, 2003),
- isoforms expressed at very low levels may not have been detected.
In conclusion, Morita and colleagues (1999) state that there is a:
“complex involvement of several phospholipases A2 in normal gastrointestinal tissues”.
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Chapter 4 Introduction
A summary of the research conducted on the topic of phospholipase A2 involvement in gut
pathology is presented in the following tables:
91
Chapter 4 Introduction
Table 4.1. Phospholipase A2 in Intestinal Disease Research.
Group Year Disease Studygroup
Conclusions
Otamiri et al 1987 Ischaemia/reperfusion injury
Rat Increased levels of lysophosphatidylcholine in ischaemic gut mucosa compared with non-ischaemic control. Increased lysophosphatidylcholine or PLA2 may mediate mucosal injury.
Otamiri, Lindahl & Tagesson
1988 Ischaemia/reperfusion injury
Rat Mucosal injury and increased mucosal permeability seen in I/R is prevented by inhibition of PLA2.
Otamiri & Tagesson
1989 Ischemia/reperfusion injury
Rat PLA2 increased in mucosa during I/R, and was significantly different to control mucosa.
Koike et al 1992 Ischemia/reperfusion injury
Rat Gut PLA2 was increased. Inhibition of PLA2 reduced distant organ injury by preventing PMN influx, PMN superoxide production and lung leak.
Minami et al 1993 Crohn’s disease Human Levels of PLA2 type II (mRNA and protein) were higher in ileal than cecal mucosa. Increased serum levels of PLA2 seen in Crohn’s patients may be due to leakage of protein from the gut mucosa.
Minami et al 1994 Crohn’s diseaseand ulcerative colitis
Human Actively inflamed mucosa of Crohn’s disease showed higher levels of PLA2 activity and type II immunoreactivity. Severely inflamed mucosa of ulcerative colitis patients showed higher levels of PLA2 and type II immunoreactivity than control mucosa.
Koike et al 1995 Ischemia/reperfusion injury
Rat PLA2 activated by I/R. Remote organ damage caused by I/R could be reduced with a PLA2 inhibitor
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Chapter 4 Introduction
Lilja et al 1995 Crohn’s disease Human Increased levels of PLA2 may contribute to
inflammation in Crohn’s disease and in recurrent Crohn’s after resection. Increased levels of mRNA of type I, II and cytosolic PLA2 observed, but type II was predominant.
Lilja et al 1995 Crohn’s disease Human Increased levels of PLA2 may contribute to inflammation in Crohn’s disease and in recurrent Crohn’s after resection. Increased levels of mRNA of type I, II and cytosolic PLA2 observed, but type II was predominant.
Nevalainen, Gronroos & Kallajoki
1995 Crohn’s diseaseand ulcerative colitis
Human Paneth cells were the only cells that showed mRNA for type II PLA2. Type II immunoreactivity in blood vessel walls likely to reflect transport of the protein rather than synthesis.
Qu et al 1996 LPS infusion Rat PLA2 release from Paneth cells was able to be triggered by LPS.
Sonnino et al 1997 Graft preservation Rat Calcium dependent, secretory PLA2 accumulated rapidly during preservation. PLA2 likely to be proinflammatory and a priming agent for intestinal tissue damage.
Arcuni et al 1999 Graft preservation Rat PLA2 released in response to other proteins, perhaps cytokines, but not due to cell death. PLA2 inhibition improves the outcome of preservation.
Haapamaki et al 1999 Crohn’s disease
Human Columnar epithelial cells in Crohn’s mucosa responsible for the synthesis of PLA2-II in colon and small intestine, but not in control mucosa.
Morita et al 1999 Inflammatorybowel disease
Human Isoform hydrolysing PE more closely related to inflammation than the PC hydrolysing PLA2 which was found irrespective of inflammation.
Table 4.1 continued. Phospholipase A2 in Intestinal Disease Research.
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Chapter 4 Introduction Table 4.1 continued. Phospholipase A2 in Intestinal Disease Research.
Koike et al 2000 Ischemia/reperfusion injury
Rat PLA2 activity in portal circulation was 10 fold higher than systemic circulation, suggesting that the serum PLA2 activity arises from ischaemic gut.
Weifeng et al 2001 Ischaemia/reperfusion injury
Rat Inhibition of PLA2 early in the ischaemia/reperfusion process decreased bacterial translocation, endotoxin release, pro-inflammatory mediator and cytokine levels. This reduced the injury to remote organs.
Thomas, Karnik & Balasubramanian
2002 Surgicalmanipulation of intestine.
Rat Inhibitors of PLA2 were protective against lung damage which may be due to reduced neutrophil infiltration, reduced oxidative stress or reduced permeability.
94
Chapter 4 Introduction
95
Results
Chapter 4: Phospholipase A2 activity in ischaemic/infarcted human bowel:
Previous research had detected phospholipase A2 in bowel and had identified the
major isoform as type IIA (Kiyohara et al, 1992, Morita et al, 1999, Nevalainen,
Gronroos & Kallajoki, 1995). There is some evidence that suggests that a complex
pattern of phospholipase proteins in the bowel (Morita et al, 1999, Scott, Graham &
Bryant, 2003). As a preliminary control experiment, immunodepletion was
conducted to remove the phospholipase A2 isoform type IIA and its closely related
isoform type V from a recombinant type IIA standard (Figure 4.3). Immunodepletion
of phospholipase A2 types IIA and V from human bowel showed that types IIA/V
were present in infarcted human bowel content (Figure 4.4), but did not account for
all of the phospholipase A2 activity (Figure 4.5).
The phospholipase A2 activity was measured in several patients with matched tissue
homogenate and bowel lumen content (Figure 4.6). These results are expressed as
specific activity of phospholipase A2 due to the large variation of total protein
between these samples. Phospholipase A2 activity was significantly increased in the
lumen content (Fisher’s exact test, p=0.048), compared with the matched tissue
homogenate. On one occasion ischaemic (tissue damage rating 2-3) and infarcted
tissue (tissue damage rating 3) were obtained from the one patient, and the infarcted
tissue showed significantly higher phospholipase A2 activity (p<0.0005), (Figure 4.7).
The tissue damage rating and the PLA2 activity were compared in several samples of
bowel tissue homogenate (Figure 4.8). The next section details the purification of the
protein responsible for this phospholipase A2 activity (Chapter 5).
96
97
Type IIA standard
0102030405060
Non depleted 4A1 10B2
PLA 2
act
ivity
, %
hyd
roly
sis
Figure 4.3 Immunodepletion of PLA2 from recombinant PLA2-IIA
protein with antibodies 4A1 (type IIA specific) and 10B2 (type IIA+V
specific). Results are expressed as the mean +/- standard error of
triplicate assays. The recombinant protein was a generous gift from
Dr. Kieran Scott.
98
Protein size, (kDa)
1 2 3 4
22
98148
Albumin
36
16 sPLA2
Figure 4.4 Silver stained gel of the antibody-bead-PLA2 complex after
immunodepletion from bowel samples. SDS-PAGE gel: 4-20% Tris Glycine
gel, Novex.
Lanes:
1- See Blue Plus2 markers.
2- immunodepletion with 4A1 antibody.
3-immunodepletion with 10B2 antibody.
4-recombinant sPLA2-IIA standard.
99
Mar
kers
sPLA
2
type
IIA
imm
un
odep
lete
d
Rep
eate
d de
plet
ion
53
34
23
17
0
5
10
15
20
25
Before depletion After depletion
PLA 2
act
ivit
y,
% h
ydro
lysi
s
Figure 4.5 Western blotting of immunodepleted bowel autolysis
sample, and phospholipase A2 activity before and after
immunodepletion of phospholipase A2 isoforms IIA and V.
The arrow indicates the immunoreactive protein of interest.
100
0
500
1000
1500
A B C D EPatient
Spec
ific
activ
ity (%
hy
drol
ysis/
min
/mg
prot
ein)
T issue homogenate Bowel lumen content
Figure 4.6 PLA2 activity in matched homogenised bowel tissue and
lumen content. These results are significantly different, Fisher’s
exact test, two tailed p=0.048. Fisher’s exact test was chosen due to
the small number of samples. These results are presented as the
mean +/- standard error of triplicate assays.
101
0
12
34
56
7
Infarcted tissue Ischaemic tissue
PLA 2
act
ivit
y (%
hyd
roly
sis)
Figure 4.7 Two homogenised bowel tissue samples from same
patient. The results are presented here as the mean +/- standard error
of triplicate assays. The tissue damage rating for these tissues was 3
for the infarcted tissue and 2-3 for the ischaemic tissue, using the
scale published by Sun et al, 1997.
102
020406080
100
a b c d e f g
PLA
2 ac
tivity
, %
hy
drol
ysis
1
2
3
Tiss
ue d
amag
e ra
ting
phospholipase activityTissue rating
Figure 4.8 Tissue damage rating of Sun et al, 1997 and corresponding
PLA2 activity of various homogenised bowel tissue samples. These
results are presented as the mean +/- standard error of at least triplicate
assays.
Discussion
Chapter 4 Discussion:
Type IIA PLA2 had been described in the bowel by many other groups, using a
variety of techniques (immunohistochemistry, in situ hybridisation, differential
display PCR). Type V PLA2 is another secretory PLA2 isoform and shares very
similar enzyme characteristics to type IIA. Type V PLA2 has been detected at low
levels in normal human small bowel by Northern blotting (Cupillard et al, 1997). In
this study, we chose to investigate phospholipase A2 at the protein level, as we wanted
to focus on the activity of the enzyme and were concerned about mRNA degradation
that may occur as a result of bowel injury. Immunoprecipitation of secretory
phospholipase A2 types IIA and V from bowel samples was conducted. The
immunoprecipitation experiment was verified by duplicating the same set of
conditions using recombinant human type IIA PLA2 as the enzyme source. The
antibodies 4A1 (IIA specific) and 10B2 (cross reactive with types IIA and V) were
used. As expected, phospholipase A2 types IIA/V were found to be present in human
bowel samples as shown by silver stained gels of the proteins attracted to the resin-
immobilised antibodies. Also as expected, type V PLA2 is likely to be a minor PLA2
isoform in human bowel, as the difference between the enzyme activity remaining
after depletion with either antibody 4A1 or 10B2 was very similar. The more
surprising but interesting result was that removal of types IIA/V from the bowel
samples failed to abolish all of the phospholipase enzyme activity. An average of
approximately one third of the phospholipase activity remained after removal of types
IIA/V phospholipase A2. This result was not simply due to an inadequate amount of
antibody, as when an additional ten fold excess of immobilised antibody was used, the
enzyme activity remained. The immunoprecipitated samples were analysed by
Western blotting and the remaining immunoreactive band appeared to be of low
molecular weight. The gels for Western blotting were run under non-reducing
conditions, as the epitope recognised by the antibody 4A1 is reported to be lost upon
reduction (Rice et al, 1998). A protein purification process was then undertaken with
the aim of identifying the protein responsible for this unexpected phospholipase
activity (Chapter 5).
We were fortunate to have access to human bowel excised during surgery to examine
the phospholipase activity. Access to human bowel tissue in suitably good condition
can be difficult due to post mortem autolysis. We had access to infarcted small
103
Discussion
bowel tissue and lumen contents, and also to normal small bowel from patients
undergoing right hemicolectomy. The normal bowel samples were always from the
terminal ileum. The samples of infarcted bowel were obtained from a number of
regions along the length of the resected small bowel. As phospholipase activity is
reported to increase along the length of the small intestine (a study in rats, Sonnino et
al, 1996) we did not try to compare all of the absolute results between samples.
Samples of tissue and lumen content were frozen as soon after surgery as practical.
Phospholipase activity has been reported to remain constant with freezing and
extended storage. For example, the phospholipase A2 from peritoneal exudate was
reported to be stable for four months at 4oC (Chang et al, 1987). Our investigations
also verified that the phospholipase activity was stable with storage at –80oC for
several months and also with repeated freeze-thaw cycles (results not shown).
The study of phospholipase A2 in the bowel has generally focussed on normal rodent
bowel tissue. Human bowel phospholipase A2 research has typically focussed on
normal tissue, at the RNA level, due to the difficulties obtaining sufficient diseased
tissue for protein studies (for example, biopsy samples), and the complexity of this
protein family. Few publications, particularly in these past decade, have been
reported that combine:
- human bowel tissue
- phospholipase at the protein level
- diseased tissue
- bowel ischaemia/reperfusion injury.
We have chosen to investigate phospholipase enzyme activity, at the protein level in
normal and ischaemic/infarcted human bowel with the aim of better understanding the
role of this disease associated protein.
On a number of occasions, matched infarcted bowel tissue and lumen content were
obtained from the same patient. The phospholipase activity in the lumen was
significantly higher than its corresponding tissue homogenate. This may reflect the
release of the enzyme into the bowel lumen from a storage site in the tissue, and may
provide a route for entry into general circulation. This idea is supported by evidence
that intestinal permeability increases in response to ischaemia and infarction, and
results in the release of gut derived factors into circulation (Kong et al, 1998).
104
Discussion
Alternatively, the enzyme may be actively synthesised and released into the lumen in
response to the ischaemic damage and may subsequently enter circulation. The
alternate possibility is that the presence of increased phospholipase activity in the
lumen simply reflects cell death and therefore leakage of the protein. A study
investigating the release of phospholipase into bowel preservation fluid concluded
that the release of the enzyme was not due to leakage of protein from dying cells
(Sonnino et al, 1997). This group provided data showing the absence of release of
lactate dehydrogenase to support their argument (Sonnino et al, 1997). An approach
like this would be valuable to assess whether the increased phospholipase activity is
due to cell death, or active release, and requires further elucidation.
The increased phospholipase activity observed as a consequence of tissue injury,
infection or inflammation may be a beneficial response to the patient in a number of
ways. Phospholipase A2 is known to have anti-bacterial properties (Nevalainen,
Haapamaki & Gronroos, 2000). Phospholipases are likely to aid in the removal or
rebuilding of peroxidised or damaged phospholipids (Vadas et al, 1993).
Conversely, phospholipase hydrolysis products have many tissue and cell damaging
effects, and inflammatory properties, as discussed in the introduction to this chapter
(Figure 4.2). For example, lysophospholipids are cytotoxic (Schoenberg & Beger,
1993). The roles of phospholipase A2 and its corresponding hydrolysis products in
inflammation, infection and tissue injury require detailed investigation. Conceivably
the important concept is the balance between the beneficial roles and damaging
effects of phospholipase during disease development.
On one occasion two bowel tissue samples were obtained from one patient, each
showing a different level of ischaemic damage. It is not unusual for the bowel to
show areas of severe damage and areas of mild damage in close proximity because of
the arrangement of the blood supply to the bowel. The infarcted tissue was given a
tissue damage rating of 3, according to the scale proposed by Sun and colleagues
(1997). The infarcted tissue showed significantly higher phospholipase activity than
the adjacent ischaemic tissue (tissue damage rating of 2-3). This is an interesting
finding (even though this is sample size of only one) as there may be a positive
relationship between the severity of tissue damage (from normal to severe infarction)
and the phospholipase activity of the tissue homogenate. Many biomarkers of bowel
105
Discussion
infarction have been found to appear too late to be clinically useful, or decrease
before the condition has been rectified.
106
Chapter 5 Introduction
Chapter 5: Protein purification.
Purification of non-pancreatic Phospholipase A2 from mammalian tissue:
The primary structure of pancreatic phospholipase A2 was reported by Puijk, Verheij
& de Haas (1977). Venom and pancreatic tissue contain high amounts of
phospholipase A2 (typically 1-10% of total protein) (Verheij, Slotboom & de Haas,
1981). In 1982, a phospholipase A2 of similar size to the pancreatic isoform was
isolated from the ileum of pigs, and was found to have distinctly different
characteristics (N-terminal amino acid sequence, isoelectric point, high proportion of
basic to acidic amino acid residues)(Verger et al, 1982). Extracellular phospholipase
A2 found at the sites of inflammation sparked great interest. A soluble phospholipase
A2 from the synovial fluid of patients with rheumatoid arthritis was purified and its
characteristics were found to be distinct from the pancreatic isoform (Stefanski et al,
1986). Phospholipase A2 is thought to be central in the eicosanoid biosynthetic
pathway due to the release of arachidonic acid. The purification of secretory
phospholipase A2 from rheumatoid arthritis synovial fluid (to obtain sequence data or
amino acid composition) was achieved almost simultaneously by three groups (Hara
et al, 1989, Kramer et al, 1989, Seilhamer et al, 1989). The latter two groups also
reported the identification of the N-terminal amino acid sequence:
Group: Source of PLA2: Manuscript
submitted:
Amino acid sequence
published:
Kramer et al,
1989
Platelets and
Rheumatoid
arthritis synovial
fluid
December 20,
1988
NLVNFHRMIKLTTGKEAAL
Hara et al, 1989 Rheumatoid
arthritis synovial
fluid
September 28,
1988
NO SEQUENCE DATA.
Seilhamer et al,
1989
Rheumatoid
arthritis synovial
fluid
December 27,
1988.
?LVNFHRMIKLTTGKEAAL
107
Chapter 5 Introduction
Type II phospholipase A2 has been purified to homogeneity and at least partial amino
acid sequence determined from:
Normal animal tissues/fluids:
Porcine ileum (Verger et al, 1982).
Stimulated rat platelets (Hayakawa et al, 1987).
Rat liver (Aarsman et al, 1989).
Rat spleen (Ono et al, 1988).
Normal human tissues/fluids:
Platelet concentrate (Kramer et al, 1989).
Spleen (Kanda et al, 1989).
Ileal mucosa (Minami et al, 1993).
Diseased animal tissue/fluids:
In models of peritonitis:
-rabbit ascitic fluid treated with glycogen IP (Forst et al, 1986).
-rat peritoneal fluid treated with casein IP (Chang et al, 1986, Chang et al,
1987).
Diseased human tissue/fluids:
Synovial fluid from rheumatoid arthritis patients (Lai & Wada, 1988).
Synovial fluid from arthritis patients (Kramer et al, 1989).
The purification schemes used in the research cited above generally involved the use
of a series of different chromatography columns (ion exchange, hydrophobic
interaction, gel filtration, affinity, RP-HPLC). The purification processes have
monitored phospholipase A2 using activity assays of column fractions at all stages of
the purifications. These purification schemes are laborious, time consuming and
expensive.
The purification of phospholipase A2 from animal tissues or fluids can be difficult due
to the low abundance of the protein in samples and unusual characteristics such as
tendency to adhere to glass and some column packings. The number of isoforms,
difficulty in differentiating isoforms and lack of many antibodies further compound
these problems. Phospholipase A2 enzymes have some characteristics that are
advantageous during purification, such as heat and acid stability. Phospholipase A2
108
Chapter 5 Introduction
enzymes often have characteristics that are quite different to the majority of proteins
in a sample, such as isoelectric point. The majority of proteins have acidic to neutral
isoelectric points (pI 4-7) whereas many phospholipase A2 isoforms tend to have basic
isoelectric points.
Heparin Sepharose affinity chromatography:
Heparin sepharose chromatography can be used to purify several proteins types based
on affinity binding and ion exchange interactions. Heparin affinity is exhibited by
many coagulation factors, enzymes, growth factors and receptor proteins (reviewed by
Farooqui, Yang & Horrocks, 1994). Affinity chromatography is advantageous due to
its ability to quickly bind proteins of interest and elute the majority of contaminating
proteins. The specifically bound proteins can be washed extensively and eluted in
suitable buffers. The affinity of a protein to a ligand such as heparin can be quite
specific and slight changes in the elution conditions can allow very narrow sample
fractions, and therefore high purity fractions.
Heparin is a group of naturally occurring glycosaminoglycans. The heparin members
vary greatly in size (5 to 40 kDa) but share characteristics such as being highly
charged, highly sulphated, and composed of disaccharide repeating units of
glucuronic acid and D-glucosamine (Dua & Cho, 1994). In vivo, heparin occurs only
in mast cells (Farooqui, Yang & Horrocks, 1994).
The attraction of proteins to immobilised heparin in a chromatography situation is
based on either specific interactions or electrostatic interactions (Farooqui, Yang &
Horrocks, 1994). It has been proposed that the specific binding of a protein to heparin
is due to a cluster of cationic residues on the outer surface of an amphiphilic α helix
(Dua & Cho, 1994). Heparin can be immobilised on resin such as sepharose or
agarose, and packed in columns for affinity purification of proteins. The proteins
with an affinity for the heparin are retained on the column and the rest of the material
is washed through. The proteins with heparin affinity can then be eluted with an
excess of heparin or changing the elution buffer composition with salts or denaturants
(Farooqui, Yang & Horrocks, 1994). There are some considerations for the use of
heparin in protein purification, firstly the affinity of a particular protein to the heparin
109
Chapter 5 Introduction
may be too high, meaning that protein is lost with each chromatographic run.
Secondly, heparin is known to inhibit some classes of protein, though this is generally
reversible. Heparin (at levels used for anticoagulation) reversibly inhibits
phospholipase A2 activity. For this reason, serum is preferable in assays when
undiluted samples are used (such as antibacterial plate assays). In highly sensitive
assays where diluted samples are used, both plasma and serum are suitable
(Weinrauch et al, 1998). Affinity columns such as heparin columns can become
fouled with protein build-up, but columns can be cleaned with a variety of agents to
prevent this. Eluted fractions can contain high concentrations of salts and may
require dialysis before further purification steps.
Heparin sepharose has been used in the purification of a variety of lipases, kinases
and phospholipases (reviewed by Farooqui, Yang & Horrocks, 1994). In the case
of phospholipase A2, the enzyme can be eluted from the column with increasing
concentrations of salts (KCl, NaCl) and thus retain native confirmation and enzyme
activity. Several of the secretory isoforms of phospholipase A2 have high affinity for
heparin (IIA, IID, IIE), while others have less affinity (IB, IIF, V), (see Table 5.2).
The strength of the affinity of phospholipase A2 isoforms generally follows the degree
of basicity, therefore in general isoforms with high pI values have higher heparin
affinity (Murakami et al, 2001), see Figure 5.7. Heparin binding of phospholipase A2
is due to a cluster of 5 to 7 C-terminal amino acid residues (Arni & Ward, 1996,
Murakami, Nakatani & Kudo, 1996), see Figure 5.8. The cluster of amino acids
responsible for heparin binding may only be evident on examination of the three
dimensional structure (Koduri et al, 1998). The heparin binding site is also a
substrate binding region, but is independent of active site residues (Dua & Cho,
1994).
Purification schemes
Several phospholipase A2 purification schemes have employed a heparin sepharose
column, usually preceeded by a crude sample clean-up step and followed by one or
more RP-HPLC columns.
110
Chapter 5 Introduction
Table 5.1 Purification of PLA2 by Heparin Affinity Chromatography:
Author(s) Year Source of PLA2 Purification scheme
Hara et al 1989 Human rheumatoid
arthritis synovial fluid
Heparin sepharose.
Hydrophobic interaction.
RP-HPLC.
Diccianni et al 1991 Porcine pancreatic PLA2
(Sigma)
Heparin Affigel.
Kim, Kudo &
Inoue
1991 Rabbit platelet enriched
plasma, cytosolic PLA2
Heparin sepharose.
Ion exchange.
Hydrophobic interaction.
Ion exchange.
Gel filtration.
Murakami et al 1998 Culture supernatent
CHO/HEK stable
transfectants.
Heparin sepharose.
Shimbara et al 1999 Baculovirus derived
recombinant rat and
mouse PLA2.
Heparin Sepharose.
Anti-sPLA2-IIA
immunoaffinity column.
111
Chapter 5 Introduction
Table 5.2. Heparin affinity of Phospholipase A2 Isoforms: Phospholipase A2 isoform Heparin affinity
(Concentration of NaCl
required for elution)
Isolectric point (Human
isoform), calculated from
the Swiss Prot database)
IB No binding 7.95
IIA 0.8M 9.38
IID 40% recovery at 1M 8.75
IIE 1M 8.53
IIF No binding 4.51
III* Unknown 9.1
V 0.4M 8.72
X No binding 5.1
XII Unknown 6.3
The Rotofor ® preparative isoelectric focussing system:
The BioRad Rotofor® cell is a liquid phase preparative isoelectric focussing
apparatus characterised by the short length of time required for separation
(approximately 4 hours for an average analysis). The Rotofor® cell has been
successfully used in the study of proteome of cerebrospinal fluid, specific enzymes in
whole plasma, purification of haemoglobin variants A, C and F, and separation of
peanut lectin isoenzymes.
Liquid based isoelectric focussing such as the Rotofor® cell establish a pH gradient
with carrier ampholytes in a liquid medium containing a low concentration of
detergent. Urea and denaturants such as DTE can be added as required. Urea can be
added to aid in the prevention of precipitation. Glycerol can be added to maintain
solubility and stability of proteins. A constant current is then applied which causes
proteins and ampholytes to migrate toward the anode or cathode according to their net
charge until they reach their pI. The liquid fractions are collected simultaneously and
quickly to avoid loss to the pH gradient using vacuum. The fractions can then be
*Mammalian sPLA2 type III occurs as a central PLA2 like domain flanked by two protein domains of unknown function. The central sPLA2 like domain has no heparan sulphate proteoglycan binding (Scott, Graham & Bryant, 2003).
112
Chapter 5 Introduction
analysed for pH, total protein, a specific target protein or run on one or two
dimensional gels. The use of a liquid phase for the protein separation is
advantageous as there is no need for elution from a solid medium. Another
advantage of liquid based IEF is that toxic polymerisation agents are avoided and
ampholyes could be omitted in favour of creating the pH gradient by ‘seeding’ the
IEF fluid with a pure protein of interest. This may have potential in the
pharmaceutical industry for production of purified proteins (Righetti, Wenisch &
Faupel, 1989).
Advantages of Preparative IEF:
The advantages of preparative isoelectric focussing include the retention of biological
activity, a high degrees of purification in a single step and flexibility in terms of
sample type and size. Isoelectric focussing is capable of high resolution separations,
is able to concentrate the protein of interest and defines a physical parameter of the
protein. The use of the Rotofor ® as a prefractionation step before 2DE has been
reported to improve the mass spectrometry sequence coverage in comparison to 2DE
alone and aids in the identification of low abundance and post translationally modified
proteins (Westman-Brinkmalm & Davidsson, 2002).
Disadvantages of Preparative IEF:
The introduction of ampholytes can be disadvantageous. Ampholytes can be difficult
to remove (Bloomster & Watson, 1983) and can interfere with post separation
analysis. Ampholytes can be removed with dialysis, gel filtration, ultrafiltration, ion
exchange chromatography or protein precipitation, though removal may be
incomplete. It has been suggested that ampholytes may bind irreversibly with
proteins, but Baumann and Chrambach (1975) found that this was not the case.
Considerations for the use of Preparative IEF:
The pH gradient in a liquid phase isoelectric focussing apparatus is achieved with a
mixture of carrier ampholytes. Ampholytes are small, amphoteric molecules that
have a high buffering capacity centered at their pI. Because ampholytes behave in a
similar fashion to proteins or peptides, care must be taken to avoid interference in
protein analysis. For example, carrier ampholytes will interfere with total protein
113
Chapter 5 Introduction
assays and give inflated estimates of protein concentrations (Bloomster & Watson,
1983). Ampholytes are incompatible with the commonly used total protein assays
bicinchoninic acid (BCA) (Brown, Jarvis & Hyland, 1989), Bradford and Lowry
assays (Guttenberger, Neuhoff & Hampp, 1991). The precipitation of the protein or
immobilisation of the protein on a membrane can overcome the interference by
ampholytes.
Detergent is added to the isoelectric focussing buffer to solubilise the proteins and to
prevent aggregation. The detergent used must carry no net charge (non-ionic or
zwitterionic) to enable the detergent to remain in the system under high currents.
Detergents may interfere with some biological assays, for example total protein assays
or those based on lipid or membrane substrates. The interference of detergent in total
protein assays can be overcome by precipitating or immobilising the protein and
thoroughly washing the sample. The interference in other assays can be more
difficult to overcome.
Some proteins are inherently unstable and may not tolerate complicated, multiple step
purification procedures. The isoelectric focussing procedure can lead to protein loss
for the following reasons:
-proteins may be unstable at their pI and may precipitate
-proteins may not tolerate very dilute solutions
-the detergent, reductants etc may alter the biological activity or structure of the
protein
114
Results
Chapter 5: Protein purification:
The protein of interest was purified from bowel tissue rather than from blood because
the tissue showed higher specific activity for phospholipase A2, was available in
larger quantities and was more likely to express a tissue specific protein. The
separation techniques were chosen because they retained the native enzyme activity,
exploited an unusual protein characteristic and removed large amounts of
contaminating protein at each step. The purification progress was monitored by
analysis of all fractions for PLA2 activity with a 14carbon labelled phospholipid mixed
micelle assay.
Unfortunately, a number of preliminary purification processes, each with different
separation steps, resulted in the identification of haemoglobin as the protein of
interest. These purification schemes are summarised in Figures 5.1 to 5.3. To verify
that haemoglobin was not responsible for the phospholipase A2 activity, a variety of
concentrations were assayed for activity (Figure 5.4), and, as expected, none showed
PLA2 activity. The absence of PLA2 activity in haemoglobin was verified by
comparison to ultrapure water, the diluting buffer used (acetate) and an equivalent
concentration of albumin (Figure 5.5). To show that haemoglobin did not
synergistically enhance phospholipase A2 activity, various amounts of haemoglobin
were incubated with pancreatic phospholipase A2 and then assayed for enzyme
activity (Figure 5.6).
Heparin sepharose affinity chromatography was performed using a step gradient of
sodium chloride to elute bound proteins. The major peak of total protein was eluted
from the chromatography column with 0.05M sodium chloride (Figure 5.9 and 5.10).
The majority of phospholipase A2 enzyme activity required 0.5M sodium chloride for
elution (Figure 5.10). The fractions with the highest phospholipase A2 activity were
then pooled for the next purification step. A step gradient of the elution buffers was
used, and 20 column volumes were routinely employed due to the low contaminating
protein levels that resulted (Figure 5.11). Due to the high salt concentration of the
heparin sepharose column fractions, the pooled fractions were dialysed against
phosphate buffer to reduce the molarity to 10mM. The dialysis step did not reduce
the enzyme activity of the samples (Figure 5.12).
115
Results
Preparative isoelectric focussing was performed using the Rotofor® system. A
preliminary experiment was conducted to confirm that the isoelectric focussing buffer
(detergent, ampholytes, glycerol) did not interfere with the phospholipase A2 activity
assay (Figure 5.13). The Rotofor® experiments were run until the power output had
stabilised for an hour (Figure 5.14). The fractions obtained from Rotofor®
experiments were analysed for pH, phospholipase A2 activity (Figure 5.15) and were
run on reducing SDS-PAGE (Figure 5.16). Five consecutive Rotofor® experiments
were conducted to investigate the reproducibility of the system, which was found to
be very good (Figure 5.17). Gels of typical fractions show that the majority of the
contaminating protein focussed in the neutral to acidic range of isoelectric points
(Figure 5.18), while the phospholipase A2 activity focussed in the basic region (Figure
5.15). The active fractions from a representative Rotofor® experiment were pooled
and re-fractionated (Figure 5.18), which allowed a better estimation of the isoelectric
point of the protein of interest (~9.7). The re-fractionation experiment extended the
number of fractions with a pH of between 7.5 and 10 (Figure 5.19). The basic
fractions of the re-fractionation experiment were run on SDS-PAGE, but no protein
band was evident of the expected size (Figure 5.18), and for this reason, a scaled up
purification was undertaken to obtain enough protein for identification .
Active fractions from a Rotofor® run were pooled and run under non-reducing
conditions, to retain phospholipase A2 activity. Proteins were electro-eluted from the
gel and analysed for phospholipase A2 activity. Pre-stained protein molecular weight
markers were run at the same time and their mobilities were used to construct a
standard curve (Figure 5.20). Enzyme activity was present in fractions 8&9,
corresponding to a protein of between 16 and 20 kilodaltons. The pH of optimal
enzyme activity was 9 (Figure 5.21). An overview of the purification scheme is
presented in Figure 5.22, and this represents an approximately 9700 fold purification.
The scaled up purification system resulted in highly purified material for protein
identification. A typical silver stained gel of active Rotofor® fractions is shown in
Figure 5.23, and this indicates that the protein was >95% pure. The fractions
exhibiting phospholipase A2 activity matched well with the fractions exhibiting a
protein band at approximately 21 kDa. The active fractions were pooled and
116
Results
concentrated by Vivaspin centrifugal cartridges. These cartridges appeared to
concentrate the protein of interest more effectively than ethanol precipitation,
although the latter was superior for removing ampholytes (Figure 5.24). The active
fractions concentrated using the Vivaspin cartridges were run on two different gel
systems, (BisTris, Invitrogen and TrisGly, Gradipore), digested with trypsin and
analysed by mass spectrometry. Positive (various protein markers) and negative (gel
pieces from no-protein lanes) controls were also digested and analysed in the same
manner. The unknown protein from the BisTris system was identified as peptidyl
prolyl isomerase/cyclophilin B (Figure 5.26). The positive control from the BisTris
system was correctly identified as lysozyme (Figure 5.26). The unknown protein
from the TrisGly system was also identified as peptidyl prolyl isomerase/cyclophilin
B (Figure 5.27). The positive control from the BisTris system was correctly
identified as restriction endonuclease (Figure 5.27). The presence of cyclophilin B in
a variety of human samples was then investigated (Chapter 6).
117
Results
118
118
Bowel content samples
Ammonium sulphate precipitation
Alkaline urea gel
Gel slices assayed for PLA2 activity.
Electrotransfer of proteins to PVDF membrane.
Region corresponding to PLA2 activity submitted to N-terminal sequencing.
Results: proteins identified as Haemoglobin α and β.
Figure 5.1
Purification scheme 1.
119
Bowel content samples
Heparin sepharose chromatography
Fractions with highest PLA2activity retained.
Alkaline urea gel to verify mobility of PLA2 activity.
Concentrated ten fold (SpeedVac).
SDS-PAGE and electrotransfer of proteins to PVDF membrane.
N-terminal sequencing
Results: proteins identified as Haemoglobin α and β.Figure 5.2
Purification scheme 2.
120
Bowel content samples
Heparin sepharose chromatography
Fractions with highest PLA2activity retained.
Preparative isoelectric focussing (Rotofor®).
Fractions with highest PLA2 activity retained.
Concentrated up to three fold (SpeedVac).
SDS-PAGE, silver stained, trypsin digest of protein bands.
Nano ES/MS/MS
Figure 5.3
Purification scheme 3.
Results: proteins identified as Haemoglobin β.
121
0
1
2
3
4
5
12.5 25 50
Haemoglobin concentration (micromolar).
PLA 2
act
ivit
y, %
hyd
roly
sis.
Figure 5.4: The lack of phospholipase A2 activity of
haemoglobin. Haemoglobin (human, Sigma) was
diluted in acetate buffer (pH 5.5). These results are
presented as the mean +/- standard error of
triplicate assays.
122
0
1
2
3
4
5
Haemoglobin Albumin Water Acetate buffer
PLA 2
act
ivit
y, %
hyd
roly
sis.
Figure 5.5: The lack of phospholipase A2 activity in
haemoglobin (25µM), bovine serum albumin (25µM), ultrapure
water and acetate buffer (pH 5.5).
123
0
20
40
60
80
100
0 12.5 25 50
Haemoglobin concentration (micromolar).
PLA 2
act
ivity
, % h
ydro
lysi
s.
Figure 5.6: Pancreatic phospholipase A2 (0.1µg/mL
containing 1mg/mL BSA, 10mM Tris) activity with
various amounts of added haemoglobin.
124
R2 = 0.75
0
2
4
6
8
10
0 0.5 1 1.
NaCl concentration (M) required for elutionis
oele
ctri
c po
in5
t
R2 = 0.84
0
2
46
8
10
12
0 0.5 1 1.5
NaCl concentration (M) required for elution
pI o
f 25
C-t
erm
inal
res
idue
s
R2 = 0.77
0
2
4
6
8
10
0 0.5 1 1.5
NaCl concentration (M) required for elution
Num
ber o
f bas
ic re
sidu
es in
25
C-te
rmin
al a
min
o ac
ids
a)
b)
c)
Figure 5.7: Heparin binding of PLA2 isoforms.
The sodium chloride concentration required to elute the PLA2 isoform from
heparin sepharose shows high correlation to the isoelectric point (pI) of
the isoform (Murakami et al, 2001, graph a) and to the pI (graph b) and
number of basic residues in the C-terminal portion of 25 amino acid
residues (graph c). Characteristics of the isoforms IIA, IID, IIE, IIF, V &
X were used to construct these graphs.
125
Figure 5.8: Sequence Alignment of Secretory Phospholipase A2 isoenzymes &
Heparin binding regions. The C-terminal 25 amino acids shown in red. The
basic residues in the C-terminal amino acids are underlined. Additional
amino acids that are likely to be involved in heparin binding are shown in blue
(Koduri et al, 1998).
Isoform
IIA
NLVNFHRMIKLTTGKEA-ALSYGFYGC ALSYGFYGC--HCGVGGR-GS-
IID
GILNLNKMVKQVTGKMP-ILSYWPYGC ILSYWPYGC--HCGLGGR-GQ-
IIE
NLVQFGVMIEKMTGKS—ALQYNDYGC ALQYNDYGC--YCGIGGS-HW-
IIF
SLLNLKAMVEAVTGRSA-ILSFVGYGC ILSFVGYGC--YCGLGGR-GQ-
V
GLLDLKSMIEKVTGKNA-LTNYGFYGC LTNYGFYGC--YCGWGGR-GT-
X
GILELAGTVGCVGPRT—PIAYMKYGC PIAYMKYGC--FCGLGGH-GQ-
IIA
PKDATDRCCVTHDCCYKRLEKRG-CGTKFLSY----KF SNSGS-RITCAKQ-----
IID
PKDATDWCCQTHDCCYDHLKTQG-CSIYKDYY----RY NFSQG-NIHCSDK-G---
IIE
PVDQTDWCCHAHDCCYGRLEKLG-CEPKLEKY----LF SVSER-GIFCAGR-----
IIF
PKDEVDWCCHAHDCCYQELFDQG-CHPYVDHY----DH TIENNTEIVCSDLNK---
V
PKDGTDWCCWAHDHCYGRLEEKG-CNIRTQSY----KY RFAWG-VVTCEPG-----
X
PRDAIDWCCHGHDCCYTRAEEAG-CSPKTERY---SWQ CVNQS--VLCGPA----
IIA
DSCRSQLCECDKAAATCFA RNKTTYNKKYQYYSNKH-CRGST P---------RC
IID
SWCEQQLCACDKEVAFCLK RNLDTYQKRLRFYWRPH-CRGQT P---------GC
IIE
TTCQRLTCECDKRAALCFRRNLGTYNRKYAHYPNKL-CTGPT P---------PC
IIF
TECDKQTCMCDKNMVLCLMN—QTYREEYRGFLNVY -CQGPT P---------NCSIY EPPPEEVTCSHQSPAPPAPP
V
PFCHVNLCACDRKLVYCLKRNLRSYNPQYQYFPNIL-CS--
X
ENKCQELLCKCDQEIANCLAQ--TEYNLKYLFYPQFL-CEPDS P---------KC
126
127
00.10.20.30.40.50.6
1 4 7 10 13 16 19 22 25 28 31Fraction
NaC
l co
ncen
trat
ion
(M)
0
200
400
600
800
1000
Tota
l pro
tein
(u
g/m
L)
NaCl concentration (M) Total protein (ug/mL)
Figure 5.9: Typical elution of the total protein a homogenised
infarcted bowel sample from a heparin sepharose column. The
majority of the total protein eluted with 0.05M NaCl.
128
Phospholipase A2 activity
NaCl 0M
NaCl 0.05MNaCl 0.1M
NaCl 0.2M
NaCl 0.5M
Total protein
NaCl 0M
NaCl 0.05M
NaCl 0.1M
NaCl 0.2M
NaCl 0.5M
Figure 5.10: The elution of total protein and PLA2 activity
from a typical heparin sepharose chromatography column.
There was no detectable total protein or phospholipase A2
activity with either 2 or 5M NaCl elution.
129
Protein size (kDa)
Protein size (kDa)
200
116
97
66
55
36
31
21
14
6
3
200
116
97
66
55
36
31
21
14
6
3
Column volumes per elution buffer 15 20
Total protein in active fractions (µg/mL)
26.57 3.91
PLA2 activity, % hydrolysis. 24.5 15.6
Figure 5.11: Effect on total protein content of elution from heparin
sepharose chromatography column using either 15 or 20 column
volumes per elution buffer. The use of twenty column volumes
decreased the amount of contaminating total protein while retaining the
majority of the enzyme activity.
130
0
20
4060
80
100
Dialysed Non dialysedPhos
phol
ipas
e A 2
act
ivit
y,
% h
ydro
lysi
s.
Figure 5.12: PLA2 activity before and after dialysis against
phosphate buffer (10mM). Dialysis of the active heparin
column fractions was required before application to the
Rotofor IEF system.
131
02040
6080
100
Water Bee sPLA2 Bowel Type IIAsPLA2
PLA 2
act
ivit
y (%
hyd
roly
sis)
Water Rotofor buffer
Figure 5.13: Confirmation that the Rotofor® buffer did not
interfere with assay of PLA2 activity. Three sources of PLA2
(bee venom sPLA2, bowel homogenate and purified type IIA
PLA2) and a negative control (water) were diluted in either
water or Rotofor® buffer.
Results are expressed as the mean +/- standard error of
triplicate assays.
132
0
200
400
600
800
1000
0 0.5 1 1.5 2 2.5 3 3.5Time (hours)
Volts
0246810121416
mAM
Ps, w
atts
Volts
mAmps
Watts
Figure 5.14: The power, current & voltage changes over
the duration of a typical Rotofor® run. The system was
run until the plateau phase had been reached and
maintained for an hour. A typical run took 3.5 hours.
133
0
5
10
15
1 3 5 7 9 11 13 15 17 19
Rotofor fraction
pH
0
20
40
60
80
PLA
2 ac
tivity
,%
hyd
roly
sis.
pH PLA2 activity
Figure 5.15: The pH and PLA2 activity of the fractions of a
typical Rotofor® experiment.
134
Mark 12
stand
ards
Mark 12
stand
ards
1 2 3 4 5 6 7 8 9 10 Origina
l sample
Origina
l sample
11 12 13 14 15 16 17 18 19 20
Exhibited PLA2 activity
Ampholytes
21
14
Figure 5.16: Typical Rotofor® fractions from pooled active
heparin fractions of infarcted bowel content eluted by 0.5M NaCl
and dialysed against 10mM phosphate. Rotofor fractions 15-18
exhibited PLA2 activity.
135
02468
1012
1 3 5 7 9 11 13 15 17 19
Fraction number
pH
2.5.03
5.5.03
7.5.03
8.5.03
9.5.03
Figure 5.17: The reproducibility of Rotofor® system. The
pH of each of the 20 fractions of five consecutive
Rotofor® experiments is shown in this graph.
136
Figure 5.18: Refractionation of a typical Rotofor® experiment. No
extra ampholytes were added, and the active fractions were 10-17.
The maximum phospholipase A2 activity was observed in fraction 17
which corresponded to a pH of 9.7. The arrow points to the region of
gel where phospholipase A2 was predicted to run to, as determined by
electroelution. The red line shows the phospholipase activity in each
Rotofor fraction, while the blue line shows the phospholipase activity
in each re-fractionated Rotofor fraction.
137
02468
101214
1 3 5 7 9 11 13 15 17 19Fraction number
pH
Rorofor 1
Re-Rotofor
Figure 5.19: The pH profile of Rotofor® fractions before
and after re-fractionation.
138
0204060
80100120
0 5 10 15Electroelution fraction number.
Size
of
prot
ein
(kD
a)
Figure 5.20: A standard curve representing the electroelution of
PLA2 from active Rotofor® fractions. The sample was run on a
non-reducing gel. PLA2 activity was focussed in fractions 8 & 9,
corresponding to a protein of 16-20kDa in size.
139
0
1
2
3
4
5
6.5 7 7.5 8 8.5 9 9.5pH
PLA 2
act
ivit
y (%
hyd
roly
sis )
Figure 5.21: The pH of maximum activity of the semi purified bowel
phospholipase. Results are expressed as the mean +/- standard
error of triplicate assays.
140
0
20000
40000
60000
80000
100000
Crude samp leHeparin co lumn Ro to fo r Electroelut ion0
2000
4000
6000
8000
10000
Specific act ivity To tal p ro tein
Figure 5.22: Purification summary of a typical experiment.
This purification experiment is equivalent to an average 9700
fold purification from the crude sample.
141
31
21
14
Prote
in
markers
11 12 13 14 15 1 6 17 18 19 20
Rotofor® fraction
Exhibited PLA2 activity
Size, (kDa)
Figure 5.23: A typical result from a Rotofor® run during the
scaled up purification, as shown by a silver stained gel of
active Rotofor® fractions. The arrow indicates a protein of
approximately 21kDa which was present in the fractions with
phospholipase activity.
142
Protein of interest
Ampholytes
Ethano
l prec
ipitat
ion
Vivasp
inco
ncen
tratio
n
Figure 5.24: Gel showing the effect of concentrating
protein and removing ampholytes using either
VivaSpin centrifuge filters or ethanol precipitation.
143
Sample Top 1-2 identifications
Number of peptides
Mowsescore
Unknown protein ~20kDa.
Peptidylprolylisomerase.*Keratin.
15
8
465
414
Positive control, lysozyme.
Lysozyme (chicken). 5 204
Negative control.
Keratin. 15 588
*Proteins matching the same set of peptides:
•Cyclophilin B (ICYNA)
•Cyclophilin B (Q9BVK5)
•Human cyclophilin
Figure 5.25: Mass spectrometry of the unknown protein from trypsin
digests of gel pieces (BisTris gel, Invitrogen).
144
Sample Top 1-2 identifications.
Number of peptides.
Mowse score.
Unknown protein ~20kDa-A.
Peptidylprolylisomerase.*Keratin.
13
5
427
272
Unknown protein ~20kDa-B.
Keratin.Peptidylprolylisomerase.*
810
435386
Unknown protein ~20kDa-C.
Peptidylprolylisomerase.*Bovine cyclophilin B.
10
16
426
331
Postive control, Restriction endonuclease E.Coli.
Restriction endonuclease Bacillus sp.
48 723
Negative control Keratin. 6 333
*Proteins matching the same set of peptides:
•Cyclophilin B (ICYNA)
•Cyclophilin B (Q9BVK5)
•Human cyclophilin
Figure 5.26: Mass spectrometry of the unknown protein from trypsin
digests of gel pieces (TrisGly gel, Gradipore).
145
Figure 5.27: Final purification scheme. This protein purification scheme
resulted in sufficient purified protein for identification of the protein of interest
by mass spectrometry.
146
Bowel content samples
Heparin sepharose chromatography, 20 column volumes.
Fractions with highest PLA2activity retained.
Preparative isoelectric focussing (Rotofor®).
Fractions with highest PLA2 activity retained.
Concentrated ten fold (VivaSpin).
SDS-PAGE, TrisGlycineiGEL, trypsin digest of protein bands.
SDS-PAGE, BisTris, trypsin digest of protein bands.
LC/MS/MS
Results: proteins identified as Cyclophilin B.
Discussion
Chapter 5 Discussion:
The aim of the protein purification experiments was to identify the protein responsible
for the phospholipase activity that remained when phospholipase A2 isoforms IIA and
V were removed from human bowel. The protein of interest was purified from
infarcted gut tissue homogenate rather than blood for a number of reasons:
- a larger amount of tissue was available,
- the tissue showed higher specific activity,
- a bowel tissue specific protein was more likely to be found.
The separation techniques were chosen because they retained the native enzyme
activity of proteins and they removed large quantities of contaminating protein at each
step. The separation techniques also exploited unusual characteristics of the protein
of interest (namely heparin affinity and very alkaline isoelectric point). The progress
of the purification was monitored by analysing all fractions obtained for phospholipid
hydrolysing activity. The assay used for monitoring phospholipase activity (the
mixed micelle assay) has been well described and is very sensitive (Farrugia et al,
1993, Green et al, 1991). This assay was chosen for its sensitivity, ability to be used
with crude and purified samples and ability to detect net phospholipase activity. A
series of PLA2 isoenzyme specific assays have been published (Yang et al, 1999), but
this was not suitable as we required an assay to monitor net phospholipase activity for
the widest range of phospholipase proteins.
Because of the low abundance of non-pancreatic phospholipase A2 in biological
samples, proteins need to be purified several thousand fold (Stefanski et al, 1986). In
the past, purification of phospholipase A2 from animal tissues has involved the use of
several sequential chromatographic columns, often in conjunction with phospholipase
assays of the fractions obtained where compatible. A typical phospholipase A2
purification process may involve one or more anion or cation exchange columns, a
hydrophobic interaction column, one or more gel filtration columns, and a final
reversed phase high performance liquid chromatography (RP-HPLC) column. For
example, Minami and colleagues used a cation exchange column (run and then
repeated) followed by a gel filtration column (also run twice), another cation
exchange column and a final RP-HPLC column to purify phospholipase A2 type II
from human ileal mucosa (Minami et al, 1993). For tissues such as spleen, multiple
ion exchange columns (cation and anion), a hydrophobic interaction column and RP-
147
Discussion
HPLC column have been used (Kanda et al, 1989, Ono et al, 1988). For biological
fluids such as synovial fluid, generally fewer columns are required, for example an
ion exchange and a gel filtration column followed by RP-HPLC and PAGE (Kramer
et al, 1989) or hydrophobic interaction, HPLC and PAGE (Seilhamer et al, 1989).
The use of multiple chromatography columns is associated with the risk that
phospholipase activity may be lost because of the buffers or solvents used or the
numerous manipulations, and therefore the activity of the protein of interest may be
reduced or lost during purification. For this project we chose to employ a small
number of purification techniques to exploit the unusual characteristic of
phospholipase A2 instead of a large number of chromatography columns.
Heparin sepharose chromatography was used for a number of reasons:
- it removed a large amount of contaminating protein in one experiment, and
retained enzyme activity,
- discrete fractions could be achieved with high specific activity by using a
step gradient of salt concentrations,
- the high salt concentrations required to elute the tightly bound protein could
be reduced by dialysis without altering the enzyme activity,
- the system could be scaled up or down relatively easily.
Groups that have used heparin affinity chromatography in protein purification have
generally employed a linear gradient of salt rather than a step gradient, but we found a
step gradient useful in terms of reproducibility and obtaining highly purified fractions.
Preparative isoelectric focussing was conducted using the BioRad Rotofor®
apparatus. The buffer used in the Rotofor® system did not appear to interfere with
the PLA2 assay, which was an advantage because the fractions could be analysed
without any modification, such as dialysis. The Rotofor® system was useful for a
number of reasons:
- the system was highly reproducible,
- the fractions obtained could be analysed in a number of different ways (pH,
enzyme activity, total protein, SDS-PAGE, electroelution),
148
Discussion
- the interference of the ampholytes in total protein assays and in protein
staining in SDS-PAGE could be overcome via a number of different strategies
(Coomassie G-250 stains with or without copper and protein precipitation
prior to total protein analysis).
The majority of the publications involving the Rotofor® system have investigated the
proteins of the cerebrospinal fluid which may reflect the development of Alzheimer’s
disease (Davidsson, BioRad technote 2859). Westman-Brinkmalm and Davidsson
(2002) found that the Rotofor® apparatus could be used as a pre-fractionation step for
cerebrospinal fluid proteins, and this pre-fractionation resulted in increased sequence
coverage and greater identification of low abundance proteins. The Rotofor®
apparatus is an excellent tool in protein purification and is particularly suited to
enzymes with compatible assays. At present, such a valuable tool in protein
purification and proteomics appears to be under-utilised.
The initial purification system of heparin sepharose chromatography, Rotofor®
fractionation and Rotofor® refractionation did not provide sufficient protein for
identification so the system was scaled up approximately twenty fold. The scaled up
purification resulted in a protein that was >95% pure on a silver stained gel, and
represented a 9700 fold purification. Throughout the purification system, a number
of protein characteristics were recognized:
- the protein size appeared to be between 16 and 20kDa,
- the phospholipase activity of the protein appeared to be sensitive to reduction
by DTT,
- the phospholipase activity of the protein appeared to be calcium dependent,
- the pH of optimal phospholipase activity appeared to be approximately 9,
- the isoelectric point of the protein appeared to be between 8.6 and 9.8,
- the protein appeared to have heparin affinity up to conditions of
approximately 0.5M sodium chloride.
Many of these features were suggestive of a secretory phospholipase isoform, but no
clear match was evident. Therefore a conclusive identification was required,
preferably by mass spectrometry.
149
Discussion
Identification of the protein of interest:
Haemoglobin was identified on a number of occasions (by Edman degradation and
mass spectrometry), due to haemoglobin either:
-co-purifying with the protein of interest,
- contaminating the samples simply because it was present in high
concentration or because of its ‘sticky’ nature (Associate Professor Mark
Kirkland, personal communication).
The problems with proteins co-purifying with phospholipase A2 has been encountered
previously, for example, histone proteins co-purified with phospholipase A2-IIA from
synovial fluid (Dr. Kieran Scott, personal communication). The possibility that
haemoglobin may have intrinsic phospholipase A2 activity was dismissed by assaying
a range of haemoglobin concentrations for phospholipase activity. None of the
haemoglobin concentrations showed phospholipase activity above background levels
(similar to the background seen in ultrapure water, buffers or albumin solution). In
addition, haemoglobin did not appear to enhance phospholipase activity.
Haemoglobin was not irreversibly bound to the protein of interest, as the size of the
protein as determined by electroelution under non reducing conditions was too small
to be complexed to haemoglobin (molecular weight of ~16kDa). When a solution of
standard haemoglobin and phospholipase A2 from either pancreas or bee venom was
run on a gradient SDS-PAGE, distinct bands for each protein were visible, suggesting
that:
- if the haemoglobin binds to phospholipase A2 proteins, the association is
disrupted by SDS,
- a gradient SDS-PAGE gel was required as the final purification step to limit
the possibility of identifying haemoglobin in subsequent experiments.
The final purification system was comprised of heparin sepharose affinity
chromatography (with twenty column volume elution), preparative IEF (Rotofor®
apparatus) and a gradient SDS gel. The final identification of the protein of interest
was made using nano-LC MS/MS. The final identification was made from two
highly purified fractions, each run in a different gel system with separate positive
controls. The two different gel systems were used to add strength to the
150
Discussion
identification, and to overcome the possibility that one type of gel may be
incompatible with the mass spectrometry system. The protein of interest was an
excellent match for cyclophilin B, a protein that is also referred to as peptidylprolyl
isomerase. Cyclophilin B is an inflammatory protein (Allain et al, 2002, Carpentier
et al, 1999) that had been previously identified in rodent intestine at the mRNA level
(Hasel et al, 1991, Kainer & Doris, 2000).
Cyclophilin B is known to have heparin sulphate binding ability (De Ceuninck et al,
2003). Heparin affinity chromatography has been used to semi-purify cyclophilin
proteins previously (Carpentier et al, 1999, De Ceuninck et al, 2003, Sherry et al,
1992). Cyclophilin A (64% homology to cyclophilin B) cannot interact with
glycosaminoglycans such as heparin because it lacks the required N-terminal binding
residues, likely to involve a triplet lysine (Allain et al, 2002, Bukrinsky, 2002). The
binding of cyclophilin B with glycosaminoglycans is thought to contribute to
cyclophilin involvement in inflammation (Allain et al, 2002). The affinity of
cyclophilin B to heparin is quite strong, and this protein is not eluted from a heparin
column until sodium chloride concentration exceeds 0.6M sodium chloride
(Carpentier et al, 1999).
The size (20.289kDa, Carpentier et al, 1999), isoelectric point (9.25) and heparin
affinity (0.6M NaCl, Carpentier et al, 1999) of cyclophilin B were consistent with the
characteristics that had been determined throughout the protein purification
experiments. The next chapter details our investigations into cyclophilin B in human
bowel.
151
Chapter 6 Introduction
Chapter 6: Cyclophilin B.
Immunophilins:
The immunophilins are a family of heterogenous proteins grouped by their ability to
bind the immunosuppressive agents cyclosporin A (the cyclophilin proteins), FK506
(FK506 binding proteins) or rapamycin (target of rapamycin or Tor proteins, (Ivery,
2000)). The FK506 binding proteins have no sequence homology to the cyclophilins
(Snyder & Sabatini, 1995). Immunophilins have peptidyl prolyl isomerase activity,
which catalyses folding of proline containing proteins in vivo and in vitro. This
isomerase activity is inhibited on binding to the relevant immunosuppressive drug
(Bochelen, Rudin & Sauter, 1999). Immunophilins have the ability to interact with
many signal transduction molecules, in particular those involving calcium or
phosphorylation (Snyder & Sabatini, 1995).
Cyclophilins:
Cyclophilin was first purified from bovine thymus and human spleen (Harding,
Handschumacher & Speicher, 1986). To date, more than forty gene encoded
cyclophilins have been recognised (Fanghanel & Fischer, 2003). The major human
isoforms are described in Table 6.1. Cyclophilins have been found in all cell types
investigated (Price et al, 1991) and are highly conserved (Allain et al, 1995). The
widespread distribution and significant levels of expression suggest an important
biological role (Ivery, 2000), but the relevance of their biological activity is unknown
(Denys et al, 1998). Cyclophilins may function as catalysts of protein folding (Kay,
1996), or protein stabilisation (Galat & Metcalfe, 1995), as molecular chaperones
(Yoo et al, 1997) or cytokines (Tegeder et al, 1997).
The two major isoforms of cyclophilin, cyclophilins A and B, are only 64%
homologous (Price et al, 1991), and the major difference between the two is the signal
sequence in cyclophilin B directing it into a secretory pathway (Price et al, 1991,
Price et al, 1994). Cyclophilin A is a cytosolic protein (Price et al, 1994), and can
comprise up to 0.1-0.4% of the soluble protein in the cytoplasm (Harding,
Handschumacher & Speicher, 1986). Cyclophilin A is the most abundant cyclophilin
in human cells (Fanghanel & Fischer, 2003). It is assumed that cyclophilin A is non-
secretory, although this protein has been detected in the synovial fluid from patients
152
Chapter 6 Introduction
with rheumatoid arthritis (Billich et al, 1997). Cyclophilin A is present in leukocytes
at around 10 fold higher levels than cyclophilin B, but is not released into the plasma
(Allain et al, 1995). Cyclophilin B is a secreted protein which has been detected in
human blood (Allain et al, 1995) and breast milk (Spik et al, 1991).
Cyclophilin C (23kDa) localises to the endoplasmic reticulum (Snyder & Sabatini,
1995) and has been shown to be a secretory protein (Price et al, 1994). Cyclophilin
40 is a component of the inactive form of the glucocorticoid receptor (Synder &
Sabatini, 1995). Cyclophilin NK plays an important role in natural killer cell
cytotoxicity (Anderson et al, 1993).
153
Chapter 6 Introduction
Table 6.1. The Major Human Cyclophilins: Protein Alternate names Molecular
weight, kDa
Isoelectric
point
Signal
peptide
Abundant
sources
Cyclophilin A Cyclophilin 1
Cyclophilin 18
18 7.8 None. Cytosolic,
ubiquitous
(Hasel et al,
1991).
Cyclophilin B S-cyclophilin
SCYLP
Cyclophilin 2
Cyclophilin 20
22.74,
unprocessed
20.29
(mature)
9.25 (mature) 1-25 signal
26-208 mature
protein
Liver,
intestine and
placenta
(Hasel et al,
1991).
Cyclophilin C Not applicable. 23 8.48 None. Liver, lung,
heart,
pancreas,
skeletal
muscle
(Schneider et
al, 1994).
Cyclophilin D Cyclophilin 3
Cyclophilin M
Mitochondrial
cyclophilin.
18 8.97 1-29 potential
signal, 30-207
chain.
Muscle, heart,
liver, kidney,
brain
(ExPASY
database).
Cyclophilin
40
Not applicable. 40 6.77 None Heart, brain,
placenta,
skeletal
muscle,
pancreas, liver
(Kieffer et al,
1993).
Cyclophilin
NK
Not applicable. 150 (mature
protein),
19.5
(cyclophilin
like domain).
10.01 None. Membrane
anchored on
natural killer
cells. Spleen,
lung, liver
(Anderson et
al, 1993).
154
Chapter 6 Introduction
Cyclophilin B in human disease:
The levels of cyclophilin B have been investigated in relation to sepsis, HIV and
organ transplant in clinical settings. A 1997 study investigated the levels of
cyclophilins A and B in the plasma of patients with severe sepsis, other diagnoses and
healthy controls using western blotting and a peptidyl prolyl isomerase activity assay
(Tegeder et al, 1997). The healthy control plasma showed a weak but distinct
cyclophilin B signal, but no cyclophilin A signal. Patients with severe sepsis showed
high levels of cyclophilin B and some (6/22) patients also showed a cyclophilin A
signal. Patients with diagnoses other than sepsis showed variable levels of
cyclophilin B, but no cyclophilin A (Tegeder et al, 1997).
Organ transplant patients showed significantly elevated levels of cyclophilin B in
plasma compared with health controls (Denys et al, 1998). It was postulated that
cyclophilin B levels may effect a patient’s sensitivity to the immunosuppressive drug
cyclosporin A (Denys et al, 1998). Levels of cyclophilins A and B were found to be
increased in the serum of patients with HIV infection (Endrich & Gehring, 1998).
The cyclophilin B-Cyclosporin A complex:
The pharmacological ligand of cyclophilins, cyclosporin A is known for its
immunosuppressive effects. The binding of cyclosporin A to a cyclophilin is
required for the immunosuppressive effect associated with this drug (Gonzalez-
Cuadrado et al, 1996). The cyclophilin-cyclosporin A complex then interacts with
protein targets such as calcinuerin (Ivery, 2000), to exert the immunosuppressive
effect. All of the therapeutic and toxic effects of cyclosporin A (and FK506) have
been found to be due to the inhibition of calcinuerin (Ho et al, 1996). The
cyclophilin B-cyclosporin A complex shows the most potent binding to calcineurin of
all cyclophilins (Kay, 1996).
Cyclosporin A and cyclophilin B appear to exert effects on each other. Cyclosporin
A has been shown to increase levels of cyclophilin B mRNA in a dose dependent
manner (Galat & Metcalfe, 1995, Gonzalez-Cuadrado et al, 1996, Price et al, 1994).
Cyclosporin A has been shown to significantly increase levels of cyclophilin B
protein in plasma compared with healthy donors (Denys et al, 1998). Cyclosporin A
has been shown to increase the secretion of cyclophilin B in cultured chondrocytes
155
Chapter 6 Introduction
(De Ceuninck et al, 2003). Cyclophilin B modifies the distribution of cyclosporin A
in blood, channelling the drug into peripheral blood mononuclear cells (PBMC) or
retaining the drug-protein complex extracellularly (Denys et al, 1998). Cyclosporin
A redirects cyclophilin B from the endoplasmic reticulum into a secretory pathway
(Price et al, 1994). The CyPB-CsA complex inhibits T cell proliferation more
effectively than cyclosporin A alone (Denys et al, 1998).
Use of cyclophilin B antibodies:
Due to the high homology of cyclophilins, antibodies generated to intact cyclophilin
proteins tend to cross react with multiple cyclophilin forms. For example antibodies
raised to recombinant cyclophilin B crossreact with cyclophilin A (Allain et al, 1995).
Useful antibodies to cyclophilin B have been generated by using a 14 residue peptide
from the C-terminal, variable region, coupled to keyhole limpet hemocyanin (KLH) to
improve the immunogenicity of the peptide (Vaitukaitis et al, 1971).
Western blotting for cyclophilin B:
One study employed an antibody raised in the mode discussed above to investigate
cyclophilin B in Jurkat cells (Bergsma et al, 1991). Jurkat cells showed four
immunoreactive bands in Western blots, but only one band could be removed by
incubating the extract with an excess of the peptide used to generate the antibody
(Bergsma et al, 1991).
ELISA studies of cyclophilin B:
Another study employing a peptide derived cyclophilin B antibody devised an ELISA
to assay blood cells and serum from blood transfusion samples (Allain et al, 1995).
A modified version of this ELISA was used to investigate cyclophilin B in organ
transplant patients (Denys et al, 1998).
Cyclophilin B in gut:
Studies in rats have shown evidence of cyclophilin B mRNA transcripts in the
intestine using competitive RT-PCR (Kainer & Doris, 2000) and Northern blotting
(Hasel et al, 1991). Competitive RT-PCR of rat tissues showed that the highest
expression of cyclophilin B occurred in the kidney, followed by the ventricle, liver
and small intestine (Kainer & Doris, 2000). Northern blotting of rat tissues showed
156
Chapter 6 Introduction
that the highest expression was in the placenta and liver, followed by the intestine and
uterus (Hasel et al, 1991).
157
Results
Chapter 6: Cyclophilin B in human bowel:
Previous research had identified cyclophilin B in Jurkat cells, (a T-cell lymphoma cell
line) therefore whole cell lysate was used as a positive control in these investigations.
Jurkat cell lysate and cell culture medium were run on SDS-PAGE to estimate protein
loading (Figure 6.1). Bowel tissue from patients with surgically confirmed bowel
infarction and control patients with normal bowel all showed cyclophilin B protein by
Western blotting (Figures 6.2, 6.3 & 6.4). Plasma from patients with confirmed
bowel infarction showed variable levels of cyclophilin B, but control patients showed
no cyclophilin B (Figure 6.7). Some lumen content from infarcted bowel showed
cyclophilin B protein, though at much lower levels than tissue homogenates (Figure
6.5). Immunoprecipitation of cyclophilin B from highly purified fractions removed
all of the phospholipase activity from these samples (Figure 6.6). The phospholipase
activity was not altered by the addition of cyclosporin A (Figure 6.10).
Immunohistochemistry was performed using an immunoperoxidase method and the
commercially available polyclonal antibody raised to a cyclophilin B peptide. After
the antibody dilution had been optimised, the ubiquitous expression of cyclophilin B
was confirmed (Figure 6.9).
158
159
1 2 3 4 5 6 7 8 9
116
66.2
45
35
25
18.4
Figure 6.1: Colloidal Coomassie stained gel of Jurkat cell lysate and cell culture
medium.
Lanes 1-3 Jurkat cell lysate, increasing amounts of sample loaded
Lane 4 No protein loaded
Lanes 5-8 Jurkat cell culture medium, increasing amounts of sample loaded
Lane 9 Protein Markers
160
1 2 3 4 5 6 7 8 9 10
26kDa
0100002000030000400005000060000700008000090000
2 3 4 5 6 7 8 9 10
Net
inte
nsity
of s
igna
l
Figure 6.2: Western blotting of cyclophilin B in infarcted human bowel samples, 30µg total protein per lane.1 Markers2 Jurkat cell lysate3 Jurkat cell culture medium4 no protein loaded5 Bowel tissue A6 Bowel mucosa A7 Bowel tissue B8 Bowel mucosa B9 Bowel tissue C10 Bowel mucosa C
161
1 2 3 4 5 6 7 8 9 1026kDa
0
20000
40000
60000
80000
2 3 4 5 6 7 8 9 10
Net
inte
nsity
of s
igna
l
Figure 6.3: Western blotting of cyclophilin B in infarcted human bowel samples, 30µg total protein per lane.1 Markers2 Jurkat cell lysate3 Jurkat cell culture medium4 no protein loaded5 Bowel tissue D6 Bowel tissue E7 Bowel tissue F8 Bowel tissue G9 Bowel tissue (infarcted)10 Bowel tissue (ischaemic)
162
2 3 4 5 6 7 8 9 10
26kDa
0
20000
40000
60000
80000
100000
2 3 4 5 6 7 8 9 10
Net
inte
nsity
of s
igna
l
Figure 6.4: Western blotting of cyclophilin B in control (normal) human bowel samples, 30µg total protein per lane.2 Jurkat cell lysate3 Jurkat cell culture medium4 no protein loaded5 Control bowel tissue A6 Control bowel tissue B7 Control bowel tissue C8 Control bowel tissue D9 Control bowel tissue E10 Control bowel content
163
2 3 4 5 6 7 8 9 10
26kDa
0
40000
80000
120000
160000
2 3 4 5 6 7 8 9 10
Net
inte
nsit
y of
sig
nal
Figure 6.5: Western blotting of cyclophilin B in bowel content samples, 40µg total protein loaded per lane.2 Jurkat cell culture medium3 Infarcted bowel content A4 Infarcted bowel content B5 Infarcted bowel content C6 Infarcted bowel content D7 Infarcted bowel content E8 Infarcted bowel content F9 Infarcted bowel content G10 Infarcted bowel content H
164
05
1015202530
Rotofor fraction 7.5 Rotofor fraction 8.5
PLA 2
act
ivit
y, %
hyd
roly
sis.
Before IP After IP
Figure 6.6: Phospholipase activity before and after immunoprecipitation
(IP) of cyclophilin B from Rotofor® fractions. Results are expressed as
the mean +/- standard error of duplicate assays.
165
0
1000
2000
3000
4000
5000
2 3 5 6 7 8 9 10 13 14 15 16 17 18 19 20
Patient
Net
inte
nsity
of s
igna
l
Bowel infarction patients:Positive control 2 3 5 6 7 8 9 10
26kDa
Control patients:
Positive control 13 14 15 16 17 18 19 20
26kDa
Figure 6.7: Western blotting of cyclophilin B in plasma, 30µg total protein per lane.
Patients 1-10 had surgically confirmed bowel infarction while patients 11-20 were
control ICU patients. The positive control used in these blots was Jurkat cell culture
medium.
MKVLLAAALI
AGSVFFLLLP
GPSAADEKKK
GPKVTVK
VY
FD
LRIGDEDVG
RV
IF
GL
FG
KT
VPKTVDNFVA
LATGEKGFGY
KNSKFHR
VI
KD
FM
IQ
GG
DF
TRGDGTGGKSI
YGER
FP
DE
NF
KLKHYGPGWV
SMANAGK
DT
NG
SQ
FF
IT
TV
KT
AW
LD
GK
HV
VF
GK
VL
EG
ME
VV
RKVESTKTD
SRDKPLKDVI
IADCGK
IE
VE
KP
FA
IA
KE
Figu
re 6
.8: A
min
o ac
ids s
how
n in
blu
e ar
e th
e try
ptic
pep
tides
obt
aine
d in
this
stud
y (B
MSF
mas
s
spec
trom
etry
). A
min
o ac
ids i
n re
d: si
gnal
pep
tide
clea
ved
in t
he m
atur
e pr
otei
n.
166
167
Figure 6.9: Immunohistochemistry of cyclophilin B in normal and infarcted
human intestine. Picture a) shows the normal bowel test slide (cyclophilin B
immunohistochemistry (brown staining), counterstained with haemotoxylin
(blue staining)) . Picture b) shows the infarcted bowel test slide. Picture c)
shows the negative control slide (haematoxylin stained). These
photomicrographs were taken at a 200X magnification. The scale bars
represent 100µm.
168
a)
b)
c)
169
02468
10
Fraction 7.5 Fraction 8.5Phos
phol
ipas
e ac
tivity
, %
hydr
olys
is
Cyclosporin A No cyclosporin A
Figure 6.10: Cyclosporin A (1µg/mL, Sigma) does not affect
phospholipase activity of cyclophilin B containing samples. Results
are expressed as the mean +/- standard error of triplicate assays.
Discussion
Chapter 6 Discussion:
The unknown protein purified in this project was identified as cyclophilin B by nano-
LC tandem mass spectrometry. The accuracy of the mass spectrometry was verified
by the addition of positive controls (lysozyme and restriction endonuclease), all of
which gave very good matches upon database searching. The identification was
repeated from two different highly purified fractions, in two different gel systems.
Each of the samples from the two gel systems showed that the best match was
cyclophilin B, also referred to as peptidyl prolyl isomerase. This was an unexpected
finding because the progress of the protein purification had been monitored at each
stage by phospholipase hydrolysis assays, and cyclophilin B has not been reported to
have phospholipid hydrolysing activity. To investigate the possibility that
cyclophilin B had the ability to hydrolyse phospholipids, the cyclophilin B was
removed from highly purified fractions via immunoprecipitation with a commercially
available polyclonal antibody raised to a cyclophilin B peptide. The peptide used to
raise the antibody (194GKIEVEKPFAIAKE208) was one of the peptides identified by
the tandem mass spectrometry, which added clarity to the experiment. The presence
of cyclophilin B in the highly purified samples was verified by Western blotting of the
protein attracted to the resin immobilised antibody. This result also suggested that
the conditions for immunoprecipitation (for example: ionic strength of the buffer,
incubation times) were adequate for the removal of cyclophilin B from solution. The
samples devoid of cyclophilin B were then analysed for phospholipid hydrolysing
activity, and found to have no remaining activity. This result suggests that
cyclophilin B is able to hydrolyse phospholipid substrates. The converse experiment
(examining the possible phospholipid hydrolysing activity of a recombinant source of
cyclophilin B) would be a valuable exercise, but an application to the only known
pharmaceutical supplier of this protein was unsuccessful. To our knowledge there is
no commercial source of the recombinant protein.
A similar experiment employing the commercially available cyclophilin A protein
was not performed, as cyclophilins A and B have only moderate homology (64%) and
quite distinct roles in inflammation. The addition of cyclosporin A did not appear to
affect the phospholipase activity of the highly purified cyclophilin samples. This
suggests that amino acids involved in the proposed phospholipid hydrolysis are likely
to be separate from residues involved in cyclosporin binding.
170
Discussion
Western blotting was undertaken to assess the presence of cyclophilin B in a variety
of bowel samples. For these experiments, Jurkat cell lysate and the culture medium
form Jurkat cell culture were used as positive controls. Jurkat cells (a T-cell
lymphoma cell line) have been shown to express significant quantities of cyclophilin
B (Bergsma et al, 1991). An appropriate negative control tissue could not be found,
as the expression of cyclophilin is reported to be ubiquitous (Allain et al, 1995,
Tegeder et al, 1997). Jurkat cell extracts were also used as an internal control in the
Western blots, to control for signal intensity in the semi-quantitative ECL Western
system employed in this project. The Jurkat cell lysate and cell culture fluid were
used as the controls in blots of samples with high (eg tissue) and low (eg plasma)
levels of cyclophilin B respectively.
Western blots of cyclophilin B protein exhibited a number of non-specific bands of
different sizes than expected for cyclophilin B protein. The non specific bands could
be removed by more stringent washing procedures or stripping the membranes and re-
probing. The non-specific bands seen with this polyclonal cyclophilin B antibody
have also been shown by Bergsma and colleagues (1991). This group showed that
only one band was due to cyclophilin B by incubating the samples with an excess of
the peptide used to generated the antibody, which cleared the cyclophilin B band from
the blot (Bergsma et al, 1991). This approach would be a valuable control
experiment in future blotting experiments.
Bowel tissue homogenates from both normal bowel and infarcted bowel all showed
very strong signal for cyclophilin B of the expected size. This is not a surprising
result given the reported ubiquitous expression of cyclophilin B, and the reported high
mRNA expression in rodent gut (Hasel et al, 1991, Kainer & Doris, 2000). However,
it is supporting evidence of similar expression patterns in human tissue, at the protein
level. The infarcted bowel lumen content samples contained variable signal intensity
of cyclophilin B, but were generally lower than the signal intensity of tissue
homogenates. Although only one sample of lumen content from normal human
bowel could be obtained, this sample contained no signal for cyclophilin B. This is
an interesting finding as it supports the concept that cyclophilin B is present in large
amount in bowel tissue and may be released in response to the ischaemic conditions.
171
Discussion
In the two tissue samples obtained from one patient, the infarcted tissue showed a
higher cyclophilin B signal than the ischaemic tissue. None of the control patients’
plasma displayed cyclophilin B, while several samples of plasma from bowel
infarction patients exhibited this protein. The possible role of cyclophilin B as a
mediator, or marker of ischaemic damage to the human bowel is an interesting
concept that requires further investigation. Interestingly, this observation is in
accordance with previous reports in other human diseases.
THE ROLE OF CYCLOPHILIN B IN INFLAMMATION:
Increased levels of cyclophilin B protein have been observed in the plasma of patients
with sepsis (Tegeder et al, 1997), HIV infection (Endrich & Gehring, 1998) and organ
transplant (Denys et al, 1998). As such, it has been postulated that cyclophilin B has
an important role in inflammation (Allain et al, 2002), and this role is due in part to
the induction of chemotaxis and proliferation of T lymphocytes (Denys et al, 1997).
Chemotaxis:
Cyclophilins induce chemotactic activity with neutrophils (Billich et al, 1997), T
lymphocytes (Allain et al, 2002), polymorphonuclear leukocytes (Sherry et al, 1992)
and monocytes (Xu et al, 1992). Cyclophilin B, in particular, induces the migration
of neutrophils and T lymphocytes (De Ceuninck et al, 2003), which induces the
inflammatory cascade (Denys et al, 1997). Chemotactic inducing activity of
cyclophilins B is thought to be due to the same binding site as cyclosporin A, as the
addition of cyclosporin A abolishes chemotactic ability (Allain et al, 2002).
Cyclophilin binding sites:
Cyclophilin B shows pro-inflammatory effects and enhances integrin mediated
adhesion of CD4+ T-lymphocytes to the extracellular matrix, through interaction with
glycosaminoglycans via the type II binding site (Allain et al, 2002). The binding
sites for glycosaminoglycans and cyclosporin A are independent and located on
opposite sides of the cyclophilin B molecule (De Ceuninck et al, 2003, Mikol, Kallen
& Walkinshaw, 1994). The cyclosporin A binding site, or type I binding site is due
to a cluster of residues in the central core of the cyclophilin B molecule (De Ceuninck
et al, 2003, Denys et al, 1998). The type II binding site is responsible for the binding
172
Discussion
of glycosaminoglycans, and is due to a cluster of N terminal cationic residues (De
Ceuninck et al, 2003, Denys et al, 1998).
The possible involvement of cyclophilin B in oxidative stress:
Cyclophilin B has been implicated in the development of oxidative stress. Oxidative
stress in vascular smooth muscle cells increases cyclophilin B secretion (Liao et al,
2000). More extensive research has focussed on the role of other cyclophilins (A and
D) during oxidative stress. Cyclophilin D appears to have an important role in
mitochondrial membrane pore opening and the consequential necrosis or apoptosis
(Halestrap, McStay & Clarke, 2002). Mitochondria are known to have a critical role
in apoptosis, which is a favourable alternative to the inflammatory necrotic process.
Under normal conditions membrane pores are non permeable. Under conditions of
moderate stress membrane pores remain closed and cells are channelled into the
favourable apoptotic pathway. Under conditions of high calcium (mM
concentrations), pores become non-specifically permeable, cells swell and necrosis
ensues. Under conditions of moderated calcium (µM concentrations), in the presence
of cyclophilin D the permeable, non-specific pore opening also occurs and cells are
channelled into the necrotic pathway (see Figure 6.11). This process is greatly
enhanced by oxidative stress, such as reperfusion after ischaemic episodes. The pore
opening can be inhibited by cyclosporin A and its analogues, at similar affinities to
peptidyl prolyl isomerase activity inhibition (Halestrap, McStay & Clarke, 2002). It
is unknown whether this phenomenon is specific to cyclophilin D, or a general
cyclophilin process.
When antisense technology was used to knockout cyclophilin A, cardiac myocytes
were more sensitive to oxidative stress, but the protective action of cyclosporin A was
more pronounced (Doyle, Virji & Crompton, 1999). This study concluded that two
cyclophilin isoforms were involved in the oxidative stress response. Cyclophilin A
had a protective role, and a second, unknown cyclophilin isoform had a detrimental
effect on cells (Doyle, Virji & Crompton, 1999). The protective role of cyclophilin A
was also observed in a model of oxidative stress in vascular smooth muscle cells
where this protein prevented apoptosis (Jin et al, 2000).
173
Discussion
It is unclear whether the effects shown by cyclophilins A and D are specific
responses, or general cyclophilin responses to oxidative stress. It would be valuable
to investigate the role of each of the major cyclophilin isoforms in oxidative stress and
ischaemic episodes, and the possible protective role of cyclophilin inhibitors.
174
Discussion
Figure 6.11. Cyclophilins and Pathological Pore opening. Modified from
Halestrap A. The Mitochondrial Permeability Transition- A Pore Way for the
Heart to Die. J. Clin. Basic Cardiol. 2002; 5:29-41, with permission from the
author.
Cyclophilin B inhibition.
There are several examples of the inhibition of cyclophilins by cyclosporin A having
a protective or anti-inflammatory role, for example:
- inhibition of cyclophilin B reduced T-cell proliferation (Denys et al, 1998),
-inhibition of cyclophilin D avoided pore formation, preventing cellular
necrosis (Halestrap, 2002),
-inhibition of murine macrophage cyclophilin inhibited chemotactic induction
in monocytes (Sherry et al, 1992),
175
Discussion
- disruption of the binding of cyclophilin to a HIV capsid protein which
resulted in non infectious virions (Francke, Yuan & Luban, 1994, Thali et al,
1994),
- inhibition of chemotactic enhancing effect on eosinophils and neutrophils
(Xu et al, 1992).
Cyclosporin A can have unwanted effects such as inducing increased secretion of
cyclophilin B and has uncontrolled cellular distribution. Analogues of cyclosporin A
that specifically target one cyclophilin isoform (such as cyclophilin B) would be
advantageous, and may help to:
- prevent the drug compartmentalisation/distribution changes in presence of
cyclophilin B,
- retain the drug extracellularly (Endrich & Gehring, 1998),
- prevent the increased secretion of cyclophilin B seen when cyclosporin A is
administered (De Ceuninck et al, 2003),
- prevent the inhibition of possibly protective cyclophilin isoforms, such as
cyclophilin A (Halestrap, McStay & Clarke, 2002, Jin et al, 2000).
A synthetic analogue of cyclosporin A (SDZ-NIM811) was devised to have no
immunosuppressive effects. SDZ-NIM811 has been shown to disrupt the binding of
cyclophilin A to a critical protein domain in the HIV virion, and as a result the virion
was less infectious (Yoo et al, 1997).
The source of circulating cyclophilin B is unknown (Endrich & Gehring, 1998), and
this would be an area of importance in understanding role in inflammation. The
patterns of secretion also need to be better understood. The possible protective
features of cyclophilin B inhibitors in inflammation and infection require further
investigation.
176
Chapter 7 Introduction
Chapter 7: Alkaline Urea gels:
Traditional protein electrophoresis (SDS-PAGE) involves the separation of proteins in their
denatured (via SDS interactions) and reduced (via DTT or β-mercaptoethanol) state. The
separation of proteins in SDS-PAGE is dependent exclusively on the molecular weight of the
protein. Protein bands in SDS gels are sharply focussed, because the proteins exist as
monomers, are unfolded and interactions between proteins are not possible.
An alternate protein electrophoresis approach retains the native state of the protein (by the
omission of reducing agents), and any denaturation of proteins is either mild, and/or
reversible. Native gels are useful for investigations of protein subunit formation, protein
degradation and binding events (Alliance protein lab). Native gels are also useful when the
protein of interest has enzymic activity that can be detected post electrophoresis. Native gels
have a number of associated problems including:
- the mobility of proteins is not simply due to molecular weight*, [so protein band patterns
can be difficult to interpret (Chettibi & Lawrence, 1989)],
- bands can be indistinct, broadened or blurred due to protein-protein interactions,
polymers or multiple forms of proteins resulting from disulfide bonding.
The high concentrations of urea that are often used in native gels help to achieve high
resolution of proteins by eliminating protein-protein interactions and ensuring that proteins
exist in their unfolded state (Whittaker & Simpson, 1983). The denaturation of proteins by
urea is a reversible process, which allows for analysis of the native protein after renaturation.
High concentrations of urea can result in the formation of cyanate ions which can cause
artifactual spots or bands in gels via carbamylation of proteins (Gorg et al, 1997, Harris &
Angal, 1989) and N-terminal blocking (Harwig et al, 1993). Chemical modification as a
result of urea is a particular concern at high temperature and at alkaline pH.
Ahmad and colleagues (1994) employed a urea gel system of alkaline pH and high acrylamide
to resolve alkaline snake venom proteins, in particular venom phospholipase A2 isoforms.
Some 150 phospholipase A2 isoforms have been characterised in venoms (Valentin &
Lambeau, 2000). A single species of snake can express up to 15 distinct phospholipase A2
isoforms (Valentin & Lambeau, 2000). Phospholipase A2 is one of the most active venom
* Mobility in native gels is due to a combination of a protein’s size, shape and number of charged residues.
177
Chapter 7 Introduction
components and can exhibit a number of toxic effects including myonecrotic effects,
cardiotoxicity, anticoagulent effects, and hemorrhagic effects (Fortes-Dias et al, 1994).
Phospholipase A2 isoforms of high pI are of particular interest as it is thought that the most
potent venom toxins are either basic phospholipase A2 proteins or complexes involving
phospholipase A2 subunits (Karlsson, 1979). Ahmad and colleagues (1994) used these gels to
identify several PLA2 isoforms that had been unable to be resolved by IEF or ion exchange
chromatography. The high acrylamide content of these gels favoured the resolution of small
proteins and large peptides. These gels were particularly suited to the investigation of
phospholipase A2 because:
- they allow post separation analysis of enzyme activity,
- phospholipase A2 enzymes from snake venom are not denatured by high
concentrations of urea,
- PLA2 enzymes are stable at extreme pH, particularly high pH,
- PLA2 can be extracted at high yield (>60%) from the gels (Ahmad & Lawrence,
1993),
but most importantly:
- PLA2 isoforms show variable numbers of charged residues, particularly numbers of
acidic residues (Ahmad & Lawrence, 1993), which result in different mobilities in
these gels.
The relative mobility of phospholipase A2 isoforms in these gels is given by:
Number of Aspartic acid residues + number of Glutamic acid residues + number of
Tyrosine residues – number of Arginine residues + 1 (Ahmad, Lawrence & Moores,
1994).
These gels were also advantageous because they are rapid, inexpensive, use small amounts of
sample and allowed post electrophoretic enzyme activity analysis. These gels are also more
suited to comparative studies than IEF.
178
Results.
Chapter 7: Alkaline urea gels:
The two part alkaline urea gels devised by Ahmad, Lawrence and Moores (1993), for
the analysis of venom phospholipase A2 was investigated for use in the analysis of
mammalian phospholipase A2. A number of extensions were possible and may
broaden the applications available for these gels. Transfer of proteins from alkaline
urea gels to membranes would allow identification of proteins of interest by N-
terminal amino acid sequencing. Proteins could be transferred to PVDF membranes
from alkaline urea gels with no apparent loss of protein through the membrane
(Figure 7.1). No protein was evident in the gel after transfer (Figure 7.2). The
protein bands transferred to the membrane were sharp and very similar to the same
sample run on a gel without transfer (Figure 7.2). The transfer of small basic proteins
from alkaline urea gels to PVDF membrane was more efficient using the CAPS
transfer buffer than the Towbin transfer buffer (Figure 7.3). Proteins transferred
from alkaline urea gels to PVDF could be successfully sequenced (Figure 7.4), which
suggests that the process did not N-terminally block the proteins. Operating the gel
system in an ice bath and chilling the buffer aided in lowering the temperature of the
system (Figure 7.5), which may help to prevent protein modification. The length of
the gel could be more efficiently used by decreasing the acrylamide concentration and
decreasing the pH of the stacking gel (Figure 7.6). The protein bands in an alkaline
urea gel could be removed and run in an SDS-PAGE system to estimate the protein
size (Figure 7.7). This approach appeared to give a reasonable estimation of protein
size, and accuracy did not appear to be improved by reducing the proteins before
electrophoresis (Figure 7.8), thought this process may lead to loss of proteins.
These gels could be used to examine carbamylated and non-carbamylated protein
(Figure 7.9). These gels could also be used to examine forms of haemoglobin
(Figure 7.10). The extraction efficiency could be improved by lowering the pH of
the extraction buffer (Figure 7.11). Phospholipase A2 could be extracted from crude
tiger snake venom and analysed for enzyme activity (Figure 7.12). These gels could
also be used to examine the mobility of a variety of available of phospholipase A2
isoforms, which are very similar in size and difficult to resolve using SDS-PAGE
(Figure 7.13). These gels were able to separate the proteins with phospholipase A2
activity from normal and infarcted gut samples (Figure 7.14). The protein from
infarcted gut appeared to have higher mobility than that from normal gut. When the
179
Results.
bowel protein of interest was partially purified by heparin sepharose chromatography
it retained high mobility in these gels (Figure 7.15). The phospholipase A2 activity
from synovial fluid and normal human gut appear to run to a similar position in these
gels (Figure 7.16). The different mobilities of proteins in these gels did not appear
to be due to different salt concentrations/ionic strength of samples (Figure 7.18).
180
181
Figure 7.1: A typical PVDF membrane after blotting. The membrane surface in
contact with the gel is shown in the upper picture. The opposite side of the
membrane is shown in the lower picture. The sample shown in these pictures is
a bowel content sample, precipitated with ammonium sulphate (60%
saturation).
182
Figure 7.2: Appearance of gel (upper picture) after blotting and an equivalent gel
without blotting (lower picture). The sample shown in these pictures is a bowel
content sample, precipitated with ammonium sulphate (60% saturation).
183
1 2 3 4 5 6
1 2 3 4 5 6
Figure 7.3: Transfer of crude snake venom proteins and small basic proteins from alkaline urea
PAGE to PVDF. Transfer efficiency of CAPS buffer (upper membrane) and Towbin (lower
membrane) are compared.
Lanes 1&2 Crotalus scutulatus (Mojave rattlesnake) venom, 20µg protein.
Lanes 3&4 cytochrome c- (bovine heart) 4 µg protein. (Cytochome C- Mr= 12.33kDa, pI=10.6).
Lanes 5&6 sPLA2-III (Apis mellifera) 4 µg protein (sPLA2-III Mr=15.25kDa, pI=8.07).
184
1 2 3 4 5 6
Theoretical: IIYPGTLWCGHGN
Experimental: IIYPGTL……
Figure 7.4: N-terminal amino acid sequencing of proteins transferred from alkaline urea gels. Lanes 1&2 Crotalus scutulatus (Mojave rattlesnake), crude venom, 20µg per lane.Lanes 3&4 Cytochrome c, 4µg per lane.Lanes 5&6 Bee venom sPLA2, 4µg per lane.
185
05
1015202530
0 1 2 3 4 5
Time elapsed (hours)
Tem
pera
ture
(o C
)
Original gel
Modified gel
Figure 7.5: Running temperature of alkaline urea gels according to
the original method or the modified method. The running buffer was
replaced with fresh buffer after two hours.
186
1 2 3 4 5 6 1 2 3 4 5 6
Modified technique Original technique
Figure 7.6: Urea gels of crude snake venoms, run according to the original method or the modified method.Lanes 1&2: Agkistrodon rhodostoma (Malayan pit viper), 20µg protein per lane.Lanes 3&4: Crotulus scutulatus (Mojave rattlesnake), 20µg protein per lane.Lanes 5&6: Echis carinatus (Saw scaled viper), 20µg protein per lane.
187
Alkaline
urea PAGEProtein markers
664535
251814
SDS-PAGE
Figure 7.7: Extraction of protein bands from alkaline urea gels for SDS
PAGE size estimation. Sample used was crude venom of Crotalus
Scutulatus (Mojave rattlesnake). SDS-PAGE: 4-20% iGEL, TrisGlycine
gel (Gradipore). Protein bands could also be extracted for Tricine gel for
the estimation of size of very small proteins (results not shown).
188
1 2 3 4 5 6 7 8 9
Cyc
toch
rom
eC
Bee
ven
om
phos
phol
ipas
e A
2
116
66
45
35
25
18
14
116
66
45
35
25
18
14
1 2 3 4 5 6 7 8 9
Figure 7.8: An assessment of the accuracy of protein size determination after alkaline urea PAGE. The gel shown is an SDS-PAGE gel, 8-16% iGEL, Gradipore.Lanes:1 Protein markers2&3 Gel pieces from urea gel, reduced with method A4&5 Gel pieces from urea gel, non reduced6&7 Gel pieces from urea gel, reduced with method B (Davie, 1982)8 No prior urea gel, reduced 9 No prior urea gel, non reduced
189
1 2 3 4
1 2 3 4116
66
45
35
25
18
14
Figure 7.9: Carbamylation of bee venom sPLA2. The upper gel is an alkaline
urea gel. The lower gel is an SDS-PAGE gel of gel pieces from the alkaline
urea gel.
Lanes 1&2 carbamylated PLA2.
Lanes 3&4 non-carbamylated PLA2.
190
1 2 3 4
1 2 3 4
118
85
50
33
26
20
Alkaline urea gel
SDS-PAGEPr
otein
markers
Figure 7.10: Haemoglobin in alkaline urea gels. The upper gel strip is from
an alkaline urea gel. The lower gel is an SDS-PAGE gel (4-20% iGEL,
Gradipore).
191
0
20
40
60
80
100
Control Extraction withEthanolamine
Extraction withTris
Extraction withMilli-Q
Phos
phol
ipas
e A 2
act
ivity
, %
of c
ontr
ol
Figure 7.11: Extraction of phospholipase A2 enzyme activity from alkaline
urea gels. Results are expressed as the mean +/- std error as a percentage of
the control (no prior gel separation). The extraction buffers were
10mM ethanolamine, Milli-Q water, or 10mM Tris pH 6.8. The enzyme
sample employed was bee venom sPLA2.
192
02040
6080
100
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31
Slice number
Phos
phol
ipas
e A 2
activ
ity
Figure 7.12: Tiger snake, crude venom, run in alkaline urea gel. Gel was sliced
into 1mm sections and protein was eluted into solution. Each section assayed for
PLA2 activity. The upper picture shows the gel stained for protein and the graph
below shows the PLA2 activity in each 1mm slice.
193
0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Slice number
PLA 2
act
ivity
of
stan
dard
s
0
5
10
15
20
PLA 2
act
ivity
of
bow
el s
ampl
e
IB IIA III BC
Figure 7.13: Native urea gel of available PLA2 isoenzymes and bowel PLA2.
Type IB: purified porcine pancreatic phospholipase A2 (Sigma).
Type IIA: recombinant human phospholipase A2, (a kind donation from Dr.
Kieran Scott)
Type III: purified bee venom phospholipase A2 (Sapphire Biosciences).
194
0
2
4
6
Distance from well (mm)
Phos
phol
ipas
e A 2
acti
vity
(no
rmal
)
0
10
20
30
Phos
phol
ipas
e A 2
acti
vity
(i
nfar
cted
)
Normal bowel Infarcted bowel
Figure 7.14: The mobility of phospholipase A2 proteins from infarcted and
normal bowel in alkaline urea gels. Infarcted bowel content (precipitated
with ammonium sulphate at 60% saturation) vs normal bowel extract.
195
Heparin column:
0
1
2
3
4
5
1 5 9 13 17 21 25 29 33 37 41
Fraction
NaC
l con
cent
rati
on,
(M)
051015202530
PLA 2
act
ivit
y, %
hy
drol
ysis
NaCl PLA2
0
2
4
6
8
10
1 4 7 10 13 16 19 22 25 28
Distance into gel (mm)
PLA
2 ac
tivity
(%
hyd
roly
sis)
Alkaline urea gel:
Figure 7.15: A heparin column of crude bowel content eluted with increasing
NaCl. The fraction that exhibited the highest phospholipase A2 activity was
retained and run on an alkaline urea gel.
196
01234567
1 3 5 7 9 11 13 15 17 19 21 23 25 27
bow
el P
LA2 a
ctiv
ity
0
5
10
15
20
syno
vial
PLA
2 ac
tivity
Normal bowel Synovial fluid
Figure 7.16: PLA2 activity extracted from alkaline urea gels of
synovial fluid and supernatent from normal bowel.
197
0
0.2
0.4
0.6
0.8
1
0 0.25 0.5 0.75 1
NaCl (M)
mob
ility
Figure 7.17: Mobility of bee venom sPLA2 (10µg per lane) with increasing
concentrations of NaCl. Data represented as mean +/- standard deviation (error
bars <1%) of duplicate values. Mobility is represented as the distance that the
protein band ran from origin divided by the distance of bromophenol blue marker
dye from origin.
Discussion
Chapter 7 Discussion:
Alkaline urea gels were used in this project to investigate whether the protein
responsible for the increased phospholipase activity in infarcted human bowel had
different characteristics from normal human bowel, and other well described sources
of phospholipase A2. These gels were more useful than SDS-PAGE gels in this type
of comparative analysis because the native activity of the protein was retained. The
process of extracting the enzyme and assaying it for phospholipase activity was far
more sensitive that protein gel staining, and therefore able to specifically localise the
protein of interest within the gel. These gels were devised by Ahmad and colleagues
(1994) for the analysis of isoforms of phospolipase A2 from snake venoms. The
connection between snake venom and intestinal proteins may appear questionable, but
others have noted the:
‘suprising similarities between the intestinal phospholipase A2 and some basic
venom proteins’ (Verheij et al, 1981).
These gels would be of use in many applications where the native activity of an
enzyme could be detected after electrophoresis. The mobility of proteins in these
gels is a function of size, shape and number of charged residues. This type of native
gel is also more useful than SDS-PAGE in the study of isoforms of similar sizes. For
example, a similar native gel (contained 8M urea and no SDS) was able to resolve
variants of lysyl oxidase from bovine aorta that were unable to be resolved by SDS-
PAGE (Williams & Kagan, 1985).
Alkaline urea gels have been criticised for a number of reasons including;
- the inability to assess protein size from these gels (Chettibi & Lawrence,
1989),
- the potential risk of carbamylation which may cause N-terminal blocking,
and therefore inability to N-terminally sequence proteins of interest,
- the potential risk of artifactual protein bands.
We considered that a number of extensions may be possible to address these issues
and increase the number of applications available for these gels.
198
Discussion
Transfer of proteins to membranes:
If proteins could be immobilised on a membrane, then they may be able to be N-
terminally sequenced by Edman chemistry or proteins of interest may be detected
with Western blotting. For this to be possible two issues had be addressed:
a) The transfer of small basic proteins (which are typical of phospholipase A2
isoforms and venom proteins) can be problematic
b) It was unknown whether proteins could be electro-transferred from urea gels.
The traditional transfer solution for proteins, the Tris Glycine buffer, (or Towbin
buffer, pH 8.3) was devised for the transfer of proteins up to a pI of approximately 7
(Towbin, Staehlin & Gordon, 1970). The efficiency of transfer of proteins varies
greatly, and it thought that the transfer buffer pH should be 1 to 2 pH units above the
isoelectric point of the protein of interest. The CAPS transfer buffer (pH 11) had
been devised for the transfer of basic proteins from SDS-PAGE gels to nitrocellulose
(Szewczyk & Kozloff, 1985).
The CAPS transfer buffer was found to transfer small basic proteins and crude venom
proteins from alkaline urea gels to PVDF more effectively than the Towbin buffer,
probably because of the high pH of the CAPS buffer. The proteins transferred to the
membrane could be successfully sequenced by N-terminal amino acid determination
by Edman chemistry (Edman & Begg, 1967). This is a positive finding because it not
only extends the applications available for these gels but suggests that N-terminal
blocking of proteins by carbamylation is minimal. The use of the membranes in
Western blotting would be an interesting extension for these gels, but was not within
the scope of this project.
Carbamylation:
The risk of carbamylation increases with increasing temperature, time and urea
breakdown, therefore efforts were taken to reduce each of these factors. Firstly, as
recommended by Ahmed & Lawrence (1994), high quality urea was used and gels
were run for two hours before the addition of the protein samples. These steps were
likely to reduce the risk of urea breakdown and to remove any breakdown products if
they were present. Secondly we chose to run the gels in an ice bath to reduce the
buffer temperature. Additionally, buffers could be pre-chilled. The acrylamide
concentration and pH of the stacking gel could both be reduced which appeared to
199
Discussion
increase the mobility of the proteins in the gel. These modifications had two results:
firstly the increased protein mobility reduced the running time and secondly the length
of the gel was used more efficiently. Bee venom phospholipase A2 contains 28
potential carbamylation sites. Bee venom PLA2 was carbamylated for 48 hours at
37oC, and was then run on an alkaline urea gel and SDS-PAGE. Carbamylation adds
only 43 atomic mass units, and this small difference could not be detected in an SDS
gel, as expected. Carbamylation results in the loss of positive charges from a protein,
and therefore mobility in alkaline urea gels should theoretically be increased. A
slight increase in mobility was suggested in the alkaline urea gels.
Native gels are excellent tools for detecting protein aggregates, unfolded
conformations and binding events (Alliance Protein Laboratories, 2003). A number
of haemoglobin forms were evident in the alkaline urea gels, and the nature of each
could be estimated by determining the size of the aggregate by SDS-PAGE.
The size of the proteins of interest could be estimated by excising protein bands from
the alkaline urea gel and re-running in an SDS-PAGE gel. The protein bands were
excised easily and cleanly from alkaline urea gels stained with either Coomassie R-
250 or Coomassie G-250 based stains. The gel pieces could be stored in the freezer,
and freezing aided in the manipulation into SDS-PAGE gels. The proteins could be
reduced with either a β-mercaptoethanol/equilibration technique (Davie, 1982) or a
DTT-boiling technique (that we developed to avoid the more toxic and odorous β
mercatoethanol). The reduction step did not appear to improve the accuracy of the
size determination, but may have resulted in protein losses. The transfer of protein
from alkaline urea gels to SDS gels was advantageous in two ways, firstly the size of
proteins of interest could be estimated and the process added an extra dimension of
resolution.
The extraction efficiency of proteins from these gels was reported to be approximately
60% (Ahmad & Lawrence, 1993). We found that the extraction efficiency was
improved by lowering the pH of the extraction buffer. Active PLA2 could be
extracted from crude tiger snake venom. These gels were used to examine a number
of different purified isoforms of PLA2 that were available to our group. The proteins
200
Discussion
with phospholipase activity from normal and infarcted human bowel were compared,
and the mobility of each of these proteins was quite distinct. Not surprisingly, the
phospholipase from normal bowel appeared to have similar mobility to the
phospholipase in synovial fluid from patient with rheumatoid arthritis. Normal gut
(Cupillard et al, 1997, Nevalainen, Gronroos & Kallajoki, 1995) and rheumatoid
arthritis (Seilhamer et al, 1989) are both known to be rich sources of type IIA
phospholipase A2. The infarcted gut protein with phospholipase activity had high
mobility in alkaline urea gels, and partially purified protein showed similar mobility.
This confirmed that the unusual protein was being followed successfully by the
protein purification process. The isoelectric point of a protein is dependent on the
environment (pH, ionic strength etc), so it was possible that different mobility in
alkaline urea gels was not due to protein differences but the ionic strength or pH of
the protein solution. To discount this possibility a series of protein solutions were
prepared containing 0 to 1M sodium chloride. Even under conditions of 1M NaCl,
the protein maintained the same mobility. This was a reassuring finding because
different protein mobility strongly suggests different protein composition. The ions
present in protein samples are likely to have high mobility under the running
conditions and are likely to be lost quickly.
201
Conclusions and Future Directions.
Chapter 3, Plasma Proteomics:
The proteomic investigation undertaken as part of this project identified a number of
proteins that appeared to be increased in the plasma of patients with confirmed bowel
infarction compared with control patients. The aim of the investigation was to assess
whether a tissue damage marker specific to bowel was present in high abundance in
plasma. The proteins that appeared to be the most differentially expressed were
variants of haptoglobin (in the region of pH 4-7) and serum amyloid A (in the region
pH 7-10). These two proteins are well recognised as being disease associated
proteins and have been shown to be present in high concentrations in inflammatory
diseases, infection and trauma. The elevated levels of these two proteins in disease
suggest that they may have a role in host defence, tissue repair and inflammation.
The existence of several highly homologous variants of serum amyloid A and lack of
variant specific assays makes the study of this protein difficult. The commercially
available serum amyloid A ELISA (TriDelta) detects the total acute phase serum
amyloid A (SAA 1 & 2 subtypes), with a good limit of detection and reproducibility.
However, if subtle patterns of SAA subtype levels exist that are indicative of a
particular disease, an ELISA such as this would not detect these patterns. At present
there are no serum amyloid A subtype specific antibodies available. It may be
interesting to probe two dimensional gel electrophoresis membranes (pH 3-10) for
SAA variants using Western blotting to assess protein changes due to disease states.
At present, the assay of haptoglobin and serum amyloid A for diagnostic purposes is
not a clinically attractive prospect because of their likely non-specific expression in
response to inflammation, injury or infection. There may be haptoglobin or SAA
subtype specific increases that are indicative of bowel infarction, but these cannot be
studied without subtype specific techniques such as subtype specific ELISA.
202
A proteomic approach for diagnostic marker identification is useful, but a number of
modifications to the techniques such as those used in this project are required to
improve the chance of finding a useful diagnostic protein. These modifications
include pre-fractionation of samples prior to two dimensional electrophoresis, or an
alternate technique such as the isotope coded affinity tag (ICAT) system.
Firstly, pre-fractionation of plasma (for example, by preparative isoelectric focussing
or sub-proteomics) would allow the generation of a set of sub samples. The sub
samples could each be run in a two dimensional electrophoresis gel system. Pre-
fractionation is likely to allow the identification of lower abundance proteins, which
are more likely to be disease associated proteins or markers of specific tissue damage.
Secondly, an approach such as the ICAT (isotope coded affinity tag) system may
provide very interesting insights into protein differences between plasma from bowel
infarction and control patients. The ICAT technique was developed to achieve
quantitative protein profiling to examine differences between the proteins from a
matched pair of samples. Briefly, this technique involves the labelling of the cysteine
residues in sample ‘A’ with a ‘light’ (hydrogen, d0) biotin linked reagent and those in
sample ‘B’ with a ‘heavy’ (deuterium, d8) biotin linked reagent. Samples A and B
are then mixed, trypsin digested and the ICAT peptides isolated by avidin affinity
chromatography (Gygi & Aebersold, 2000). The peptides are then identified by LC-
MS/MS and quantified by the ratio of the ICAT labelled peptide pairs. The
preliminary ICAT experiments focussed on the protein differences between yeast
grown with a variety of carbon sources (Gygi et al, 1999). Recent ICAT publications
have reflected the diversity of potential applications (cortical neurons, wheat and
membrane proteins).
The advantages of the ICAT technique include the highly specific and robust labelling
process and the ability to use a wide variety of protein sources and quantities. Other
separation techniques could be added to the ICAT procedure to identify lower
abundance proteins (Gygi, Rist & Aebersold, 2000). The disadvantages of the ICAT
technique include some difficulty in database searching because of the modification
of peptides by the tag, and some peptides are missed because they contain no cysteine
residues (~8% of proteins in yeast contain no cysteine residues, (Gygi, Rist &
203
Aebersold, 2000)). In this type of project, the ICAT approach would be
advantageous because large amounts of protein could be analysed which would
increase the likelihood of identifying a low abundance, disease associated protein.
The ICAT technique also allows the identification of minor protein differences
between two states, in this case bowel ischaemia/infarction and control plasma.
In a different issue, proteomics may be a valuable tool to identify protein changes
associated with the development of remote organ injury that may be seen as a
consequence of bowel injury, either in blood or in mesenteric lymph. It would be of
great interest to study protein factors in mesenteric lymph that may be:
- mediators of remote organ injury
- potential therapeutic targets for remote organ injury
- expressed at different time points during the development of remote organ
injury
-involved in the induction of related processes such as increased gut
permeability, neutrophil priming and intestinal mucosal damage.
The study of lymph would be potentially less difficult than plasma, as lymph has only
half the total protein of plasma (Anderson & Anderson, 2002). Previous research
into the potential mediators of tissue/cellular damage carried in the mesenteric lymph
has generally been conducted in rodent models of hemorrhagic shock. Bacteria and
endotoxin are unlikely to be important factors, as these could not be detected in the
post shock lymph (Deitch et al, 2001, Magnotti et al, 1998). The potential
importance of a neutral lipid (rather than protein or carbohydrate) factor was
highlighted by another study (Gonzalez et al, 2001). It would be of great interest to
identify the nature and source of this lipid factor, and the enzyme(s) responsible for its
production.
204
Conclusions and Future Directions.
Chapter 4, Phospholipase Activity in Mesenteric
Ischaemia/Infarction:
Increased mucosal phospholipase A2 activity associated with gut ischaemia
reperfusion injury had been previously reported in rat models (Koike et al, 1992,
Koike et al, 1995, Otamiri et al, 1987, Otamiri, & Tagesson, 1989). The involvement
of phospholipase A2 in the pathogenesis of ischaemia/reperfusion injury had also been
proposed by several groups, based on the protective qualities of phospholipase
inhibitors (Koike et al, 1992, Koike et al, 1995, Otamiri et al, 1987, Otamiri, Lindahl
& Tagesson, 1988). Many groups had identified the presence of the isoform of
phospholipase A2 type II, in normal gut tissue using a variety of techniques
(immunohistochemistry, in situ hybridisation, PCR), but few studies have investigated
PLA2 isoforms in diseased tissue. We were in the unusual but fortunate position to
have access to human bowel tissue from both bowel infarction and control patients.
In this project, early experiments suggested that while type IIA phospholipase A2 was
present in injured human bowel, this isoform was not responsible for all of the
phospholipase activity in these samples. Phospholipase activity was significantly
higher in the lumen of infarcted human bowel than in the corresponding bowel tissue.
This finding suggests a possible route for the protein to enter general circulation. It
would be a valuable future direction to examine the synthesis and/or release of this
protein in response to the development of the ischaemic injury. The possibility that
the protein is simply being leaked from dying cells needs to be eliminated. This
could be achieved by measuring the rate/percentage of cell lysis by assaying
cytoplasmic proteins or cell membrane integrity. An in depth investigation of the
hydrolysis products of phospholipase activity and their effects during
ischaemia/reperfusion injury and remote organ injury would also be valuable.
It would also be interesting to examine the phospholipase enzyme activity in matched
human portal and systemic circulation, ischaemic and infarcted tissue, to examine the
205
relative enzyme activity at each stage of the possible release into circulation. It may
also be a valuable exercise to use a labelled protein to assess intestinal permeability
changes in response to ischaemic injury, perhaps at the same time as the examination
of phospholipase activity. In a rat model of ischaemia/reperfusion, phospholipase
activity was ten fold higher in portal than systemic circulation, and this suggested that
the circulating phospholipase activity arose from the injured gut (Koike et al, 2000).
Our results showed that phospholipase activity was significantly higher in infarcted
than matched ischaemic tissue. Although this was only a sample size of one, this
result may be of significance if a spectrum of phospholipase activity reflects a
spectrum of tissue damage. In turn this may be of relevance in the development of a
diagnostic test, as the level of the protein in circulation may reflect the severity of the
condition. In future investigations, many more matched tissue samples would assist
in answering these questions. Many potential diagnostic markers of bowel infarction
have not found clinical utility because they either:
- decrease before the condition has been rectified (Bounous et al, 1984,
Jamieson et al, 1982).
- continue to increase even after the condition is rectified or
- cannot distinguish between ischaemia and infarction (Gearhart et al, 2003).
A suitable animal model could assist in examining the correlation between the degree
of tissue damage and protein response. However, there are significant species
differences in the distribution of phospholipase A2 isoforms making an animal model
unreliable, however this may not be an issue with other potential markers of bowel
injury. A time course study of protein changes at the various stages of the disease
development would also be a valuable future direction, and an animal model would be
essential for this investigation.
206
Conclusions and Future Directions.
Chapter 5, Protein Purification:
The protein purification experiments in this project were undertaken to identify the
protein with phospholipase activity that was increased as a result of infarction.
Immunodepletion results suggested that the well described phospholipase A2 isoforms
of type IIA and V did not account for all of the phospholipase activity in infarcted
human bowel. The protein purification techniques used in this project were chosen
because of their ability to exploit an unusual characteristic of the protein of interest,
and in doing so, remove large amounts of contaminating protein at each step. In
addition, each purification technique used in this project retained the native enzyme
activity of the protein of interest, and therefore the protein could be tracked at each
stage. A number of preliminary purification attempts identified the purified protein
as haemoglobin. It was quickly confirmed that haemoglobin does not possess
phospholipase activity, nor does it enhance the activity of low levels of
phospholipase. It was concluded that haemoglobin was likely to have been co-
purifying with the protein of interest. To overcome this problem, a greater number of
column volumes were employed with the heparin affinity chromatography column.
The purification system was also scaled up approximately twenty fold, and this is
likely to have assisted in targeting the protein of interest for identification. The
scaled up purification with more stringent washing steps allowed the isolation of
several highly purified fractions, each with sufficient protein content for identification
by mass spectrometry.
The protein purification techniques used in this project (heparin affinity
chromatography, preparative isoelectric focussing with the Rotofor® apparatus) were
very useful for this application. Heparin affinity chromatography can be used to
easily semi purify a protein with heparin binding ability, whilst generally retaining the
native activity of the protein. It would be of interest to automate this system, which
may result in higher reproducibility and larger quantity purifications while decreasing
the currently highly labour intensive nature of this technique. It would be of interest
207
to optimise the protein desalting and concentration steps that are required at the
interface of the heparin column and the next technique (in this case, preparative
isoelectric focussing). The optimal desalting/concentration step would remove all of
the interfering salts or other contaminants and the majority of the water without any
protein alteration or loss. The dialysis step used in this project was time consuming
and did not concentrate the protein which resulted in the need for several Rotofor®
runs of dilute solutions, instead of one run of a pooled and concentrated sample.
The final identification of the protein of interest as cyclophilin B was made by nano
LC-MS/MS. An unusual aspect of the identification of cyclophilin B was that the
purification progress had been monitored at every stage by phospholipid hydrolysis
assays, but cyclophilin B was not known to possess phospholipase activity. A set of
experiments were undertaken to assess the possibility that cyclophilin B possessed
phospholipid hydrolysing ability. Immunodepletion of cyclophilin B from the highly
purified final fractions removed all of the phospholipase activity. This indirect
evidence was interesting and supported the idea that cyclophilin B had phospholipase
activity, but the direct investigation would have been preferable. However, an
application to the only known supplier of human cyclophilin B protein was
unsuccessful. In the future it would be of interest to produce recombinant human
cyclophilin B to examine the enzyme characteristics in detail, and to investigate its
pro-inflammatory properties. Recombinant human cyclophilin B has been produced
previously (Bergsma et al, 1991, Price et al, 1991, Spik et al, 1991) and these
methods would form the basis of this future direction.
208
Conclusions and Future Directions.
Chapter 6, Cyclophilin B:
Cyclophilin B had been reported to be a ubiquitously expressed protein as detected by
northern blotting of mouse tissue panels (Hasel et al, 1991). Cyclophilin B had been
observed previously in rodent intestinal tissue at the mRNA level (Hasel et al, 1991,
Kainer & Doris, 2000). Very little research has focussed on cyclophilin B at the
protein level in tissues, but some work has detected cyclophilin B protein in cell
culture (Bergsma et al, 1991).
In this project, western blotting of cyclophilin B was performed using a commercially
available polyclonal antibody. Immunoreactive cyclophilin B protein was detected in
all bowel tissues investigated, from both control and bowel infarction patients.
Immunohistochemistry results supported the western blotting results, and showed
widespread distribution of cyclophilin B in many cell types in both normal and
infarcted tissue sections. Future directions in this area include the investigation of
cyclophilin B protein in a variety of normal and diseased human tissues via
immunohistochemistry, and the corresponding plasma by ELISA.
Another future direction for this project may include the use of recombinant human
cyclophilin B in the development of a sensitive and specific ELISA that is suitable for
clinical samples. To our knowledge, two cyclophilin B ELISAs have been
developed. These two ELISA protocols used the same cyclophilin B peptide
generated polyclonal antibody and constructed a standard curve using recombinant
cyclophilin B protein.
Allain et al, 1995, published a cyclophilin B ELISA with the following format:
-anti-cyclophilin B coated plates
-anti-cyclophilin B peroxidase conjugate
-O-phenylenediamine substrate
209
The limit of detection of this ELISA was reported to be 10ng/mL and the average
normal plasma level of cyclophilin B was found to be approximately 150ng/mL.
Denys et al, 1998, published a modification of the above technique with the following
format:
-anti-cyclophilin B coated plates
-anti-cyclophilin B biotin conjugate
-avidin-peroxidase conjugate
-O-phenylenediamine substrate
This ELISA was reported to have better sensitivity than the above method, because of
the likely biotin signal amplification effect. This system would be a valuable starting
point for the development of an ELISA in this project. Average normal plasma levels
were reported to be approximately 87ng/mL.
A sensitive and specific ELISA for cyclophilin B would allow the large scale
screening of patients samples and this may lead to a better understanding of the
correlation between increased cyclophilin B levels and disease states. In this project,
plasma cyclophilin B was not detected in any control patients, but several samples of
plasma from patients with confirmed bowel infarction contained cyclophilin B (via
Western blotting). An ELISA with a very low level of detection may be able to
establish typical ranges for plasma levels of cyclophilin B in bowel infarction, healthy
volunteers and control ICU patients. At present, this is not an option because
recombinant protein could not be obtained, and therefore a standard curve could not
be constructed.
Another future direction would be the development of novel cyclophilin B inhibitors
that blocked the inflammatory activity of this protein without altering the distribution
of cyclosporin A in blood or extracellular fluid or initiating the expression of more
cyclophilin B. It would also be of great interest to examine the potential role of
cyclophilin B as a mediator of inflammation or tissue injury, perhaps with an
emphasis on bowel ischaemia or infarction. Furthermore, an investigation into the
protective roles of cyclophilin B inhibitors in inflammatory conditions, oxidative
stress and ischemia/reperfusion injury could be extended to include bowel conditions
such as bowel ischaemia/infarction.
210
Conclusions and Future Directions.
Chapter 7, Alkaline Urea PAGE:
The alkaline urea gel system of Ahmed, Lawrence & Moores (1994) was found to be
useful in this project, to examine differences in protein characteristics between a
control and disease state. This application was more suited to an alkaline urea gel
system than SDS PAGE because:
-the protein of interest was present in very low abundance and could not be
detected by protein staining, but could be detected by extracting enzyme
activity.
-the proteins from normal and healthy tissue were of similar size, and
therefore unable to be resolved by SDS PAGE, but as mobility in alkaline urea
gels is due to a number of variables, this technique could resolve these
proteins.
These gels were modified to allow greater mobility of proteins, and therefore greater
use of the entire length of the gel. The modified alkaline urea gel showed that the
proteins with phospholipase activity from normal human bowel and rheumatoid
arthritis synovial fluid (both well described and abundant sources of type II PLA2)
were similar and focussed in a region close to the site of application. In contrast, the
protein with phospholipase activity from infarcted human bowel lumen content had
distinctly different, and higher mobility. It would be of interest to use these gels to
examine mammalian phospholipase from normal and disease state samples.
A number of limitations were associated with these gels and attempts were made to
overcome these problems in order to extend the applications available. We have
proposed a method to estimate protein size of the proteins of interest from alkaline
urea gels. This technique resulted in a reasonable estimation of protein size which
may give information about a protein of interest and may allow a suitable purification
process to be planned (for example the use of gel filtration chromatography or
211
preparative PAGE). An additional, but unexpected, advantage of this technique was
the extra dimension of resolution provided by this step. For example, one protein
band observed in an alkaline urea gel of a crude snake venom resolved into two
protein spots when run in an SDS PAGE gel. This may be advantageous in very
complex protein samples, in a similar fashion to two dimensional gel electrophoresis.
Future directions for this area include an investigation of the suitability of western
blotting as an extension for these gels. Proteins separated by alkaline urea gels can
be successfully transferred to PVDF membrane, and it may be possible to probe these
membranes for immunoreactive proteins including venom toxins. This approach may
be useful for the identification of isoforms of proteins of interest that may share
similar size and therefore may be unable to be separated by SDS PAGE based western
blotting.
212
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