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RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN
-A: A STRUCTURAL AND FUNCTIONAL STUDY
Agatha Lau
A thesis submitted in conformity with the requirements for the Degree of Master of
Science in the Graduate Department of Labonatory Medicine and Pathobiology,
University of Toronto
Q Copyright by Agatha Lau, 1999
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RNA-BINDING OF LC3 TO THE AU-RICH ELEMENT OF FIBRONECTIN
mRNA: A STRUCTURAL AND FUNCTIONAL STUDY
Master of Science, 1999
Agatha Lau
Department of Laboratory Medicine and Pathobiology, University of Toronto
ABSTRACT
During neointima formation in the Fetal ductus arteriosus (DA) and in diseased coronary arteries,
migration of smooth muscle cells (SMC) is modulated by upregulation of fibronectin (FN). This
involves increased translation of FN mRNA when microtubule-associated protein 1 light chain 3
(LC3) binds an AU-rich element (ARE) in the 3' untranslated region (32JTR) of FN rnRNA. We
now investigate how LC3 binds to the ARE by generating LC3 peptides and mutant LC3, followed
by northwestern (NW) immunoblotting and electrophoretic mobility shift assay (EMSA). The
positive charge of 3 arginine residues (the arginine-nch motif)(ARM) appears critical for LC3-ARE
binding. The significance of this motif in regulating FN mRNA translation is dernonstrated by
stably-transfecting wild-type (WT) vs mutant LC3 into LC3-nul1 HT1080 human fibrosarcoma
cells and observing increased fibronectin with wild-type LC3 only, associated with an elongated,
slower growing phenotype.
First of d l , 1 would like to express my deepest gratitude to my supervisor, Dr. Marlene Rabinovitch, for her tireless support and encouragement in these past two years. Her enthusiasm and talent in science, as weli as her determination and perseverance, have inspired me enormously in shaping my future path in iife and career. It has been an invaluable experience to pursue science under her supervision.
1 would also like to thank the members of my advisory cornmittee for contributing their precious time and scholarly advice to this work Dr Philip Marsden, Dr Fred Keeley, Dr Mario Moscarello and Dr Emil Pai.
1 am indebted to the members in Dr Rabinovitch's laboratory and the Division of
Cardiovascular Research for their help, consideration and humor. Special and most sincere thanics to Dr Bin Zhou, who is always so eager and patient to help and taught me al1 those
speciai 'tricks' in doing experiments. Also, 1 sincerely thank Dr Hassan Zaidi and Dr Haisong Ju, the most comprehensive and user-fnendiy RNA and Cellular Biology
'Dic tionaries' ; Dr Catherine Mason, the Northem 'superstar'; Claire Coulber and Alecia Lennard, my cells' most caring 'baby-sitters' while 1 was away; Joan Jowlabar, our wonderful secretary, who helped me with ail those letters and slides; and Dr Pascale Dufourcq, who kindly granted me the pnvilege to stay in her apartment while she was away, saving me from commuting during al1 the hazards of snow storms and TTC strike!
It was also my fortune to have met, worked and become fkiends with al1 these excellent people: Dr Peter Jones, Andrea Burry, Stacy O'Blenes, Caroline Fallery, Sandra Demaries,
Kyle Cowan and Arian Khandari (a very special one h m ET!)
Lastly, 1 would like to dedicate this thesis to my beloved parents, Francis and Cecilia Lau, for their unconditional and unlimited support and care, as well as endurance and understanding in the past 25 years.
TABLE OF CONTENTS
ABSTRACT
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF FIGURES
LIST OF ABBREVIATIONS
INTRODUCTION
Overview
1. Fibronectin and Intima1 Cushion in the Developing Ductus Arteriosus
Fibronectin and Srnooth Muscle Cell Migration
Regulation of Fibronectin Synrhesis
Light Chain 3 (LC3) Upregulates Fibronectin mRNA Translation
Microtubule Involvement in LC3-mediated Enhunced FN M A Translation
Phosphorylation of LC3 Enhmrces LC3-mediated FN Translational Upregulatron
Cell Motiliv is Relared to Other LC3-reguiafed Genes Bedes Fibronectin
II. Fibronectin in Post-Cardiac Transplant Coronary Arteriopathy
Neointinml Formation in Posr-Cardiac Tramplant Coronary Aneriopathy
Fibronectin and Migration of Inflammatory Cells und Smooth Muscle Cells
Coronary Artery Smooth Muscle Cells
iii
viii
III.
IV.
Fibronectin Reverses the Transformed Phenotype of HTlOSO, a - -
Human Fibrosarcoma Ceïï Line
Dom-regulatr8n of Fibronectin in Oncogenic Trmisformuhon
Fibronectin Suppresses the Transfomd Phenotype of HTIOBO Cells
Role of EGR-2 in FN Upregulation and Tumor Suppression
Stable Transfection of LC3 in HT1û80 Cells Upregulates FN Syntheis
Role of Protein-RNA Interactions in mRNA Translation
Role of 3' Untranslated Region ( 3 ' m ) AU-Rich Element (ARE) in mR Translation
ARE-binding Proteins mtd Their Functiom
RNA-binding Motvs
HYPOTHESES and OBJECTIVES
MATERIALS AND METHODS
Ce11 Culture
Expression of Recombinant LC3
Partial Proteolysis of LC3 by Endoproteinases
5' end-radiolabeling of RhrA Probe
Zn vitro Transmption of RNA Probe
North western (NW) Blot Analysis
Site-directed Mutagenesis
Gel Mobility Shrfi Assays
Stable TrCUZSfection
Western Immunoblot Analysis
Cell Growth Curves
Indirect Immunofluorescence
FN Biosynthesis
RNA Isolation and Northern Blot AnalysiS
RESULTS
LC3 Binds the ARE of FN mRNA at the IOkD N-terminal Region
LC3 Binds Specificaily to ARE of FN mRNA
Arginine-Rich Motif (ARM) is Cntical in LC3 Binding to ARE of FN rnRNA
LC3 Buidhg to ARE via ARM Codbned in Stably-transfected HTl080 Ceils
Effect of WT and Mutant LC3 on FN Synthesis
LC3 Regulates Ceii Growth and Morphology via ARM-ARE Binding
Subcellular Locahatîon of WT and Mutant LC3 in Stable- transfectants
DISCUSSION
FUTURE STUDES
APPENDIX I
APPENDIX II
LIST OF FIGURES
Schematic summary of FN regdation by IL-1p. TNF-a and EDP as described in the text-
Figure 1
Figure 2
Figure 3
Figure 4
Schematic ribbon drawing of the hnRNP C RBD (residues 2-94).
Amino acid sequence of rat LC3.
Northwestern (NW) blot anaiyses show LC3-ARE binding localizes to a 10 kD N-tenninal region on LC3.
Figure 5 Arnino acid sequence of rat LC3 showing the 2 candidate RNA binding sites.
Fi,oure 6
Figure 7
Figure 8
Sumrnary diagram of NW biot analysis of LC3-ARE binding.
RNA binding of LC3 protein and lOkD peptide is preferential for the ARE.
LC3 binds ARE at the arginine-rich motif (ARM) via a charge-charge interaction.
Figure 9 Gel mobility shifi assay on HTlO8O cytosolic extracts with stably transfected WT and mutant LC3 confirms the significance of the ARM in AREcbinding.
Figure 10 Tmmunofluorescence labeling of ceil swface FN deposit in WT and mutant LC3 transfected HT1080 celis.
Figure I l FN expression in specific clones of WT and mutant LC3 stably-transfected HT1080 cells.
Figure 12 Steady state levels of FN mRNA in WT and mutant LC3 stably-transfected HT1080 ceiis.
TransIational efficiency of FN mRNA in WT and mutant LC3 stably- transfected HTlO8O cells.
Figure 13
CeU count of WT and mutant LC3 transfected HTlOSO celis. Table 1
Figure 14
Figure 15
Figure 16
Growth curves of WT and mutant LC3 transfected HTlO8O cells.
Effect of WT and mutant LC3 expression on HTlO8O cell morphology.
Immunofluorescence labeling of tubulin in WT and mutant LC3 transfected HTlO8O ceus.
Figure 17 hunofluorescence labeling of WT and mutant LC3 in distinct subceliular locations in stably-transfected HTlOSO cells.
Figure 18 Quantitative analyses comparing the subceiiuia. distribution of WT and mutant LC3 in stably-transfected HT1080 cells by western immunoblot.
vii
LIST OF ABBREVIATIONS
A
Ao
D
ARE
ARM
ATP
AUBF
BSA
C
CA
CD
cDNA
CNBr
cpm
CS
CS 1
CS-RBD
DA
DAPI
d m
DNA
Dm
EC
ecNOS
EBP
aàenosine
aorta
apoiipoprotein D
adenosine uridine nch element
arginine-nch motif
adenosine S'-triphosphate
adenosine uridine binding factor
bovine serum aibwnin
cytosine
coronary artery
caihepsin D
complementary deoxyribonucleic acid
cyanogen bromide
count per minute
chondroitin sulphate
connecting segment 1
consensus RNA-binding domain
ductus artenosus
4', 6-diamidino-2-phenylîndole
deoxycytidine S'-triphosphate
deoxyribonucleic acid
dithiothreitol
endothelid ceils
endotheiial constitutive nitric oxide synthase
elastin-binding protein
ECM
EDP
EDTA
EGF
e h
EMEM
EMSA
EPAN
EPLC
F
FBS
FN
FP
G
GAG
GAPDH
GM-CSF
GST
h
HA
HC1
Hel-N1
hnRNP
H R P
IgG
IL-f p
IL-3
extracellular rnahix
eiastinderived peptide
ethylenediamine te-tic acid
epidermal growth factor
embryonic lethal abnormal vision
Eagle's minimal essential medium
electrophoretic mobility shift assay
endoproteinase Lys-C
pheny lalanine
fetal bovine serum
fibronectin
free probe
guanosine
glycosaminoglycan
gl yceraldeh y de 3-phosphate dehydrogenase
granulocyte macrophage colony-stimulating factor
glutathione S-transferase
hour
hyaluronan acid or hemaglutinin
hy drochlonc acid
human elav-like neuronal protein 1
heteronucleoprotein
horseradis h peroxidase
immunoglobulin G
interleukin-1 $
interleukin-3
N O S
m-'Y IPTG
K
KCI
kD
KH
k-EL
LC3
LPS
M
MAP
MHC
Mgcl?
min
mRNA
N W
n
NaCl
nNOS
NO
NOS
P
PA
PAGE
PBS
PCR
uiduciile niûic oxide synthase
in terferon-y
isopropy l-PD-thiogalactopyranoside
lysine
potassium chloride
kilodaiton
K homology
kappa-elastin
light chah 3
lipopolysaccharide
methionine
microtubule-associateci protein
major histocompatibilty complex
magnesium chloride
minute
messenger ribonucleic acid
northwestem
number of experiments or samples
s o d i u m chloride
neuronal nitric oxide synthase
nitric oxide
nitric oxide synthase
phosphate
pulrnonary artery
pol yacry lamide gel elecirophoresis
phosphate buffered saline
polymerase chain reaction
PCTCA
PDGF
Phe
PI
PNK
PVDF
Q R
RBD
RER
RGD
RGG
rl?m
RNA
RNP
RNP-CS
RRM
rRNA
SD
SDS
SEM
SMC
snRNA
TBE
TBS-T
TCA
TGF-B
post-cardiac trausplant coronary aaaiopay
platelet derived growth factor
p hen y lalanine
isoelectric point
polynucleoti& kinase
pol yvinyldifiuoride
glutamine
arginine
RNA binding domain
rough endoplasmic reticulum
arginine-giycine-aspartate
@nine-gl ycine-gi ycine
revolutions per minute
ri bonucleic acid
ri bonucleoprotein
ribonucleoprotein consensus sequence
RNA recognition motif
ribosomal ri bonucleic acid
standard deviation
sodium dodecyl sulphate
standard emr of the mean
smwth muscle cells
small nuclear ribonucleic acid
tris-hm&-EDTA
tris buffered saline-tween 20
trichloroace tic acid
transfonning growth factor-$
tumor necrosis façt0r-a
transfer ribonucleic acid
3' untranslated region
uridine
uridine S'-triphosphate
ultraviolet
very l ate antigena
wild type
xii
INTRODUCTION
Overview
Upregulation of fibronectin 0 is associated with the smooth muscle cell (SMC)
migratory phenotype contributhg to intimai cushion formation in the closure of the ductus
arteriosus @A) in development and to the formation of the neointima in the post-cardiac
transplant coronary arteriopaîh y (PCTCA) and other occlusive vascular diseases. Studies
on the mechanism of FN upregulation in vascular diseases led to the discovery that hght
chain 3 (LC3) of microtubule-associated protein LA and 1B binds to the AU-rich element
(ARE) in the 3' untranslated region (3'UTR) of the FN mRNA and upregulates its
translation via ribosomal recniitment. This thesis investigates the peptide sequence in LC3
which binds to the ARE of the FN mRNA, and examines its role in regdating FN mRNA
translation using the HTLOSO human fibrosarcoma ceil line. The transformed phenotype of
HT1080 ceils stably transfected with wild type (WT) and mutant LC3 cDNA is dso
examined.
The following Introduction therefore reviews the role of FN in intimai cushion formation
during the closure of the DA as well as in PCTCA, outlining the effect of FN on SMC
migration, and rnechanisms involved in FN upregulation with emphasis on the roles of
LC3 and microtubules. The data supporting the role of FN in reverting the transformed
phenotype of HT1080 cells is then addressed. This is followed by a review of RNA-
protein interactions which modulate mRNA translation, focusing on the importance of
ARE, the function of other ARE-binding proteins, and common RNA-binding motifs.
1. Fibronectin and Intimal Cushion in the Developing Ductus Arteriosus
Ductus Arîen'osus
The ductus artenosus (DA) is a large fetal shunt comating the pulrnonary artery with the
aorta and allowing the majonty of the right ventricular cardiac output to bypass the
unexpanded lungs. The ductus closes at birth as the lungs expand and blwd oxygen
tension nses. The onset of breathing increases artenal oxygen tension mggenng the strong
vasoconstriction of the ductus. This process is mediated by a cytochrome-P450-dependent
mechanism (Coceani et al., 1988) which results in the release of a potent vasoconstrictor
endothelin-1, from endotheLial and smooth muscle cells (Coceani and Kelsey, 1992).
However, the functional closure of the DA is highly dependent on the prior formation of
the intimal cushions, a process which is initiated around 100 days of a 145-&y gestation
period in the fetal lamb DA, and is more or less completed by day 138. The formation of
intimal cushions is initiated by the accumulation of extracellular matrix @CM) in the
subendothelium, the region separating the endothelial cells (EC) from the intemal elastic
laminae- Smooth muscle cells (SMC) f'm the rnuscular media of the vesse1 wall migrate
in to the rnatrix-enric hed subendothelid region.
Extracellular Matrix-Ce22 Interactions in IntimaZ Cushion Formation
Studies from our iaboratory have shown that a sequence of cell-matrix interactions plays a
criticaï role in the remodeling process associated with the formation of intimal cushions.
The increased production of glycosaminoglycans (GAGS) specifically hyaluronan acid
(HA) by EC and chondroitin sulfate (CS) by SMC leads to their accumulation in the
subendothelium (Boudreau and Rabinovitch, 199 1). The hydrophilic properties of HA
allow it to bind large arnounts of water which causes expansion of a tissue space and
physically facilitates ce11 movement (Toole et al., 1984). The increased production of CS
causes shedding of the 67 k D elastin-binding proteins h m SMC surfaces, leading to the
impaired assernbly of elastin fibers (Hinek et al., 1991) and to the increased production of
elastin peptides, al1 of which favor the unrestricted movement of SMC into the
subendothehum. The major elastin peptide producecl was identified as the 52 k D muicated
form of tropoelastin (EGnek and Rabinovitch, 1993). This peptide cannot be insolubilized,
but is stable and a potent chernotactic factor to SMC. This elastin peptide can also directly
influence the migration of SMC by inducing their production of the matrix glycoprotein
fibronectin (FN) (Hinek et al-, 1992).
Fibronectin and Smooth Muscle Ce12 Migration
Fibronectin is a homodimenc glycoprotein composeci of subunits of 220-250 kD linked by
disulfide bonds close to their carboxy-terminal ends. It is present in the plasma or
associated with cells or their ECM. It influences ce11 adhesiun, migration, proliferation,
differentiation, cytoskeletal organization and apoptosis (Hynes, 1990; Hynes and Lander,
l992), depending on its interaction wi th the heteroàimeric transmembrane ECM binding
proteins, integrins. A specific RGD (arginine-glycine-aspartate) sequence located in the
type III repeat of FN is recognized by the aspl and a& integins (Pierschbacher and
RuosIahti, 1984; Rouslahti and Pierschbacher, 1987). Studies on SMC adhesion and
migration using blocking antibodies specific to different integrin complexes showed that
SMC adhesion to FN depends exclusively on hinctioning fil integrins while SMC
migration depends on both the UV pj and a4$, integrin receptors (Clyman et al.. 1992;
Molossi et al., 1995~).
FN has been implicated in SMC migration by modulating SMC from a 'contractile' to a
'synthetic' phenotype. In response to FW, cultured rat aortic SMC exhibit ce11 adhesion
and spreading, loss of myofilaments, cessation of the ability to contract, formation of
extensive rough endoplasmic reticulum (RER) and golgi complexes, increased RNA and
protein synthesis, as well as the ability to replicate DNA, divide and produce ECM
components in response to platelet-derived growth factor (PDGF) and other mitogens
(Hedin and Thyberg, 1987; Hedin et al., 1988). The minïmaI cell-attachment sequence on
FN, RGDS, was found to be responsible for the FN-mediatecl SMC dedifferentiation by
interacting with the B intefins (Hedin et ai., 1989). This FN-mediated-phenotypic change
also likely contributes to the enhanced SMC migration observed in intimai cushion
formation in the DA. In a parallel study comparîng cultured SMC h m the DA, aorta (Ao)
and pulmonary artery (PA) of lûû-day fetal lambs, DA SMC demonstrated a 2-fold
increase in FN synthesis compared to Ao and PA celis (Boudreau and Rabinovitch, 1991).
This upregulation of FN appears to contribute to the spindle-iike etongated morphology of
the DA SMC, as well as theîr enhanced migration in three-dimension collagen gels, since
RGD peptides and antibodies against M can convert DA SMC to a flattened, stellate
morphology and inhibit their migration to a level similar to Ao SMC (Boudreau et al.,
199 1).
In addition to directly altering SMC phenotype, increased production of FN leads to the
stimulation of migration dong a FN gradient as has been demonstrated in chick embryonic
precardiac ceiis duing heart formation. This directional migration can also be abrogated by
an antibody to FN or RGD peptides which interact with integrins and this results in
inhibition of cardiac development (Linask and Lash, 1998a; Linask and Lash, 1998b).
That FN-mediated change in phenotype and directional migration were critical to the infiux
of SMC into the subendothelium which leads to intima1 cushion formation in the DA, was
demonstrated by Mason et al (Mason et al., 1999a), based upon manipulation of the
regulatory mechanism of FN synthesis in fetal lambs.
Regulation of Fibronectin Synthesis
Upregulation of FN synthesis occurs at both transcriptional and pst-transcriptional levels.
Growth factors and cytokines such as epidermal growth factor (EGF), transfonning
growth factor-6 (TGF-fl), platelet-derived growth factor (PDGF) and interferon-y (IFN-y)
activate FN gene transcription (Blatti et al., 1988; Chen et al., 1977, Diaz and Jimenez,
1997). Interleukin- l$ (IL- 1 f3) upregulates FN gene transcription in vascular SMC
(Clausel1 and Rabinovitch, 1993; Molossi et al., 1995a) whereas hmor necrosis factor-a
(TNF-a) which cooperatively interafts with IL16 appears to influence FN synthesis at a
post-transcriptional level. This will be discussed in the next section. A number of
cytokines and growth factors have been shown to upregulate FN expression at a post-
transcriptional level by modnlatmg mRNA splicuig or stability or translational efficiency.
For example, TGF-8 increases transcription of FN mRNA in fibroblasts Qgnotz et al.,
1987), alters the splicing pattern of FN mRNA in cuitured normal human fibroblasts (Borsi
et al., 1990) and increases FN mRNA stability in cultured human dermal fibroblasts
(Raghow et al., 1987). In contrast, while IFN-y can upregulate FN gene transcription, its
ability to destabilize the FN mRNA and repress FN mRNA translation by inhibiting the
elongation steps results in an overall downregulation of FN synthesis in cultured human
and murine fibroblasts (Diaz and Jimenez, 1997; Levine et al., 1990).
Boudreau et al. found that the increase in FN synthesis in DA compared to aorta SMC was
not associated with an increase in steady state levels of FNA mRNA or in mRNA stability
or in differences in FN mRNA splicing (Boudreau et al., 1992). suggesting that enhanced
translational efficiency of FN mRNA in DA SMC results in the observed FN upregulation.
Zhou et al. (Zhou et al., 1997) later identified a protein which binds the adenosine-uridine
rich element (ARE) of the 3' untranslateci region (UTR) of the FN mRNA and upregulates
its translation through enhanced ribosome recruitment. This RNA-binding protein was
identified as the light chain 3 (LC3) of the microtubule-associated protein w) 1A and
1B.
Light Chain 3 (LC3) Upreguhtes Fibronectin mRNA TransZation
Light chah 3 (LC3), a 16.4 kD protein enriched in rat brain and CO-purified with
microtubules, was first identified by J. Hammarback as a subunit of the neuronal
~crotubule-associated proteins (MAPs), MAPlA and MAPlB (Mann and Hammarback,
1994). It was thought to play a role in regulating the microtubule binding activity of
MAPlA and MAPlB. Zhou et al. purified LC3 from the DA and Ao SMC as a
cytoplasmic factor which binds the AU-rich element (ARE) of the 3' UTR of the FN
rnRNA (Zhou et al., 1997). It was expressed at higher levels in the cytosolic extracts from
the DA compared to the Ao SMC, and was associateci with increased ARE-binding activity
and increased FN mRNA translation in the DA SMC. Its role in pst-transcriptionai
upregulation of FN, was further established by demonstrating that overexpression of
recombinant LC3 in Ao SMC, resulted in enhanced FW mRNA translation to Ievels
observed in the DA SMC without altering FN mRNA levels (Zhou et al., 1997). ïhis
LC3-mediated upregulation of FN mRNA translation is also dependent on nitric oxide
(NO), which is induced in the DA compared to the Ao associated with both increased
expression of neuronal NO synthase (nNOS) and endothelid constitutive NOS (ecNOS).
Increased NO results in enhanced phosphorylation and binding of LC3 to the ARE of the
FN mRNA (Mason et al., 1999b). Since LC3 is CO-p&ed with microtubules in vivo and
associated with microtubules assembled from purified tubulin in vitro (Mann and
Hammarback, 1994), microtubules appear also be involved in iranslational regulation of
FN (Zhou et al., 1998).
Microtubule Involvement in LC3-mediafed Enhanced FN mRNA Translation
MicrotubuIes have been implicated in the storage, sorting and translational control of
mRNAs (Singer, 1992; St Johnston, 1995). However, most of the studies about
microtubule-mediated translationai regulation or localization involve mRNAs encoding
cytoskeletal or cytoplasmic proteins, whereas little is known about how microtubules
regulate translation of mRNAs encoding secreted pmteins such as F N Zhou et al. (Zhou
et al., 1998) inves tigated the role of microtubules in influencing LC3-mediated FN mRNA
translation. Since FN is a secreted ECM glycoprotein synthesized at the rough
endoplasmic reticulum (RER) bound to polysomes and then packaged into secretory
vesicles for later release, it was hypothesized that microtubules may help target FN mRNA
to membrane bound polysomes for translation via LC3. When cultured DA SMC were
treated with colchicine to disrupt microtubules, FN mRNA translation was inhibited,
concomitant with the decreased association of FN mRNA and LC3 protein with the RER
(heavy pdysomes in sucrose &nsity gradient) (Zhou et al., 1998).
Phosphorylation of LC3 Enhances LC3-mediafed FN Translational
Upregulation
Consistent with a dual function as both the microtubule-binding protein and the RNA-
binding protein, LC3 was found to distribute between 2 functionaiiy distinct pools within
the ceII: the unphosphoryfated, translationdy inactive form binds the microtubuIes while
the phosphorylated, translationally active form associates with the FN mRNA and the other
components of the translational machinery such as 60s ribosomal units. Phosphorylation of
LC3 has been demonstrated to shift LC3 fimm the microtubules towards the membrane-
bound polysomes probably by enhancing the LC3-ARE binding activity, associateci with
induction of FN mRNA translation (Mason et al., 1999b).
Cell Motiliry is Related to m e r Genes Besides Fibronectin
Studies in DA SMC and in CA SMC in post-cardiac transplant coronary arteriopathy
(PCTC A) (detailed discussion in next section) show a correlation between FN upregulation,
ce11 motility and migratory phenotype. However, other genes in both DA and CA SMC
may also contribute to this phenotype as a 'consteiiation' regulated at a pst-transcriptional
level by the LC3 switch. Using an LC3 protein affinity column and incubating it with
RNA harvested from adult rat brain (a nch source of microtubule assocîated proteins), a
bound transcript was identified which encoded the 3'UTR of apolipoprotein D (apo D),
which also contains an ARE-like element (UUAUCTUCUU)(Burry, Andrea, M.Sc Thesis,
Department of Lab Medicine and Pathobiology, 1998). Apo D was increased in DA
compared to Ao SMC, and in migratory compared to non-migratory cells. suggesting that it
might be necessary for the motile SMC phenotype.
II. Fibronectin in Post-Ciràiac Transplant Coronary Arteriopathy
Neointimal FoIlll(lfi0on in Post-Cardiac Transpiant Coronary Artenoopathy
Post-cardiac transplant coronary arteriopathy (PCI'CA) is a major complication affecting
the long-term survival of cardiac transplant recipients (Uretsky et al., 1992). It is
characterized by occIusive neointimal formation in the donor heart coronary arteries. It
represents an ongoing immune-inflammatory reaction in the vesse1 wali with activation of
endotheliai cells expressing major histocompatibilty complex W C ) II antigens which
s tirnulate 1 y mp hoc yte binding and proli feration, and sustained released of growth factors
and cytokines, causing proliferation and migration of SMC into the subendothelium and
accumulation of ECM, notably EN, contributing to intimal thickening (Solaman et al.,
199 1; Clausel1 et al., 1993). The latter is similar to the intimal cushion formation in DA:
Increased fragmentation of the interna1 elastic laminae is also observed in PCTCA
associated with increased activity of a serine elastase measured in donor coronary arteries
following heterotopic heart transplant in piglets (Oho and Rabhovitch, 1994).
Fibronectin and Migration of Inflmmaiory Cells und Sntooth Muscle Cells
Cytokine-mediated FN upregulation in donor coronary EC and SMC in PCTCA was
responsible for recruiting inflamrnatory cells, through interactions of the CS 1 and RGD
motifs on FN with inflammatory ce11 surface integrins, &pi and asBi respectively. In a
porcine endothelial-smwth muscle celi co-culture system, IL-l&stimulated EC and SMC
FN s ynthesis and induced transendothelial lymphocyte migration, which was blocked by
CS 1 and RGD synthetic peptides and FN antibodies (Molossi et al., 199Sb). Blockade of
the FN binding VLA4 (a&) integrins on lymphocytes with CS1 peptides in rabbits
following heterotopic cardiac transplantation resulted in reduced infiltration of T cells, l a s
accumulation of FN and a >50% decrease in the incidence and severity of donor coronary
artery intimal thickening (Molossi et al., 1995~). These studies provided further evidence
that increased expression of FN was not only critical in fafiüiatiag migration of SMC in the
developing intima1 cushion of the ductus but also in the pathological formation of the
neointima in vascular disease.
Coronary Artery Smooth Muscle Cells
The mechanism responsible for the upregulation of FN synthesis in donor versus host heart
coronary artery (CA) SMC was specificaily investigated, In piglets after heterotopic
cardiac transplantation, the early development of a cmnary arteriopathy is charactenzed by
increased immunostaining for FN and IL-Ip in the vesse1 wall (Clausell et al., 1993).
Further investigation using cultured donor and host CA SMC demonstrated an increased
steady state level of M mRNA in donor cells, which could be reduced to host ce11 levels
by neutralizing antibodies to IL-1s associated with a decrease in FN synthesis to host cell
levels. This supported ILI$ upregulation of M synthesis by inducing FN gene
transcription (Clausell and Rabinovitch, 1993). On the other hand, TNF-a induces FN
synthesis in CA SMC without increasing M mRNA levels, implying that the regulation
may be at a post-transcriptional level (Molossi et al., 1995a). This might explain the
reciprocd interaction of IL-ID and TNF4 in modulating FN synthesis, as illustrateci in Fig
1.
IL- 1 f3 induces its own gene transcription (DinarelIo, 199 1; Wamer et al., 1987) as well as
the transcription of the TNF-a gene by activating the transcription factor NF-- via a
protein kinase C-dependent pathway (Bethea et ai.. 1992). Similarly, TNF-a induces its
own mRNA translation (Clausel1 et aZ., 1994) dong with the induction of the IL-1$ gene
transcription by the activation of IL4 transcription factor NF-- (Kruppa et al., 1992).
TNF-a also stabilizes IL-1$ mRNA via pmtein kinase C activity (Gomspe et al., 1993). It
is proposed that TNF-a upregulates FN synthesis via the upregulation of IL-@, but also
Figure 1. Schematic summary of FN regulation by IL-lp, TNF-a and EDP
as described in the text.
IL-1p gene TNF-a gene
via another pathway leading to the post-transcriptional upregulation of FN. This
phenornenon was demonstrated by the ability of both TNF-a antibodies and IL-lp
antibodies alone to block FN synthesis induced either by IL-1s or TNF-a (Molossi et al.,
1995a).
IL- 1 may also regulate FN synthesis at a pst-tranmiptional level by inducing elastase
activity and the production of elastin-derived peptides (EDP). EDP, in the form of kappa-
elastin @-EL), augments IL-l$ upregulation of FN synthesis in CA SMC (Cowan et al.,
1999, rnanuscipt submitted). It is postulated that EDP causes a conformation change in
the ce11 surface elastin-binding protein (EBP), facilitating the binding of IL-lp to type 1
surface receptor, a process which can be blocked by IL-@ receptor antagonists.
Al ternativel y, EDP, like TNF-a, increases the efficiency of FN mRNA translation,
following enhanced transcription by IL-1$. When only exogenous EDP is added to CA
SMC, FN spthesis can also be induced without a corresponding increase in mRNA levels
(Co wan et al., 1999, manuscript submitted).
Besides regulating Llf5 expression, TNF-a also upregulates FN synthesis via an NO-
dependent pathway. TNF-a causes an induction of NO production in cultured CA SMC,
resulting in an increased binding of LC3 to the ARE in the 3' UTR of the M mRNA,
thereby increasing its translational efficiency (Mason et al., 1999, manuscript in
preparation). A similar NO-dependent LC3-mediated upregulation of FN mRNA
translation has also be demonstrated in intima1 cushion formation in DA (Mason et al.,
1999b) which has been discussed above.
III. Fibronectin Reverses the Tradormed Phenotypc of HT1080, a Human
Fibrosarcoma Ceii Line
Do wn-regdation of Fibronectin in Oncogenie Tronsformotr-on
Loss of FN fkom the ce11 surface has been show to be closely associated with malignant
transformation of cells, and has been related to the decxeased cellular adhesion and
increased metastasis of tumor cells (Hynes, 1973; Gahmberg and Hakomori, 1973). The
tumongenic down-regulztion of FN has been exclusively studied in N-tas-transfomd
human fibrosarcorna HT1080 ceils, which express low levels of FN and lack cell surface
FN matrix deposits (Oliver et al., 1983; Dean et al., 1988), in response to the N-ras
oncogene (Brown et al., 1984; Paterson et al., 1987). The effect is post-transcriptional
(Chandler and Bourgeois, 1991), and was attributed to a reduction in nuclear processing or
stability of processed FN mRNA (Chandler et al., 1994), causing a shorter half-life of
about Il hours compared to 70 hours in nonnal fibroblasts. The end result is a 50 fold
decrease in basal levels of FN compared to normal fibroblasts. For this m o n transformed
ce11 lines have been commonly used to study the role of FN in ceil growth, differentiation
and adhesion, as well as the different regulatory mechanisms of FN gene expression.
Recent studies suggested that the adhesion of cells to ECM proteins, particularly FN,
through the integrin famil y of adhesion receptors transduces si p a l s that regulate ce11
proliferation, differentiation and apoptosis (Juliano and Haskill, 1993; Miyamoto et al.,
1995).
Fibronectin Suppresses the Transfomard Phenotgpe of HT1080 Cells
To elucidate the role of FN in modulating malignant ce11 phenotype, Akamatsu et ai.
overexpressed a full-length cDNA encoding plasma-type FW in HTlOSO human
fibrosarcoma cells. This resulted in a more flattened morphology, deposition of a
moderately developed FN rnatrix, reduced ceU motility on the substratum and poor growth
when these cells were injectai S.C. into nu& mice. Overexprcssion of FN also suppressed
the ability of the tumor cells to proliferate in soft agar, which couid be reversed by RGD
peptides and antibodies against FN (Akamatsu et al., 1996). These resuits indicated that
increased deposition of FN in the pericellular matrix per se can suppress the motility and
growth potential of mmor cells through interaction with RGD-recognizing or aspi
integins. Moreover, the binding of the integrin %pl to substrate-adsorbed FN has been
reported to inhibit DNA synthesis in ~ 1 0 8 0 cells (Wang et al., 1995).
Besides overexpressing recombinant FN by transfection, upregulation of FN synthesis in
HT 1080 cells can also be achieved by inducing endogenous FN production at different
molecular levels. For example, while dexamethasone (a synthetic glucocorticoid)
upregulates FN expression by augmenting the steady state level of unspiiced FN tranmipts
and increasing the mRNA half-iife from -11 to 26 hours, it has no effect on either the
morphology or the growth rate of the cells (Ehretsmann et al., 1995; Dean et al., 1988).
Forskoiin (an activator of adenylate cyclase) and transforming growth factor (TGF-$)
both increase the rate of FN gene transcnption. While TGF-p has no effect on the
morphology and the growth rate of the cells, Forskolin causes cells to become more
elongated with numerous projections and significantly decreases their growth rate (Dean et
al., 1988). These studies, in contrast to Akamatsu's mentioned above, suggested that
upregulation of FN is not exclusively related to the revertant phenotype of HT 1080 cells.
Instead, concomitant regulation of other genes or proteins, might be important in reversing
the transformed phenotype of HT 1080 celis.
Role of EGR-I in FN Upreguiation and Tumor Suppression
EGR-1, a transcription factor, is a member of the immediate early growth response gene
family that shares close homology in its DNA-binding domain with a well-known tumor
suppressor gene, WT-1. Its expression level is significantly reduced in human breast
tumor ce11 lines (Huang et al., 1997) and in the HT1080 human fibrosarcoma ceil Line
(Huang et al., 1995). Transfection of EGR-1 into HT1080 cells leads to FN upregulation
as well as tumor suppression. Transfected EGR-1 increases FN secretion via 2 pathways:
a direct induction of FN gene transcription by binding to a known positive transcription
activation site in the FN prornoter; and an indirect mechanism associated with the
upregulation of TGF-$ (Liu et al., 1999) which enhances FN gene transcription (Dean et
al., 198 8). EGR- 1 exerts its tumor suppressing ability probably by downregulating Bcl-2
expression, since overexpression of Bcl-2 in EGR-1 msfected cells restores the
transformed phenotype to these fibrosarcoma cells (Huang et al-, 1998). This m e r
confirms a dissociation between FN upregulation and tumor suppression observed in
HT 1080 ceils. That is, increased FN may be necessary but not sufficient to revert the
maiignant HT1080 phenotype.
Stable Transfection of LC3 in LIT1080 cells Upregdàtes FN Synthesii
LC3 increases FN expression in cultured DA SMC by binding to the ARE in the 3'UTR of
the FN mRNA thereby upregulating its translation (Zhou et al., 1997). To elucidate the
detailed mechanism involved, HTlOSO cells, with no detectable LC3 expression and
negligible FN ce11 surface deposits, were stably transfected with an LC3-encoding plasmid
to study the regulation of FN synthesis (Zhou et al., 1999, manuscript submined). LC3
expressed in the HT1080 cells localizes to the perinuclear region, where most of the
translational machinery (polysomes) are localized. It also distributes dong the microtubule
filaments as granular particles, consis tent with its property as a microtubule-associated
protein. Synthesis and ce11 surface deposition of FN is selectively induced by the
expression of LC3, together with a morphologicd change from a rounded to a flattened cell
shape consistent with a 'revertant' phenotype (Paterson, 1987), a re-arrangement of
microtubules, and a slower growth rate (Zhou et al., 1999, manuscript submitted). While
there is no difference in FN mRNA levels in LC3 and vector transfected cells, polysome
proNe analysis in LC3 transfected ceiis &rnonstrated that FN mRNA is more concentrated
in heavy polysornes where LC3 and ribosomai subunits are co-dïstributed. The finding
that LC3 also binds 40s and 60s ribosomai subunits in vitro (Zhou et al., 1999, manuscn'pt
submined) leads us to propose that LC3 upregulates tranlsation of FN mRNA by ribosome
recruitrnent .
IV. Role of Protein-RNA Interactions in mRNA Translation
Role of 3' Untrcrnsloted Region (3VTR) AU-Rich Eknrent (ARE) in mRNA
Translation
AU-rich elernents are common regdatory sequences within the 3' untranslated regions of
mRNAs encoding infiammatory mediators or immediate early response genes such as
cytokines, oncogenes, and signaling molecules involved in cell growth and differentiation,
as well as FN. Thes elements modulate both mRNA stability and translational efficiency.
Caput et al. found that the octanucleotide sequence, UUAUUUAU, exists as one or
multiple copies, and is important for these hinctions (Caput et al., 1986). Its role as an
rnRNA destabilizing elernent was first discovered in c-fos (Treisman, 1985) and GM-CSF
(Shaw and Kamen, 1986). Insertion of the 3'WR from these 2 genes into globulln
mRNA resulted in the destabilization of the message. Since then, the presence of the ARE
has been shown to destabilize a large number of labile mRNAs, including c-myc (Jones
and Coles, 1987), IFN-$ (Whittemore and Maniatis, 1990) and IL-3 (Wodnar-Filipowicz
and Moroni, 1990).
Ln addition to their function as destabilizing elements, ARES can also modulate translational
efficiency. To date, most of the studies show that ARES inhibit mRNA translation. In the
case of IFN-p, the presence of the ARES in the 3'UTR of the mRNA greatly suppressed
mRNA translation in both Xenopus oocytes and in the reticulocyte lysate, whereas
removing the ARES from the 3'UTR increased the IM-f3 translation up to 100 fold in
Xenopus oocytes and 10 fold in reticulocyte lysates without affecting mRNA stability
(Kruys et al., 1987; Kmys et al., 1988). ARES from c-fos and GM-CSF have also been
shown to have the similar inhibitory effects on mRNA translation as on mRNA stability
(Kniys et al., 1989).
There are a number of proposed mechanisms for the inhibitory effects of ARES on mRNA
translation. Kmys et al. demonstrated that less wild-type IFN-p mRNA associated with
polysomes (the translationai machinery) compared to mutant IFN-p mRNA without ARE,
suggesting that the ARE inhibits IFN-$ mRNA translation via decreased recniitment into
polysomes (Kniys et al., 1990). On the other han& Sachs et al showed that increasing
poly A tail length of 1 . - $ mRNA significantly decreased its translational efficiency in
reticulocyte lysates while shortening the poly A tail or removing the ARE fiom the 3'UTR
enhanced translation (Sachs and Davis, 1989; Tanin and Sachs, 1996; Tanin and Sachs,
1995; GraFr et al., 1993). This suggested that ARES might bind the poly A tail and inhibit
its interaction with poly A binding proteins which are required for translational initiation.
Alternatively, the opposite might occur, i.e. binding of specific trans-acting factors on
ARES might facilitate the release of poly A tail, allowing for the binding of poly A binding
proteins and translation initiation.
Thus, AREs are not always associated with mRNA translational inhibition. Han et al.
reported that lipopolysaccharide (LPS) induced TNF-a expression by enhancing mRNA
translation (Han et al., 1990a; Han et al., 1990b; Han et al., 1990c) associated with
complex formation beniveen TNF-a mRNA and the RND-binding protein TIAR (Gueydan
et al., 1999). Zhou et al. also showed that FN upregulation in DA SMC was due to
translational enhancement of the ARE-containing mRNA (Zhou et al., 1997). Instead of
selectively modulating mRNA stability or translational efficiency, in some cases these 2
properties of ARES are actually interdependent. Studies have shown that c-fos and GM-
CSF AREs rely on ongoing translation to exercise their mRNA destabilizing functions,
probably related to the translational-dependent assernbly of a >20s degradation complex
(Savant-Bhonsale and Cleveland, 1992; Aharon and Schneider, 1993; Winstall et al.,
1995). These leads to the hypothesis that ARES can either reduce or increase mRNA
translation, depending on their interaction with tissue-specific cytoplasmic proteins.
ARE-binding Proteins and Their Functions
Since AREs have been demonstrated to be important in modulatuig mRNA stability and
translational efficiency, numemus cytoplasmic proteins that bind ARE-containing 3'UTRs
have been identified in a variety of cells or tissues. These ARE-binding proteins can be
functionally assigneci to two categories: the fmt group contains proteins such as adenosine-
uridine binding factor (AUBF) (Malter, 1989) and embryonic lethal abnormal vision
(Ela)-like proteins such as Hel-N1 and Hu-R (Levine et al., 1993; Chung et d., 1996; Ma
et al., 1996) whose ARE-binding activities are associated with stabilization of labile
-As as well as enhanced mRNA translation; the second group includes RNA-binding
proteins sucn as AUFl (Zhang et ai., 1993) whose ARE-binding activities correlate with
rapid mRNA decay that may or may not depend on ongoing translation.
AUBF was fmt identified by Malter as an ARE-binding protein in lymphocyte cytoplasmic
extracts (Malter, 1989). Further studies showed its binding to ARE-containing labile
mRNAs including GM-CSF, IL-3, IFN-y, c ~ u s and v-myc. AUBF binds specificaily to
the destabilizing motif AUUUA of mRNA. Mutations within the AUUUA motifs
demonstrate that both nucleotide sequence and s e c o n d q structure are important in
AULTUA.AUBF RNA complex formation (Giliis and Maiter, 199 1). Binding of AUBF to
AUUUA stabilizes the ARE-containing labile mRNAs. In vitro decay assay of GM-CSF
mRNA demonstrated that depletion of AUBF binding activity led to the accelerated decay
of the GM-CSF mRNA and its decrease in half-life fiom 90 to 20 minutes (Rajagopalan
and Malter, 1994).
Another RNA-binding protein, AUF1, was initially identified through its involvement in c-
myc mRNA degradation in a cell-free mRNA decay system. AUFl facilitates the
association of c-myc with polysomes (Brewer and Ross, 1988) and accelerates c-myc
mRNA turnover in vitro (Brewer, 1991). In vivo studies have also demonstrated that
AUFl targets decay of ARE-containing mRNAs. Upregulation or downregulation of
AUFl has been associated with increased and decreased decay of ARE-containing RNAs,
such as GM-CSF (Pende et al., 1996; Buzby et al., 1996). Recently, Laroia et al has
found that the dec2y of ARE mRNAs is associated with the displacement of elF 4G fro,
AUF1, the ubiquitination of AUFl and the degradation of AUFl by pmteosomes (Lamin et
al., 1999). Therefore, there is strong experimental data supporting AUFl as one of the
ARE-binding factors facîiitating mRNA decay via the 3 W ï R ARE, but whether this factor
can also influence mRNA translation is unproven-
ARE-binding proteins are only one class of RNA-binding proteins that regulate post-
transciptional gene expression. Many other RNA-binding proteins that have been
identified, including those binding to pre-mRNA, pre-ribosomai RNA (rRNA) or s m d
nuclear RNA (snRNA) are involved in capping, pre-mRNA spiicing and polyadenylation.
The characterization of these different RNA-binding proteins has led to the identification of
several RNA-binding motifs and studies of their interactions with RNA.
RNA-binding Motifs
Based on the negative-charge nature and specific conformation of RNA, most of the
protein-RNA interactions involve charge-c harge interactions such as hydrogen bonds and
specific secondary structures such as beta-sheets. The most cornmon RNA-binding motifs
identified include the RNP (ribonucleoprotein) consensus motif (or RBD motif), the
Arginine-rich motif (ARM), the RGG Box and the KH (K homology) motif.
The RNP motif is by far the most widely found and bat-characterized RNA-binding
motifs. It is also referred to as the RNA recognition motif (RRM)(Kenan et al., 1991;
Query et al., 1989), RNP consensus sequence (RNP-CS)(Swanson et al., 1987; Dreyfuss
et al., 1988), and consensus RNA-binding domain (CS-RBD)(Bandziulis et al., 1989),
and is commonly found in riboaucleoproteins such as heterogeneous nuclear RNP C
(hnRNP C). It consists of a 90-amui0 acid sequence containhg 2 short highly conserved
sequences, RNPl (an octapeptide) and RNP;! (a hexapeptide), interspersed with a number
of other, mostly hydrophobie conserved amino acids (Swanson et al., 1987; Dreyfuss et
al., 1988;BandWulis et al., 1989;Kenan et al., 199 1). The three-dimensional structures of
RNP motifs in U1 snRNP A (U1 A) and hnRNP C have been determined and appear to be
very similar (Fig. 2)(Nagai et al., 1990; Hotlinan et al., 1991; Wittekind et al., 1992). The
$aBBaP secondary structural elements of the RNP motif fomis a four-stranded antiparaliel
sheet. The RNPl and RNP;! are located on the cenaal 83 and p l strands respectively.
The charged and aromatic side chains of these 2 sequences are solvent exposeci, aiiowing
them to make direct contact with bound RNA, pmbably through hydrogen bonds and ring
stacking (Nagai et al., 1990; Gorlach et al., 1992). Although the highl y conserved RNPL
and RNP2 are crucial for RNA binding, the highly variable regions, particularly in the
Ioops and the termini, contain the major determinants of RNA-binding specificity. In U1
70K, U1 A and hnRNP C, the amino acids on the immediate COOH-terminal of their RNP
motifs determine the RNA-binding specificity (Query et al., 1989; Gorlach et al., 1992;
Scherly et al., 1991).
The arginine-rich motif (ARM) is usuaiiy short, about 10-20 arnino acids long. Other than
the preponderance of arginine residues, the different ARMs show little sequence identity
(Lazinski et al., 1989), and the structures are diverse as well, including stem-lmps in W-
proteins', interna1 Ioops in 'Rev' or bulges in Tat' (Malim et al., 1989; Dayton et al.,
1989; Gorlach et al., 1992; Iwai et al., 1992). Despite the unconserved secondary
structure for the ARM, the structun, rather than particular sequence, appears to be the
major binding determinant. Interaction with RNA involves both the phosphonbose
Figure 2. Schematic ribbon drawing of the hnRNP C RBD (residues 2-94).
The arrows represent the 4 antiparalleI$ strands, and the curled ribbons represent the 2 a
helices. The labels pl-84 indicate the 4 saands of the sheet; the labels al and a2
indicate each a h e h . The RNPI and RNP;- consensus sequences are juxtaposed on the
adjacent central antiparallel strands (fi sîrands 3 and 1, respectively). N and C denote the
arnino and carboxyl tennini of the domain, respectively (Modifieci h m Dreyfuss G., et al.,
Annu Rev Biochem 62:289-321, 1993).
backbone (Bartel et al., 1991) and the bases (iwai et al., 1992) of the RNA. Compared to
other charged residues, such as lysine, arginine residues have more potential for forrning
hydrogen bonds. The positive charges probably increase nonspecific affinity for RNA.
This initiates a t tachent and facilitates searching for a higher afflnity and more specific
binding site (Calnan et al., 1991; Weeks and Crothers, 1991; Tan et al., 1993).
The RGG box was first identified as an RNA-binding domain in hnRNP U (Kiledjian and
Dreyfuss, 1992). It is about 20- to 25-amino acid long, consists of closely spaced Arg-
Gly-Gly (RGG) repeats interspersed with other, often aromatic, amino acids. The nurnber
of RGG repeats varies between proteins, with six in hnRNP A l and eighteen in yeast
GARI. These repeats are often found in combination with other RNA-binding domains
(Kiledjian and Dreyfuss, 1992). In nucleolin which contains 4 RNP motifs and 1 RGG
box, specific binding to pre-ribosomal RNA requires the 4 RNP motifs and the RGG box
increases the overall RNA affinity by IO-fold (Ghisolfi et al., 1992). This might suggest
that RNA binding of RGG box is relatively nonspecinc but it plays a more important role in
facilitating the RNA binding of other RNA-binding domains. However, in the case of
hnRNP U, the RGG box is the only RNA-binding element and is able to discriminate
between different RNA sequences (Kiledjian and Dreyfuss, 1992). The binding affinity of
RGG box to RNA can be modulated by methylation of arginine residues within the RGG
box. hnRNP A l has been shown to be pst-translationally arginine-methylated in vivo
within the RGG box (Kim et al., 1998). Its binding property to single-stranded nucleic
acid is significantly reduced subsequent to methylation, suggesting that pst-translational
methyl group insertion to the arginine residues reduces protein-RNA interaction, perhaps
due to interference of hydrogen bonding between guanidino-nitrogen arginine and
phosphate RNA.
The K homology (KH) motif was initially identified in the human hnRNP K protein and
was Iater found in other RNA-binding proteins in other orgaaisms as well (Siomi et al.,
1993a; Siomi et al., 1993b). It is a stretch of about 45 amino acids characterized by a core
sequence VIGXXGXXI flanked by few interspersed conserved residues. By NMR
spectroscopy, the secondary structures of the KH motif were identified to consist of 3
stranded fi-sheets connected by 2 helical regions (Castiglone Morelli et al., 1995). In
hnRNP K, 3 K H motifs are present, each of them contributing to RNA binding. In
neuronal proteins Nov-l and Nov-2, 3 KH domains are also identified (Lewis et al.,
1999). The 2 KH domains in human FMR proteins play an important rote in the funcion of
the protein. Mutations at these domains are associated with fragile X syndrome, the most
common inherited cause of mental retaradation (Musco et al., 1996).
RATIONALE
Previous studies in our laboratory &monstrateci that LC3 binds the AU-rich element (ARE)
at the 3' untranslated region of the FN mRNA and upregulates its translation through
ribosome r e d t m e n t (Zhou et al., 1997; Zhou et al., 1998). In view of the importance of
RNA-protein interaction in regulating gene expression, this thesis investigates the
significance of this LC3-ARE interaction using both structural and functional appmaches.
LC3 is a basic protein, with a pI value of 9.2 (Mann and Hammarback, 1994). From the
amino acid sequence of the molecde, we note that most of the positively charged residues
are Iocated in the N-terminal haif. Since charge interaction plays a critical role in RNA-
protein binding, the RNA-binding site is therefore more likely located in the N-terminal half
of the molecule, as shown in Fig 3. Art arginine-rich motif (ARM) which might represent a
high affinity binding site is present toward the end of the N-terminal half.
The HTlOSO human fibrosarcorna cell line, which is LC3-nul1 in nature, has k e n used to
study the functional significance of LC3-ARE interaction. Stable transfection of IlTl080
cells with LC3-encoding plasmids increases FN mRNA translation through ribosome
recruitment, the same mechanism as in DA cushion foxmation. Flattened cell morphology
and decreased ce11 growth is also observed. Therefore, stable transfection of HT1080 cells
with wild type and mutant LC3-encoding plasmids can be performed to elucidate the direct
correlation between LC3-ARE binding and FN mRNA translation, as well as the cell
morphology and ce11 growth.
Figure 3. Amino acid sequence of rat LC3.
Positively charged residues (shown as bold) are concentrateci at the N-tenninal half of the
protein. ARM, representing arginine-nch motif, is a common RNA-binding motif.
....,.... 1....,....2....,.~.œ3~.œœ,œœoœ4œœœœ,œœoœ5o.o.,.~~. 6 MPSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNM
....,....7....,....8...., . . . . 9 . . . . , . . . . 10 ...,.... 11 ...,.... 12 SELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESERDEDGFLYMWASQETFG
ARM
. . o . , . . . O 13 . . . , . . . . 14. TALAVTYMSALKATATGREPCL
1. LC3 binds to the AU-rich elernent (ARE) in the 3' untranslated region (3'UTR) of the
FN mRNA via RNA-binding motif(s) a . the N - t e e a l .
2. LC3-ARE interaction via the LC-3 motif(s) identified is criticai for the upregulation of
FN synthesis in HT 1080 cells and this property is duectly related to slower ceil growth
and reorganization of microtubules.
OBJECTIVES
1. Determine the amino acid sequence in LC3 which binds to the ARE region of the 3 U ï R
in FN mRNA, using proteolytic cleavage and site-directed mutagenesis.
2. Determine the effect of LC3-ARE binding through this motif on FN synthesis by stably
transfecting wild type (WT) and mutant LC3 into LC3-nuii HTlOSO human fibrosarcorna
ce11 line.
3. Determine whether LC3-mediated FN upregulation contributes to the decreased growth
rate and changes celi shape in HTlO8O celis.
MATERIALS AND METHODS
CelZ Culture
The HT1080 human fibrosarcoma ceii iine was purchascd from American Type CeU
Culture (ATCC) and cultured with Eagle's minimal essential medium 0 (GIBCO
BRL, Buïlington, ON) containing 10% fetal bovine senun (FBS) (Intergen, mirchse,
NY), 1 96 antibioticslantimycotics (GIBCO) and 0.2% gentamicin (GIBCO)(for cells with
stable transfection only). For ail comparative snidies, cells were passageci at the same tirne
and plated at the sarne density. To assess morphologie differences, the cultures were
photographed with a phase-contrast microscope (Nikon Inc. Garden City, NY).
Expression of Recombinant LC3
pGEX-LC3 vector (produced by Zhou) was synîhesized by cloning the LC3 coding region
(produced by J. Hammarback) into pGEX-2T vector (Pharmacia Biotech.) at a 5' BamHI
site and a 3' EcoRl site in the plasmid The plasmid was transformed into DH5a E.coli
competent cells (GIBCO). Positive clones were confirmed by Mini-preps and restriction
enzyme digestions. The plasrnid-containing E-coli was amplified by growing ovemight in
2xYTA medium at 37"~, followed by induction with isopropyl-fl-D-thiogalactopyranoside
(IPTG) for 4 h. Total protein was extracteci and the glutathione S-transferase (GST)-LC3
fusion protein was purified using glutathione sepharose 4B beads (Sigma Chemical Co.).
The recombinant LC3 was eluted from the beads after ovemight incubation with thrombin
(Sigma or Phannacia) which cleaves at the GST fusion site with the recombinant LC3. The
punfied recombinant LC3 protein was confirmed by SDS-PAGE and western
immunoblotting.
Generation of LC3 Pepîïdes
1. Carhepsin D/ Endopoteinase Asp-N/ EnaOproteeulare Lys-C Digesrion
Lyophilized recombinant LC3 was suspended in buffers containing endoproteinases at
3 7 " ~ for specific t h e penods {C'hepsin D digestibn (cleave specijically beîween Phe-Phe
linknge) : LC3 was suspended in 50mM ammonium acetate pH3.5 at a ratio of lpgllpl of
buffer con taining Cathepsin D (Sigma C hemical Co.)(enyme:substrate= 1 :5O) for 30 min;
Endoproteinase Asp-N Digestion (cleave specificoly at the N-teminal end of Aspartare
Acid residues): LC3 was suspended in SOmM sodium phosphate pH 8.0 at a ratio of
l ~ g f l p l of buffer containing Endoproteinase Asp-N (Boehnnger Mannheim)
(enzyme:substrate=l : 100) for 3 h; Endoproteinose L y s 4 Digestion (cleave specificaly a?
the C-terminal of Lysine residues); LC3 was suspended in 1% (w/v) ammonium
bicarbonate at a ratio of Ipg/Lpl of buffer containing Endoproteinase Lys-C (Boehringer
Mannheim)(enzyme:substrate=1:50) for 5 h). The reaction was stopped by freezing the
samples at -70"~. Different enzyme concentrations and incubation times were tested and the
optimal condition was determined by assessing the amount and the integrity of peptide
produced with no degradation as shown by SDS-PAGE followed by silver staining.
However, in al1 cases, complete digestion of LC3 cannot be achieved because longer
incubation time leads to the degradation of the peptides.
II. Cyunogen Bromide (CNBr) Digestion
CNBr was dissolved in 70% formic acid to a concentration of 50 m g M . Lyophilized
recombinant LC3 was suspended in CNBr/70% formic acid at a ratio of 1pg:lpl. The
reaction mixture was incubated at room temperature for 30 min. 10 fold volume of water
was added to stop the reaction. Complete cleavage cannot be achieved even if incubation
leaves ovemight. Therefore, 30 minute-time point was chosen to preserve the intergrity of
the generated peptides h m further non-specific degradation.
5' end-radiolabelhg of RNA Probe
18-mer RNA oligonucleotides containing either the wild type consensus sequence
(UUAUüUAU) of the AU-rich region element (ARE) of the FN mRNA, or the mutant
consensus sequence (GGAGGGAG)(synthesized by Biotechnology Centre, University of
Calgary, Calgary, Alberta) were radiolabeled with ~y-3~P] ATP using T4 pol ynucleotide
kinase (PNK) (Pharmacia Biotech.). 50 ng of RNA oligonucleotide was incubated with
150 K i of [y-32~1 ATP(3000 Ci/mmol), 9.5 U of T4 PNK and 10x PNK B a e r to a total
volume of 20 pl- The reaction mixture was incubated at 37°C for 1 h. The enzyme was
then inactivated by heating the sample at 95°C for 2 min or by adding 1 pl of 0.M EDTA
pH8.0, followed by incubation on ice. The probe was purified using a NucTrap Probe
Purification Column (Strategene). The radioactivity of purified probe was determined by
liquid scintillation spectrometry.
In vitro Transcription of RNA Probe
The full-length 3WïR of the rat FN mRNA containing wild type or mutated ARE (al1 U's
mutated to G's) were subcloned into pBSKS4 vector (performed by Zhou). Plasmids were
linearized with XbaI. RNA was transcribed with T3 RNA Polymerase in the presence of
[ 3 2 ~ ] - U T P for 1 h at 37°C. DNA templates were removed with DNase 1 digestion for 15
min at 3 7 " ~ , and the full-length probes were obtained by 6% acrylamide/8M urea gel
purification.
North western (NW) Blot Analysis
Northwestem blot analysis was carried out as previously described (Chen, 1993) with
modifications. Enzyme-digested and control undigested wild type or mutant recombinant
LC3 samples, 2.5 pgllane, were resolved under reducing conditions on a 10-20s tricine
gel (Novex) and electroblotted ont0 a 0.2 Pm nitroceiiulose membrane. The LC3 peptides
were allowed to renatwe in RNA-binding buffer (lOmM Tris-Cl, pH7.5, 50mM NaCl,
ImM EDTA, and l x Denhardî's solution) overnight at 4 ' ~ . Rior to RNA binding, proteins
were blocked with RNA-binding buffer containing 100 pg/d tRNA for 1 h at room
temperature. The radiolabeled ARE of the rat FN mRNA at a concentration of 107 c p d d
was also incubated with RNA-binding b m e r containing 100 pghl tRNA for 1 h at room
temperature. The blot was theu incubated with the radiolabeled RNA mixture at room
temperature for 1 h, and finally washed with RNA-binding buffer twice for 5 min. The
dried nitrocellulose membrane was exposexi to X-ray film for autoradiography at -70°C.
Site-directed Mutagenesis
Three mutant pGEX-LC3 vectors were generated by PCR to produce mutant recombinant
LC3 for NW immunoblotting analysis : i, pGEX-LC3/R68-70Q, ii, pGEX-LC3/R68-70K,
and iii, pGEX-LC3lF79-80A. Three pairs of oligonucleotides carrying mismatched bp
corresponding to the speciiic mutations were used The upper and lower primers were: i,
5'-ATTCAACAGCAACTGCAGCTCAAT-3' and S-CAGTTGCTGTTGAA'ITATC-
TTGAT-3'; ii, 5'-ATTAAAAAGAAACTGCAGTCAAT-3' and 5'-CAGTTTCTTTTT-
AATTATCTTGAT-3'; iii, GCCGCGGCCCTCCTGGTGAATGGG-3' and 5'-
GAGGGCCGCGGCITGGTTAGCATT-3' (nts corresponding to the mutated amino
acids shown in bold). A S'-end primer, 5'-CGGGATCCCATATGCCGTCCGAG-
AAGACC-3', located upstream of the mutated site and a downstrearn 3knd primer, 5'-
CTGGATCCGAATTCAAGCATGGCTCTCITCC-3', were also used. Three 450-bp
products with specific mutations were generated by PCR and subjected to restriction
enzyme digestions. Three 435-bp Ba--EcoRI fragments containing the mutated sites
were then used to replace the corresponding fragment within @EX-LC3. These mutant
pGEX-LC3 vectors were transformed into E-coli and expressed as mutant recombinant
LC3 as mentioned above.
Another three mutant pCR3-LC3 vectors containing the full length LC3 sequence were
generated by the same method for stable transfection into HTlO8O human fibrosarcoma
cells: i, pCR3-LC3/R68-70Q. ii, pCR3-LC3/R68-7OK, and iii, pCRÎLC3/F79-8OA. The
same sets of p h e r s carrying the specific mutations were used. The S'-end primer and 3'-
end primer flankuig the mutated site w m SIGAGCKGGATCCACTAGTCCAGTGTG-
GTGG-3' and 5'-GTCACCGCCGGCGAGCTCAGATCTCCCGGG-3'. Three 976-bp
products with specific mutations were generated by PCR and subjected to restriction
enzyme digestions. Three 963-bp BamHl-XbaI fragments containing the mutated sites
were then used to replace the corresponding fragment within wild type pCR3-LC3. Al1
constnicts were confirmed by restriction enzyme mapping, and the mutations were verined
by DNA sequencing.
Gel Mobility Sirift Assays
10 pg of cytoplasmic extracts from WT and mutant LC3-transfected ElTl080 cells was
incubated with 1x1@ cpm of the 3WïR of the rat FN mKNA in RNA-binding buffer
(1 5rnM Hepes, pH 7.9, lOOmM KCl, 5mM MgCL2, 10% giycerol, 0.2mM DTT) in a total
volume of 20 pl containing 2 pg of tRNA for 30 min at 3 0 " ~ . 0 . N of RNase A and 400U
of Rnase Tl were added to each sample and incubated for 15 min at 3 7 " ~ . Samples were
then run on a 6% native polyacrylamide gel in 0 . 2 5 ~ TBE (Tris-borate-EDTA) buffer
(90rnM Tris, 90mM br ic acid, 2mM EDTA) at 250V on ice, dried and exposed to X-ray
film for autoradiography at -70°C. For cornpetition and specificity studies, 500x of
unlabeled RNA probes containing either WT or mutant ARE were incubated with
cytoplasmic extracts for 10 min before adding the labeled RNA transaïpts. (Note: 0.2mM
DTT were added to the RNA-binding buffer to reduce the disulphide bonds between
individual LC3 proteins to generate monomeric LC3 with higher ARE-binding affinity, as
shown in UV cross-linking analysis in a previous published paper (Zhou er al., 1997).
However, the potentiai inactivation of RNase A by DTï was not observed, probably
because the disulphide bonds in RNase A do not contribute to its RNA digestion activity.)
Stable Transf ection
24 h prior to transfection, HTlO8O ceUs were plated at a density of 10s cells 1100-mm dish.
HT1080 cells were stably transfected using SuperFect Reagent (QIAGEN Inc. Valencia,
CA). 10 pg of empty vector (pCR3), wild type (pCR3-LC3/WT) or mutant (pCR3-
LC3/R68-70Q, pCR3-LC3/R68-70K, and pCR3-LC3IF79-80A) LC3 plasmids were used
to transfect each dish for 3 h. The cells were then fed with fresh complete medium
containing the aminoglycoside G418 (200 pg/ml, GIBCO). The media were changed
every two days with gradually increasing concentrations of G418 up to 800 pglml. 8
clones transfected with empty vector and 24 clones each transfected with pCR-LCJ/WT,
pCR3-LC3/R68-70Q, pCR3-LC3R68-70K and pCR3-LC3#9-80A were selected on the
basis of resistance to G418 (800 pglml) by selective trypsinization and screened for LC3
expression using westem immunoblot analysis. 4 clones each were venfied to express
WT-LC3, R/K-LC3 and F/A-LC3, while 3 clones were verified to express R/Q-LC3.
These clones, together with 3 vector-transfected clones (which were shown to express no
LC3 by westem inimunoblot), were expanded individually and passaged at least 3 times
before use.
Western Irnmunoblot Analysis
Confluent cells were harvested by scraping into phosphate-buffered saline (FBS) and spun
at 3,000 rpm for 10 min at 4°C. Pellets were resuspended in twice the volume of
h ypo tonic bu ffer (30 mM Tris-Cl, pH7.9) with proteinase inhibi tors aprotinin, pepstatin
and leupeptin (1 pg/d each) and lysed by 3 cycles of freeze-thaw, followed by a 1-h
centrifugation at 16,000g at 4°C Cytosolic extracts (S) were isolated and pellets were fmt
digested with RNase-free DNase 1 (Phannacia Biotech) for 30 min at 37°C. Supernatant
(PI) was extracted after a 10-min spin at 16,000g at m m temperature. Pellets were M e r
extracted by resuspending in 2 peliet volumes of 1% SDS, and supernatant (Pz) was
isolated &ter a 10-min spin at 16,000g at RT. Protein concentrations in supernatant (S)
and pellets (Pl and Pd were measured by the BCA protein assay kit (Bio-Rad Laboratories,
Hercules, CA) followed by spectrophotometry at 562 nm. Rotein samples were separated
by reduced sodium dodecyl sulfate (SDS)-PAGE (10-20% tricine gel, NOVEX) and
electrotransferred onto a PVDF membrane. The membranes were bloçked for 1 h at m m
temperature in TBS-T (Tris-buffered saline with 0.5% Tween-20) containing 5% non-fat
milk and then probed with rabbit-anti-LC3 antismim (1:2Oûû dilution in TBS-T) (kindy
supplied by Dr. J. Hammarback, Department of Neurobiology and Anatomy, Bowman
Gray School of Medicine, Winston-Salem, NC) or mouse monoclonal anti-tubulin IgG
(12000 dilution in TBS-T, Sigma) overnight at 4°C. The membranes were washed 4 x 5
min with TBS-T followed by incubation with HRPconjugated goat-anti-rabbit or donkey-
anti-mouse IgG (1:3000) (Amersham. Buckinghamshire, England) for 1 h at room
temperature, and then washed 4 x 5 min with TBS-T and developed using an enhanced
chemiluminescence western blotting detection reagents (Arneaham). For the quantitative
analysis of LC3, the 16kD immunoreactive band for each sample was assessed by
densitometric analysis.
Cell Growth Cumes
HT1080 cells stably expressing blank vector, wild-type (WT) or mutant (WQ, R/K and
F/A) LC3 were plated on 6-well dishes at a density of 1 x 105 cells/well. The media
containing G418 were changed every day. Cells were trypsinized every 24 h and the ce11
number was determined using an improved Neubauber hemacytometer (Amencan Optical
Scientific Instrument Division, Buffalo, NY) by taking the average of two separate counts
for each well.
Indirect Immunofluorescence
HTlO80 cells were plated on 2.2 cm* coverslips at a density of 105 cells/well and cultured
for 3 days. Cells were fixed in 100% methanol at -20°C for 3 min and allowed to air dry.
Following rehydration in phosphate-buffercd saiine (PBS) for 30 min, celi were blocked
with PBS containing 1 % normal goat s e m and O. 1% bovine senmi albumin @SA) for 1
h at room temperature. For immunofluorescence staining of fibronectin 0, tubulin and
LC3, cells were probed with a monoclonal rabbit anti-FN IgG (1:100 dilution;
Neomarker), a monoclonal mouse anti-tubulin IgG (1: 1000 dilution; Sigma Chemicai Co.)
or a rabbit anti-LC3 antisenun (1:100 diiution) in PBS containing 0.1% BSA oveniight at
4°C. After 3 x 5 min washes with 0.1% BSA/PBS, cells were then incubated with
secondary antibodies: fluorescein-conjugated goat-anti-mouse IgG for FN and tubulin
staining and fluorescein-conjugated goat-anti-rabbit IgG for staining of LC3 (aii dilutions at
150) for 30 min at room temperature, foiiowed by 3 x 5 min washes and mounted with
antifade reagent (Molecular Robes Inc., Eugene, OR). Cell nuclei were stained with 1: 150
diluted DAPI (Sigma) in distilled water for 30 min a€ter the incubation with secondary
antibody. For negative controls, normal mouse IgG or 1% normal goat semm were used
instead of the primary antibodies.
FN Biosynthesis
HT1080 cells individually expanded from vector-transfected, wild-type (WT) or mutant
(WQ, R/K and F/A) LC3-transfected clones were plated on 6-well dishes at a density of
5x 1 d cells/well. After 24 h, cells were labeled with OsSI-methionine (10 pCi/ml) for 5 or
20 h (as indicated) in 2 ml media rnixed with 3 vol of methionine-free and 1 vol of complete
EMEM containing 20% FBS. The conditioned media were collected for analysis of FN
protein. Triplicate assessments of total protein synthesis were obtained from 50p1 aliquots
of culture medium precipitated in 1% BSA/15% aichloroacetic Md (TCA) and analyzed by
liquid scintillation spectmmetry. Measurement of FN protein production was perfonned by
incubating the conditioned media containing equal counts of total TCA precipitated protein
with 50 pl of Gelatin 4B-Sepharose (Pharmacia Biotech Inc., Pixataway, NJ) overnight at
4 " ~ . The FN retained on the beads after 3 washes with 1 ml of TBS containing 0.5%
Tween-20 was eluted by boilïng for 5 min in 60 pl of nducing SDS-sample bmer (5%
mercaptoethanol, 2% SDS, 10% glycerol, 62.5mM Tris-AC1 pH6.8) and resolved by 6%
SDS-PAGE. Gels were fmed in 5% acetic acid1096 mthanol for 30 min and prepared for
fluorography by treatment with ~ n f ~ a n c e (DuPont-NEN, Boston, MA), dried, and
exposed to the film. Using the autoradiograph as a template, the corresponding bands were
cut h m the gel and the radioactivity determineci by a liquid scintillation counter.
RNA Isolation and Northern Blot Anabsis
Total RNA was extracted from the cells using QIAGEN RNA extractkg kit (QLAGEN)
following manufacturer's instructions. lOpg of total RNA from each sample was resolved
on a 1% agarose gel, transferred ont0 a Hybond-N membrane by capillary elution
overnight, and fixed by W-irradiation. After blocking, the membranes were probed with
a [ 3 2 ~ ] - d ~ ~ ~ random labeled human FN cDNA (106cpm/ml) overnight at 5 0 " ~ . followed
by 2 washes with 2x SSC/O.l% SDS at 55°C for 30 min and 2 washes at 6 5 " ~ for 1 h.
Autoradiographs of northem blots were analyzed by relative densitometry. Ethidium
bromide staining of 28s and 18s ribosome RNAs served to control for loading conditions.
RESULTS
LC3 Binds the ARE of FN mRNA at the lOkD N-terminai Region
When LC3 binds to the ARE at the 3'UTR of the FN mRNA, the efficiency with which
this mRNA is translated to protein is increased. To identiQ the ARE-binding site(s) on
LC3, LC3 peptides were generated by enzymatic or non-erizymatic cleavage of recombinant
LC3. Their differential ARE-binding activity was then assessed by northwestem (NW)
blot analysis. The endoproteinases used incluàe Cathepsin D, which cleaves spcificall y
between Phe-Phe Linkage; Endoproteinase Asp-N, which cleaves specifically at the N-
terminal of Aspartate acid residues; and Endoproteinase Lys-C, which cleaves specificdy
at the C-terminal of the Lysine residues. Cynogen Bromide cleaves by reacting specifically
with methionine residues to produce peptides with C-terminai homoserine lactone residues
and new N-terminal residues. The peptides generated were separated by SDS-PAGE (Fig
4A), transferred ont0 nitroceilulose membranes, and probed with a [32P]-radiolabeled RNA
probe containing the FN ARE. ARE-binding activity of these peptides was detected by
autoradiography (Fig 4B).
Cathepsin D (CD) cleaves specifically between Phe-Phe residues at amino acid 79 and 80
of LC3, generating 2 peptides: the 8.9 kD N-terminal peptide and the 7.1 kD C-terminal
peptide (Fig 4Aa). Two Iower bands around 7 kD were observed. They might represent 2
different C-terminal peptides due to a post-translational modification near the C-terminal
end of the protein, or one of these peptides might represent the degradation product of the
N-terminal peptide or the whole LC3 proteins. The [ 3 2 ~ ] - ~ ~ ~ was able to bind to the
undigested 16kD recombinant LC3, but none of the CD-generated peptides (Fig 4Ba).
Complete CNBr cleavage should generate 5 peptides, al1 smailer than 6.8 kD. However,
incomplete cleavage led to the production of 2 bands above 7 kD and a smail band about 4
kD (Fig 4Ab). The highest molecular weight 10 kD peptide showed strong binding to the
Figure 4. Northwestern (NW) blot analyses show LC3-ARE binding
localizes to a 10 kD N-terminal region on LC3.
Recombinant LC3 was enzymaticdly or non-enzymah'cdy digested into discrete peptides,
and their binding activity to the radiolabeled ARE was compared by northwestem (NW)
analycir. A. Silver staining of the SDS-PAGE shows the LC3 peptides generated by the
cleavage of recombinant LC3 by Cathepsin D (CD)(a), Cyanogen Bromide (CNBr)(b),
Endoproteinase Asp-N (EPAN)(c) and Endoproteinase Lys-C (EPLC)(d), as well as the
control undigested LC3 protein (control). B. Representative autoradiographs demonstrate
the binding activity of the [32P]-radiolabeled FN ARE to the LC3 peptides and undigested
LC3 proteins. ARE bound to al1 undigested 16kD LC3 proteins. Note the absence of
ARE-binding in peptides generated by CD (a), EPAN @) and EPLC (c), while strong
binding is observed in the -1OkD peptide generated by CNBr indicated by arrows in both A
and B. Since this lOkD peptide was confirrned to contain the N-terminal sequence, this
suggests that the 1 OkD N-terminal region of LC3 possesses ARE-binding activity .
C D-d iges ted
con t rol
CNBr-digested
cont r d
EPAN-digested
control
control
EPLC-digested
a control
probe, as marked by an amow, while the middle (-7kD) and the lowest peptides (-4kD)
showed no binding at ail (Fig 4Bb). These upper 2 bands were sent for amino acid N-
tenninal sequence analysis. (The sequencing report for l O k D and 6.8kD peptides are
shown in appendix 1 and II respectively). The major peptides corresponded to the N-
terminal of LC3 although the minor peptides generated did not correspond to any part of
LC3. The higher band, about 10 kD, therefore represents the N-terminal peptide spanning
the sequence from P2 to M88, while the lower band about 6.8 kD, represents the N-
terminai peptide spanning the sequence from PZ to M60. This suggested that the RNA
binding site(s) was within the C-terminai portion of the 10 kD peptide, from residue S61 to
M88. This region (as shown in boid in Fig 5) contains 2 possible RNA-binding sites: the
positively-charged Arginine-Rich Motif (ARM) and a predicted Psheet region (p). Since
the conformation of the protein plays a significant role in RNA-protein interaction, Circular
Dichroism can be perforrned to examine the secondary structure of the peptide, and
determine whether the CNBr cleavage alters the conformation of the peptides and results in
the increased ARE-binding.
To further confirm the ARE-binding activity of the region S61-M88, endoproteinase AspN
(EPAN) digestion was performed to generate peptides spanning residues D56 to R103,
w hich contain the speculated ARE-binding region (S6 1 -M88) (Fig 4Ac). However,
without the N-teminal sequence, none of the peptides showed ARE-binding (Fig 4Bc).
Endoproteinase Lys-C (EPLC) digestion was also perfomed to generate a 7.5 k D peptide
which contained the speculated ARE-binding region plus the C-terminal of LC3 (Fig 4Ad).
Again, no ARE-binding was observed (Fig 4Bd). Therefore, these results suggested that
while the region S61-M88 contained the ARE-binding site(s), the N-terminal region of LC3
was also mandatory for RNA-binding, either by influencing the charge andfor the
secondary structure of the protein. A sumrnary of the northwestern analyses is shown in
Fig 6.
Figure 5. Amino acid sequence of rat LC3 showhg the 2 candidate RNA
binding sites.
The bold region spanning residues 61 to 88 represents the speculated RNA binding region
circled in Fig 6. It contains 2 candidate RNA binding motifs: the arginine-rich motif
(ARM) and a predicted bsheet region @).
. . . . , . . . . 1....,....2....,....3....,....4...., . . . . 5 . . . . , . . . . 6 MPSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNM
u ARM
O... , o . . . 13 . . . , . . . . 14. TALAVTYMSALKATATGREPCL
Figure 6. Summary diagram of NW blot analysis of LC3-ARE binding.
The bars represent the whole LC3 amino acid sequence while the saipped regions represent
the regions of the peptides generated by enzymatic or CNBr cleavage using listed on the
Ieft. The numbers below the bars represent the N- and C-terminal residues of the peptides
and the arrows above represent the LC3 cleavage sites used by the various reagents. The
size and ARE-binding property of each peptide are Listeci on the right. The lOkD CNBr-
generated peptide bound ARE while the 6.8 kD peptide did not, leading to the speculation
that the C-terminal region of the lOkD peptide (circled) contains the RNA binding site(s).
This circled region represents the bold sequence as shown in Fig 5.
REAGENTS USED DIGESTED LC3 FRAGMENTS
Cathepsin D
Endoproteinase
Endoproteinase
FRAGMENT BINDING(Y1N) SIZES
LC3 Binds Specifically to ARE of FN mRNA
To confirm the specificity of LC3 peptide binding to the ARE in the FN 3'UTR, NW
analyses were carried out using both wild type (WT) and mutant ARE. Equal
countsfamounts of wild type and mutant probes (U's in ARE mutated to G's) were
incubated with blots containing equal amounts of transferred protein. Both blots were then
exposed on the same film. The autoradiograph is shown in Fig 7. In this way, any
difference in the intensity of bands wouId likely reflect the differential afnnity of the probes
for the proteins. By comparing the densitometric analysis of the binding intensity of the
16k.D protein and the l O k D peptide to the WT and mutant ARE, the respective affinity to
the WT ARE was about 6 times and 2 thes higher than that of the mutant ARE (Fig. 7).
Although we cannot conclude that LC3 binds exclusively to the ARE, the binding to ARE
is preferentïal compared to 'poly G' in mutant probe. The binding between the LC3
peptide and the mutant ARE might be nonspecific in nature, based on solely charge
interaction. The difference between the binding of the WT compared to the mutant ARE
may reflect the sequence specificity of the protein-RNA interaction, and this can be m e r
tested using other mutants or mutant oligonucleotides as cornpetitors. Interestingly, 16kD
whole LC3 protein showed a bwer ARE-binding affinity compared to the lOkD peptide.
There are 2 possibilites contributing to this differential binding: fmt, there might be an
ARE-binding inhibitory element located at the C-terminal half of the protein. The absence
of this element in the lOkD peptide therefore enhances its ARE-binding afftnity; second, the
conformation of the l O k D peptide rnight be different from the N-terminal region of the LC3
protein, which somehow favors the ARE-binding. We can further investigated these
possibilities by circular dichroism or mass spectroscopy.
Figure 7. RNA binding of LC3 protein and l O k D peptide is preferential for
the ARE.
The specificity of ARE-LC3 binding was investigated by NW analyses using WT or mutant
ARE-RNA oiigonucleotide probes. Representative autoradiographs show stronger binding
of 16kD and 1OkD LC3 proteins and peptides to the WT compared to the mutant FN ARE
probes. Bottom shows the corresponding densitometric analysis comparing the binding
afinity of WT and mutant FN ARE region to the 16kD LC3 protein and 10kD peptide.
lOkD CNBr-generated LC3 peptide
Arginine-Rich Motif (ARM) is Critical in LC3 Binding to ARE of FN
mRNA
To M e r examine the importance of the 2 candidate RNA-binding regions within thel0kD
N-terminal LC3 peptide, we used site-directed mutagenesis to mutate these 2 regions.
Arginine nch motifs (ARM) are important in RNA-binding since they increase the non-
specific affïnity of proteins for RNA (Tan et al., 1993) and make specific hydrogen
bonding networks with the RNA sugar-phosphate backbone and bases (Iwai et al., 1992).
We therefore rnutated the 3 arginines (R) in the ARM either to 3 glutamines (Q) to examine
the significance of positive charge, or to 3 lysines (K) to examine the significance of
structure. Phenylalanines play a cntical role in RNA binding in hr&V A l by forming
stacking and hydrogen-bonding interactions with the guanine nucleotides (Merill et al.,
1988; Ghetti et al., 1990; Ishida et al., L986). We therefore also mutated the 2
phenylalanines (F) at the predicted beta sheet strtucutre to 2 alanines (A), to determine
whether the 2 benzene rings in phenylalanines are involved in ring stacking interaction with
the ribose component of the RNA.
Site-directed mutagenesis was performed using PCR, followed by subcloning the mutated
LC3 into the @EX-2T vector to generate GST-fusion proteins as described ir, detail in the
Methods. Wild type (WT) and mutant LC3 were subjected to N W analysis as above, and
results are shown in Fig 8. Comparable amounts of WT and mutant recombinant LC3
(though somewhat less in IUQ LC3) were loaded ont0 SDS-PAGE and transferred, as
illustrated by silver staining in Fig 8A. Densitornetric analyses of the autoradiograph
representing the NW analyses (Fig 8B) normalized to the amount of proteins loaded were
shown in Fig 8C. These quantitative results demonstrated that WT LC3 showed the
strongest ARE-binding compared to al1 other mutant LC3. LC3 carrying an R to K
substitution (RK) in the ARM and an F to A substitution (HA) in a possible sheet
structure showed about two-thirds of the ARE-binding affinity as compared to the WT
Figure 8. LC3 binds ARE at the arginine-rlch motif (ARM) via a charge-
charge interaction.
A. Silver staining of the SDS-PAGE shows the amounts of WT and mutant recombinant
LC3 proteins loaded and transferred B. Representative autoradiograph shows the NW
analyses of WT and mutant recombinant LC3 binding to the [32~]-radiolabeled ARE probe.
C. Densitometric analyses normalized to the amount of proteins tested (as shown in A)
demonstrated the strongest binding of WT LC3 to ARE, while mutant LC3 carrying the R
to Q substitution in the ARM (R/Q) showed lower binding to the ARE compared to the WT
and other mutant LC3 species. LC3 with an R to K substitution in the ARM (WK) and a F
to A substitution in the potential fl sheet structure @/A) showed about two-third of binding
affinity to ARE compared to WT LC3. Therefore, the 3 arginine residues constituting the
ARM are critical for LC3-ARE binding, probably via a charge-charge interaction.
WT R/Q Wb: FIA
WT R/Q R/K FIA
LC3, while LC3 carrying the R to Q substieon (R./Q) in the ARM showed a significantly
lower affinity to the ARE compared to the WT and other mutant LC3. Therefore. the
positive charge of the 3 arginine residues in the ARM is aitical for ARE binding of the
LC3. This RNA-binding is more lîkely a chargecharge interaction which is not sequence
specific, because the substitution of arginines to lysines, which is another positively
charged residue, does not significantly lower ARE-binding affinity.
The Importance of A R M in LC3-ARE Binding Confirmed in Stably-
transfected HTlOSO Cells
One of the disadvantages of N W analyses is that proteins have been denatured, transferred
renatured during the process, and their ability to retain their original conformation is
therefore questionable. Since secondary and tertiary structure might play a cri tical roIe in
RNA-protein interaction, it is very important to maintain the original conformation of the
proteins when investigating their RNA-binding activity. To c o n f i our NW results, we
therefore carried out electrophoretic mobility shift assay (EMSA) using the [ 3 2 ~ ] -
radiolabeled rat FN whole 3'UTR under non-denaturing conditions and cytosolic extracts
from H T I O S O cells expressing stably-transfected WT and mutant LC3.
The H T l O S O human fibrosarcoma cells, with undetectable LC3 expression and very low
levels of FN synthesiç, were stably transfected with a PCR mammalian expression vector
carrying WT or mutant LC3 sequences. Positive clones were selected by increasing
concentration of neomycin G418 up to 800 pg/ml and selected clones were confirmed by
western immunoblot for the expression of transfected LC3, while vector-transfected clones
were confirmed for the absence of LC3 expression. Binding reactions were canied out
with 10 ~g of HT1080 cytosolic extract and 105 cpm radiolabeled FN 3'UTR with intact
ARE. The results are shown in Fig 9. When extracts obtained from HT1080 cells with
stably-transfected WT, R/K and F/A LC3 were added to the radiolabeled FN 3WïR, three
Figure 9. Gel mobility shift assay on HTlOSO cytosolic extracts with
stably-transfected WT and mutant LC3 confiims the significance of the
ARM in ARE-binding
A. Representative autoradiograph of gel mobility shift assay shows the binding between
the [32~]-radiolabeled FN 3'UTR and the cytosolic extracts from HT1080 cells stably
transfected with wild type and mutant LC3-encodîng plasmids. No complex formation was
observed with only FN 3'UTR (FP). Three binding complexes were formed in ceil
extracts with stably-transfected WT, R/K and F/A LC3 (indicated by arrows), while the
lowest complex was absent in ceil extracts with empty vector and WQ LC3 (indicated by
*). These top 2 complexes were specific for ARE, which could be partially competed by
500x cold FN 3WïR with intact ARE (cold FN) but not mutated ARE (cold FNA). B.
Densitometric analysis of the lowest complex illustrates complex formation between FN
3'UTR and WT, R/K and F/A LC3-containing ce11 extracts, but not R/Q LC3 or empty
vector transfec ted ce11 extrac ts. Compiex formation in WT and R/K LC3 can be competed
by cold FN with intact ARE (cold FN) but not mutant ARE (cold FNA), while FIA was
only slightiy competed by both.
FP Vect FIA
d -3 z 7 Z Z k iL Cr, CL
FP Vect WT WQ R/K FIA
binding complexes were formed (indicated by amows). The Iowest complex was absent in
cell extracts with empty vector and WQ LC3 (indicated by *). Since the upper two bands
were present in d l WT and mutant LC3 transfected HTlOSO cells as well as empty vector
transfected cells, they might represent complex formation between FN 3'UTR and other
proteins endogenously expressed in HT1080 celis. These 2 complexes seemed to be
specific for the ARE since they could be partially competed by 500x cold FN 3'UTR with
intact ARE (cold FN) but not with mutated ARE (cold FNA). On the other hand, the
lowest band appears to represent the complex fomied between LC3 and FN 3'UTR, since
it is absent in vector transfected controls. Its absence in R/Q LC3 transfected celis was
consistent with the NW results mentioned above, and m e r confirmed the significance of
ARM in LC3-ARE binding via chargecharge interaction. Demsitometnc analysis was then
performed on the lowest complex formed between FN 3'UTR and WT, R/K and FIA LC3
(Fig 9B). Interestingly, when we compared the specificity of ARE-binding between WT,
R K and F/A mutant LC3 by cornpetition studies, the lowest complexes in both WT and
R/K LC3 could be partïaily competed by 500x cold FN 3- with intact ARE (cold FN)
but not with mutated ARE (cold FNA), while the complex fonned between F/A LC3 and
FN 3'UTR could only be slightly competed by both cold WT and mutant FN 3'UTR,
leading to the speculation that F to A substitution downstream of the ARE-binding site
results in the loss of ARE binding specificity. The RNA binding specificity imposed by
sequences flanking the RNA binding site has also been shown in hnRNP A l (Burd and
Dreyfus, 1995) and nucleolin (Senin et al., 1997). These flanking sequences might
contribute to RNA binding specificity by increasing the structural stability of the RNA-
protein complex or by increasing the contact surface between the protein and the RNA.
Though the ARE-binding specificity of LC3 is removed by the F/A mutation, the ARE-
affmity of LC3 is probably contributeci by the ARM since the inmase in affinity of LC3 to
the WT FN 3'UTR Vs mutant was sirnilar when comparing the lowest complex fomed
with W T and R K mutant,
Effect of WT and Mutant LC3 on FN Synthesis
Previous studies show that IlTl080 cells synthesize low level of FW and do not exhibit ceil
surface FN matrix deposits, a feature associated with the tumorigenicity of the cells.
However, the transformeci phenotype of the cells can be reverted by the upregulation of FN
synthesis induced by different mechanisms of FN gene regulation. For example,
dexarnathasome upregulates FN expression by increasing FN mRNA stability while TGF-
$ increases FN gene transcription (Dean el al., 1988). Previous work in our laboratory
shows that FN can be upregulated in HT1080 cells by stable transfection with LC3 which
enhances translational efficiency of FN mRNA (Zhou et al., 1999, manuscript submirted).
To elucidate if LC3-ARE binding plays the key role in the LC3-rnediated upregulation of
FN synthesis, WT and mutant LC3 constructs were stably transfected into HT1080 cells
and their FN biosynthesis was compared.
Indirect immunofluorescent staining of FN showed a filamentous pattern on the ceil surface
of WT, R/K and F/A LC3 transfectants (Fig lob, d and e), and FIA LC3 showed the most
intense staining. R/Q LC3 transfectants showed diffuse cytoplasmic staining instead of
punctate surface staining (Fig lOc), indicating that less FN was deposited on the surface of
R/Q transfectants compared to WT and other mutant transfectants. Vector transfectants
showed no punctate staining at al1 (Fig lOa), which was consistent with the previous
finding that HT1080 cells show no surface FN matrix deposits.
To confirm that increased FN surface deposition correlates with increased FN biosynthesis
in LC3 transfectants, cells were metabolically labeled with [3SS]-methionine for 5 h and
newly-synthesized FN secreted into the cultured media were measured. Determined by
TCA precipi tation, equal amounts of proteins in cuitured media were incubated with gelatin
4B sepharose beads to purify FN, which was then eluted and resolved by SDS-PAGE. As
Figure 10. Immunofîuorescence Iabeling of ceIl surface FN deposit in WT
and mutant LC3 transfected HTlOSO cells.
Vector transfectants (a) showed weak FN sîaining while WT (b). R/K (d) and F/A (e) LC3
transfectants showed a punctate and filamentous pattern of FN staining on the c d surface.
F/A LC3 (e) showed the most intense staining among the three. In contrast, WQ LC3
transfectants (e) showed a diffuse cytoplasmic staining of FN instead of a punctate surface
staining. ( f ) corresponds to negative control with secondary antibody alone.
shown in Fig 1 1, FN synthesis in LC3 WT transfectant clones was increased by about 3
fold when compared with vector transfectants. WQ transfectants did not show an increase
in FN synthesis when compared to vector transfèctants, but with the R/K mutant clone,
synthesis was increased about 4 fold (not significantly different h m wild type LC3). In
the FIA transfectant, FN synthesis was increased by about 5 fold compared to the vector
transfectant and was significantiy higher than in WT LC3 transfectants. This demonstrates
that the F to A substitution in the f3 sheet region enhances the LC3-mediated FN
upregulation. The clones used in assessing FN synthesis were expanded from those used
in the gel shift assays.
WT LC3 upregulates FN synthesis by enhancing FN mRNA transiation, since no
difference was observed in the steady state level of FN mRNA in LC3 compared to vector-
transfected HTlOSO cells and LC3 shifted FN mRNA ont0 heavy polysornes (Zhou et al.,
1999, manuscript submifted). To exclude the possibility that the mutant clones were acting
at different levels of regulation (e.g., mRNA transcription or stabili ty), northem blot
analyses were performed to measut the steady-state level of FN mRNA as shown in Fig
12. No significant difference was observed when comparing FN mRNA leveIs in the
different transfectants, supporting the proposal that RIQ and F/A LC3 regulates FN
synthesis by acting on the translation of FN -A. Translational efficiency, as defined
by the ratio of FN synthesis to FN mRNA Ievel, was compared among different
transfectants in Fig 13. The F/A transfectant was about 4 times more efficient than vector
transfectant in FN mRNA transIation, while the WT and WK transfectant were about 3 and
3.5 times more efficient than vector transfectant. The R/Q transfectant showed a àecrease
in mRNA translation compared to the WT LC3 transfectant, to a level similar to vector
transfectant. When the ARM, the component critical to LC3-ARE binding, is mutated as in
R/Q LC3, FN synthesis decreases probably by failing to help dock FN mRNA ont0
ribosomes for transIation. In contrast, FIA LC3 enhances FN synthesis probably by
Figure 11. FN expression in specific clones of WT and mutant LC3 stabiy-
transfected HTlO8O cells.
FN synthesis by specifc clones, whose ARE-binding affinity has been c o n f i e d by gel
mobility shift assays, is shown. A. An autoradiograph shows 5-h [35S]-methionine
labeled newl y synthesized FN from culture medium of vector-transfectants, WT LC3
transfec tan ts, R/Q LC3 trans fectants, R/K LC3 transfectants and F/A LC3 transfectants
containing equal total TCA-precipitated proteins counts. B. A graph shows a significant
increase of FN synthesis in WT, R/K and F/A LC3 transfectants cornpared to vector and
R/Q LC3 transfectants (*P<0.05 compared to vector). F/A LC3 transfectant also shows a
significant increase in FN synthesis compared to WT LC3 transfectant (+P<O.OS compared
to WT LC3). n= 4 for each transfectant. Bars reflect SEM.
Vect WT R/Q R/K FIA
Vect WT R/Q R/K FIA
Figure 12. Steady-state levels of FN mRNA in WT and mutant LC3 stably-
transfected HTlOSO cells.
A. A representative autoradiograph of northem blot shows a comparable amount of
steady-state FN mRNA in vector (vect), WT, RIQ, R E and FIA LC3 transfected cells.
GAPDH serves as a positive control and ethidium bromide staining of 28s and 18s
ribosome RNAs serve as controls for loading conditions. B. A graph of relative
densitometric units of FN mRNA normalized for 18s ribosomal RNA confîrms similar
levels in al1 transfectants. n=3 for each transfectant. Bars represent SEM and significance
was tested by Student t-test.
Vect WT R/E F/A
7
Vect
Figure 13. Translational efficiency of FN mRNA in WT and mutant LC3
stably-transfected HTlOSO cells.
Using the ratio of FN protein synthesis / FN mRNA, the increase in LC3 WT Vs vector is
not apparent with the R/Q mutants but is observeci with R/K and F/A mutants.
Vect WT R/Q R/K FIA
LC3
façilitating the dockïng of FN mRNA onto translational machinery, via inmasecl affinity :O
ribosomes or other ribosornal proteins. However, increased affinity to FN mRNA was not
observed in NW or gel shift analyses.
LC3 Regulates CeU Growth and Morphology via ARM-ARE Binding
Upregulation of FN in tumor cells has been reported to revert the transforrned phenotype
by enhancing adhesion of cells to the substrate, changing the morphology of the celis from
rounded to a more spreading shape as well as decreasing growth rate (Akamatsu et al.,
1996). Previous studies have show that WT LC3 transfected HT1080 ceUs adopted a
more flattened cell shape and slower growth rate compared to vector transfectants (Zhou et
al., 1999, manuscript submiîîed). To elucidate whether these phenotypic changes are
related to LC3-ARE interaction and upregulation of FN synthesis, ce11 morphology and
growth rate of vector, WT and mutant LC3 transfectants were examined Each transfectant
was plated at the same density in 6-weil dishes and the number of cells per well was
counted every 24 hours. Ce11 counts were shown in Table 1 and growth curves were
plotted in Fig 14. After 4 days, WT and F/A LC3 transfectants showed significantly
slower growth compared to vector, RIQ and R/K LC3 transfectants, while R/Q showed the
highest growth rate even compared to the vector transfectant Therefore, LC3 inhibits
HTlOSO ce11 growth probably via its interaction with the ARE of FN mRNA, resulting in
the upregulation of FN synthesis which in turn reverts the transformed phenotype of these
tumor cells. It is also possible that LC3 suppresses cell growth via binding to the ARE of
other mRNAs w hic h encode growth-regulatory elements.
The morphology of these different transfectants was compared 24 h after plating at the
sarne density. Representative phase contrast photornicrographs are shown in Fig 15. WT
and FIA LC3 transfected celIs (Fig 1% and f) were more e l o n g d , spread better and were
less phase-dense compared to the non-transfected and vector-transfected cells (Fig 15a and
Table 1. Ce11 count of WT and mutant LC3 transfected HTlOSO ceiis.
Values are mean f SEM. n= number of clones. *Pd .05 compared with the vector.
Vector (n=3)
WT LC3 (n=4)
iUQ LC3 (n=3)
R/K LC3 (n=4)
N A LC3 (n=4)
Day 1 Day 2 Day 3 Day 4
Figure 14. Growth curves of WT and mutant LC3 transfmted ET1080
cells.
Growth curves of empty vector, WT and mutant LC3 transfected HTIOSO cells were
plotted with the average number of cells per weii in 4 consecutive days (as s h o w in Table
1) after plating at the same density on &y O. Significantly reduced growth of WT and F/A
LC3 transfectants compared to vector and other mutant LC3 transfectants is observed 4
days after plawig. (*Pc0.05 compared to the vector-transfected cells.)
Figure 15. Effect of WT and mutant LC3 expression on HTlOSO eell
morphology.
A representative phase-contrast photomicrograph showing the WT and mutant LC3
transfected HTf 080 cells 24 hours after plating. Non-tïansfected (a) and vector-transfected
(b) cells were more phase-dense and showed a rounded ce11 shape compared to the
elongated and flattened ce11 shape of the W T (c) and F/A (f) LC3 transfected cells. R/Q (d)
and R/K (e) msfected cells showed an intermediate phenotype between the rounded and
the fi attened celi shape.
b), while WQ and WK transfectants (Fig 1Sd and e) showed an intermediate phenotype
between the vector and WT LC3 transfectants. These morphological differences were
further examined by indirect immunofluorescence of mimtubules with an anti-tubulin
antibody (Fig 16). Consistent with the previous photomicmgraph (Fig 15), WT, F/A as
weii as WK LC3 transfected cells (Fig 16b, e and d respectively) showed an eiongated cell
shape with densely organized microtubule arrays. WQ LC3 transfectants were similar to
vector transfectants (Fig 16c and a), with less well organized microtubules. Therefore,
besides decreasing celi growth, L a - A R E interaction also induces a flattened ceU shape in
HT 1080 cells, probably by influencing microtubule organization.
SubcelIular Localization of WT and Mutant LC3 in Stable-transfectants
Since LC3 bas a dual role as both a microtubule-binding protein and an RNA-binding
protein, its subcellular distribution might affect its different functions in regulating
microtubule dynamics and FN mRNA translation, or the coordination of both. From the
RNA binding studies above, WQ LC3 shows lower RNA-binding activity compared to WT
or other mutant LC3. It would be interesting to see if the decrease in RNA-binding of WQ
LC3 would shift the distribution of the protein away from the FN mRNA and translational
rnachinery, resulting in increased LC3 CO-localization with mimtubules.
Subcellular distribution of LC3 was first examined by indirect immunofiuorescence of LC3
with an anti-LC3 antiserurn as s h o w in Fig 17. Al1 WT and mutant LC3 transfectants
were expressed along the microtubules as granules as well as in perinuclear region
associated with ribosomes. WT LC3 transfectants showed very intense granular staining in
the perinuclear region (Fig 17a) evident as bright yellow dots while al1 mutant transfectants
showed less intense perinuclear staining (Fig 17c. e and g). Interestingly, at a different
focal plane, al1 W T and mutant aansfectants showed a network pattern of intranuclear
staining (Fig 17b, d, f and h), most prominent in F/A and R K transfectants (Fig 17h and f
Figure 16. Immunofluorescence labelhg of tubuiin in WT and mutant LC3
transfected HTlOSO cells.
Consistent with the celi morphology shown in Fig 15, vector-msfected (a) and WQ LC3
@) transfected cells showed a rounded cell shape and less organized tubulin staining, while
the WT (c), R K (d) and FIA (e) LC3 transfected cells showed a more elongated shape with
densely organized fdamentous microtubule staining. (f) corresponds to negative conml
with secondary antibody alone.
Figure 17. Immunofluorescence Iabeihg of WT and mutant LC3 in distinct
subcellular locations in stably-transfected HTlOSO cells.
Al1 WT (a) and mutant LC3 (R/Q(c), R/K(e) and F/A(g)) transfectants showed dual
staining in the cytoplasm dong the microtubules as granules as well as in the pennuclear
region associated with ribosomes. WT LC3 aansfectants showed a very strong granular
staining in the perinuclear region as bright yellow dots while al1 mutant transfectants
showed a less intense perinuclear staining. Interestingly, at a different focal plane on the
nght column (b, ci, f and h respectively), al1 mutant transfectants showed a network pattern
of intranuclear staining, most prominent in F/A (h) and WK ( f ) transfectants. Thus
intranuclear LC3 might be associated with some unknown nuclear cytoskeletal components
or with the nuclear membrane dong with pretranslational machinery. (i) and (j) correspond
to vector-transfectants and negative control with secondary antibody alone.
respectively). This intranuclear LC3 might be associated with some unknown nuclear
cytoskeletal component or with the nuclear membrane dong with pretranslational
machinery. This should be further investigated by comparing LC3 concentration in
purified nuclear extracts €rom these cells. However, there is no notable
immunohistochemical difference in LC3 distribution when comparing WT and mutant LC3-
transfected cells.
To quantify the differential distribution of LC3 associated with the microtubules or the
ribosomes respectively, the supernatant and peilet fractions of the ceil lysate fiom the empty
vector, WT and mutant LC3-transfected HT1080 cells were assessed for LC3 by western
immunoblot. Since microtubules are mostly concentrated in the supematant fraçtions, as
confirmed by western imrnunoblot using anti-tubulin antibody as shown in Fig 18A, LC3
present in the supematant represents the fraction both free in the cytoplasm and bound to
the microtubules, while that present in the pellet represents the fraction in the nucleus or
bound to membranes including the translational machinery, ribosomes and B A .
Twenty pg of protein from the supernatant and each of the 2 pellet fractions were loaded
ont0 the gels and transferred ont0 membranes. For the cell lysate from empty vector
transfected HT LOS0 cells, no LC3 was detected (data not shown); whereas for the WT and
mutant LC3 transfectants, more LC3 was found in the 2 pellet fractions compared to the
supernatant (Fig 18A). Densitometry was performed on the LC3 immunoreactive bands to
quanti fy the difference between the supernatant and the pellet. The pellet/supematant (PIS)
ratio was obtained by dividing the sum of the densitometric units from the 2 pellet bands by
the densitornetric unit of the supernatant band. As shown in Fig 18B, there is no
significant difference in the P/S ratio amring the WT and the mutant LC3, even the R/Q
tC3, illustrating that the lack of ARE-binding does not unload RIQ LC3 from the
translational machinery or the ribosomes, probably kcause of the interactions between
LC3 and other membrane-bound components besides FN mRNA. From our previous
Figure 18. Quantitative analyses comparing the subcellular distribution of
WT and mutant LC3 in stably-transfected HT1080 cells by western
imrnunoblot.
A. Representative western immunoblots showing LC3 and tubulin expression in the
supernatant (S) and the 2 pellet (Pl and Pd fractions of the cytoplasmic extract from
HT1080 cells stably transfected with WT or mutant (R/Q, R/K and F/A) LC3 constructs.
Densi tome try was perforrned on the LC3 immunoreac tive bands to calculate the
pellet/supernatant ratio (P1+P2/S) as shown in B. No significant difference is observed in
the pellet~supernatant ratio of cell extracts fiom WT and different mutant LC3 transfectants.
Bars refiect standard deviations fiom n=4 WT LC3 transfectants, n=3 R/Q LC3
transfectants, n=4 R/K LC3 transfectants and n d F/A LC3 transfectants.
WT RIQ R/K FIA
FIA
studies, LC3 was found to bind 40s and 60s ribosomal subunits, suggesting that LC3
facilitates the docking of FN mRNA onto the translational machinery by acting as a iinkage
between these 2 elements (Zhou et al., 1999, manusmpt subrnitted).
DISCUSSION
In this thesis we investigated the site on LC3 which binds the AU-rich element (ARE) on
the 3' UTR of the FN mRNA, and how this specific RNA-protein interaction contributes to
the regulation of M synthesis, as well as the tumorigenicity and the morphology of LC3
stably-transfected HT1080 cells. To narrow down the potential ARE binding site to a
smaller region within the molecule, we used proteases and CNBr to generate discrete LC3
peptides from the 16 kD tecombinant LC3 protein, foliowed by NW blot analysis to assess
the ARE-binding activity of these peptides using a [32P]-radiolabeled ARE oligonucleotide
probe. A 10 kD N-terminal peptide generated by CNBr cleavage showed strong ARE-
binding activity while the 6.8 kD N-terminal peptide showed no binding. This narrowed
the ARE binding site down to residues S61 to M88, which was confirmed by proteolytic
digests using other enzymes. Since peptides containing only the S61-MS8 region show no
binding to the ARE, the upstream Pl-M60 sequence was also judged to be important for
ARE-binding, probably by contributing to the charge andor the secondary structure of the
protein. Based upon these results, we tried to generate a tmncated recombinant 10 kD N
terminal peptide, but could not show that it possessed ARE binding ability, perhaps
because it was not folded properly or not glycosylated in E. coli comptent ceils. We
therefore perfonned site-dîrected mutagenesis to determine the specific residues within
S61-M88 which bind the ARE. There are 2 possible RNA binding sites: the arginine-rich
motif (ARM) containing 3 consecutive arginine residues and a predicted sheet region
containing 2 consecutive phenylalanine residues. We mutated the 3 consecutive arginines
or the 2 consecutive phenylalanines: R to Q substitution in the ARM to alter charge
abolished the ARE binding activity upon W anaiysis, while R to K substitution which did
not alter charge, did not appear to decrease ARE binding. F to A substitution in the
predicted f3 sheet region also did not affect ARE binding. This led to our conclusion that
the positive charge of the 3 R's in ARM are critical for ARE binding of LC3, probably by
contributing to the charge-charge interaction.
Since N W analysis rnight distort the original conformation of the protein, we set out to
confirm our N W resulu by gel shift analysis in which the protein and the RNA are in intact
state. However, we were unable to produce convincing gel shift results using recombinant
intact wild type or mutant LC3 proteins with either the ARE oligonucleotide or the whole
FN 3'UTR probe. This is likely because pst-transiational modification of the recombinant
proteins might be different in E-coli compared to mammalian cells. Considering that
distinct complex formation is easily shown using the ARE oligonucleotides or the FN
3'UTR and endogenously expressed LC3 present in cultured smooth muscle cells from
sheep ductus arteriosus and aorta and piglet coronary artery (Zhou et al., 1998; Mason et
al., 1999, manuscript in preparation), we stably transfected Hi' 1080 cells with plasmids
encoding the WT and the mutant LC3, and used the cell extracts to perform gel shift
analyses. Zhou has shown by NW analysis that the 16kD recombinant LC3 expressed in
HT1080 transfected cells maintains its RNA-binding capacity with preference for ARE
(Zhou et al., 1999, manuscri' submined). From the gel shift anaiysis shown in Fig 9,
WT, RIK and FIA LC3-containing HT1080 ce11 extracts formed 3 ARE-dependent
complexes witb the [32~]-radiolabeled FN 3'UTR, whereas in cell extracts containing WQ
LC3 and empty vector, only the top 2 complexes were formed. Therefore, the lowest
complex represents the recombinant LC3 and the ARE- FN 3'UTR, while the top 2
complexes might represent cornplex formation between FN 3'UTR and other endogenously
expressed proteins in ml080 cells. Previous NW analysis using [32~]-radiolabeled FN
3'UTR and HT108O ce11 extracts containing WT LC3 or empty vector demonstrated that
beside the l6kD recombinant LC3, a 200kD and a 551d) proteins also showed ARE-
binding in both LC3 and vector transfectants (Zhou et al., 1999, munuscript submined).
These 2 proteins which in previous studies were also immunoreactive with the LC3
antibody might represent the top 2 complexes observed in our gel shift analysis. The
absence of lower complex formation when extracts h m R/Q-LC3 transfected cells were
used, further cofirmed the significance of the 3 R's in the ARM of intact LC3 in ARE-
binding.
When investigating the ARE-binding specificity of the WT and mutant recombinant LC3
expressed in HTlOSO celis by cornpetition studies, binding between FN 321TR probe and
WT or R/K LC3 could be partially competed by cold EN 3WTR with intact ARE but not
mutated ARE; whereas binding between FN 3'UTR and F/A LC3 could only be slightly
competed if at al1 by cold FN 3WïR with either intact or mutated ARE. This showed that
binding of WT and R/K LC3 to FN mRNA is ARE-specific, consistent with the NW
results in Fig 7 which showed higher affinity binding of WT LC3 to WT ARE
oligonucleotides compared to mutant ARE oligonucleotides. However, F to A substitution
in the predicted B sheet stnicture resulted in the loss of ARE-specificity in mRNA binding,
dernonstrating that the 2 phenyldanines downstrearn of the ARE-binding sites are important
in detemiinkg the ARE-specificity of LC3 in RNA binding.
The importance of sequences flanking the RNA binding domain in confaring binding-
specificity has been shown with other RNA-binding proteins. The required flanking
sequences v q h m 5 residues in Ul-A and U2-B" to 1 11 residues in La proteins (Kenan,
199 1). This irnplies that motif alone rnay not contain sufficient information to function as a
sequence-specific RNA binding domain. The requirement for flanking sequence suggests o
function important in maintaining the secondary structure of the protein which permits the
motif to form a direct contact with the RNA. In cases where flanking sequences do not
contribute to secondary structure, they may instead provide additional RNA contacts.
hnRNP A i is similar to LC3, in that it also binds to the ARE of the 3WTR of c-fos and
GM-CSF mRNA, and this property inhibits the ARE-dependent mRNA turnover of c-fos
mRNA (Hamilton et al., 1993). hnRNP A l acts as both a pre-mRNA binding protein
involved in nuclear RNA processing as well as a trans-acting factor involved in modulatuig
cytoplasrnic mRNA turnover and translation (Hamüton et al., 1993). It contains 2 RNA
recognition motifs (RRMs) tandemly arranged at the N-terminus, followed by a C-terminal
glycine-rich region (Ghetti et al., 1990). It was proposed that the first RRM binds to the
RNA target, and the interaction is stabilized by the second RRM (Burd and Dreyfuss,
1995). To better understand how LC3 interacts with the ARE of FN mRNA, it would be
very helpful to have the crystallized structure of LC3 or the FN mRNA-LC3 complex.
To study the association between ARM-ARE interaction and FN synthesis, WT and mutant
LC3encoding plasmids were stably-transfected into HT1080 cells, which are LC3-nul1 and
express low levels of FN. Previously, Zhou stably transfected HT1080 cells with
plasmids containing empty vector or WT LC3, and showed upregulation of FN mRNA
translation in WT LC3 transfectants by enhanceci ribosome recruitrnent (Zhou et al., 1999,
manuscript submined). Consistent with Zhou's nsults, we showed a 2.5 times increase in
FN synthesis and secretion into condition medium in WT LC3 compared to vector
transfectants. Consistent with the inability of R/Q LC3 to f o m a complex with the FN
3'UTR was its inability to upregulate FN synthesis compared to WT LC3. This was
evident both by immunofluorescent staining of FN deposition on the surfaces of cultured
cells and by [%]-metabolic labeling to detect newly synthesized and secreted FN. W e
confimed that the inability of R/Q LC3 to upregulate FN was not due to a decrease in FN
mRNA level or stability, leading to the conclusion that WQ LC3 is inefficient in docking
F'N mRNA onto polyxibosomes for translation. This can be further investigated by
polysorne profile andysis.
In contrast, F/A LC3 showed a significant increase in FN synthesis compared to the WT
LC3. Since the FN mRNA level is unchanged in FIA transfectants, this upregulation in FN
synthesis, manifest both as enhanced FN deposition in the ECM and secretion in
conditioned medium might be due to the ability of FIA LC3 to facilitate the docking of FN
mRNA ont0 heavy poiysomes. F/A LC3 might have a stronger affinity for ribosomes or
other ribosomal proteins, such as 40s and 60s, compared to the WT LC3. F/A LC3 might
also enhance FN synthesis via increased FN mRNA binding. Aithough binding between
F/A LC3 and ARE oligonucleotide or intact FN 3VTR is unchanged compared to W T
LC3, as illustrated in both NW and gel shift analyses, we cannot exclude the possibility
that FIA LC3 has a stronger affinity for other regions of the FN mRNA, such as the S'UTR
or the coding region. The Ioss of ARE-specificity of FIA LC3 as shown in cornpetition
studies might lead to the enhanced affinity for other regions of the FN M A , or even to
other mRNAs which might indirectly contribute to the upregulation of FN synthesis, as
well as the other phenotypic changes observed To examine the binding affinity of FIA
LC3 to ribosomal proteins, total ce11 lysates from HT1080 cells can be incubated with
agarose beads conjugated with GST-FIA LC3 fusion proteins and bound extracts can be
tested for the presence of 6ûs and 40s ribosomal subunits using 28s and 18s RNA probes
in northern blot analysis, or tested for the presence of other ribosomal proteins using
western immunoblots.
Besides acting on the translational upreguiation of FN mRNA, F/A LC3 might also
enhance FN synthesis by increasing the cytosolic pool of FN mRNA availabie for
translation. From the immunostaining pattern of LC3 in HTlOSO cells in Fig 18, al1 WT
and mutant LC3 transfectants showed intranuclear staining of LC3, but this was most
prominent in FIA LC3 transfectants. Since the FN mRNA level is unchanged in F/A LC3
transfectants, F/A LC3 does not appear to play a role in transcriptional regulation. It might,
however, interact with other nuclear proteins to form a mRNP complex that facilitates the
export of FN rnRNA into the cytoplasm for translation. To investigate the role of LC3 in
the nucleus, nuclear extracts could be obtained and we could determine using
immunoprecipitation with appropriate antibodies whether LC3 forms a complex with other
nuclear proteins.
FN has k e n shown to revert the transformed phenotype of tumor cells by enhancing
adhesion of these cells to the substrate, changing the morphology of the cells h m rounded
to spread and decreasing growth rate (Akamatsu et al., 19%). Zhou showed that by
stably-transfecting HT108O cells with WT LC3 which induces FN mRNA translation, he
was able to revert the transformed phenotype of the HTlOSO cells in a similar manner as
was s h o w in other studies by overexpressing FN cDNA. Here, we further showed that
LC3-mediated FN synthesis and the resulting reversion of transformed ~ 1 0 8 0 phenotype
depends on the ARM-ARE interaction. WQ LC3 transfectants, which failed to upregulate
FN synthesis due to the mutated ARE-binding site, were also unable to decrease the growth
rate of the cells or revert them to a flattened shape. They behaved Iike empty vector
transfectants. In contrast, the FIA LC3 mutant, acted like a "super" LC3 and both in
upregulating FN synthesis and in reducing growth rate and reverting cell phenotype.
While, these data directly implicate FN in reverting the transfonned phenotype of the
HT1080 cells, LC3 rnight also bind to other mRNAs which contribute to the slower growth
rate and flattened ce11 shape. To investigate whether FN alone is necessary for this
antitumor effect, we can transfect these LC3 stably-transfeçted ceils with cDNA encoding
antisense FN mRNA to see if the revenant phenotype can be overcome. It would be
interesting to fuxther investigate the significant anti-tumor effect of FIA LC3 in animals
where we could detenaine the impact of gene transfer of this mutant on fibrosarcoma
growth.
In this thesis, we show that LC3 upregulates FN mEWA translation via the contribution of
the arginine-rich motif (ARM) to the LC3 binding to the AU-rich element (ARE) in FN
rnRNA. By site-directed mutagenesis, mutant LC3 with R to Q substitution in the ARM
was unabie to bind to the ARE of the FN mRNA and upregulate its translation. Steady
state levels of FN mRNA in HTlO8O cells transfected with RiQ LC3 are unchanged
compared to WT LC3 transfectant, confirming the significance of ARE-ARM interaction at
the level of mRNA translation, without any effect on transcription, mRNA stability or
splicing. On the other hand, the upregulation of FN mRNA translation imposed by FIA
LC3 also acts at the level of mRNA translation with no notable change in FN mRNA
compared to WT transfectants. Therefore, polysome profile analysis wiil be critical in
determining more precisely whether the EUQ mutant fails to move FN mRNA into the heavy
polysome fractions and the F/A mutant does so even more efficiently than WT LC3. By
comparing the distribution of FN mRNA with that of the WT or mutant (R/Q LC3 or FIA
LC3) we will get a better idea about whether LC3 affects the docking of FN mRNA on the
tram lational machinery .
In addition to binding FN mRNA, LC3 also appears to directly bind to ribosomd subunits
(both 40s and 60s)(Zhou et al., 1999, manuscript submitted), and may also bind other
proteins important in translation initiation. It would be interesting to Qtennine whether the
R/Q and F/A mutations influence also these protein binding properties of LC3. Previously,
Zhou used agarose beads conjugated with GST-LC3 fusion protein to extract any bound
ribosomal proteins from the ceil extracts. However, considering that there might be a
ciifference in the confoxmation and pst-translational modification of fusion LC3 compared
to the endogenously expressed protein, that might influence protein-protein interaction, it
would be more convincing to study LC3-protein interaction by immunoprecipitating
endogenously expressed LC3 h m stably-transfected HT 1080 cells. While the affinity and
specificity of the currently available LC3 peptide antibodies does not permit
immunoprecipitation, this may be overcome by producing antibodies to the intact molecule
or by epitope-tagging LC3. HA-tagging would aüow the immunoprecipitation of LC3 by
using some commercially available antibodies towards the HA tag. Since in the HT1080
cells, additional proteins are recognized by the U33 antibody, epitope-tagging of LC3 could
facilitate the intracellular localization and compa.rtmentaüzation of LC3. This would help
address the circumstances under which LC3 might be prescrit or tramlocate to the nucleus,
and associate with other mRNAs or proteins to form a RNP cornplex- Other possibilities
include conditionally expressing LC3 tagged to green fluorescent proteins (GFP) to more
precisely follow the intraceilular dynamics of this protein.
Other mRNAs such as that for apolipoprotein D have dso been isoiated using an LC3-GST
column. The ability to imrnunoprecipitate LC3 could ailow for more direct intracellular
detection of mRNAs with which this protein associates. Yeast two hybrid systems couid
also be used to detect other proteins with which LC3 interacts. A more recent strategy
would suggest that LC3 tagged with a PKA site could be used to screen a cDNA
expression library, for protein-protein interactions.
Microtubules have k e n shown to play a critical role in FN mRNA translation. When DA
SMC are treated with colchicine which disrupts the microtubule organization, they showed
a decreasc in FN mRNA translation, associated with less FN mRNA present in the
polysome fractions compared to the untreated DA SMC. Therefore, it would be interesting
to investigate if R/Q and F/A mutant LC3 affect FN mRNA translation by altering its
association with the microtubules. This can be achieved by both in vitro and in vivo
microtubule-binding assays. Ultimately, it would be very important to identify the
microtubule binding site(s) on LC3, which also play a significant role in regulating FN
mRNA translation. This will M e r elucidate the novel mechanism of how microtubdes
regulate niRNA translation.
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BlOTECHNOLOGY SERVICE CENTRE DEPI:. OF CUMCAL BIOCHEMISTRY
100 COLLEGE ST, ROOM 351 TORONTO. ONTARIO M5GiLS
P E m E SEOUENCE ANAI-YSIS FACILlTY Phone: (416) 978-5554
. - &K. J UY Date: ...............................~.
- / s- Hfle User: .............................~......................................................................
......................................................................................... COMMENTS: .............. ..-. . ~ c 7 z y k . . xt.. .z&ifi!. œfle~~
BIOTECHNOLOGY SERVICE CENTRE Dqt Crinicd Biochuniztxy 100 CoiIege S t. Room 351
Toronto. Ontario MSG L I 3
/Yb-,- d ? / p DATE: -----.,-...-..-.-.--.-. --- /G' /CD @7~@30s5/" SAMPLE: ...-œ..-œ-. -.-. .-œ..-œœœ. ...œ-œ.o-CODE: .,..., ..-,.., ,,,....,,,, ,.,
F i le=c:\porton\datal\G9803WP.01R froin 5-50 to 26-00 min, Low seaie = 41.5ïS1 M. nigh scale = CC-5751 av,
+***+*********++++ HSC Biotechnology Service Centte **t*+++**+tt*t**+* * * Pept ide ~equencing Facility t
* pl&R 4, 1998 18:14:37 * * * CYCLE NUMBER: 1 * * lOKD * *****************+**************************************************** Peak Ret-Time Amino Peak Amount
# ( m i n - 1 A c i d Area moles) Comments 1 6-00 3398 4-48 ' 2 6.33 181108 238 &O 3 7-12 ASP 6404 7.24 4 8 - 5 7 SER 6022 18-95 5 9.17 THR 2379 4-76 6 9 - 7 5 GLY 16481 24-07 7 12-88 ALA 57940 60-20 8 15-37 TYR 3304 3.65 9 18-10 DTT 599238 77.52 10 18.68 PRO 6646 7.67 11 21.02 DPTU 50320 2.93 12 21-90 64531 85.12 13 23-77 XLE 7458 8.70 14 2 5 - 4 3 1968 2 - 6 0
F i Le=c:\porton\datal\C98030ZP ,02R f rom 5 -50 to 26-00 min, Lou s c a k = 41,5223 rn- Nigh scate = 44-5223 W.
Peptide Sequencing Facility MAR 4, 1998 19:02:07
* CYCLE NUMBER: 2 1, * lOKD * ***************+****************************************************** Peak P
1 2 3 4 5 6 7 8 9
R e t . Time ( m i n . 1
6.35 7.12 8 .55 9 - 7 3 12 87 18-10 21.02 21-88 23.73
Amino A c i d
ASP SER GLY ALA D m DPTU
ILE
Peak Area 196308
2775 4644 3737
34272 430328
38346 49450
6383
Amount moles 1 Comments O
258.95 - 3.14
1 4 - 6 1 .
5-16 35-61 55.67 2.23
65.23 7.45
F i te=c:\portori\datal\C080U1C~.03R frairi 5.50 to 26-00 min, LOU scate = 41,4245 mv- nigh scate = 44,424s H,
Peptide Sequencing Facility MAR 4, 1998 19:49:36
* CYCLE NUHBER: 3 * * lOKD * ********************************************************************** Peak Ret.Time Amino Peak Amount # ( m i n , 1 Acid Area t~molesl Comments 1 6 - 2 7 185842 2 4 5 - 1 5 - 2 10-15 HIS 11546 14 r 0 4 3 1 2 - 8 2 ALA 29729 30.89 4 18-07 D!FT 380345 4 9 - 2 1 5 2 1 - 0 2 DPTU 32511 1 - 8 9 6 21 -87 58306 76.91 7 23 243 1506 1 - 9 9 8 25.42 2520 3.32
F i le=c:\porton\datal \C9803(UP.OCR f rom 5-50 CO 26-00 min- L w scak =
*******+******+*++ HSC Biote&nology service C e n t r e *+***********f+t** * f
* Peptide Sequencing Facility O
* MAR 4 , 1998 20:37 :06 O
* f
r~ CYCLE NüMBER: 4 O
* lOKD f
********************************************************************** Peak Ret.Time Amino Peak Amount
# ( m i n . 1 A c i d Area ( V ~ O ~ S ) C o m m e n t s O
1 6.32 185890 245.21 - 2 8.55 SER 3588 11.29 3 10.22 HIS 1760 2.14 4 12.87 ALA 27598 28.67 5 18.08 DTT 386597 50.01 6 19.32 MET 3419 3.65 7 21-02 DPTU 26178 1.52 8 21.88 49301 65.03 9 23-73 ILE 4832 5.64 10 25.42 2891 3.81
F i ~e=c:\porton\&tal\G9CU)30CP,05R froni 5-50 to 26-OO min. L w scale = 41,4392 nv, H i g h scate = 44.4392 mv,
***+t***********t+ HSC Biotechnology Semice Centre ************++**** * 4 * Peptide Sequencing Facility * HAR 4 , 1998 21:24:35 * * # * CYCLE NUEIBER: 5 4 * lOKD * ********************************************************************** Peak Etet-Time Amino Peak Amount
pl ( m i n - 1 A c i d Area moles) Comments 8
1 6-33 186944 246.60 - 2 9 - 1 7 THR 2557 5.12 3 12-88 ALA 32278 33 54 4 1 8 - 1 0 DTT 436769 5 6 - 5 0 5 21.03 DPTU 29173 1.70 6 2 1 - 9 0 53260 70.26 7 2 3 - 7 8 ILE 7344 8 - 5 3
**************+*** HSC Biotechnology Semice Centre ****t*********+*t*
* a * Peptide Sequencing Pacility * * MAR 4, 1998 22:12:04 * * * * CYCLE -ER: 6 * * lOKD * **********************************************************************
Peak R e L T i m e Amino Peak Amount # (min. 1 A c i d Area moles 1 Comments . 1 6.33 190281 251 -01 - 2 10.18 H IS 1304 1.59 3 12.88 ALA 33647 34.96 4 18.10 DTT 436416 56-46 5 21.02 DPTU 27745 1 .61 6 21.90 49359 6 5 - 1 1 7 23.22 P m 3394 4 . 4 0 8 23.75 ILE 6732 7.85
BIOTECHNOLOGY SERVICE CENTRE DEPT. OF CLiNïCAL BIOCHEMISTRY
100 COLLEGE ST., ROOM 351 TORONTO, ONT"RI0 MSG I I 5
PEPTIDE SEO1 IMCE ANALYSIS FACiLITY Phone: (416) 978-5554
. Falrnmile: (416)978-8802
Date:
-HD ........................... ......................................... S ample: .œœo.o....CODE: G%f=@~ -7- 9- Pkpe User: ....................................................................................................
COMMENTS: ....................................................................................... fl&/e. . . d y .&. +2.jpi3&&Kœ .. A%&?* 0. de~dca' . ........ ................
BIOTECHNOLOGY SERVICE CENTRE Dept Cüniui B i o d i d w 100 Coiiegc Sr. Room 351
Toconm. Onrario M5G LL5
41,3%7 rn, tligh scale = GC.3967 nu,
*********++*+**t+t HSC Biotechnology Service Centre ****+***+**++**t*+ * * * Peptide Sequencing Facility * * MAR 4, 1998 12:08:22 t * * * CYCLE NüMBER: 1 * * 6-8KD * ********************************************************************** Peak Ret.Time Amino Peak Amount
# (min. ) A c i d Area (~molesl Comments .. 1 5.98 9592 12-65 - 2 6.33 218576 288 -33 3 7.15 7359 9.71 _ 4 7.87 10361 13.67 5 8.55 SER 7344 23.10 6 9.73 GLY 40310 58-87 7 12.87 ALA 67366 49-99 8 15.32 TYR 1766 1.95 9 18.08 DTT 729758 94.41 10 18.67 PRO 13745 15.85 11 21.00 DPTU 48491 2.82 12 21.88 73148 96.49 13 23.77 ILE 8399 9.80
C l ,4855 riva H i g h scale = CC.CUSS rn,
Peptide Sequencing Facility MAR 4, 1998 12:55:52
* CYCLE NUMBER: 2 * r~ 6.8KD a ********************************************************************** Peak Ret.Time Amino Peak Amount # ( m i n . I A c i d eh bmolesl Comments 1 6 - 3 3 198195 2 6 1 - 4 4 - 2 7 - 1 2 ASP 3178 3-59 3 8 . 5 5 SER 7799 2 4 - 5 3 _ 4 9 - 7 5 GLY 4125 6-02 5 12-88 ALA 30942 32-15 6 13.98 5062 6-68 7 15.67 4333 5.72 8 18-08 DTT 419459 54-27 9 21-02 DPTU 28943 1-68 10 21-88 40005 52 -77 11 2 3 - 7 5 ILE 3422 3-99
*+***++*++**t**+** HSC Biotechnology service Centre ************+**t** * * * Peptide Sequencing Facility * * nAR 4, 1998 13:43:20 * * * * CYCLE NUMBER: 3 * 6.8KD * ********************************************************************** Peak Ret-Time Amino Peak Amount
# ( m i n . 1 A c i d Area (~molesl - Comments 1 6-33 204739 270 - 08 - 2 9 - 7 7 GLY 1637 2 - 39 3 10-18 HIS 26951 32 - 77 4 12-88 ALA 26143 27.16 5 18.10 DTT . 375674 4 8 60 6 21-02 DPTU 27785 1-62 7 21.90 38473 50.75 8 23.75 ILE 5558 6 - 4 8
~ilc=c:~rtori\&tal\C98030C-OCR fran 5-50 to 26-00 min. LW scalt = 41,JQbl mr- H i g h scalc = CL.3961 H,
t Peptide ~equencing Facility t MAR 4, 1998 14:30:50 * * CYCLE NUMBER: 4 * * 6.8KD * ********************************************************************** Peak f
1 2 3 4 5 6 7 8 9
R e t . Time ( m i n , 1
6.35 10.15 12.87 18-08 19-28 21.02 21-90 23.70 23-98
Amino A c i d
HIS ALA DTT MET DPTU
ILE LYS
Peak Amount 2irea f ~molesl Comments . 196019 258.57,
5201 6.32 21229 22.06 -
346530 44.83 17412 18.60 27049 1.57 34407 45.39 4869 5.68 8531 14.83
41,3748 RI- H igh scale = 44,3748 rv,
*****+*+*****t++*t HSC Biotechnolw service C e n t r e +**++t*+*i**+**t**
* * * Peptide Sequencing Facilfty f
* HAR 4, 1998 15:18:19 * * * * CYCLE NOMBER: 5 * * 6.8KD * **********************************************************************
Peak Ret.Time Amino Peak Amount # ( m i n . A c i d Area moles 1 Comments .. 1 6.33 194769 256-92 _ 2 9.17 THR 6676 13.37 3 12-87 AtA 21509 22.35 4 18.10 DTT 346120 4 4 - 7 8 5 18.67 PRO 7836 9.04 6 21 .02 DFTU 23149 1.35 7 21 .90 3 1965 4 2 - 17 8 23.75 ILE 4459 5.20
File=c:\portor\\datal\t98030S,06R fran 5-50 to 26-00 min. Lou scrlc = C1,429!5 W . High scale =
Peptide ~equencing Facility MAR 4, 1998 16:05:49
* CYCLE NUMBER: 6 * * 6.8KD 4 ***************+****************************************************** Peak Ret-Time Amino Peak mount # (min ) A c i d Area (~nroles 1 Comments 1 6.32 198036 261.23 2 12.87 A U 20697 21.50 - 3 13.92 1574 2.08 4 18.10 ûTT 331341 42.87 5 19.85 1843 2 .*4 3 6 21.02 DPTU 22309 1-30 7 21.90 37841 49.92 8 23.18 PHE 6374 8.27