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Aberrant Glycosylation in HEMPAS Patients
Homa Kameh
A thesis submitted in conformity with the requirements for the degree of Master of Science
Ins titute of Medical Sciences University of Toronto
O Copyright by Homa Kameh 1997
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Aberrant Glycosylation in HEMPAS Patients Homa Kameh Master of Science, 1997 lnstitute of Medicai Sciences, University of Toronto
ABSTRACT
HEMPAS (hereditay erythroblastic mdtinuclearity with positive acidified serurn
lysis test) is a rare congcnital anernia. A group of enzymatic lesions affecting the
biosynthesis of N-linked oligosaccharides has been reported in HEMPAS. This study was
conducted to investigate the molecular basis of KEMPAS in a group of Ontario patients.
Lectin binding and endoglycosidase digestions were utilized to determine the nature of
abnormal oligosaccharides on two erythrocyte membrane giycoproteins, Bands 3 and 4.5.
The results suggest a hybrid or high mannose N-glycan in place of poly N-
acetyUactosamine on these proteins, consistent with a defect in a-mannosidase II. A
reduction in the enzymatic activities of GnT II or P4GdT. as reported in some HEMPAS
patients, was not detected in the cdtured EBV-tnnsformed lymphobiasts of Our patients.
Moreover, poly N-acetyllactosamine was present on proteins in these cells. The alteration
in the oligosaccharide moiety of Band 4.5 did not interfere with its glucose transport
ACKNO WLEDGMENTS
My greatest appreciation goes to my supervisor, Dr. Reinhart Reithrneier, for
believing in me. Without his guidance and support this thesis would not have been
possible. His knowledge and admirable personality offered me the best example to follow
dunng my graduate program. I thank Dr. Harry Schachter for his CO-supervision, his
patient sharing of his expertise in glycobiology and for ail those sessions of "clariQing
things". I also thank my supervisory cornmittee member, Dr. Robert Murray for his
constructive comrnents which helped me to improve rny work.
I thank my parents for their support and patience even though they could not
elaborate on what their girl was doing. Bahnm and Pari be thanked for their understanding
and their constant presence beside me.
My thanks to Mark Gettner, Dr. Vieth and Dr. Jahromi for their inspiring words
and for wnting those recommendation Ietters. I thank Dr. Whiteside for her willingness to
offer expert comrnents in the annual meetings.
Dr. Carolina Landolt-Marticorena and Lisa Tam are thanked for their generosity
with their knowledge and for helping me get started. 1 thank Dr. Ieffrey Chanik, Dr. Tip
Loo, Dr. Ienny Tan, Dr. Gary Song, Bryan Loo, Denise Eskinazi and Anita Nutikka for
their technical assistance. 1 also thank my labrnates Milka, John, and Jing for sharing the
rare and joyful moments of "some good results"!
Last but not least 1 would like to thank rny friends: Susan, Valerie, Martine, Simin,
Emily, Mark, Michael and H a s for their heart warming words and being always there for
me. They ail share this degree with me. 1 especiaily thank E d y for moral support and her
"emo tional intelligence".
TABLE OF CONTENTS
I . INTRODUCTION ....................... ..... ....................................*...................*..................... 1
I . I PROTEIN-LINKED OLIGOSACCHARIDES ........................................................................ L 3 ......................................................................................... . i 1.1 Biosynthesis of N-giycans -
1.1.1.1 Synthesis of dolichoi-iinked sugar precursor ...................................................... 2 ................................................................................ 1.1.1.2 Oiigosaccharyi transferase 6 1.1.1.3 Trimming of the N-linked high mannose precursor ............................................. 6
................................................................... 1.1.1.4 Glycosyitransferase characteristics 1 O .................................................. 1.1.1.4.1 Domain structure of giycosyltransferases 1 O
............................... 1.1.1.4.2 Retention and targeting of the glycosyiation enzymes 1 2 ....................................................................................... 1.1.2 Poly N-acetyiiactosamine 1 3 ............................................................... 1.1.2.1 Poiy N-aceryi factosamine synthesis 1 3 1.1.2.2 Distribution of poiy N-acetyliactosamine .................................................. 1 6 1.1 .2.3 Poiy N-acetyllactosamine as blood group antigens .......................................... 1 9
1.1.3 Characterizarion of oligosaccharides ...................................................................... 20 1.1.3.1 Lectins .............................................................................................................
3 7 ........................................................................................... 1.1 .3.2 Endoglycosidases .- - 1.2 THE ROLE OF CARBOHYDRATES ON MEMBRANE GLYCOPROTEINS ........................... 24
1.7.1 Eariy foiding and protein stubiiiry ......................................................................... 24 ................................................................................. 1.2.2 Chaperon- facil itated foiding -25
.................................................................. 1.2.3 Sorting and rargeting of glycoproteins -26 ........................................................................... 1.2.4 Clearance of serum glycoproteins 2 7
............................................................................................... 1.2.5 Celi-cell interactions 2 8 ....................................................................... 1.2.6 Funclional roie of oiigosaccharides 2 9 I.3 ERYTHROCYTE MEMBRANE GLYCOPROTEINS ............................................................ 40
.......................................................................................................... 3.1 Giycoplio rin 4 . - L . .
1.3.2 Band 3 ................................................................................................................... 42 ............................................................................................... 1.3.3 Glucose transporter -44 1.4 ER YTHROPOIESIS ........................................................................................................ 45
..................................................................................................................... 1.5. HEMPAS 50 1.5.1 Ciinicai features ..................................................................................................... 52 .......................................................................................... 1.5.2 Hemato logical jèatur es. 5 - 2 A ................................................................................................ 1.5.3 Sero fogicai features
. 3 3
........................................................................................................... 1.5.4 Biochemistry 5 4 ............................................................................................................... 1.5.5 Treatment 5 5 ..................................................................... 1.5.6 Moiecular abnormalities in HEMPAS 5 5
.............................................. 1.5.7 Abnomalities in glycosyiation enqmes in HEMPAS -5 8 1.5.8 HEMPAS and other "diseases of aberrant giycosyiation " ....................................... 6 1
1.6 RESEARCH PROJECT AND HYPOTHESIS ...................................................................... 6 3
. II MATERIALS AND METHODS ....................................... ...................................m..... *64
11.1 MATERIALS ................................................................................................................ 64 11.2 METHODS .................................................................................................................... 67
11.2.1 Preparation of ghost membranes ........................................................................... 67 ................................................................................... 11.2.7 Prepomtion of cell extracts 67
............................................................... 11.2.3 Enqmatic digestion of oiigosaccharides 67 ........................................................................................................... 11.2.4 L e c h Biots 68
11.2.5 Western Biots ........................................................................................................ 69 .............................................................................................. 11.2.6 Staining procedures 69 .................................................................................. .......... 11.2.7 Enzyme . assqs . .......... -6 9 ................................................................................................ 11.2.8 Giycol~p~d analysis 70 .................................................................................... 11.29 Glucose transport assay -7 L 11.2.10 Preparation of g lycopeptide from HEMPAS erythrocytes ................................... 72
......................................................................................... 11.2.1 1 Atialytical procedures 7 3
III. RESULTS ......... ................ .....~..... ............................................................................. 74
III . I heter III . 2 111.3 111.4 I I I . 5 III . 6
Polyacrylamide gel electrophoresis of etythrocyte membrane proteins from normal. .............................................................................. ozygotes and HEMPAS patien fs -74
Enzymatic deglycosylation of normal and HEMPAS eryrhrocyte membrane proteins74 Lectin bittding of erythroid membrane gfycoproteins ............................................... 77
.............................. Diagnosis of HEMPAS heterorygotes by iectin binding atiaiysis 82 ............................................... Staining of erythrocyte membrane sialoglycoproteins 89
............................................................. Studies on EB V-transformed lymphoblnsts -89 ................................................................................................. 111.6.1 Lecrin bbiding 89
111.6.2 Enryme assays ................................................................................................. 92 ..................................................................................... 111.7 Culriued erythroid ce11 fine 97
.......................................................... 111.8 Ftinctional strldies on HEMPAS erythrocyres 97 ............................................................................................ 111.9 HEMPAS glycolipids 102
.......................................................................... 111.10 HEMPAS g fycopeptide analysis 105
IV . DISCUSSION .......................................................................................................... *..IO9
V . CONCLUSIONS AND FUTURE DIRECTIONS ........................................................ ***124
REFERENCES .~.~............~~...~..s......s...~..~....~...........................................................~.......... 129
LIST OF TABLES
TABLE 1.1 The specificities of some plant lectins
TABLE 1.2 The specificities of endoglycosidases
TABLE 1.3 Inhibitors of the gl ycosy lation enzymes
TABLE 1.4 Summary of roles of oligosaccharides on some transporters and channets
TABLE 4.A ATPases
TABLE 4.B Channels
TABLE 4.C Transporters
TABLE 4.D Antiporters
TABLE 4.E Neurotransmitter tmsporters
TABLE 4.F Na-dependent symport
TABLE 4.G Glucose transporters
TABLE 1.5 Classical features of the three types of congenital dyserythropoietic anemia
(CDA) TABLE 1.6 Summary of reported glycosylation defects in HEMPAS patients
TABLE 1.7 Summary of known diseases of aberrant glycosylation
LIST OF FlGURES
Figure 1.1 Structures of the three types of N-linked oligosaccharides 3 Figure 1.2 The phosphodolichol pathway 5
Figure 1.3 The processing pathway of N-linked oligosaccharides 7
Figure 1.4 Domain organization of the glycosyltransfenses 1 1
Figure 1.5 Structure of poly N-acetyllactosamine oligosaccharide on adult Band 3 15
Figure 1.6 The structures of A, B and H antigens 19
Figure 1.7 Structure of Lewis"LeX) and sialyl-Lewis' oligosaccharide determinants 29
Figure 1.8 Folding model for the membnne domain of human Band 3 (a) and a model of
the human erythrocyte glucose transporter (GLUTI) (6) 43 Figure 1.9 Stages of the erythroid differentiation from a stem ce11 to an erythrocyte 49
Figure 2.2.1 Erythrocyte membnne protein profile of control, heterozygotes and HEMPAS patients 75
Figure 2.2.2 Enzymatic deglycosylation of erythrocyte membrane proteins of control and
HEMPAS patients 78
Figure 2.2.3 Enzymatic digestion of poly N-acetyllactosamine sugar chah and tomato lectin
blot of control and hempas erythrocyte membrane proteins SO
Figure 2.2.4 Endoglycosidase H treatment and Con-A blot of control and HEMPAS erythrocyte membrane proteins 83
Figure 2.2.5 ECA blot of control and HEMPAS erythrocyte membrane proteins 85
Figure 2.2.6 Tomato lectin binding analysis of control, heterozygotes and homozygote of
HEMPAS 87
Figure 2.2.7 Stains-al1 staining of erythrocyte membnne glycoproteins of control,
heterozygote and HEMPAS patients 90
Figure 2.2.8 Tomato lectin blot analysis of EBV-transformed lymphoblast extncts of control
and HEMPAS patients 93
Figure 2.2.9 Glycosyltransferase enzyme assays on control and HEMPAS EBV-transformed
Iymphoblast extracts 95
Figure 2.2.10 Immunoblot and tomato lectin overlay of extracts from cultured hurnan
erythroid ceils 98
Figure 2.2.11 3 - 0 - m e t h ~ l - [ ~ ~ ~ ~ ~ l u c o s e uptake by control and HEMPAS erythrocytes 100
Figure 2.2.12 Immunoblot analysis of control and HEMPAS erythroid membranes with anti-
glucose transporter antibody 103
Figure 2.2.13-a Thin-layer chromatogram of erythrocyte 106
Figure 2.2.13-b The results of corresponding scanning densitometry 106
LIST OF ABBREVIATZONS
P6GlcNAcT
CDA
CDGS II
CHO
Con-A
EBV
ECA
E m A
Endo H
ER
Fuc
Gal
GaiNAc
Glc
GlcNAc
GnT
GP
Lac-Cer
LAMF'
Man
Neu
PBS PNH
PP-Do1
SA (NeuNAc)
SDS-PAGE
TBS
TM
p 1-3 N-acety lglucosaminy 1 tram ferase
p 1-4 Galactosyltransferase
Congenitai dyserydiropoietic anernia
Carbohydrate-deficient glycoprotein syndrome type II
Chinese hamster ovary ce11
Concanavalin A
Epstein-Barr virus
Erythrina ciestagalli agautinin
Ethy lenediaminetetraace tic acid
Endo-P-N-acety lglucosaminidase H
Endoplasmic reticulum
Fucose
Galactose
N-acetylgaiactosamine
Glucose
N-acetylglucosamine
Wace ty lglucosaminy ltransferae
Glycophorin
Lac tosy lceramide
Lysosomal associated membrane protein
Mannose
Neuraminic acid
Phosphate buffered saline
Paroxysmal nocturnal hemoglubinuîa
Dolicholpyrophosphate
Sialic acid (N-acety lneuraminic acid)
Sodium dodecyl sulphate-polyacrylamide p l electrophoresis
Tris buffered saline
Trans-membrane
I. INTRODUCTION
Glycoproteins exist in al1 eukaryotic cells and indeed mos t
integral membrane proteins are glycosylated or are associated w i t h
glycosylated subunits. Although most protein- and lipid-linked
sugars are localized at the ce11 surface or luminal face of cellular
cornpartments, there is also evidence for existence of cytoplasmic
and nucleoplasmic glycoproteins (Hart et al.. 1989). A unique feature of
carbohydrates which distinguishes them from other biological
macromolecules is the complexity of their structure. They can b e
made from different monosaccharide residues, while the linkage
between two residues can be shaped in two isomeric forms (a & P)
and the anomeric carbon can be linked to three or four different
positions of the hydroxyl groups. For this reason carbohydrates, in
contrast to proteins or nucleic acids, may consist of complicated
branched structures.
1.1 Protein-linked oligosaccharides
Protein-linked oligosaccharides are classified as O-linked (O-
glycan), attached to the hydroxyl group oxygen of serine o r
threonine, and in collagen to hydroxylysine, and N-linked (N-glycan),
attached to the amide nitrogen of asparagine. Many glycoproteins
contain both O-linked and N-linked oligosaccharides. An ab u n d a n t
type of O-glycan in animal cells is the mucin-type gIycan in which
the oligosaccharide c h a h starts with an N-acetylgalactosamine l inked
to serine or threonine. The biosynthesis of O-glycans does no t
include the preassembly and processing steps required for N-glycans.
This thesis will concentrate on N-glycans and their alteration in
HEMPAS.
There are three types of N-glycans: high mannose, hybrid a n d
cornplex (Figure 1.1). Al1 three types consist of a common "core"
structure which is Mana 1 -6(Mana 1 -3)ManB 1 -4GlcNAcp 1-4GlcNAc-
Asn. High mannose sugars may have up to six additional mannose
residues. In complex chains Galp 14GlcNAc (N-acetyllactosamine) i s
attached to the outer two core mannose residues. In a hybrid chain,
the al-3 mannose is linked to N-acetyllactosamine, while the a 1-6
arrn carries additional mannose residues.
I. 1. f Biosynthesis of N-gfycans
This topic has been reviewed by Fukuda (Fukuda and Hindsgaul,
1994), Montreuil (Montreuil et al., 1995), Komfeld (Komfeld and Komfeld, 1985)
and Hubbard (Hubbard and Ivatt, 1981).
1.1.1.1 Synthesis of dolichol-linked sugar precursor
Protein N-glycosylation starts cotranslationaily in the ER b y
transfer of a preformed oligosaccharide from a ER-ernbedded lipid,
dolicholpyrophosphate (PP-Dol), to the glycosylation acceptor site of
a nascent polypeptide. Dolichol contains a hydrophobic
polyisoprenoid chain which c m span the ER membrane four or five
times. Structural features of dolichol, namely chain length, cisltrans
pattern and a-saturation, have been listed as critical factors for t he
efficient functioning of dolichol as an oligosaccharide donor.
The oligosaccharide precursor contains three glucose, nine
mannose and two N-acetylglucosamine residues. Its synthesis is
Man a l L 0''
3 6 M a n al+î-Man al Man p t j4GlcNAc
3
High mannose type
(Man a1 +2)
Hybrid type
Man a l -
I
Complex type
Figure 1.1 Structures of the three types of N-linked oligosaccharides
catalyzed by glycosyltransferases residing in the membrane of the
ER. Figure 1.2 summarizes this synthetic process known as the
phosphodolichol pathway. In the first step, N-acetylglucosamine
phosphate is transferred to phosphodolichol at the cytoplasrnic face
of the ER by the enzyme N-acetylglucosaminyl phosphate
transferase. This reaction can be inhibited by tunicarnycin. The
enzyme has been cloned from mouse mammary gland and the gene
is located in chromosome 17. The amino acid sequence of this
enzyme suggests ten TM segments spanning the membrane of the ER
The sequence contains two potential dolichol recognition seq ue nces
(PDRS) and a conserved putative catalytic site. Other pro teins
carrying PDRS include yeast glycosyl transferases and ribophorin-I.
The addition of a second N-acetylglucosamine and the first five
mannose residues coming from üDP-GlcNAc and GDP-Man
respectively occurs at the cytoplasmic surface of the ER, while al1
subsequent additions of mannose and glucose occur on the luminal
face, requiring the flip-flop of the Man,GlcNAclPPDol. Mannosyl a n d
~lucosyl phosphodolichol, which provide the sugars for the luminal P
extension of the oligosaccharide, probably undergo the same trans-
membrane movement. The sugar precursor is then transferred to
the amide nitrogen of an asparagine located in a tripeptide
recognition sequence, Asn X Ser or Asn X Thr (where X is any amino
acid except praline), by oligosaccharyl transferase on the luminal
side of the ER membrane.
Figure 1.2 The phosphodolichol pathway (adapted from Montreuil et al.,
1995)
1.1.1.2 Oligosaccharyl transferase
Oligosaccharyl transferase activity in canine p anc reas
microsomes is mediated by an oligomeric cornplex composed of
ribophorin I and II and a 48 kDa subunit (Kelleher et ai., 1992). This
complex is a component of the mammalian transiocon. The
translocon, an assembly of peripheral and integral membrane
proteins, contains a gated protein channel which allows the
translocation of newly-synthesized peptides across the RER
membrane. It can also open laterally into the lipid bilayer to
accommodate integration of membrane proteins. This corn plex
includes signal recognition particle receptor (SRPR), Sec 6 1 p comp lex,
translocating chain associated membrane protein (TRAM), signal
peptidase and oligosaccharyl transferase complex (Gilmore, 1993). 1 t
has been proposed that the translocation cornplex also accommodates
glucosidase I and II, as well as the membrane-bound rnolecular
chaperone, calnexin (Chen et al., 1995). These proteins are involved in
mediating the folding of the translocating polypeptide.
1.1.1.3 Trimming of the N-linked high mannose prectrrsor
Al1 three types of N-linked sugars are formed by processing of
the Glc,Man,GlcNAc, precursor by glycosidases a n d
glycosyltransferases as the glycoproteins pass through the
endomembrane system (Figure 1.3). Al1 endomembrane
compattments (ER and Golgi complex) are coanected through
peripheral vesicles that originate from one cornpartment and fuse
with the membranes of another organelle. The trirnming starts b y
removal of three terminal glucose residues. Two membrane proteins
of the ER, glucosidase I and II, cleave off the a l-2-linked and two
a 1 -3-linked glucose residues, respectively. Both activities can b e
inhibited by castanospermine and deoxynojirimycin.
Many of the N-linked oligosaccharides are posttranslationally
reglucosylated after losing their original glucose residues by an ER
glycosyltransferase; however the enzyme utilizes only denatured O r
misfolded glycoproteins as its substrate. Monoglucosylated
glycoproteins associate with the ER chaperone, calnexin, and a r e
retained in the ER until proper folding has been attained. Release of
fully folded glycoproteins from calnexin requires the removal of t he
remaining glucose by glucosidase II (Hebert et al., 1995). According to
the model proposed by Helenius (Helenius, 1994) the ER contains a
unique folding and quality control machinery in which calnexin
serves as a retention factor and a chaperone, glucosidase I acts as a
signal activator and glucosidase II as a signal modulator which
regulates binding to calnexin. In this model glucos yltrans ferase
functions as a quality control factor.
After removal of glucose residues, four al-2 mannose residues
are trimmed sequentially by ER and cis-Golgi a-mannosidase
enzymes. These two enzymes have different specificity and inhibitor
sensitivity; the ER enzyme which removes only one a 1-2-linked
mannose is not sensitive to the inhibitor of cis-Golgi a-mannosidase
(a-mannosidase 1), deoxymannojinmycin. The resulting Man,GlcNAc,
chah is the substrate for N-acetylglucosaminyltransferase I (GnT 1)
which starts the antenna on the a 1-3 mannose arm in the medial-
Golgi. GnT 1 is the crucial enzyme for the synthesis of hybrid a n d
cornplex N-glycans. In the absence of this enzyme none of t he
modifications by a 1-316 mannosidase (a-mannosidase II), GnT I I ,
GnT III, GnT IV and core a l - 6 fucosyltransferase can take place. A
Chinese hamster ovary (CHO) cell line (Lec 1) deficient in GnT I does
not synthesize complex N-linked carbohydrates and accumulates
Man,G1cNAcl high mannose oligosaccharide (Kumar et al., 1990). Mice
lacking GnT I activity due to disruption in the encoding gene (Mgat-
1) do not survive to term (Metzier et al., 1994). Mgat- 1-" embryos a r e
developmentally retarded especially in neural tissue (Ioffe and Stanley.
1994). It seems that heterozygotes with about 50% GnT f activity
develop normally.
The product of GnT 1 can serve as the substrate for GnT III and
Golgi a-mannosidase II. Depending on the relative activities of these
two enzymes, the product can be a hybrid containing a bisecting
GlcNAc Linked to the P mannose or a potential complex chain
respectively. The modification by GnT III disqualifies the sugar chain
as a substrate for a-mannosidase II. Consequently the bisected chain
can only be elongated on the a 1-3 arm leading to a hybrid t y p e
sugar. However if the activity of cc-mannosidase II is higher than
GnT III, two mannose residues on the a 1-6 Man arm are removed
and GnT II can then initiate the a 1-6 antenna. a-mannosidase U can
be inhibited by a plant alkaloid swainsonine. Other medial-Golgi N-
acetylglucosaminyl transferases, namely GnT N, V and VI, may ad d
extra antennae to the core. The antennae are usually elongated a n d
terrninated by addition of galactose, N-acetylglucosamine, N-
acetylgalactosamine, fucose and sialic acid residues. In addition,
non-carbohydrate groups like sulfate and phosphate can be added.
The diversity of complex glycans derives from their number of
antennae, the pattern of branching and the number and type of
monosaccharides forming the antennae.
The sugars required for the above glycosylation reactions a r e
donated from nucleotide sugars synthesized in the cytosol (UDP-
GlcNAc, UDP-GalNAc, UDP-Glc, UDP-Gal, GDP-Man, GDP-FUC) or in th e
nucleus (CMP-SA). Dedicated antiports mediate the transport of
these nucleotide sugars into the ER and Golgi apparatus and remove
the produced nucleotides from these compartments by an exchange
mec hanism.
Biosynthesis of glycoconjugates is regulated by the level of
activity of glycosyltrans ferases and the availability of n u c leo tide
sugars (Gahmberg and Tolvanen. 1996). In addition, the accessibility of t h e
glycoprotein to the modifying enzymes determines the extent of t he
oligosaccharide processing (Gibson et ai., 198 1).
1 . i 4 Glycosyltransferase characteristics
1.1.1.4.1 Domain structure of glycosyltransferases
Al1 cloned mammalian glycosyltransferases are type 1 I
membrane proteins (cytoplasmic N-terminal and luminal C-terminal
domains) and consist of the same domain arrangement (Figure 1.4)
as follows: 1- a short cytoplasmic N-terminal domain, 2- a
hydrophobie transmembrane domain and 3- a long C-terminal
catalytic domain in the lumen of the ER or Golgi complex. Ln bovine
p 1-4 galactosyltransferase (P4GalT) these domains contain 24, 20 and
358 amino acids respectively (Fukuda and Hindsgaui, 1994). The luminal
domain includes a flexible stem region distal to the trans me rnb rane
domain with low amino acid conservation among species.
Lumen 0
Membrane O
O Cytoplasm
Figure 1.4 Domain organization of the glycosyltransferases. 1 ) Cytoplasmic
N-terminal domain, 2) Hydrophobie transmembrane domain. 3) Stem re g i O n
including the protease sensitive site (arrow) and 4) C-terminal catal y tic
domain.
The stem region is usually rich in proline and glycine residues a n d
consensus N-glycosylation sites. The catalytic domain c m be cleaved
at the stem region by proteases to produce a soluble active enzyme.
The soluble catalytic domain of p4GalT was purified from bovine
milk and showo to be enzyrnatically active (D'Agostaro et al., 1989)
suggesting that transrnembrane and cytoplasrnic domains are no t
required for the catalytic Furiction of glycosyltransferases. The
transmernbrane domain plays a role in the targeting and retention of
enzymes to the endomembrane compartments (Colley et al., 1992).
1.1.1.4.2 Retention and targeting of the glycosylation enzymes
Although newly-synthesized proteins destined for secretion O r
for plasma membrane insertion are transported from the ER through
the Golgi complex by "bulk flow", it seems that ER and Golgi resident
proteins require a signal to withhold them from this default path
(Rothman and Orci, 1992). It has been suggested that t ransmembrane
domains play a central role in the Golgi retention by providing a si te
for interrnolecular interaction leading to oligomers or aggregates too
large to be transported. Replacement of the transrnembrane domain
of vesicular stomatitis virus (VSV) G protein with a transmembrane
domain of a Golgi-resident protein results in aggregation and
retention of the chimera in the Golgi (Pelham and Munro, 1993). However,
in the case of GnT 1 the transmembrane domain is not sufficient for
complete medial-Golgi retention; cytoplasmic and luminal domains
clearly contribute to this event (Burke et al., 1994). It is possible that ail
three domains are involved in the lateral interactions leading to
aggregate formation and enzyme localization.
Protein oligomerization or aggregation may happen b e t ween
different enzymes residing in the sarne compartment. Addition of a n
ER retention signal to GnT I c m also partially retain a-mannosidase
II in the ER. Therefore, enzymes in the same compartment may
interact specifically with each other to form heterocomplexes (Nilsson
et ai., 1993).
An alternative mode1 suggests that localization of the enzymes
rnay be a consequence of favorable interactions between their
transmembrane domains and the lipid bilayer in which they a r e
inserted. The length of the transmembrane dornain seems critical
since the replacement of this domain in sialyltransferase with 1 7
leucines does not interfere with its retention in the Golgi, while a
transmembrane segment of 23 leucines results in cell surface
expression (Pelham and Munro, 1993). The relatively s ho r t
transmembrane domains of Golgi proteins may interact with the low
cholesterol bilayer of the Golgi and never reach the thicker post-Golgi
membranes (Bretscher and Munro, 1993). In the case of GnT I t h e
transrnembrane segment, being 25 arnino acids long, does not m e e t
these criteria, suggesting that other energetically favorable pro te i n-
Iipid interactions cannot be excluded. The replacing of the 25-amino
acid transmembrane domain of GnT I with that of the transferrin
receptor (28 amino acid) does not abolish Golgi localization (Burke et al.,
1994), emphasizing the involvement of other domains and re ten tio n
factors in targeting.
1.1.2 PoQ N-aceryllactosamine
1.1.2.1 Poly N-acetylloctosamine synthesis
Poly N-acetyllactosamine chains were first described in glycans
present on human erythrocytes (Knisius et al., 1978). These long
complex N-linked oligosacchuides are composed of repeating units of
Galp 1 -4GlcNAcp 1-3, which are synthesized by the alternative action
of p 1-4 galactosyltransferase (B4GalT) and J3 1-3 N-
acetylglucosaminyltransferase (B3GlcNAcT).
P4GalT, the first mammalian glycosyltransferase cloned, h a s
two forms which are different in the length of their amino-terminal
cytoplasmic domains. The two f o m s are the result of two
transcription promoters in the P4GalT gene (Shaper et al., 1988).
Although the functional role of the two transcripts has not b e e n
established, the longer form may be preferentially targeted to t h e
ce11 surface, while both forms are targeted to the Golgi complex (Shur,
199 1; Evans et al., 1993). Surface galactosyltransferase molecules O n
embryonic carcinoma cells have been reported as receptors for poly
N-acetyllactosamine chains (Shur, 1982). In addition to synthesis of N-
acetyllactosarnine, P4GalT combined with a-lactalbumin catalyses th e
biosynthesis of lactose in the lactating mammary gland.
P4GalT is expressed in most tissues and cells, showing
properties of a housekeeping gene; therefore, P3GlcNAcT (extension
enzyme) is the key enzyme for the synthesis of N-acetyllactosamine.
Both lactose and Galpl-4GlcNAc can be used as a substrate for
P3GlcNAcT; however, the latter is a better acceptor (Piiier and Cmron,
1983).
The poly N-acetyllactosamine c h a h may be branched, as in t h e
adult form of Band 3 (erythrocyte anion exchanger) (Figure 1.5)
(Fukuda et al., 1979). The branching enzyme, p 1-6 N-ace ty lglucosamin y 1
transferase (P6GlcNAcT), which acts on GlcNAcp 1-3Galp 1-OR
(R=sugar) has no action on acceptors having terminal galactose
residues (Piller et al., 1984), irnplying that the branching occurs only
after the substitution of the nonreducing terminal galactose by p 1 -
3GlcNAc (Komfeld and Kornfeld, 1985). However, Gu et al (Gu et ai., 1992)
proposed a second pathway for the synthesis of b ranched
lactosaminoglycan, which involves a novel P6GlcNAcT that can a d d
GlcNAc branch on the penultimate Gd in GalP 1 -4GlcNAcB 1 -3Galp-R.
Repeating N-acetyllactosamine
tri and te traantennary N-linked
biantennary chains (Komfeld and
units are present more often on
oligosaccharides cornpared to
Kornfeld, 1985) and there is a
preferential attachment of poly N-acetyllactosamine chains to t h e
GlcNAcPl-6 antenna of the al-6 arm (van den Eijnden et d., 1988).
Polylactosamine chains may be sialylated by terminal a2-3 (or less
often, a2-6) sialic acid (Fukuda and Hindsgaul, 1994). In a te t r a m te n nary
poly N-acetyllactosarnine chain a2-3 sialyltransferase pre fers
terminal galactose residues on the antennae of the a l - 6 a m , while
cr2-6 sialylation takes place preferentially on the 1-2 antenna of the
a 1-3 arm ("branch specificity") (Fukuda and Hindsgaul, 1994). The chai n
may also be terminated by a 1-3 fucose or a 1-3 galactose. Evidence
suggests that poly N-acetyllactosamine generally is a better accep tor
su bstrate for glycosyltrans ferases than the single N-
acetyllactosarnine unit (Fukuda and Hindsgaul, 1994).
l. 1.2.2 Distribution of poiy N-acetyilactosamine
The poly N-acetyllactosamine structure is the major
carbohydrate moiety on human erythrocytes. It is carried by two
erythroid membrane proteins, Band 3 (anion exchanger) and Band
4.5 (glucose transporter) and by glycolipids (neolacto series).
Neutrophilic granulocytes contain both a significant amount of
neutral lactosaminoglycan with the GalP lg4(Fuca 1-3)GlcNAc
structure and also sialylated tetra antennary poly N-
acetyllactosamine (Fukuda et d., 1984b). This structure has also bee n
found on Chinese hamster ovary cells and thyroid cens (Fukuda et al.,
1984a). In addition, lysosomal membrane proteins, LAMPs, contain
this type of sugar (Fuhda and Hindsgaul, 1994). LAMP 1 and LAMP 2
have 18 and 16 N-glycosylated sites respectively. Some of these
sites are selectively modified by poly N-acetyllactosamine. The po 1 y
N-acetyllactosarninyl chains of LAMPs purified Rom human chronic
myelogenous leukemia cells contain only two N-acetyllactosamine
uni ts (Carlsson and Fukuda, 1 WO), w hile mature granulocytes (HL-60)
contain more repeats. In the latter, it has been proposed (Lee et al.,
1990) that differentiation be associated with an increase in t h e
number of N-linked sites modified by poly N-acetyllactosamine a n d
the number of the poly N-acetyllactosamine side chains in one
oligosaccharide. This may result in longer half Life of LAMPs a n d
higher stability of lysosomes.
Secretory proteins usually contain none or a small amount of
this sugar. Interestingly, the fusion of the transmembrane a n d
cytoplasmic domains of vesicular stornatitis virus G protein w i t h
human chorionic gonadotropin (hCG) a chah was able to alter t h e
modification of the N-glycan from a typical short complex type to a
poly N-acetyllactosamine form when expressed in COS-1 cells (Fukuda
et al., 1988).
There is some evidence for N-acetyllactosarnine repeats w hic h
are O-glycosidically bound to peptides. They exist on the hurnan
leukocyte commoo antigen present on B and T lymphocytes (Chiids et
al.. 1983) and on a bovine erythroid membrane protein (Gp2) (Suzuki et
al., 1985). In the latter case the sugar mediates the interaction with
the pathogen, Mycobacteriurn pneumoniae.
Two critical factors have been proposed for the formation of
poly N-acetyllactosamine sugar on a protein (Fukuda and Hindsgaul, 1994).
The first is the tertiary structure of the protein which mus t
accommodate the elongating enzymes. It seems that mu1 tispanning
membrane proteins are preferable acceptors for the poly N-
acetyllactosamine chain, possibly because anchoring to t he
membrane increases the accessibility of the growing oligosaccharide
to poly N-acetyllactosamine forming enzymes. The second factor is
the rate of protein movement through the Golgi complex, which mus t
be slow enough to provide sufficient contact with the Golgi enzymes
(Wang et ai., 1991). A mutant of Band 3 carrying a deletion of nine
amino acids at the boundary of the cytoplasmic domain and the first
predicted transmembrane segment lacks the normal poly N-
acetyllactosamine sugar (Sarabia et al., 1993). Altered tertiary s truc ture
may account for this event. Studies on Madin-Darby canine kidney
(MDCK) cells demonstrated that the Golgi transit time of LAMP 2
might regla te the extent of modification by poly N-
acetyllactosamine (Nabi and Rodriguez-Boulan, 1993). Longer resident t i me
in this cornpartment allows efficient action of the elongating
enzymes. Consistent with this hypothesis, in Mk Mk (glycophorin A -
and B-deficient) erythrocytes a delay in the translocation of Band 3
to the ceIl surface caused by lack of glycophorin A has been
suggested as the reason for an increase in the number of N-
acetyllactosamine repeats and the subsequent increase in the M, of
Band 3 (Bruce et al., 1994).
1.1.2.3 Poly N-acetyllactosamine as blood grorip an tigens
Protein-linked poly N-acetyllactosamine carries AB0 blood
group antigens. In O-type blood group individuals expressing H
antigen, a l - 2 fucosyltransferase substitutes the terminal galactose of
poly N-acetyllactosamine with a 1-2 fucose to fonn Fuca 1 -2Galp 1-4
(or 3) GlcNAc-R (H antigen). cDNA for the enzyme has been cloned
and the gene was located on chromosome 19. H antigen can b e
converted to A and B blood group antigens by action of a 1-3 N-
acetylgalactosaminyltransferase (A-enzyme) and a l - 3
galactosyltransferase (B-enzyme) respectively. The structures of
these antigens are shown in Figure 1.6.
Fuc al 4 2 ml PL 4 1 3 G l c N A c pl +R
FUC a 1 c2 Fuc al f12
Figure 1.6 The structures of A. B and H antigens
A and B transferases differ only in four amino acids, which may
dictate their specificities. O aIlelic cDNAs carry a single base deletion
resulting in a shift in the reading frame of the A and B
glycosyltransferases and a nonfunctional enzyme (Yamamoto et al., 1990).
Poly N-acetyllactosamines also serve as I/i blood group
antigens; Bands 3 and 4.5 are major carriers of these antigens
(Hakomori, 1981). The conversion of the fetal linear poly N-
acetyllactosamine (i) to the adult branched form (1) (Figure 1.5) is a
major developmental change in erythrocytes (Fukuda et al., 1979) whic h
occurs within the first few months after birth; fetal and neonatal
erythroid cells supposedly lack the P6GlcNAcT. The i antigenic
determinant has been shown to be a linear hexasaccharide of the N-
acetyilactosarnine type, while the 1 determinant has the branch point
Ga1 PI-4GlcNAcPl-6 (Gooi et al., 1984). The I/i antigens are not
restricted to protein-linked poly N-acetyllactosamine and are also
carried by lipids (Fukuda et al., 1984~). Rare adult individuals with a
genetic defect cannot develop 1 blood group antigen (Weiner et al., 1956).
1.1.3 ChnracteBzation of oligosaccharides
1.1.3.1 Lectins
Lectins are proteins with multiple binding sites for specific
sugars. Lectin column chromatography has been widely used to
isolate and fractionate oligosaccharides and glycopeptides based o n
carbohydrate binding specificity. Moreover, glycoproteins sep ara ted
by SDS gel electrophoresis and transferred to nitrocellulose c m b e
probed by lectins to provide information about the type of glycans
present.
Sorne lectins interact with the core portion o f N-linked sugar
chains while others are specific to the outer antennae. An example
of the former is concanavalin A (Canavalia ensiformis, Con A) which
reacts with sugar c h a h containing two non-substituted or C-2
su bstituted a-mannopyranosyl residues (Fukuda and Kobata, 1993).
Therefore, biantennary cornplex, high mannose and hybrid t y p e
sugar chains bind to Con A but with different affinities. High
mannose and hybrid type sugar chains bind to the lectin strongly
due to their multiple binding sites. A bisecting GlcNAc changes the
conformation of trimannosyl core, resulting in a weak interaction
(Fukuda and Kobata, 1993). An a 1-3Fuc at GlcNAc of the Galp 1-4GlcNAc O r
Neua2-6 at the GlcNAc of Galpl-3GlcNAc group in the outer chain
interferes with the Con A binding (Fukuda and Kobata, 1993). Con-A binds
strongly to the ER and cis-Golgi forms (high mannose type) of
glycopro teins (Tartakoff and Vassalli, 1983).
Tomato lectin (Lycopersicon esculentum, LEA) is an example of
a lectin that interacts with the poly N-acetyllactosamine structure. Tt
binds to sugars containing three or more N-acetyllactosaminyl
(Galp 1 -4GlcNAcp 1-3) repeats (Merkie and Cummings, 1987). lnhibi tion
studies have shown that tomato lectin is specific for oligomers of p l -
4-linked N-acetylglucosamine even if they are not consecutive.
Tomato lectin is a po tent hemagglutinin; neuraminidase-treated cells
are agglutinated with the lectin more readily than untreated cells.
Erythrina cristagalli agglutinin (ECA) is ano ther lectin with b inding
activity for N-acetyllactosamine; however, the presence of more than
one N-acetyllactosamine unit is not required for the binding to this
lectin. The lectins referred to in this thesis are Iisted in Table 1.1.
L e c f i n S p e c i f i c i t y
LEA (Tornato) OIigomers of P 1 -3-Iinked N-
( L y c o p e rsicon esculentrcrn) acety lglucosamine (>3 units)
Concanavalin A (Con-A) Two non-substituted or C-2 substituted
(Canavalia ensiformis) a-mannopyranosyl residues
ECA N-acetyllactosamine
(Erythrina crysragalli)
Table 1.1 The specificities of some plant lectins
1.1.3.2 Endogiycosidases
Endoglycosidases are enzymes which can cleave an in ternal
linkage between two sugars in an oligosaccharide, w hile
exoglycosidases cleave the sugars from the nonreducing terminal.
Since digestion of the carbohydrate moiety usually changes t he
mobility of the glycoprotein on SDS-PAGE, glycosidases can be used
to study the existence of certain structures in a glycoprotein. The
degee of hydrolysis by these enzymes depends on the size a n d
conformation of the peptide portion and the number of
oligosaccharide chains on a polypeptide. The specificities of so me
endoglycosidases are summarized in Table 1.2.
The enzyme peptide N-glycosidase F (peptide-Na-[N-acetyl P-
glucosaminyl] asparagine amidase) ac ts on the P- aspartylglycosylamine linkage and releases oligosaccharide, leaving
an aspartic acid residue at the glycosylation site. This enzyme
cleaves ail types of N-linked carbohydrates including - i - , and
tetraantennary complex sugars. However, some restrictions appl y:
the enzyme rnay not function if the carbohydrate-linked asparagine
is on the amino or carboxyl end of a peptide (Maley et al., 1989).
Endo-P-N-acetylglucosaminidase H (endo H), an enzyme which
cleaves fi 1-4 linkage in the chitobiose unit (GlcNAcpl-4GlcNAc) of N-
linked oligosaccharides, requires a tetrasaccharide, Mana 1 - 3 M a n a 1-
6Manp 1-4GlcNAc as its specific substrate (Fukuda and Kobata, 1993). The
most important sugar is the cc-mannosyl residue at the non-reducing
terminal; the sugar chah can still be hydrolyzed if the C-2 position of
this residue is substituted. Therefore, the enzyme can hydrolyze al1
high mannose and most hybrid type chains. Since complex chains d o
not have the outer Mana 1-3, they are resistant to endo H even af t e r
cheir outer chain moieties are removed. This enzyme is widely u s e d
to follow the synthesis of glycoproteins in the endomembrane system
because after leaving the media1 Golgi, complex glycoproteins become
resistant to endo H.
E n z y m e S p e c i f i c i t y
Peptide N-glycosidase F R-GlcNAc---Asn (R = Any type of N-giycan)
Endo H R-GlcNAc---GlcNAc-Asn (R = Wybrid and high mannose N-glycrins)
Endo -p-galactosidase F R-GalB L -4GlcNAc-R' (R & R' = Sugar)
Table 1.2 The specificities of endoglycosidases (--- indicates the site o F
cleavage)
Endo- P-galactosidase F, isolated originally from E. freundi, i s
specific for P-galactosidic bonds in which galactose is attached t O
glucosamine or N-acetylglucosamine. The cleavage is slower if t h e
invoived galactose is a branch point (Fukuda et al., 1978). Substitution of
galactose by sulfate, as in some glycoprotein hormones, m a y
interfere with cleavage by endo-P-galactosidase F.
1.2 The role of carbohydrates on membrane glycoproteins
Most proteins in nature are glycosylated (Edge et al., 1993) and t h e
glycosylation process, specifically N-glycosylation, is very conserved
in eukaryotes (Parekh, 1991). In a survey of the SWISS-PROT database
(Gahrnberg and Tolvanen, 1996) from 1823 animal pro tein entries w i t h
reported extracellular features (potential membrane proteins) 9 L .7%
were described as glycoproteins. In addition, representation of the
potential N-glycosylation tripeptide in protein extracellular domains
is more than should be found by chance. Therefore, it seems tha t
there is an evolutionary tendency towards protein glycosylation
suggesting significant biological functions for pro tein-lin ked
carbohydrates. Some of these functions are discussed below. It is
important to mention that, although many properties of proteins a r e
affected by glycans, these effects cannot be generalized and must b e
studied in the context of each glycoprotein.
1.2.1 Enriy fokiing and protein stability
Nascent polypeptides are glycosylated cotranslationally while
they are folding. It seems that early folding of proteins assists t he
prevention of nonproductive side reactions. The presence of
carbohydrates on folding intermediates enhances their solubility a n d
facilitates the folding process by preventing protein aggregation.
Oligosaccharides rnay also be needed to stabilize folded domains. The
extent of the stabilizing effect depends on the amino acid sequence of
the protein. The synthesis of G-protein from two strains of vesicular
stomatitis virus (VSV) in the presence of tunicamycin (inhibitor of N-
glycosylation) led to different degrees of intracellular pro tei n
aggregation, which was explained by the diversity of the amino acid
sequence of the peptides (Gibson et al., 198 1).
Moreover, glycosylation may have a general stabilizing effect
against proteolytic and denaturing agents (Wang et al., 1996).
Oligosaccharides increase the resistance to proteases by shielding th e
glycoprotein. The removal of carbohydrates Rom heavily O- or N-
glycosylated proteins results in their thermal instability (Wang et al.,
1996).
1.2.2 Chaperone-facilitateci folding
Glycoproteins undergo glucose trimming immediately af te r
glycosylation in the ER. At least two ER molecular chaperones,
calnexin and calreticulin, bind to the monoglucos y lated produc t O f
glucosidase I and II (Gahmberg and Tolvanen, 1996; Ware et al.. 1995). This
association can be inhibited by castanospermine (glucosidase
inhibitor) resulting in folding perturbation (Chen et al.. 1995). Consistent
with this. tunicarnycin causes incorrectly-folded polypeptides,
protein degradation and mis-location of newly synthesized pro teins
(Gahmberg and Tolvanen, 1996). However, it seems that there are p ro te ins
( e g . rat liver ~ansferrin, chick ernbryo fibroblasts procollagen, HLA-
DR B ce11 antigen) that do not need N-linked sugars during the folding
process (Helenius, 1994).
In contrast to other chaperones, calnexin binding is linked to
the composition of the oligosaccharides rather than the conformation
of the polypeptide (Helenius, 1994). Since calnexin is a type 1 ER
membrane protein (Bergeron et al., 1994), its binding to t he
oligosaccharide "appendages" keeps glycoproteins fixed in place w i t h
minimal interference in the folding process. Moreover, calnexin m a y
bind to more than one N-linked sugar, facilitating protein folding b y
bringing protein domains together. Fully deglucosylated Folded
proteins dissociate from calnexin and move to the Golgi, while
incorrectly folded proteins are reglucosylated and retained in the ER
(Tatu et ai., 1995).
1.2.3 Sorting and targeting of giycoproteins
A conventional example of glycan-mediated sorting is th e
targeting of hydrolytic enzymes to the lysosome. As lysosomal
enzymes travel through the Golgi cornplex, they are p hosp horylated
on some of their mannose residues . First, N-
acetylglucosaminylphospho transferase catalyzes addition of N-
acetylglucosamine 1 -phosphate to selected mannose residues on t he
lysosomal enzymes and then N-ace tylglucosamine- 1-phosp hodies ter
a-N-acetylglucosaminidase removes the N-acetylglucosamine residue
(Kornfeld et al., 1982). The resulting mannose 6-phosphate (M-6-P)
tagged enzymes bind to receptors (P-type lectins) which a r e
probably located in the tram-Golgi cornpartment. M-6-P receptors
(MPRs) are type I membrane proteins with a cornmon
extracytoplasmic motif presenting once in the calcium-dependent
form and fifteen times in the calcium-independent Form. The former
is a dimeric receptor while the latter is a monomer. The complex of
the receptor and the lysosomal enzyme exits the Golgi in vesicles,
which then fuse with an acidic prelysosomal cornpartment (Komfeld et
al., 1982). In an inborn error in which the M-6-P tag is not made on
the enzymes (1 cell disease), lysosomal enzymes are exported to t h e
extracellular environment.
N-linked oligosaccharides have been suggested as signals for
the migration of glycoproteins to the ce11 surface. As mentioned
above, most membrane proteins are glycoproteins. The introduction
of N-glycosylation sites to a chimera of rat growth hormone and a
membrane-anchored domain results in the ce11 surface presentation
of the chimera (Gahmberg and Tolvanen, 1996).
In addition, plycans are involved in sorting and delivery of
secreted proteins. In polarized kidney epithelial ce11 trans fec ted
with growth hormone, the nonglycosylated form does not show a n y
preference for secretion from either the apical or bas0 latheral
surface, while the glycosylated protein is secreted from the apical
side (Scheiffele et al., 1995).
1.2.4 Clearance of serrim glycoproteins
The serurn survival t h e of serurn glycoproteins can b e
regulated by alterations in their carbo hydrates. Non-reducing
terminal sialic acid seems essential for continued viability of t h e
glycoproteins in the circulation. Removal of the sialic acid b y
neuraminidase exposes galactose residues of the o l igosacch~des t O
bind with hepatic ce11 surface receptors (C-type lectin) (Ashwell and
Morell, 1974). Desialylation seems essential but not sufficient for
clearance of serurn glycoproteins, since transferrin survival is
unaffected by complete desialylation. On the other hand, t he
presence of sialic acid on hepatic receptors is required for t he
binding activity leading to internalization and catabolism of se ru m
glycoproteins in the lysosome (Ashwell and Morell, 1974).
1.3.5 Cell-ce11 interactions
The specific interaction of carbohydrates with lectins is the
underlying mechanism for many biological phenomena such as cell-
ce11 adhesion, sperm-egg interaction and bacterial adhesion.
A family of mammalian lectins known as selectins are involved
in adhesion of Ieukocytes to vascular endothelium. Secretion of
cytokines during inflammation stimulates the expression of t w O
types of selectins, E- (endothelial) and P- (platelet) selectin, on the
endothelial surface (Fukuda and Hindsgaul, 1994). The interaction of
monocyte and neutrophil carbohydrates with these selectins slows
down the cells, in an event called "leukocyte rolling". This is followed
by the release of chemoattractants and activation of integrins which
leads to attachment, penetration and extravasation of leu koc ytes .
The carbohydrate ligands for E- and P-selectins are Lewis" (Lex) a n d
sialyl-Lewiss oligosaccharides, shown in Figure 1.7. For a complete
inflammatory response the rolling stage mediated by carbo hydrate-
selectin interaction is critical.
A similar interaction facilitates "lymphocyte homing". This is
when circulating lymphocytes leave the vessels in the lymphatic
organs and cross the lymphatic parenchyma to return to t h e
circulation via the lymphatic system. This journey provides t h e
opportunity for exposure of lymphocytes to foreign antigens.
Homing of lymphocytes to peripheral lymph nodes in part is
mediated by carbohydrate-dependent mechanisms. L- (1 y mp hocy te)
selectin recognizes a sialic acid containing oligosaccharide moiety O n
post capillary venular endothelium in lymph node (Fukuda and Hindsgaul,
1994). Some of the ligands for L-selectin are O-linked oligosaccharides
carried by mucin type glycoproteins termed GlyCAM (glycosylation-
dependent ceil adhesion molecule); sulfate and sialic acid s e e m
essential for GlyCAM binding. Since L-selectin is displayed O n
neutrophils, monocytes and eosinophils, it also contributes to
"leukocyte rolling" activity.
SAa243Gal P 1 4GlcNAc 4 R 3 T
Fuc a1
Sialyl-Le *
Figure 1.7 Structure of Lewisx (Lex) and sialy 1-Lewisx O ligosaccharide
determinants
1.2.6 Functional role of oligosaccharides
As has been discussed above, oligosaccharides play important
roles in biosynthesis and sorting of glycoproteins. However, it seems
that the role and the degree of indispensability of these
oligosaccharides are variable in different glycoproteins. In addition,
many of the functions of ce11 surface protein-linked oligosacc harides
are physiologically important, but not always essential for the
protein function (Gahmberg and Tolvanen, 1996).
Transporters and channels constitute a large group of ce11
surface glycoproteins. There is a great body of evidence describing
the significance of glycosylation in the function of these membrane
glycoproteins. Table 1.4 sumrnarizes some of these Cindings. The
third column of this table indicates methods used to modify o r
eliminate the normal glycosylation pattern. These include: 1) si te-
directed rnutagenesis, 2) treatment with glycosylation inhibitors
(Table 3 , 3) enzymatic deglycosylation and 4) expression i n
glycosylation mutant ce11 lines defective in glycosylation enzymes.
I n h i b i t o r E n z y m e
Tun icamyc in N-acetylg lucosaminylphosphotransferase
Castanospermine Glucosidase I and II
Deoxy nojirimycin
Deoxymannoj i r imycin Cis-Golgi a-mannosidase (a-Man 1)
Swa inson ine a-mannosidase II ( a-Man II)
Table 1.3 Inhibitors of the glycosylation enzymes
Oligosaccharides may serve a role in the functional expression
of transporters and channels by maintaining a stable form of t he
protein. For example, nonglycosylated N d K ATPase shows increased
sensitivity to trypsin digestion (Table 1 -4.a). Since the function of
oligosaccharides in this case is possibly non-specific, any kind of N-
linked sugar can mediate it (Zamofing et d., 1988). However, a more
complex oligosaccharide seems to shield the ATPase be tter agains t
ATPase
NdK ATPase (P) Alboint et al., 1992
Cell line
Xenopris ooc y ic Ia &B) Microsoriiül nieinhrüne
2ulturcd chick scnsory i c u r a n
- Method of m o d i f i c a t i o n Tunicaniycin
Tunicaniycin
P r o p e r t i e s
Reduced: Oiiabain binding to 60% Rb upiake io 60% Ern, Ep (no1 due IO reduçed protein synihesis or iniraccllular Na+)
Normal: protein synihesis Reduced: Trypsin resisiance (a, p) Ncwly synihcsizcd P & u ATPasr: activity
Normal : p & a synihesis Trypsin sensiiiviiy ATPase aciiviiy
Normal: ATPase üciiviiy Ouahain binding R h iranspori
N o r m a l : Asscnibly Cell surfücc çxprcssioii ratc b g r ü d a l i o ~ l Reduced: 60% pproiin syiithesis
- -
R o l e
Required for çompleie surface expression
-In siabilizing ihe subunits
- A non coniplex chain riiay hc sufficient; however, coniplexiiy incrcases the irypsin rcsistance
No1 required for ihe t ranspori
Nol in intraccllular ~ransloçütion or üssenibly
Table 1.4 Suii~iaüry O C ralcs of oligiisüccliarides (iii soiiic transporters and clianiicls Tuble 4.a AïPases
A n t i p o r t c r
N ~ ' / H ' antiporter Cnirnillori et rd., 1994
Ylrsirfi et nl., 1988
Band 3 (Ery throcytc anion c x c h a n g c r ) Groves & Trmaer, 1994
Ce11 linc
rransfccicd PS 120 hariisier ~ihrahlüsi wiih IINHE-1
Rot rcnai hrush horder
hlethod af m o d i f i c a t i o n N75D
Swüinsoiiine ia vivo
N o r m a l : Aniiloridç sensiiivity Yield of Ht suicide-surviving cells
R e d u c e d : Amiloride sensitive Na uptükc Vm, N o r m a l : K m
Rcduccd : Rüic of Nat/Hi cxchünge V m * , No cffect
R e d u c e d Band 3 rnediatcd Cl- influx
N o r m a l 2' siruciurc Proicrise sensiiivity Inliihiior hinding I n c r c a s c d Rüic of aggregüiion
R o l e
Noi in cc11 surface expression or f u n c t i o n
Complex iype N- linked sugars on the membrane direcily or indirectly influence transport act iv i iy
ln correct folding of Band 3 wiih the highest activiiy
Not in ihe transport f u n c î i a n
Tablc 1.4 (Coni i i iucd) l'ahlc 4,d Aniiporicrs
T r a n s p o r t e r
Na/Glucose symport Wir & Lever 1994
N at/Pi syniport
Rat kidney cortex (NaY,-2) H [ J ~ ~ ? S el cri., 1994
Cell line
LLC-pKI
BBMV
Method of 1 P r o p e r t i e s
PNGF
N o r m a l : K m Glucose uptake Proieiii synthesis Ce11 surface expression Lack of: NaIGlucose syniport üciivity
-- -- -
N o r m a l : Transport aciiuiiy
I
N248Q R e d u c e d : Na/glucose syriiport (30%)
Tunica tnyc in
N32SQ Double
. -
R e d u c e d : Methy lglucose upiake
Functional transporter is cxpressed
N o r m a l Apparcrit üffiniiy for P, or Na' pH depciidencc R c d u c e d Trünspori raie I n c r c ü s e d Intracellulür localization
R o l e
Not required for lransport funciion or membrane i n s e r l i o n
Contributes io ihe functional integrity
No! in iransport aci iv i ty
Not required for the func t iona l express ion
Surface delivery
Table 1.4 ( C o n t i n u c d ) Tuhle 4,f Na-dcpciidcnt sy iiipori
p. . - - --
T r a n s p o r t e r
GLUT 1 A'rrnrtc~cti et al., 1 9 9 1
Astrrro et (il . , 1993 Astr~io et tri,, 1 9 9 1
Ferr~eas et a l . , 1 9 9 1
1Yhecler d Hir ik le , 1 9 8 1
-
Ccll line
~r&fccied rai adipose cc l l s
Endoiheliuin of the hrnin cap i l l a r i e s
Epithcliurii of the clioroid plexus
CHO cclls
Rcconstiiuicd in liposome üfter trcütrricnl
Mcthod of m o d i f i c a t i o n N57Q
Diffcrential N- glycosy la t ion
Exoglycosidüscs
PNGF
P r o p e r t i e s I R o l e
Rcduccd : Express ion
Norriiül expression
Imporlani i n biosynthcsis of iransportcr in physiological ly relevant tissues Subce l lu la r localization (apical vs büsolateral)
Reduced: Cell surface expression Hülf lire Affiniiy for glucose I n c r e a s e d : K m
-1n t racc l lu la r t a r g c t i n g Protein stabiliiy
-Maintaining a high affinicy binding site
Nornial: A minimum c h a h is Zero-tram influx required for Rediiced: irünsport aciivity Viiiiix (25%)
Rcduccd: Requircd for Vniüx (50%) t r a n s p o r t Upiüke act iv i iy Sninll incrcasc in Kiii
Normal : The whole complcx Nci upirikc of glucose c h a h is not
r e q u i r e d
proteolysis. In the above study cells are treated with a glycosylation
inhibitor, tunicamycin, which has a global effect on al1 cellular
glycoproteins. Some of these glycoproteins may be involved in
folding and trafficking of transporters. Therefore, the indirect effect
of inhibitors should be considered while interpreting the results of
such studies. Tunicamycin is also known to interfere with protein
synthesis (Tamkun and Fambrough, 1986).
Although oligosaccharides do not usually affect the affinity of a
transporter for binding to a ligand, they may influence the properties
of the transport function. The inhibition of glycosylation in the
nucleoside transporter (Table 1.4.c) alters the kinetics of uridine
transport; however, it does not abolish the function (Hogue et al., 1990).
In the rat kidney K channel (Table 1.4.b), the oligosaccharide
stabilizes the open conformational state of the channel (Schwalbe et al.,
1995). It is important to mention that in studies involving
glycosylation site-mutated proteins, the possibility of carbohydrate
independent conformational changes cannot be eliminated. S ugars
may also influence channels' properties by creating a specific
microenvironment in the vicinity of the ligand binding site. This
seems to be the case for the Kchannel in the rat brain (Table 1.4.b).
The sialic acids present on this channel influence the local electric
field and their removal leads to a change in the voltage dependence
of the activation.
In many cases, deglycosylation of a transporter reduces V,,, of
transport without a change in its K. Usually this is due to t h e
decreased expression of the nonglycosylated transporter at the ce11
surface. For instance, in GLYT1, the glycine transporter (Table 1 . 4 . ~ ) ~
a reduction in V,,, is accompanied by an elevation in t he
intracellular amount of the transporter (Olivares et al.. 1995), w hic h
suggests the impairment of nonglycosylated transporters i n
translocating to the ce11 surface. The sarne effect is seen in the
surface expression of NdCl dependent serotonin transporter u p O n
tunicamycin treatment or mutagenesis (Table 1.4.e).
Only in rare cases do the data suggest that the carbohydrate
moiety is directly involved in ligand binding. The transporter of the
organic cation, tetraethylarnmonium (TEA), expressed in the presence
of tunicamycin shows lower affinity for TEA and reduced TEA up take
compared to the glycosylated form (Table 1.4.c). Asano (Asano et al.,
1991) proposed that the oligosaccharide on GLUTl (erythrocyte
glucose facilitator) is also required to maintain a high affinity binding
site; however, this has not been supported by other works (Table
1.4.g). A more popular view is that a minimum chah, possibly
containing the core and a few more residues, is sufficient for normal
transport activity of GLUT 1 (Table 1.4.g). This is consistent with the
view that mammalian cells require only simple oligomannosyl N-
linked structures for survival (Stanley and Ioffe, 1995). The role of N-
glycosylation of GLUTl is further examined in this thesis.
Since a srnall subset of the glycosyltransferase genes is
expressed in each ce11 line (Stanley and Ioffe, 1995), different cells exhibit
different patterns of glycosylation. This may Iead to some
controversial results: the expression of Na/glucose symporter in COS-
7 cells in the presence of tunicamycin results in a reduction in the Na
dependent glucose uptake. while in LLC-pK1 celI line the uptake
stays normal (Table 1.4.f). Besides, glycosyIatioo may only be
important in the biosynthesis of transporters in the physiologically
relevant tissues (hg et al., 1996). The expression of glycosylation s i te-
mutated GLUT4 is reduced in rat adipose cells, while it is not affected
in COS-7 cells (Table 1.4.g).
In conclusion, the role of oligosaccharides on t ranspor ters
mimics their general function in the biosynthesis of o the r
glycoproteins. They are involved in protein foldinp and a r e
important for optimal ce11 surface expression. In some specific cases
sugars influence the kinetic properties of transporters. The
involvement of oligosaccharides in maintaining a high affini ty
substrate binding site has also been proposed, although it is
evidently not well supported.
1.3 Erythrocyte membrane glycoproteins
The erythrocyte membrane is the most well-characterized ce11
membrane. After removal of hemoglobin by hypotonic lysis, t h e
remaining membrane is called a "ghost" and coosists of 52% protein,
40% lipid and 8% carbohydrate. The cytoskeleton of erythrocytes
uniquely forms a fibrous shell beneath and attached to the p lasma
membrane, providing the flexibility and stability required for t h e
erythrocyte's function in the circulation. Protein elements of
erythrocyte membrane are of two types, integral (intrinsic) a n d
peripheral (extrinsic). The cytoskeleton is mainly cornposed of
peripheral proteins which are attached to the membrane through
integral proteins. Spectrins (a and P) are the major components of
erythrocyte cytoskeleton and are aligned side by side to form a
dimer; the dimers can then combine head-to-head to form an a2P, -
tetramer.
Actin (Band 5) filaments, made of thirteen actin monomers
and a molecule of the fibrous protein, tropomyosin, bind to the N-
terminus of p spectrin. The state of actin polyrnerization seems
important to rnaintain membrane flexibility, since compounds t h a t
inhibit its polymerization increase membrane flexibility. Two O ther
peripheral proteins, Band 4.1 and adducin, bind to this cornplex. A
non-muscle myosin is also associated with actin; the amount of t h e
myosin is higher in neonatal erythrocytes.
The binding of the cytoskeleton to the plasma membrane is
mediated by the protein, ankyrin. Ankyrin consists of two domains;
one dornain binds to P spectrin, while the other is attached to t he
cytoplasmic domain of Band 3, the anion exchanger. Band 3 a n d
glycophorin are two major erythrocyte integral m e m b r an e
glycoproteins and both have exposed regions on the ce11 surface.
L 3.1 Giycop ho rin
Glycophorin (GP) is a general term for erythrocyte
sialylglycoproteins. Glycophorin A is the most abundant species a n d
constitutes 85% of erythrocyte sialylglycoproteins by weight. There
are about one million copies of glycophorin per cell. Glycophorin A
is a type 1 membrane protein and carries fifteen extracellular O-
linked and one N-linked oligosaccharide. The negative charge of
sialic acids carried by glycophorïn may prevent adherence of cells to
each other and to the endothelium. This glycoprotein serves as t h e
M M blood group antigen; the determinants are a combination of two
amino acid residues (1 and 5) and sorne O-linked oligosaccharide
moieties.
Glycophorin B (GPB) is almost identical to the N blood group
form of GPA for the first 26 amino acid residues, although its
cytoplasmic domain is distinct from GPA. The gene for GPB is located
on the same chromosome as GPA and is believed to be a product of
gene duplication. Glycophorins C and D have similar domain
structures to GPA but the genes are located on a different
chromosome. Both GPC and GPD are translated from the s ame
mRNA. GPD lacks the N-terminal region of GPC. GPC is not erythroid-
specific and is expressed in many tissues.
1.3.2 Band 3
Band 3 (AEI), a member of the anion exchanger (AE) family, is
responsible for the electroneutral exchange of bicarbonate for
chloride across the red cell membrane (Cabantchik and Rothstein, 1974).
This perrnits release of bicarbonate synthesized by ery throc y te
carbonic anhydrase into the plasma and increases the CO, carrying
capacity of the blood. There are 1.7 million copies of Band 3 in each
erythrocyte.
Human Band 3 is a 911-amino acid protein with a single site of
N-linked glycosylation at Asn-642. Two domains of the glycoprotein
can be segregated by trypsin cleavage at Lys-360. The transport
function is provided by the carboxyl-terminal membrane domain,
which traverses the membrane up to 14 times (Figure 1.8-a). The
"anion passage" may be partly located between two Band 3 molecules
forming a dimer (Reithmeier et al., 1996). The amino-terminal c ytosolic
Figure 1.8 Folding model for the membrane domain of human Band 3 ( a )
(the first 358 N-terminal amino acids are not shown) and a model of the hurnan
erythrocyte glucose transporter (GLUTI) (b) (adapted from Baldwin, L993 w i t h
some modifications). N642 and N45 are the sites of N-linked glycosylation O n
Band 3 and GLUTL respectively.
domain is the site of binding to the cytoskeleton elements (ankyrin,
Band 4.1 and Band 4.2), hemoglobin and glycolytic enzymes.
Band 3 is glycosylated on the extracellular loop between
transmembrane segments 7 and 8. There are two types of complex
N-linked oligosaccharide on Band 3 molecules; one has a poly N-
acetyllactosamine structure (shown in Figure 1.5) with variable
lengths, while the other is a short complex sugar chain. Al1
oligosaccharides present on Band 3 contain galactose, mannose, N-
acetylglucosamine, fucose and very little sialic acid (Tsuji et al., 1980).
The major developmental change of carbohydrate structure in t he
erythrocyte membrane has been shown to be the branching of poly
N-acetyllactosamine at C-6 of some galactose residues (Fukuda et al.,
1979). Neither deglycosylation of Band 3 by enzymes (Casey et ai., 1992)
nor mutation of the N-glycosylation site (Groves and Tanner, 1994)
significantly interferes with the function of the exchanger.
1.3.3 Glricose transporter
Band 4.5 (GLUTL) is a member of the GLUT family a n d
facilitates passive transport of glucose into erythrocytes (Carnithers,
1990). Apparently it has a broad specificity, transporting aldoses
(pentoses and hexoses) but with a Low affinity for fucose. The
protein contains 492 amino acids with high sequence identity among
mammals. There are 0.5 million molecules of Band 4.5 in a red cell,
constituting about 6% of the total membrane protein.
The hydrophobic membrane domain of the glucose t ranspor ter
is mainly a-helical with 12 predicted transrnembrane segments
(Figure 1.8-b). The carboxy- and amino-termini are located in t h e
cytoplasm as well as a large central loop between TM 6 and 7 .
Existence of an aqueous channel within the protein has been
pos tulated.
In intact erythrocytes the transport is passive, bidirectional
and saturable. To enter the cell, glucose first binds to an outward-
facing site, as do some disaccharides which inhibit the transport;
however, they cannot be transported by GLUTl (Baldwin. 1993). T w O
kinetic models have been proposed for glucose transport. In the
single site mode1 one glucose binding site is capable of being exposed
to cytoplasmic and extracellular faces at different times. Based on a
multiple site mode1 simultaneous influx and efflux sites exist on the
transporter. Cytochalasin B, a fungal metabolite, inhibits glucose
transport by binding to an intracellular moiety of GLUTI.
Band 4.5 contains an N-linked glycosylation site at Asn-45, i n
the first predicted loop, which contains poly N-acetyllactosamine
oligosaccharide. Although there is some evidence suggesting a
functional role for this sugar (Asano et ai., 1991), the exact poly N-
acetyllactosamine structure may not be an absolute requirement
(Feugeas et al., 199 1; Wheeler and Hinkle, L98 1).
1.4 Erythropoiesis
This topic has been reviewed by Handin (Handin et al., 1995) a n d
Jmd (Williams et ai., 1990).
Blood ce11 formation in rnammals begins in the fetal y o k sac
(blood islands) and, in humans, shifts to the fetal liver at about 6
weeks. This is accompanied by a switch in globin chah production
from primitive to fetd globins. By the end of the first trimester i n
human fetuses, the major hemoglobin produced is derived from
gamma gene expression (hernoglobin F). The final anatomic site of
hematopoiesis during ontogeny is the medullary cavity of bone
marrow which becomes the major site of hematopoiesis at about 5
months of gestation. The bone marrow of the fetus has little capacity
to respond to stress and relies on extramedullary hematopoiesis b y
liver and spleen. The level of fetal hemoglobin drops at birth a n d
gradually falls over the first year after birth, maintaining a low b u t
detectable level in most adults.
The life span of most functional blood cells is short: for
example, red blood cells survive 100-120 days with a daily
replacement of 1%. Therefore, bone marrow needs to continu0 usly
produce different types of blood cells. Pluripotential stem cells make
this possible. These cells have the capacity of self-renewal a n d
clonal proliferation. Randomly, or due to the inductive effect of their
microenvironment, some stem cells generate progeny that enter a
differentiation pathway (progenitors), while others produce more
stem cells. Progenitors have reduced self-renewal capacity and a r e
committed to ultimately develop into the mature cells of a single
lineage. Progenitors are assayed by their capacity to form colonies i n
vifro. These cells include early and late erythroid burs t-forming
units (BFU-E) and more mature erythroid colony-forming units (Cm-
E). BFU-E give rise in culture to rnulti subunit colonies of
norrnoblasts that derive their name from their characteristic b urs t-
like rnorphologic appearance and seemingly explosive production of
large number of cells from a single cell. CFU-E have very limited
capacity for proliferation and differentiate into precursors (Figure
1.9), which are the most abundant cells in bone marrow; erythroid
precursors constitute 20-30% of bone marrow cells.
The first identifiable erythroid Iineage precursor,
pronormoblast, has a darkly basophilic cytoplasm. This ce11 m a tu re s
to polychromatic normoblast and ultimately to the last nucleated
erythrocyte precursor, orthochromatic normoblast containing a full
amount of hemoglobin. These cells, after extrusion of the nucleus,
become reticulocytes, which are slightly larger than rn a t u re
erythrocytes. Reticulocytes continue their maturation in the
circulation to become biconcave nonnucleated ery throcy tes.
Reticulocytes exist in small numbers in the peripheral blood of
healthy individuals. However, in hemolysis and blood loss, more
reticulocytes are released prematurely into the circulation. Normal
destruction of erythroid precursors in the bone marrow is less than
10% of the developing cells, while this increases in ineffective
erythropoiesis caused by hemoglobinopathies or congenital
dyserythropoietic anemias (CDAs).
There are 4.8-5.4 x 10'' red blood cells IL in peripheral blood.
They cootain hemoglobin which carries oxygen from the lungs to the
tissues and carbon dioxide in the reverse direction. The adult form
of hemoglobin is a tetramer of two a and two P chahs, attached to a
prosthetic group called heme, formed by protoporphyrin iX
complexed with an iron rnolecule. The iron molecule is in ferrous
form in a functional hemoglobin. Iron is absorbed in the duodenum
by endocytosis or specific recep tor-transport mechanisrn and
transported in plasma bound to transferrin. Plasma delivery of iron
to cells is mediated by ceU surface transferrin receptors. Lron is
stored in hepatocytes and macrophages in the form of Eerritin.
Macrophages in bone marrow, liver and spleen acquire most of their
iron from senescent erythrocytes. While hemoglobin is catabolized,
the iron is oxidized to the trivalent state (methemoglobin) a n d
liberated from heme by heme oxygenase. This iron is then deposited
in femtin preventing damage from uncontrolled oxidation. Herne in
the circulation is bound to albumin or hemopexin and is cleared from
plasma by the liver. Globin produced from hemoglobin degradation
is metabolized to amino acids in the liver and spleen.
The proliferation, differentiation, and survival of hematopo ie tic
progenitor cells is achieved by a number of different glycoproteins,
the hematopoietic growth factors. Erythropoietin, the Eirs t
hematopoietic growth factor to be identified and characterized, is a
34 kDa acidic glycoprotein which promotes erythropoiesis by acting
strictly on late erythroid progenitor cells in adult marrow. In t h e
adult erythropoietin is produced by the kidney in response to low
oxygen tension in arterial blood (hypoxia). The receptors For
erythropoietin are present on the surface of relatively mature
erythroid progenitors. Binding of erythropoietin activates tyrosine
phosphorylation of a number of substrates like Janus kinase 2 (Jak2)
leading to transcription activation and thus expressing t he
erythropoietin biologic functions (Shivdasani and Orkin, 1996).
Pluripotential stem c e k 1
Progenitor cells I
I Reticulocyte Bone mi
1 - circulation
1 Monocyte 1 Eosinophil
Figure 1.9 Stages of the erythroid differentiation bom a stem ceIl to an e r y t h r o c y t e
I.S. HEMPAS
Crookston was the first to propose (Crookston et al., 1969) the te r m
HEMPAS for the congenital dyserythropoietic anemia type I I ,
characterized in five patients by erythroblastic rnultinuclearity i n
bone marrow, ineffective erythropoiesis and positive acidified s e ru m
(Ham) test. HEMPAS is an acronym for Hereditary Erythroblastic
Multinuclearity with a Positive Acidified Serum test used to
distinguish this anemia from the other kinds of congenital
dyserythropoie tic anemias.
Congenital dyserythropoietic anemias (CDAs) are a
heterogeneous group of rare anemias classified into type 1, II and I I I
based on the hematological and serological findings (Heimpel and Wendt.
1968). Table 1.5 shows a surnmary of these features.
Acidified-
serum lysis test
Type 111
Multinuclearity
Gigantoblasts
Erythrocytes
EM
Anti-I
agglut inat ion
Type 11
Mu1 tinuclearity
Karyorrhexis
Characreristics
Bone marrow
Type 1
Megaloblastic
Chromatin
br idging
Macrocytosis
Nuclear pore
Autosomal
agglut inat ion
l n h e r i t ance
recessive recessive dominant
Normocytosis
Double membrane
Table 1.5 Classical features of the three types of congenitd
Macrocytosis
CIefts in nuclei
Autosomal
dyserythropoietic anemia (CDA)
Autosornai
There are only a few hundred reported cases of CDAs, although it is
likely that many others have been misdiagnosed or unrecognized
(Marks and Mitus, 1996). In addition to the classical types, variants a n d
cases with overlapping features have been described.
A group of type II serological variants described be t w een
1973- 1982, who mostly demonstrated the morphological
characteristics of HEMPAS, was reviewed by Boogaerts a n d
Verwilghen (Boogaerts and Verwilghen, 1982). Surprisingly, these patients
did not have a positive acidified serum lysis test. Therefore it is not
clear if these patients were affected by HEMPAS or other types of
C'DA. Interestingly, they also did not show agglutination with anti-i
antibody. One of the patients (G. K.) who tested negative with more
than 30 sera suffered from additional clinical complications (gout)
and the membrane abnormality was seen in his granulocytes a n d
platelets in addition to erythroblasts (Lowenthal et al., 1980).
An extensive smdy on 39 HEMPAS patients further established
the clinical and hematological features of this disease (Verwiighen et al.,
1973). This study showed that the anemias previously diagnosed and
termed erythroblastic endopolyploidy (De Lozzio et al., 1962), hemolytic
anemia with multinucleated normo blasts (Roberts et ai., 1962) a n d
ery thropolidiscariosis hemolitico esplenornegalica (Grignaschi, f 970)
were the s m e as HEMPAS.
CDA II (HEMPAS) is the most common type of CDA (Marks and
Mitus, 1996). Morphological abnormali ties of the bone m a r r O w
erythroblasts combined with the serological tests have been used to
diagnose HEMPAS patients from many countries and both sexes
(Fukuda, 1990). However in the Verwilghen snidy al1 of the patients
except one were Caucasians. A male HEMPAS case suffering f r o m
iron overload was diagnosed at the age of 69, although the diagnosis
is usually made before the age of thirty (Greiner et al., 1992).
1.5.1 Clinical feutures
Mild to severe chronic anemia is the most common feature of
HEMPAS. Episodic jaundice is common among these patients and
biopsy of the liver showed hepatic cirrhosis andlor hemosiderosis i n
those who were tested. This can be explained in part by iron
therapy and blood transfusion but it is not always iatrogenic'. Some
patients suffer from gall Stones or enlargement of the liver and
spleen (hepatosplenomegaly). Widening of the diploic space a n d
mental retardation are seen in a few patients.
1.5.2 Hernatological featiwes
In peripheral blood, red cells appear with abnormal shape
(poikilocytosis) and size (anisocytosis). There is no marked increase
in the number of reticulocytes.
In bone marrow, erythroid hyperplasia is significant, while
there is no abnormality in granulopoiesis and thrornbopoiesis. In t he
bone marrow of five patients studied by Crookston, 20-36% of the
late erythroblasts were multinucleated or contained multilobulated
or fragmented nuclei (karyorrhexis). Late po lychromatophilic
ery thro blas ts were more abnormal compared to earlier s tages
(proerythroblasts).
' Describing a condition that has resulted from treatment as either a n unforeseen or inevitable side-effect,
There was also a high proportion of damaged cells (smudged
cells) and phagocytic reticulum cells containing nuclear debris a n d
hemosiderin granules (Crookston et al.. 1969). Marrow iron stores w e r e
increased and iron granules were visible in the cytoplasm of some
erythroblasts (Venvilghen et al., 1973). In general, the rnorphology of th e
bone marrow provided evidence for the intramedullary des truc tion
of erythroblasts.
Electron microscopic studies revealed an additional s t ructure
lying parallel with and inside the plasma membrane, appearing like a
"double membrane" in the majority of normoblasts (Wong et al.. (972).
The same membrane abnormality was seen with less frequency in a n
isolated form (cisterna) in mature red cells. The "double membrane"
effect is also present in cultured erythroblasts from bone marro w
and peripheral blood (Fiorensa et al., 1994). It is known that t he
additional membrane derives from the ER, since immunogold
electron microscopy on red blood cells has shown localization of a n
ER marker, protein disulfide isomerase, in the lumen of the cisternae
(Aiioisio et ai., 1996).
1.5.3 Serological features
HEMPAS erythrocytes demonstrate 5-25% lysis with ten out of
thirty compatible normal sera in an acidified pH; no lysis occurs i n
the patient's own serum. This implies that KEMPAS red cells ca r ry
an antigen not detectable on normal cells and that some normal s e r a
contain the corresponding antibody (Verwilghen et al., 1973). This
antibody c m be removed from normal sera by patient's cells but not
by normal cells. The acidified condition enhances the activation of
the complement system.
The only other disease in which erythrocytes show sensitivity
to acidified serum lysis is paroxysmal noc turnal hemoglo b inuria
(PNH). The erythrocytes of PNH are hemolyzed even in acidified
homologous serum because of their deficiency in plycosyl
phosphatidylinositol- (GPI) anchored proteins that regulate
cornplement activation (Rosse, 1990).
HEMPAS erythrocytes show high agglutination with anti-i
antibody and undergo lysis with anti-1 and anti-i antibodies
(Verwilghen et al., 1973). Increased agglutinability by anti-i may be d u e
to ineffective erythropoiesis, as an increase of i antigen can b e
induced by marrow stress caused by repeated phlebotomies (H ihan
and Giblett, 1965). However, clinically normal relatives of HEMPAS
patients who appear to be heterozygote carriers also have increased
agglutinability to anti-i (Marks and Mitus, 1996). There is some evidence
of increased sensitivity to complement in some HEMPAS patients,
consistent with Tomita's view that the complement regulation is
aberrant in HEMPAS erythrocytes, probably due to the defec tive
glycosylation of glycophorin A (GPA), which was proposed to serve as
a cornplement regulatory protein (Tomita and Parker, 1994).
1.5.4 Biochemists,
The level of unconjugated bilirubin is increased in HEMPAS
sera consistent with the destruction of erythroblasts and pro bably
the short-life-spanned circulating red cells (Verwilghen et al., 1973).
Studies with 59Fe showed rapid clearance of iron from plasma,
increased plasma iron turnover and slow incorporation of iron in
hemoglobin (Crookston et al.. 1969), which are characteristic of ineffective
erythropoiesis.
Plasma lipid and vitamin E levels were low in sorne patients.
Since lack of vitamin E can cause ineffective erythropoiesis
associated with multinucleated erythroblasts in animals, vitamin E
was administered to two patients without any hematological
improvement (Verwilghen et al.. 1973).
1.5.5 Treatment
Some HEMPAS patients are sufficiently anemic to need
repeated transfusions, while some may undergo splenectomy w i t h
clinical improvement; in these patients peripheral hemolysis m a y
play a predominant role in the pathogenesis of anemia (Barosi and
Cazzola, 1979). Other treatments with hematinics like Bl2 , folic acid,
pyridoxin, and corticosteroid have not been effective. Iran the r ap y
is contra-indicated because of the patient's risk of developing iron
overload. An iron chelating agent, deferoxamine, was used to reduce
iron overload with little effect. Regular phlebotornies, the mo s t
effective method for reducing body iron stores, are performed for
some patients if the hemoglobin levels allow.
1.5.6 Molecular abnonnalities in HEMPAS
The first molecular defect characterized in HEMPAS patients
was the reduced sialic acid content of erythrocytes and consequent
reduction in the negative surface charge (Gockerman et ai., 1975). This
was consistent with the altered electrophoretic mobility of
glycophorin, the prominent carrier of sialic acid on ery thro id
membranes, reported later (Anselstetter et al., 1977). However this w a s
known not to cause HEMPAS directly, since erythrocytes exist despite
the absence of glycophorin (Gahmberg, 1976). Reduction in sialyiation of
glycophorin A may be a part of a general glycosylation defect
occurring secondary to the bone marrow stress accompanying
anemia (Mawby et al., 1983).
Two-dimensional polyacrylamide gel electrophoresis of
erythroid membrane proteins revealed an altered protein pat tern
including faster migration of Band 3, in four HEMPAS patients
(Anselstetter et al., 1977). The abnormality was not seen in CDA I,
autoimmune hemolytic anemia, hereditary hemolytic anemia a n d
normal erythrocytes. It was extended to more membrane protein
components in the patient G. K., consistent with the more severe
clinical/morphological characteristics in this variant (Harlow and
Lowenthai, 1982). Baines (Baines et al., 1982) was the first to propose t h a t
the rapid migration of HEMPAS Band 3 in SDS-gei electrophoresis is
the result of altered glycosylation. The amino acid compositions of
the peptides derived from proteolysis of the extracellular domain of
Band 3 from intact KEMPAS red cells were normal, excluding a defect
in the peptide sequence. Radiolabeling of erythrocyte membranes
with galactose o x i d a s e / ~ a ~ [ 3 ~ ] 4 showed reduced 3~ incorporation
into Band 3 and 4.5, revealing under-glycosylation of the two
glycoproteins (Scarteuini et al., 1982). It was concluded that features of
an erythroblastic membrane may remain in mature HEMPAS
erythrocytes.
Treatment of the EEMPAS red cell membranes with endop-
galactosidase did not affect the pattern of labeled glycoproteins,
while it abolished the labeling of a low molecular weight protease-
resistant species (Fukuda et al., 1984~). This HEMPAS-specific
glycoconjugate, termed "HEMPAS glycan", is composed of poly N-
acetyllactosaminylceramide and may accumulate in HEMPAS
erythrocyte membranes (Fukuda et al., 1986a). Sorne evidence suggests a
general enhancement in the synthesis of glycolipids in HEMPAS
erythrocytes (Bouhours et ai., 1985; Joseph et al., 1975). In normal
granulocytes fucosylated unbranched poly N-acetyllactosamine exists
on both proteins and lipids, implying that glycosyltransferases
involved in synthesis of carbohydrates on proteins can also modify
lipids in the same cell. Therefore, the KEMPAS detéct must affect a
protein-specific stage of glycosylation.
Immunogold electron microscopy using an anti-Band 3
antibody showed that Band 3 molecules form clusters be fore
incorporation into the plasma membrane of HEMPAS ery throc y tes
(Fukuda et al., 1986b). This may be due to under-glycosylation of Band 3,
which increases its hydrophobicity. Band 3 as an anchor of t he
erythroid cytoskeleton plays a role in maintainhg erythrocyte s hape.
The clustering of this membrane protein may cause deformation of
erythrocytes seen in the peripheral blood of HEMPAS patients. I n
addition, the clustering of Band 3 may promote the binding of
autologous antibodies to erythrocytes and trigger their removaI f rom
circulation as in senescent cells. Natural anti-Band 3 IgG binds to
senescent erythrocytes through sialylated poly N-ace tyllac tosamine
of Band 3 (Ando et ai., 1996). The ctustering of Band 3 mediated b y
denatured hernoglobin in these cells marks them as aged (Low et al.,
1985). Further studies indicated the accumulation of Band 3 and
glycophorin A in autophagie vacuoles seen in HEMPAS ery th roc y tes
(Fukuda et al., 1987b); this rnay be the mechanisrn by which defec tive
plasma membranes are discarded.
1.5.7 Abnormalities in glycosylation enzymes in HEMPAS
As can be seen from the above, a large body of evidence has
suggested abnormalities in the glycosylation pathway as the cause of
HEMPAS. The reported defects in glycosylation enzymes in HEMPAS
patients are summarized in Table 1.6 and discussed below.
H E M P A S E n z y m a t i c Cell t y p e R e f e r e n c e
p a t i e n t d e f e c t
T.O. & B. D. GnT II Lymphocyte Fukucla er ai..
1987a
G. C. Man II Cultured EBV- Fukttda et al.,
transformed lymphob last 1990
G. K.' B4GalT Mononuc lear celi Firktida et al.,
1989
Table 1.6 Summary of reported glycosylation defects in HEMPAS patients
* Atypical variant of HEMPAS
The enzymatic activity of GnT II in lymphocytes of two
HEMPAS patients (T.O. & B. D.) has been shown to be reduced to 10%
and 30% of normal, respectively (Fukuda et al., 1987a). In the same
study the structure of carbohydrates from HEMPAS Band 3 w a s
elucidated by fast atom bombardment-mass spectrometry (FAB-MS).
In the case of T. O., the structure was a tnrnannosyl hybrid sugar
containing a core structure and a NeuNacu2-6Galp 1-4GlcNACP 1-2
c h a h on the a l - 3 a m , which supported the presence of a lesion in
GnT II. However, Band 3 purified frorn patient B. D. carried b o t h
hybrid and cornplex chains. It was concluded that in the patient B.
D., the low activity of GnT II may cause a delay in synthesis of t he
a l -6 arm and a subsequent reduction of lactosaminoglycans, while i n
T. O., the low GnT 11 activity blocks synthesis of complex chains.
Different mutations in the GnT II gene were suggested as the basis of
heterogeneity among these two HEMPAS patients. The au thors
stated that "in the patient T. O. the incompleteness of the a 1-6 a r m
leads to a total failure of glycosylation by lactosaminyl repeats". This
disagrees with the result of a study (Charuk et al., 1995) done on a
patient with carbohydrate deficient glycoprotein syndrome type 1 I
(CDGS II). in the CDGS II patient even though there is no detectable
GnT II activity in mononuclear cells due to a point mutation in t he
GnT II gene coding region (Tan et al., 1996), erythrocytes contain 50%
poly N-acetyllactosamine relative to normal cells. In this case the
al-3 a m is probably modified by poly N-acetyllactosamine sugar.
CDGS II is a multisystemic congenital disease which causes
psychomotor retardation.
Another case of HEMPAS (G. C.) suffering from Liver cirrhosis
and hemosiderosis was subjected to an extensive investigation b y
Fukuda (Fukuda et al., 1990). In contrast to the previously analyzed case
(T. O.), this patient showed normal activity of GnT II but low activity
of a-mannosidase II (Man II) in cultured EB V-transformed
lymphoblasts. Structural analysis revealed a hybrid chain with f ive
mannose residues as the major glycan of G. C. erythrocyte membrane
glycoproteins. The retention of the two uncleaved mannose res idues
on the Man a 1-6 a m is consistent with the low activity of Man II.
Northern blot analysis of poly(A)' mRNA extracted from transformed
lymphoblasts of G. C. showed that the expression of Man II mRNA is
reduced to less than 10% of normal, while it was normal for two
other unrelated HEMPAS patients. Therefore, it seems that HEMPAS
may be associated with more than one lesion in glycosylation
enzymes.
In the variant of HEMPAS, G. K., the enzymatic defect has b e e n
determined to be in membrane-bound galactosyl transferase (GalT),
since its activity is 24% of normal in mononucleated rnicrosomal
membranes (Fukuda et al., 1989). Carbohydrate analysis suggests a high
mannose type sugar on erythroid membrane glycoproteins and also
on some serum glycoproteins. In contrast to membrane-bound GdT,
activity of GalT in the serum of G. K. is higher than normal which
raises the possibility of a mutation in the transrnembrane or s t e m
region of the enzyme, enhancing its proteolysis or secretion. Since
GalT is involved in the synthesis of poly N-acetyllactosamine on both
protein and Lipids, a defect in Gd T explains why patient G. K. canno t
make "HEMPAS glycan". In addition, a lesion in GalT blocks synthesis
of poly N-acetyllactosamine on a l l antennae; consequently it affects
tri- and tetra-antennary structures as much as biantennary.
Probabfy th i s is the reason for the observation of membrane
abnormality in G. K.'s granulocytes, which normally present tri a n d
tetra-antennary poly N-acetyllactosamine chains.
In typical cases of KEMPAS, ce11 lines other than erythroid a r e
not usually affected, probably because they contain sugars with
different structures compared to erythrocytes (Fukuda, 1990).
However, analysis of carbohydrates from HEMPAS (G. K.) s e rum
transferrin shows altered structures which may play a role in t he
occurrence of liver cirrhosis in HEMPAS patients (Fukuda et al., 1992).
1.5.8 HEMPAS and other "diseases of aberrant glycosylation"
Recently Koscielak (Koscielak, 1995) has proposed to class ify
HEMPAS and al1 other diseases related to defects in the metabolism
of glycoconjugates as "diseases of aberrant glycosylation". This
includes HEMPAS, carbohydrate-deficient glycoprotein s yndro me
(CDGS), 1-ce11 disease, galactosemia in subjects on galactose-free diet,
variants of leukocyte adhesion deficiency, and of Ehlers-Danlos
syndrome, paroxysrnal nocturnal hernoglobinuria (PNH) and T n
syndrome. Some of the characteristics of these diseases a r e
summarized in Table 1.7.
1.6 Research project and hypothesis
As discussed above, HEMPAS is associated with a group of
lesions affecting the synthesis of the poly N-acetyllactosaminyl
oligosaccharide. Since the nature of the lesion varies among patients,
it is essential to establish the molecular basis of HEMPAS in each case
separately . In addition, it has been recently shown that
carbohydrate deficient glycoprotein syndrome (CDGS) type II is
associated with a point mutation in the catalytic dornain of the gene
encoding GnT II (Tan et ai., 1996), leading to over 98% reduction in the
activity of GnT II in fibroblasts and mononuclear cells (Jaeken et al.,
1994; Chanik et al., 1995). The reduced enzymatic activity of GnT II ha s
been also reported in lymphocytes of two HEMPAS patients (T.O. & B.
D). That the reduced GnT II activity can apparently give rise to two
different clinical manifestations, namely HEMPAS and CDGS II ,
further attracted our interest in studying the properties of HEMPAS
in a group of known patients in Ontario. The purpose of this thesis is
to investigate: 1-The alterations in the red ce11 membrane protein-
and lipid-linked oligosaccharides in this group of patients, 2-The
effects of these alterations on the transport properties of the
erythrocyte glucose transporter, 3-The nature of the enzymatic
defect present in these patients.
II. MATERZALS AND METHODS
ZI.1 Materials
Materials were purchased from the following suppliers: e n do - P-galactosidase and endoglycosidase H frorn Boehringer Mannheim
and peptide N-glycosidase F from New England Biolabs; oc tae thylene
glycol mono-n-dodecyl ether (C,,E,) from Nikko Chemicals;
biotinylated lectins and Vectastain kit from Vector Laboratories Inc.;
Cherniluminescent kit and BCA protein assay reagents from Pierce;
SpinBind DNA extraction unit from M C Corp.; UDP-[~~CIG~CNAC a n d
3-0-rneth~l-['~~]~lucose frorn NEN Research Products; U D P - [ ~ H ] G ~
from American Radiolabeled Chemicals Inc.; Di-N-ace tylchi to biose
from Seikagaku Corporation; Phenylmethylsulfonyl fluoride (PMSF),
GlcNAc, Triton X-100, and Cytochalasin B frorn Sigma Chemical Co.;
MES and Pronase from Calbiochem-Novabiochem Corporation; S ep-
Pak Cl8 from Millipore Corporation; AGLX8 resin from Biorad;
Sephadex G-50 and Con-A Sepharose from Pharmacia; scintillation
fluid from ICN; autoradiography film from Dupont; TLC-plastic s hee t s
silica gel 60 from EM Industries Inc.; neutral glycosphingolipid
standards from Matreya Inc.; D20 from Aldrich Chemical Company,
Inc. GlcNAcMan3-octyl was synthesized in Dr. H. Schac hter' s
laboratory by Dr. Fokert Reck.
Blood samples were donated by M. D. (HEMPAS patient). A. S.
and D. B. (offspring of M. D.). Units of blood were obtained from tw O
HEMPAS patients, C. L. and L. F. (siblings), for therapeutic p urposes
by Dr. D. Levy in Guelph and sent to Toronto on ice. Normal controls
were provided by H. S., H. K. and the Blood Bank, Hospital for Sick
Children, Toronto.
EBV-transformed lymphoblas ts were cultured in the Tissue
Culture Service, Department of Metabolic Genetics, Hospital for Sick
Children, Toronto. Blood samples were collected in tubes containing
acidlcitrateldextrose and diluted 1: 1 with RPMI (Roswell Park
Mernorial Institute) 1640 medium. 10 ml of blood was layered on 3
ml of Ficoll and centrifuged (2,000 rpm, 30 min, room temperature,
Beckman benchtop). The interface was removed and washed two
times with 10 ml of RPMI 1640 medium, with centrifugation at 1,000
rpm for LO min. The pellet was incubated in 1 ml of RPMI 1 6 4 0
containing O. 1 1 mgfml pyruvate, 15% fetal calf serurn and 0.3-0.5 m 1
of filtered supernatant of EBV-infected marmoset ce11 Line B95/8, i n
the presence of cyclosporin A at 37°C until the cultures turned
yellow. The resultant EBV-transformed lymphoblasts were grown i n
RPMI 1640 medium with pyruvate (0.11 mglml) and 1 5 8 fetal calf
serum at 37°C in 5% carbon dioxide in 25-cm' Falcon tissue culture
flasks. Cells were grown in suspension to 106 cells/ml and colIected
by centrifugation (1,000 rpm, 10 min). Cells were washed with PBS
three times and stored at -20°C.
The erythroid cultures were prepared in Dr. A. Axelrad's
laboratory, Department of Anatomy and Cell Biology, University of
Toronto. Mononuclear cells in 20 ml fresh heparinized
(preservative-free heparin, 15 Ulml) peripheral blood diluted 1 : l
with a-minimal essential medium (a-MEM) were separated by Ficoll-
Hypaque density gradient centrifugation at 400 g for 30 minutes.
The interface was removed and washed with a-MEM containing 0.1%
fatty acid-free and globin-free BSA and cultured in flat-bottomed
(1.5 x 1.0 cm) plastic wells. 1.5 x los cells were cultured in 0.7 ml of
serum-free liquid culture medium composed of Basal SeroZeroTM
Stem Ce11 Medium, IL-3 (10 ng/mi), erythropoietin (3 Ulml), s t em
cell factor (50 nglml), hemin (100 PM) and al1 trans-retinoic acid
(ATRA) (30 mM). The Petri dishes containing the wells we re
incubated at 37°C in a humidified atmosphere and 5% CO2 for L 4
days. Cells were collected from 22 wells by centrifugation (400 g, 1 0
min) and the pellet was washed with cold PBS and used to prepare
cell extracts.
I I Methods
11.2.1 Preparation of ghost membranes
All steps of ghost preparation were performed at 0-4°C. To
remove white cells, blood was mixed with 0.9% NaCl and centrifuged
at 3,000 x g for 10 min. The upper buffy coat and the wash solution
were removed by gentle aspiration. Ghosts membranes w e re
prepared by hypotonic lysis according to Casey and Reithmeier (Casey
et al., 1989). Erythrocytes were lysed with 10 volume of ice-cold 5 m M
sodium phosphate, pH 8.0 (5P8) in the presence of 1 m M
phenylmethylsulfonyl fluoride (PMSF). Hernolysate was removed b y
aspiration and washes proceeded using 5P8. The ghost membranes
were then collected by centrifugation at 30,000 x g for 20 min.
11.2.2 Preparation of ce11 extracts
Cultured human EBV-transformed lymphoblasts (= 1 x 107
cells) or cultured erythroid cells (= 3 x 106) were washed with PBS,
dissolved directly in Laemmii sample buffer, boiled for 3-5 min a n d
filtered in the SpinBind DNA extraction unit to remove DNA. The
resulting ce11 extracts were used for the enzymatic deglycos y lation
and subsequent SDS-PAGE.
112.3 Enzymatic digestion of oligosaccharides
Ghost membranes (2-5 mg/ml) were treated overnight w i th
peptide N-glycosidase F1 (5- 10 UIpg of total protein), endo-p-
galactosidase' (0.06 mU/pg of total protein) or endoglycosidase H3
(50 U/pg of total protein) at room temperature in the presence of 1%
(vlv) octaethylene glycol mono-n-dodecyl ether (C 12Eg). Laemmli
sample buffer (2x) was then added. Control samples were incubated
under the same condition without enzymes.
11.2.4 Lectin Blots
Proteins in the ghost membrane preparations (4-25 vg),
cultured human lymphoblast ce11 extracts (15 yg) or erythroid celI
extracts (= 2 x IO6 cells) were resolved by SDS-PAGE a n d
electrophoretically transferred over 12 h at 50 V to a nitrocellulose
membrane. After blocking the membrane with 0.25% gelatin in 10%
ethanolamine and 0.1 M Tris, pH 9.0 (blocking buffer), transblots
were incubated overnight with 0.5 pg/ml biotinylated tomato lectin
or Concanavalin A or ECA in antibody buffer (0.25% gelatin, 0.05%
Triton X-100, 0.15 M NaCl, 5 m M EDTA, 50 m M Tris, pH 7.5). Bound
lectin was detected by avidin and biotinylated horseradis h
peroxidase reagents (Vectastain) ( 1 : 10,000 dilution) and w e r e
visualized using a cherniluminescent detection system consisting of a
luminol/enhancer reagent and a peroxide solution (1: 1). Al1 s teps
were done at roorn temperature. Blots were exposed to a n
' One unit is defined as the amount of enzyme required to remove >95% of t h e carbohydrate from 10 pg of denatured RNase B in a 10 pl reaction at 37°C in I h o u . ' One unit is the enzyme activity, that releases 1 pmol of reducing sugar p e r min (measured as galactose) from keratan sulfate at 37°C and pH 5.8.
One unit is the enzyme activity which hydrolyzes I vmol d a n s y l - Asn(GLcNac),(Man), within 1 min nt 3 7 T and pH 5.5.
autoradiography film.
11.2.5 Western Blots
Ghost membrane proteins (10 pg) or human cultured erythroid
ce11 extracts were resolved by SDS-PAGE, transblotted and blocked as
described above. Membranes were incubated overnight with a
rabbit anti-human GLUTl (1 : 10,000) or anti-human Band 3 ari tibody
(1:5,000) in the antibody buffet at room temperature. Biotinylated
anti-rabbit IgG was used as the secondary antibody, which was then
detected in the cherniluminescent reaction as above.
11.2.6 Staining procedures
Proteins were resolved by SDS-PAGE and stained in a solution
of 0.05% Coomassie Blue, 25% isopropanol and 10% acetic acid. To
destain the gel, a mixture of 5% methanol and 10% acetic acid w as
used. For Stains-all, gels were fixed in 30% isopropanol overnight
with a change in the morning. Staining was done in a solution of
0.025% Stains-al1 (Kodak), 25% isopropanol, 7.5% formamide and 3 0
m M Tris-base pH 8.8, in the dark.
11.2.7 Enzyme assays
PBS-washed cultured human lymphoblasts were homogenized
manually in 0.25 M sucrose on ice in 1.5 ml-Eppendorf tubes using a
Kontes Pellet Pestle Mixer. GnT LI activity was measured in a
radiochernical assay using UDP-[~~C]GICNAC (38 m o l , 1 8 3 7
dp rnhmol) and GlcNAcP 1-2Mana 1-3 [Manal-61ManP-octyl
(GlcNAcMang-octyl) (40 nmol) as substrates and 10-20 pg of ce11
homogenate protein in a total volume of 40 pl. Di-N-acetylchitobiose
(80 nmol) and U D P - ( ~ H ] G ~ (40 nrnol, 2182 dpm/nmol) were
sirnilady used to measure GalT activity. The reaction mixtures (40 pl)
also contained MES, pH 6.5 (125 mM), MnCl, (12.5 mM), Triton X- LOO
(5%), GlcNAc (5 pmol, only for GnT II assay) and AMP (0.2-0.5 pnol) .
After incubation at 37 OC for 1 h, the reactions were stopped b y
addition of 0.5 ml ice-cold water.
The radioactive product of GnT 11 assay was obtained b y
adsorption to a Sep-Pak C l 8 cartridge and elution with 3 ml
methanol. For Ga1 T assay the product was separated from other
radioactive cornpounds by elution through 0.2 ml of AGI-X8 resin
(chloride form) equilibrated with water. Radioactivity was me as u re d
by scintillation counting and values were corrected for controls
incubated without the acceptor substrates. Product formation w a s
proportional to time of incubation and the amount of the ce11
homogenate used (data not shown).
11.2.8 Glycolipid analysis
Lipid extraction was done on ghost membranes ( 5 mg total
protein) using 20 volumes of chloroformlrnethanol (2: 1) at room
temperature. The extract was filtered through a medium speed Filter
paper into a separatory funnel and the volume was adjusted to pive
a Folch partition: chloroform/methanol/water (2: l:O.6). The lower
phase which contains oligoglycosyl ceramides was dried on a rotary
evaporator and reconstituted in 200 pl chloroform/methanol (2: 1).
Samples (6- 10 pl) were loaded on a silica-covered plastic TLC plate
and resolved in chloroform:methanol:water (60:40:9). The TLC w as
sprayed with 0.5 % orcinol in 3 M sulfuric acid and heated at 100°C
for a few minutes. The densities of bands corresponding to
glycosylceramides were assessed by scanning using a W P scanner
and UVP-Grabit software program.
11.2.9 Glucose transport assay
Glucose transport by red cells was measured according to May
(May, 1988). 3-O-methy l-[14~]glucose transport was initiated b y
adding 50 pl of PBS-washed erythrocytes at 20% haematocrit to 20 pl
of ice-cold PBS containing 0.125 pCi 3-O-rnethyl-[14C]glucose (specific
activity of 315 Ci/mol). The mixture was then incubated for 30 s o r
35 min on ice. The assay was terminated by addition of 1.2 ml of
ice-cold PBS containing 10 yM cytochalasin B (stop solution). The
suspension was then centrifuged for 5 s in a microfuge and the
supernatant was removed by aspiration. The pellet was resuspended
in another 1.2 ml of stop solution followed by centrifugation. The
second wash was aspirated and the pellet was resuspended in 0.2 rn 1
of ice-cold PBS and 1 ml of 6% trichloroacetic acid was added wi th
vortexing. After centrifugation 0.5 ml of the supernatant was used
for scintillation counting. Correction for trapped extracellular label
was made by subtraction of the count obtained for cells incubated
with stop solution prior to the addition of the Labeled sugar (zero
time). The 3-0-rneth~l-[14~]~lucose uptake in 30 s was expressed a s
the fraction of the equilibriurn value (35 min incubation). The
uptake was linear for the first 2 min (data not shown).
11.2.10 Preparafion of glycopeptide from HEMPAS erythrocytes
Ghost membranes were prepared as described above from
HEMPAS (L. F.) biood. 30 ml of phost suspension ( 1 10 mg total
protein) was delipidated with 10 volumes of chloroform:methanol
( 2 1 ) while stirring for 2 h. The mixture was separated into two
phases using a separatory funnel. The upper aqueous phase was
centrifuged to collect the insoluble protein pellet. Pronase digestion
was performed on the pellet in 110 ml 0.1 M Tris-HCI buffer at pH
7.8 containing 2 m M calcium chloride and 20 mg pronase. The digest
was incubated at 37 O C for 24 h. LO mg pronase was added every 2 4
h for a total incubation tirne of 96 h.
The top layer (the non-digested protein settled naturally) ( 10
ml) of the pronase digest (after adjusting the pH to the column pH)
was loaded on a 200 ml Sephadex G-50 column equilibrated with 0.1
N acetic acid at room temperature. The column was run in 0.1 N
acetic acid. Collected fractions were tested for the presence of hexose
with the phenol-sulfuric acid method: 0.2 ml of 5% phenol was
added to 0.1 ml of each fraction diluted 1:l with water. Following
mixing, 1 ml concentrated sulfuric acid was added to the top of t he
solution and left at room temperature for 10 min. The assay was
then mixed and incubated at 30°C for 20 min. The absorbante was
read at 490 nm.
Those fractions containing sugar were pooled (13 ml), adjusted
for pH and salts (to the same values as the Con-A column) and loaded
on a 5 ml Con-A Sepharose column equilibrated with TBS-azide (0.0 1
M Tris-HC1, pH 8, 0.15 M NaCl, 1 m M CaCI,, 1 m M MgCI2, 0.02%
sodium azidej. The column was washed with 30 ml TBS-azide and
eluted with 30 ml of 10 rnM a-methylmannoside and then with 1 5
ml of 0.1 M a-rnethylmannoside in TBS-azide.
The two eluates were pooled and lyophilized and reconstituted
in a small volume of water. A Sephadex G-50 column equilibrated in
water was used to remove salts and a-methylrnannoside. Fractions
containing sugar detected by phenol-sulfuric assay were pooled,
lyophilized and prepared for NMR studies. The sample was dissolved
twice in 0.5 ml of D,O (99.9% D) with intermediate lyophilization,
allowing 6 hr for exchange prior to each lyophilization. A third
reconstitution was done in 0.5 ml 99.96% 40 and the sample w as
transferred into a 5 mm diameter NMR tube. NMR analysis w as
performed at the NMR Centre, University of Toronto.
11.2.11 Annlytical procedures
Protein measurements were performed according to Lowry
(Lowry et al., 1951) except for the glycosylation enzyme assays for wh ich
the BCA kit (Pierce) was used. SDS-PAGE was done according to
Laemrnli (Laemmli, 1970). The scanning of blots was done by a CS 3 0 0
Linear gel scanner (Hoeffer Scientific hst.) and Macintegrator I
software.
III. RESULTS
III . I Polyacrylamide gel electrophoresis of erythrocyte rn e rn b rune
proteins f o m normal, heterozygotes and HEMPAS patients
Erythrocyte membranes were prepared by hypotonic lysis a n d
proteins were resolved by SDS-PAGE. Normal Band 3 contains a
heterogeneous carbohydrate chah which accounts for its diffuse
pattern on SDS-polyacrylamide gels (Figure 2.2.1, lanes 1 & 2). The
heterogeneity is due to the variable number of Galpl-4GlcNAc units
on each molecule of Band 3. However, HEMPAS Band 3 is a sharper
band (lanes 5 & 6), which implies a more uniform nature for the
oligosaccharide chain. Apparently Band 3 molecules containing long
chahs of sugars are missing in the erythrocytes of HEMPAS patients.
Two HEMPAS heterozygotes, who are offspring of the patient M. D.,
show an intermediary phenotype in terms of the size and
heterogeneity of their carbohydrates (lanes 3 & 4).
Additional lower rnolecular weight bands in lanes 5 and 6 a r e
probably the result of proteolysis, to which HEMPAS membranes a r e
more sensitive. HEMPAS membrane proteins dissolved in Laemmli
sample buffer degraded faster than normal controls; however, boiling
the sample could reduce the extent of proteolysis (data not shown).
111.2 Enryrnatic deglycosylation of normal and HEMPAS e r y t h rocyte
membrane proîeins
The enzyme peptide N-glycosidase F hydrolyzes the N-
dycosidic linkage between peptides and a l l three types of N-linked b
Figure 2.2.1 Erythrocyte membrane protein profile O f
control, heterozygotes and HEMPAS patients
Erythrocyte ghost membranes were prepared as described i n
Methods; proteins (10 pg of total proteinllane) were resolved b y
SDS-PAGE on a 7.5% polyacrylamide gel and stained with Coomassie
Blue. lanes 1 and 2, normal controls; lanes 3 and 4, heterozygotes
(A.S. & D.B.); lanes 5 and 6, HEMPAS patients (M.D. & C.L).
Spectrins +
Band 3 + Band 4.1
Band 4.2 +
Actin
oligosaccharides. Treatment of solubilized membranes with th is
enzyme resulted in an increase in the mobility of Band 3 on the gel
(Figure 2.2.2, lane 2). Although the shift in HEMPAS Band 3 is
smaller than normal Band 3 (lane 4), enzymatic deglycosylation of
HEMPAS Band 3 produces a protein with the same electrophoretic
mobility as the deglycosylated normal Band 3. This result indicates
that the different electrophoretic pattern seen in normal a n d
HEMPAS Band 3 can be attributed to an alteration in the
carbohydrate moiety. The band in lanes 3 and 4 at about 56 kDa is
probably a proteolytic product of Band 3.
111.3 Lectin binding of erythroid membrane glycoproteins
Tomato lectin, which specifically binds to poly N-
acetyllactosamine, can recognize normal Band 3 and Band 4.5 (Figure
2.2.3-8, lane 1). Pretreatment of solubilized membranes with endo-P
galactosidase, an enzyme which specifically cleaves the Gd 1 -4
linkage of polylactosamine oligosaccharide, abolishes the binding of
Band 3 and 4.5 to tomato lectin (lane 2). There is no noticeable
binding of tomato lectin to HEMPAS membrane proteins (lane 3) ,
which is consistent with its resistance to endo-p galac tosidase
digestion (Figure 2.2.3-A, lane 4).
On the other hand, Con-A, a lectin specific for hybrid and high
mannose N-glycans, can bind to HEMPAS Band 3 (Figure 2.2.4-B, lane
3), while no binding is seen in the normal control (lane 1). The
binding is sensitive to endoglycosidase H (lane 4), which cleaves t he
linkage between the first two N-acetyglucosamine residues in t h e
Figure 2.2.2 Enzymatic deglycosylation of e r y t h roc y t e
membrane proteins of control and HEMPAS patients
Erythrocyte ghost membranes were subjected to deglycos y lation
with peptide N-glycosidase F as described in Methods. Samples ( 10
pg of total proteinllane) were resolved by SDS-PAGE on a 10%
polyacrylamide gel and stained with Coomassie Blue. Lanes 1 and 2 ,
normal controls; lanes 3 and 4, HEMPAS patient (L. F.); Lanes 2 and 4
after treatment with 5 Ulpg of peptide N-glycosidase F (PNGF) for 1 6
h at room temperature; Lane 5, SDS-PAGE standards.
PNGF
Figure 2.2.3 Enzymatic digestion of poly N - acetyllactosamine sugar chain and tomato lectin Mot O f
control and HEMPAS erythrocyte membrane proteins
Erythrocyte ghost membranes were solubilized and treated with 0.06
mUlpg of endo-B-galactosidase (Endop) for 16 h at room temperature
as described in Methods; proteins were resolved by SDS-PAGE on a
10% polyacrylamide gel (4 kg total proteinhne). Tornato lectin blo t
was performed and visualized as described in Methods. Panel A ,
Coomassie blue stained gel; panel B, corresponding tomato lectin blot.
Lanes 1 and 2, normal control; Lanes 3 and 4, HEMPAS patient (L.F.);
Lanes 2 and 4, after enzymatic treatment; Lune 5, SDS-PAGE
standards.
Band
Band 4.5 +
core of a high mannose or a hybrid oligosaccharide. Treatment with
this enzyme causes a small shift in HEMPAS Band 3 mobility (Figure
2.2.4-A, lane 4). Based on this result Band 3 contains either a hybr id
or a high mannose oligosaccharide in HEMPAS patients.
ECA, a lectin specific for N-acetyllactosamine disaccharide, do e s
not bind to HEMPAS Band 3 and Band 4.5 (Figure 2.2.5, lanes 3 & 4),
while i t binds to these glycoproteins in normal control (lanes 1 & 2).
This suggests that in HEMPAS, Bands 3 and 4.5 do not contain even
short units of N-acetyllactosamine. However, since sialic acid m a y
inhibit the binding of ECA to glycoproteins, the presence of sialic acid
on altered HEMPAS oligosaccharides could be another explanation for
the lack of binding to ECA.
111.4 Diagnosis of HEMPAS heterozygotes by lectin binding annlysis
The relative tomato lectin binding to Band 3 from two offspring
of the HEMPAS patient (M. D.) was assessed by scanning of the lectin
overlay (Figure 2.2.6). The intensity of lectin binding was directly
proportional to protein loading, as determined in a separate
experiment (data not shown). Based on reported family studies
HEMPAS is an autosornal recessive disorder. That Band 3 of these
putative heterozygotes shows approximately 50% reduction in t h e
tomato lectin binding supports the above mode of inheritance. These
preliminary results suggest that lectin binding analysis can be used
as a sensitive tool for diagnosis of clinically healthy heterozygotes of
HEMPAS.
Figure 2.2.4 Endoglycosidase H treatment and Con-A blot o f
control and HEMPAS erythrocyte membrane proteins
Erythrocyte ghost membranes were solubilized and treated with 5 0
U/pg of endoglycosidase H (Endo H) for 16 h at room temperature a s
described in Methods and were resolved by SDS-PAGE on a 10%
polyacrylamide gel (10 pg total proteinllane for the gel and 4 pg total
protein/Iane for the blot). Con-A lectin blot was performed a n d
visualized as described in Methods. Panel A , Coomassie blue stained
gel; panel B, corresponding Con-A blot. Lanes 1 and 2, normal control;
Lanes 3 and 4, HEMPAS patient (L.F.); Lanes 2 and 4, after enzymatic
treatment; Lane 5, SDS-PAGE standards.
- 2 12.0 kDa
Band 3 + - 97.2
- 66.4
- 55.6
Band 3 +
Figure 2.2.5 ECA blot of control and HEMPAS e r y t h r o c y t e
membrane proteins
Erythrocyte membrane proteins were resolved by SDS-PAGE on a
10% polyacrylarnide gel. ECA lectin blot was performed a n d
visualized as described in Methods. Lanes 1 and 2, normal control;
Lanes 3 and 4, HEMPAS patient (L.F.); Lanes I and 3, 12 kg total
proteinllane; Lanes 2 and 4 , 25 pg total proteidlane.
Band 3 + Band 4.5 -
Figure 2.2.6 Tomato lectin binding analysis of C O n trol,
heterozygotes and homozygote of HEMPAS
Tomato lectin blot (10 pg total proteinhne) performed as described
in Methods. Relative amounts of lectin binding to Band 3 were
estimated by scanning densitometry . Panel A , tomato lectin blot.
Lnnes 1 and 2, normal control; lanes 3 and 4, heterozygotes (A.S. &
D.B.); lane 5, HEMPAS patient (M.D.). Panel B, the results of
corresponding scanning densitometry.
Band 3 -m
Band 4.5 -+
111.5 Staining of erythrocyte membrane sialoglycoproteins
To investigate if another major erythrocyte glycoprotein,
glycophorin A (GPA), is affected by the HEMPAS condition,
erythrocyte membrane proteins resolved by SDS-PAGE were s t ained
with Stains-al1 (Figure 2.2.7). By this method, highly acidic
compounds like sialoglycoproteins stain blue while other proteins
stain pink and lipids stain yellow. The lower blue band (PAS I I )
(below actin) is the major erythrocyte sialoglycoprotein, Glycophorin
A, and the higher blue band (PAS 1) overlapping the leading edge of
Band 3 is the dimer of GPA. The results show that GPA is expressed,
dimerized and sialylated in the HEMPAS patient (M. D.) and t he
heterozygote (A. S.). A difference neither in the electrop horetic
mobility of GPA nor in the intensity of the band is observed in
HEMPAS patient (M. D.) or heterozygote A. S. compared to normal
conuols. This may suggest that Glycophorin A is not affected b y
HEMPAS condition in this patient. The sharpness of HEMPAS Band 3
(lane 4) compared to normal controls (lanes L & 2) is also observable
by this method of staining.
111.6 Studies on EBV-transformed lymphoblasts
111.6.1 Lectin binding
In the search for a cultured ce11 hne that presents the HEMPAS
defect and thereby will enable study of the glycosylation pathway,
whole ce11 protein extracts from cultured EBV-transformed
iymphoblasts were subjected to SDS-PAGE and lectin blotting. CeU
extracts €rom four HEMPAS patients showed a protein pattern t h a t
was similar to the normal control (Figure 2.2.8-A). The proteins
Figure 2.2.7 Stains-al1 staining of erythrocyte m em b r ane
glycoproteins of control, heterozygote and AEMPAS patients
Erythrocyte ghost membranes were prepared as described i n
Methods; proteins (20 pg of total proteinllane) were resolved b y
SDS-PAGE on a 7.5% polyacrylamide gel and stained with Stains-all.
lunes 1 and 2, normal controls; lane 3, heterozygote (A.S.); lane 4,
HEMPAS patient (M.D.).
Spectrins +
Band 3 +
Actin +
+- PAS I (GPAdimer)
+ PAS IT (GPA)
were able to bind to tomato lectin in an endo-p galactosidase-
sensitive fashion (Figure 2.2.8-B). The tomato lectin-positive bands
include LAMPs and other poly N-acetyllactosamine-containing
proteins. This result indicates that the synthesis of polylactosamine
in HEMPAS EBV-transformed lymphoblasts is not impaired by t he
HEMPAS condition and that the defect may be restricted to t he
erythroid ce11 lineage. The consistent dark band with a rnolecular
mass of 60 kDa (shown by arrow), which is not sensitive to endo-p
galactosidase, is an endogenous biotinylated protein.
111.6.2 Enzyme assays
N-acetylglucosaminyl transferase II (GnT II) and P4 galactosyl
transferase (GalT) enzyme activities were measured on EBV-
transformed lymphoblast extracts from a normal control, four
HEMPAS patients and a carbohydrate-deficient glycoprotein
syndrome type II (CDGS II) patient with a known mutation in t he
gene encoding GnT II. No reduction was observed in the enzyme
activities in any of the HEMPAS patients (results shown in Figure
2.2.9). The activities appeared elevated in HEMPAS patients,
although further assays musc be performed to confirm this. GnT I T
activity in the CDGS patient is very low as expected. Mononuclear
cells from M. D., L. F. and C. L. also showed normal levels of GnT II i n
a separate experiment (Ch& et al., 1995). These experiments show
that the HEMPAS patients tested were different from previously
characterized patients, T. 0. and B. D., with decreased levels of
lymphocyte GnT II activity (Fukuda et al., 1987a) and the HEMPAS
Figure 2.2.8 Tomato lectin blot analysis of EBV- transformed
lymphoblast extracts of control and HEMPAS patients
Cultured EBV-transformed lymphoblasts were dissolved directly i n
Laemmli sample buffer, boiled for 3 min and filtered in a DNA
extraction unit to remove DNA. The extracts (5-10 pg total
proteinlpl) were treated with or without endo-p-galactosidase (50
pU/yg) in the presence of 2% C,?E,. After overnight incubation a t
room temperature, 3 volumes of Laemmli sample buffer were a d d e d
and proteins were separated by SDS-PAGE on a 7.5% pol yacry lamide
gel (10 pg total proteinllane for the gel and 15 pg for the blot).
Tomato lectin blot was performed as described in Methods. Panel A ,
Commassie Blue stained gel; panel B, corresponding tomato lectin
blot. Lnnes 1 and 2, normal control; lanes 3/4 5/6, 7/8. and 9/10,
HEMPAS patients (L.F., M.D., M.K. and C.L. respectively); Lanes 1.3,5,7
and 9 , before and lanes 2, 4, 6, 8 and 10, after endo-P-galactosidase
treatment. The band shown by the arrow is an endogenous
biotinylated protein.
Figure 2.2.9 Glycosyltransferase enzyme assays on con trol
and HEMPAS EBV-transformed lymphoblast extracts
The ce11 extracts were prepared and enzyme activities we re
measured as described in Methods. Panel A, GnT II assay ( i n
duplicates); panel B, GalT assay (error bars represent s tandard
deviations). Cnt: normal control; L.F., C.L., M.D. and M.K.: f o u r
KEMPAS patients; J.V.: patient with carbo hydrate-deficient
glycoprotein syndrome (CDGS) type II.
variant G. K. with decreased level of GalT activity in mononuclear
c e k (Fukuda et al., 1989).
111.7 Czrltrrred erythroid cells
Erythroid cells from normal human peripheral blood cul tured
in serum-free liquid media for 14 days were determined by Giemsa
staining to be mostly nucleated normoblasts. These cells contain a
protein (= 90 kDa) which can react with anti-Band 3 antibody (Figure
2.2.10-A) and tomato lectin (Figure 2.2.10-B). This protein, in spi te
of its sharpness on a polyacrylamide gel, is likely to represent Band
3. That the expression of 1 antigen (branched poly N-
acetyllactosamine) increases during the differentiation may explain
the sharpness of Band 3 at this pre-reticulocyte stage. However, the
possibility that the band is simply a biotinylated erythroid protein
cannot be ruled out. In the future, it would be important to examine
the presence of Band 3 and poly N-acetyllactosamine in cultures of
HEMPAS normoblasts.
111.8 Functional strrdies on HEMPAS erythrocytes
The glycosylation defect in HEMPAS is not restricted to Band 3.
Band 4.5, the erythrocyte glucose transporter is also under-
glycosylated. This condition provides a good mode1 to investigate t he
role of oligosaccharide in the transport function of Band 4.5, since t h e
oligosaccharide has been implicated in the transport activity of
GLUTl (Asano et al., 1991; Feugeas et al., 1990). The uptake of radioactive 3 - O-methylglucose was measured in normal and HEMPAS intact
erythrocytes as an indicator of glucose transporter function. The
Figure 2.2.10 Immunoblot and tomato lectin overlay o f
extracts from cultured human erythroid cells
Normal cultured erythroid cells (= 3 x I O 6 ) washed with PBS were
dissolved in Laemmli sample buffer, boiled for 5 min and filtered in
a DNA extraction unit. One third of the sample was subjected to SDS-
PAGE on a 7.5% polyacrylamide gel, transblotted and probed with a
rabbit antibody against human Band 3. Biotinylated anti-rabbit IgG
was used as the secondary antibody, which was detected in a
cherniluminescent reaction. Proteins in the remainder of the s amp le
were similarly resolved on a 7.5% polyacrylamide gel and subjected
to tomato lectin blot as described in Methods. P a n e l A , immunoblot
with anti-Band 3 antibody. Panel B, corresponding tomato Lectin blot.
Lane 1, normal cultured erythroid extract; lune 2, SDS-PAGE
biotinylated standards. The doublet shown by the broken a r row
probably represents endogenous biotinylated proteins.
LOO
Figure 2.2.11 3 - 0 - m e t h y l - [ ~ ~ ~ ] g l u c o s e uptake by control
and HEMPAS erythrocytes
The glucose uptake was measured as described in Methods a n d
expressed as the fraction of maximum uptake (35 min incubation).
Error bars represent standard deviations.
results are similar for the uptake of glucose in the control and the
patient (Figure 2.2.11).
To assess the relative expression of the transporter in HEMPAS
and normal erythrocytes, ghost membranes were subjected to
Western blot analysis using a polyclonal rabbit antibody raised
against the C-terminal peptide of human GLUT1. As shown in Figure
2.2.12 normal Band 4.5 is a broad band at around 45-60 kDa,
however, in HEMPAS it is sharper and rnigrates faster than normal.
The removal of the oligosaccharide by peptide N-glycosidase F
norrnalized this difference. Scanning of the corresponding blo t
showed no reduction in the amount of Band 4.5 in HEMPAS compared
to the normal control. Based on this result Band 4.5 poly N-
acetyllactosamine is not directly involved either in the transport
function or the expression of the transporter. This supports the idea
that a minimum oligosaccharide chah is sufficient for the function of
the glucose transporter (Feugeas et ai., 199 1).
111.9 HEMPAS g&yco&ipids
The polylactosamine moiety of normal Band 3 and Band 1.5
serves as blood g o u p antigen I/i (i: fetal form, 1: adult form).
Although HEMPAS glycoproteins do not carry polylactosamine, an ti-
I/i antibodies can effectively agglutinate HEMPAS ery throc y tes
(Verwilghen et ai., 1973). This strongly suggests the existence of
polylactosamine on other membrane elements, perhaps glycolipids.
The expression of oligoglycosylceramides was compared i n
KEMPAS and a normal control by thin layer chromatography. The
results indicate a higher yield of oligoglycosylceramides f ro m
Figure 2.2.12 Immunoblot analysis of control and HEMPAS
erythroid membranes with anti-glucose transporter
ant ibody
Erythroid ghost membranes were treated with or without peptide N-
glycosidase F (10 U/pg) as described in Methods and were resolved
by SDS-PAGE on a 10% polyacrylamide gel (10 pg total proteinflane) .
A rabbit antibody against the C-terminal peptide of human GLüTl
was used as the primary antibody, which was then detected by a
biotinylated anti-rabbit IgG in a chemiluminescent reaction. Panel A ,
anti-glucose transporter immunoblot. Lanes 1 and 2, normal control;
lanes 3 and 4, HEMPAS patient (L. F.); lanes 1 and 3 before and lanes
2 and 4 after N-glycosidase F treatrnent. Panel B. the results of
corresponding scanning densitometry of lanes 2 and 4.
PNGF
Band 4.5 +
LUU i
180 ;
Control HEMPAS
HEMPAS (L. F.) erythrocyte membranes (Figure 2.2.13-A) (similar
result was obtained in another separate experiment). The ratios of
lactosylceramide (Lac-cer) and triglycos ylceramide to
tetraglycosylceramide (the most consistent band in the two samples)
were assessed by scanning the thin layer chromatogram (Figure
2.2.13-B). These ratios are higher for the HEMPAS patient compared
to normal control, suggesting an alteration in the metabolism of
glycolipids causing an accumulation of lactosylceramide a nd
triglycosylceramide.
111.10 HEMPAS glycopeptide analysis
The nature of the structures of oligosaccharides present on
HEMPAS erythrocytes provides insights into the site of blockage i n
the glycosylation pathway. Whole erythroid membrane was used to
prepare glycopeptides and Con-A affinity chromatograp hy w as
performed to obtain high rnannoselhybrid oligosaccharides
previously detected by lectin blot analysis (see the section on lectin
binding).
A sugar peak was detected in the fractions of the first
Sephadex G-50 column between the void and total volume; however,
the yield was very low (4%) as measured by phenol-sulfuric acid
assay. This may be due to hydrolysis of the carbohydrates b y
glycosidase contamination in pronase or poor digestion of the
precipitated glycoprotein by pronase. The purification proceeded b y
affinity chromatography on a Con-A column. Quantitation at this
stage was impossible due to interference by a-methylmannoside. A
second Sephadex G-50 column was used to remove salts a n d
Figure 2.2.13-A Thin-layer chromatogram of erythrocyte
oligoglycosyl ceramides €rom normal control and HEMPAS
pat i ent
Lipid extracts of red ce11 membranes were prepared,
chromatographed and stained as described in Methods. Lune 1,
normal control (10 ylllane); lane 2, HEMPAS patient (L.F.) (6 y lllane);
lane 3, neutral glycosphingolipid standards (5 plllane), 03
(cerebrosides), LC (lactosyl ceramide), Gb, (ceramide trihexoside) a n d
Gb, (globoside) (origin is at the bottom of the chromatogram).
Doublets are formed for GC, LC and Gb, as a result of heterogeneous
fatty acid composition.
Figure 2.2.13-B The results of corresponding scanning
dens i tometry
The arnounts of lactosyl ceramide and triglycosyl ceramide relative
to tetraglycosyl ceramide were measured. The two species with
different fatty acid lengths were included in one measurement.
Gb3/Gb4 HEMPAS
methylmannoside from the eluate of the Con-A colurnn. Two sma l l
peaks of sugar, eluted prior to the large peak of methymannoside
(total volume), were prepared for NMR studies. The NMR spect ra
could not be interpreted due to the small amount of material
obtained. An alternative strategy to isolate the oligosaccharide f ro m
HEMPAS erythrocytes is required.
IV. DISCUSSION
The HEMPAS patients (L. F., C. L. & M. D.) studied in this thes is
are among the first series of HEMPAS cases described by Crookston
in 1969 (Crookston et al., 1969). They al1 demonstrate mild anemia,
abnormal bone rnarrow morphology and a positive acidified se ru m
lysis test.
In the first phase of this project, membrane proteins of
KEMPAS erythrocytes were analyzed. SDS-polyacrylamide gel
electrophoresis of membrane proteins from HEMPAS erythrocytes
shows an increase in the electrophoretic mobility of Band 3
compared to normal (Figure 2.2.1). This is consistent with the results
of early studies on HEMPAS patients (Anselstener et al.. 1977). Normal
Band 3 contains two types of oligosaccharides: a typical shor t
complex N-glycan chain (mol wt 2.000) and a heterogeneous poly N-
acetyllactosamine-containing complex N-glycan (mol wt up to 8,000)
(Fukuda et al., 1986b). On a Coomassie Blue-stained gel these two species
of Band 3 are observed as the dense leading edge and the diffuse
trailing part, respectively. The difference between HEMPAS a nd
normal Band 3 seerns to be limited to the heterogeneous poly N-
acetyllactosamine c h a h . Since the HEMPAS Band 3 is not as broad
as normal, the HEMPAS N-glycan on average should be smaller than
that of normal (termed under-glycosylation). Deglycosylation of
membranes by N-glycosidase F c m eliminate the difference in the
mobility of HEMPAS and normal Band 3 (Figure 2.2.2). Although this
strengthens the idea that the abnormality in HEMPAS Band 3 is a t
the carbohydrate level, a small change in the peptide portion of Band
3 cannot be exciuded.
Lectin binding analysis and endoglycosidase digestion w e r e
performed to obtain information about the type of abnormal sugar
on HEMPAS Band 3. Tornato lectin has been widely used to detect
glycoproteins containing three or more N-acetyllactosamine units
(Merkle and Cummings, 1987; Nabi and Rodriguez-Boulan, 1993). Normal Band 3
interacts strongly with this lectin, while HEMPAS Band 3 does not
show any binding (Figure 2.2.3). Consistent with this, HEMPAS Band
3 oligosaccharide is not sensitive to the poly N-acetyIlactosamine-
digesting enzyme, endo-b galactosidase. Normal Band 4.5 contains
the same type of oligosaccharide as normal Band 3, while HEMPAS
Band 4.5 also does not bind to tomato lectin. Therefore, a defect in
the synthesis of poly N-acetyllactosamine has been postulated to b e
present in HEMPAS. However, it is not clear if under-glycosylation of
Band 3 and 4.5 is the cause of HEMPAS pathology or is secondary to
the original defect.
It is known that the time that a glycoprotein remains in the
Golgi compartrnents is critical for the synthesis of poly N-
acetyllactosamine (Wang et d., 1991). An abnormality in this organelle
may speed up the passage of the glycoproteins (Bands 3 and 4.5) a n d
reduce the time they are exposed to the elongating enzymes (P3 GalT
and P4 GlcNAcT). Consequently, proteins may not be modified b y
poly N-acetyllactosamine to the extent that they are normally.
The translocation of Band 3 to the plasma membrane is
specifically influenced by glycoph~rin A (GPA). Several pieces of
evidence suggest that there is a molecular interaction between Band
3 and GPA (Bruce et al., 1994). Co-expression of Band 3 and GPA in
Xenopus oocytes appears to increase the rate of movement of Band 3
to the ce11 surface (Groves and Tanner, 1992). In En (a-) red blood cells
(deficient in GPA), Band 3 has an increased Mr reflecting longer N-
glycans on this protein. On the contrary, "in the Dantu and St(a+) r e d
blood ce11 variants, which effectively have more GPA than normal
because of expression of GPA hybrid proteins, the average length of
the N-glycan chah of Band 3 is reduced" (Groves and Tanner, 1992). Since
some GPA abnormalities, such as under-sialylation, have been
indicated in early studies on HEMPAS erythrocyte membranes
(Anselstetter et al., 1977), it is plausible that GPA plays a role in the under-
ulycosylation of HEMPAS Band 3. Under-sialylated GPA may be ab le D
to interact with Band 3 more readily than normal GPA a n d
subsequently increase the rate of Band 3 translocation to the ce11
surface. This ultirnately reduces the chance of the protein to b e
modified by poly N-acetyllactosamine. However, this mec hanis rn
may not be able to explain under-glycosylation of Band 4.5. 1 n
addition, staining of membrane proteins with Stains-al1 did not
reveal any noticeable change in GPA of our HEMPAS patient, M. D.
(Figure 2.2.6). Further experiments are necessary to test the above
role of GPA in targeting and translocation of KEMPAS Band 3. On the
other hand, ECA, a lectin specific for GaiPl-4GlcNAc (N-
acetyllactosarnine) disaccharide, does not bind to HEMPAS Band 3 o r
Band 4.5 (Figure 2.2.5), indicating the absence of even short repeats
of N-acetyllactosamine in these proteins. Therefore, it is unlikely
that the total absence of N-acetyllactosamine is due only to a change
in protein trafficking.
HEMPAS Band 3, in contrast to normal, binds strongly to Con-A
and is sensitive to Endo-H (Figure 2.2.4), which provides strong
evidence for a hybrid/high mannose oligosaccharide. This has led to
the view that the synthesis of complex N-linked sugars is blocked
and hybrid or high mannose sugars cannot be further processed. To
investigate the nature of this blockage, two glycosylation enzymes
were analyzed. Since ce11 compartments containinp these enzymes
are not present in erythrocytes, cultured EB V- tram formed
lymphoblasts were used for the enzyme analysis. These cell lines
have been widely used to study the mechanisms of many genetic
diseases, like ataxia telangiectasia (featuring neurodegeneration,
immunodeficiency, chromosomal instability and predisposition to
cancer) and Werner's syndrome (a progerioid' disorder w i th
premature aging phenotype). However, the findings in t h e
transformed cells may not always reflect the situation in primary
cells because of transformation.
A defect in GnT II, as previously reported in HEMPAS patient T.
O. (Fukuda et al., 1987a), was not detected in EBV-transformed
lymphoblasts of Our patients. Further, a GnT II defect can only
explain the absence of the al-6 antenna, not the total lack of poly N-
acetyllactosamine seen in our patients (L. F. and M. D.). The activity
of GnT II is almost absent in a CDGS II patient (5. V.), whereas the
Progerîa: a syndrome of uncertain generic inheritance, characterized b y precocious senility of striking degree, with death from coronary a r te ry diseasr frequently occurring before LO years of age.
poly N-acetyllactosamine content of Band 3 is 50% of normal in this
patient ( C h m k et al., 1995).
There is no evidence for a defect in P4 galactosyl transferase
activity in Our HEMPAS patients as reported in the atypical HEMPAS
case (G. K.) (Fukuda et al., 1989). Furthermore. the erythrocytes of ou r
group of patients agglutinate with anti-i/I antibodies, in spite of the
absence of poly N-acetyllactosamine on the membrane glycopro teins,
which strongly suggests the synthesis of this oligosaccharide on t h e
glycolipids. It is generally assumed that the glycosyltransferases
which synthesize poly N-acetyllactosamine on proteins also recognize
carbohydrates on lipids (Fukuda et al., 1986a); thus a defect in elongating
enzymes cannot be the case in our patients. In addition, severe
clinical and morphological features of G. K. have not been
documented in our patients.
The enzymatic activities of GnT II and GalT may be increased
in our HEMPAS patients compared to the normal control. Similarly
an elevated activity of GalT has been documented in cultured
lymphoblasts of the patient G. C. (Fukuda et al., 1990) and in mononuclear
cells from another typical HEMPAS patient (Fukuda et al., 1989).
Interpersonal variations may account for the differences b e t w ee n
our group of patients and the control. In future, it is desirable to
include more controls in this type of analysis. Studies on B
lymphocytes and EBV-transformed B lymphoblasts from patients
with rheumatoid arthritis demonstrated that EBV transformation
results in an increase in galac tos yltrans ferase activity (Wilson et al.,
1993). For this reason, it is desirable to investigate the expression of
the glycosylation enzymes in cells directly isolated from perip heral
blood with no viral transformation. Unfortunately, sufficient
amounts of fresh blood were not available from our HEMPAS patients
to conduct such studies.
Surprisingly, tomato lectin binding studies on HEMPAS C U 1 tured
lymphoblasts did not replicate the erythrocyte pattern; no significant
difference was seen between HEMPAS and normal control. These
data indicate that poly N-acetyllactosamine is present in these cells
in HEMPAS. Therefore, the defect in the synthesis of poly N-
acetyllactosamine is either restricted to the erythroid ce11 lineage o r
can be bypassed by an alternate pathway in the EBV-transformed
lymphoblasts (see below).
Our results from lectin binding analysis, combined with t h e
serological characteristics of Our patients, strongly suggest a lesion in
a-mannosidase II. Without the action of this enzyme, a hybrid N-
glycan with five mannose residues accumulates, which can react
strongly with Con-A. It seems that the a l - 3 a m of this hybrid N-
jlycan cannot readily be elongated and for this reason there is a
minimal amount of poly N-acetyllactosamine in Bands 3 and 4.5. A
similar situation has been previously reported for one KEMPAS
patient (G. C.) (Fukuda et al., 1990). In G. C., reduced expression of a-
mannosidase II mRNA and subsequent low activity of a-manno s idase
II is shown to result in a hybrid N-glycan on Band 3.
Preliminary experiments on "knock-out" mice with a nul1
mutation in the a-mannosidase II gene have shown that in spite of
the absence of a-mannosidase II activity, complex supars can b e
made in d l tissues except in the red blood cells (Chui, D. et al;
Abstract for International Symposium on Molecular and Ce11 Biology
of Glycoconjugate Expression, 1996, Switzerland). An al ternate
pathway independent of a-mannosidase II has previously been
demonstrated (Kornfeld, 1982) for the synthesis of complex N-glycans.
This pathway can be triggered by energy deprivation or glucose
starvation and leads to the formation of Man,,(GlcNAc),PPDol
structures, which can transfer oligosaccharide to the pro teins,
followed by normal processing in the absence of a-mannosidase I I
(Komfeld, 1982). Such a mechanism may be responsible for t he
synthesis of complex sugars in the tissues of the "knock out" mice
and in HEMPAS cultured EBV-transformed lymphoblasts. The reaso n
why this pathway is not triggered in erythroid cells needs fur ther
investigation.
Knowing the fine structure of oligosaccharide on Band 3 would
help in locating the specific site of the defect in the glycosylation
pathway. Attempts were made to purify glycopeptides from
pronase-treated HEMPAS erythrocyte membranes. NMR spectra d id
not reveal the presence of any oligosaccharide, probably due to low
yields during the purification procedure or hydrolysis of the sugar.
An alternative strategy involving the isolation of oligosaccharides
from purified Band 3 can be applied.
The measurement of Golgi a-mannosidase II activity involves
ceIl fractionation and preparation of a pH profile due to interference
of lysosomal a-mannosidases (Fukuda, 1990). Since the Golgi
mannosidase II activity measured by this method is low, even i n
normal cells, a more sensitive technique has been developed, Le., t h e
enzyme is immunoprecipitated by an anti-human a-mannosidase 1 1
antibody before measuring the activity (Moremen. K. W., perso na1
communication). EBV-transformed lymphoblast extracts a r e
presently being tested by this method. A iow a-mannosidase 1 I
activity in cultured EB V-transformed lymphoblasts may denote th e
above alternate pathway, whereas a normal activity may imply a r e d
cell-specific glycosylation lesion. In the latter case, it is necessary to
study the erythroid ce11 lineage.
For this purpose, in a pilot experiment normal human erythroid
cells from peripheral blood were cultured. Giernsa staining of t h e
cultured cells showed that the majority of the cultured cells were
nucleated erythroid cells. The expression of Band 3 and poly N-
acetyllactosarnine in the cultured cells was examined by anti-Band 3
antibody and tomato lectin respectively. The results show t h a t
cultured erythroblasts can synthesize tomato lec tin-detec table
oligosaccharides. To make sure that this binding is specific for poly
N-acetyllactosamine, it is desirable to test the sensitivity of this
binding to endo- P galactosidase; this enzyme should abolish th e
binding to tornato lectin. In addition, to confirm the fact that Band 3
is modified by poly N-acetyllactosamine at this stage of
differentiation, Band 3 can be immunoprecipitated by anti-human
Band 3 antibody and then be tested for the lectin binding. Although
these preliminary results suggest the expression of glycos ylated
Band 3 in the nucleated erythroid cells, the possibility of
contamination with erythrocytes and granulocytes c a n o t b e
eliminated. Studies on rabbit bone marrow erythroid cells have
shown a progressive increase in ce11 surface expression of Band 3, u p
to the reticulocyte stage (Foxwell and Tanner, 198 1). In future studies O n
HEMPAS erythroid cultures, it is important to be aware that t h e
incorporation of Band 3 into the membrane may be delayed to t h e
later stages of erythroid maturation, as evidenced in animals under
bone marrow stress (Foxwell and Tanner, 198 1).
A defect in the glycosylation of Band 3, specifically at the stage
involving a-mannosidase II, can explain some of the hematological
and clinical signs seen in HEMPAS patients. For instance, Fukuda
(Fukuda et al., 1986b) showed that under-glycosylated HEMPAS Band 3
molecules form clusters in the membrane, perhaps due to thei r
increased hydrophobicity. Since Band 3 is a major membrane
protein of erythrocytes and is attached to the cytoskeleton elements
(ankyrin, Band 4.1 and 4.2), its clustering has a great impact on t he
shape of red blood cells and may cause the macroscopic membrane
abnormalities seen in HEMPAS (Fukuda et al., L986b). In typical cases of
HEMPAS, other types of blood cells usually do not demonstrate
abnormality, either reflecting an erythroid-specific defect O r
indicating that the under-glycosylation in other cells does not affect
the organization of the cytoskeleton. For instance, granulocytes ca r ry
tri- and te tra-antennary poly N-acetyllac tosamine N-glycans a n d
attenuation of the al-6 antema may not impose the same effect as in
a biantennary poly N-acetyllactosamine structure.
Splenomegaly is a clinical sign of HEMPAS. The spleen, as O ne
of the hematopoietic organs, is involved in the filtration of blood,
fetal hematopoiesis and terminal differentiation of blood cells. Only
normal, deformable erythrocytes pass through the inter-endo thelial
slits of splenic sinuses. Abnormally-shaped erythrocytes like
spherocytes, which are poorly deformable, are likely to be retained
by the spleen. Since the membrane abnormality of HEMPAS is seen
as early as erythroblastic cells (Vainchenker et al., 1979) and reticutocytes
released from the bone marrow do not enter the circulation until
conditioned by the spleen, it is plausible to assume that clearance of
abnormal HEMPAS reticulocytes causes congestion and e nl a r geme n t
of the spleen. Barosi (Barosi and Cazzola, 1979) studied the relation
between ineffective erythropoiesis (intramedullary hemolysis) a n d
peripheral hemolysis in some HEMPAS patients. These patients
demonstrated prominent peripheral hernolysis rnediated through t h e
spleen and were markedly irnproved clinically after splenectomy.
A plasma glycoprotein, transferrin, has been shown in HEMPAS
patients to be rnodified by high mannose and hybrid chains i n
addition to the normal sialylated biantennary complex type
oligosaccharide (Fukuda et ai., 1992). Abnomally glycosylated molecules
of transferrin are removed from the circulation by mannose
receptors present in the liver. A clearance system that recognizes
non-reducing mannose or GlcNAc in sinusoidal lining cells of the liver
and isolated rat alveolar macrophages has been detected, mediating
plasma clearance of infused human placenta1 P-glucuronidase w i th
high efficiency (Achord et al., 1978). Mannose-binding pro teins h a v e
been isolated from the Liver and serum of humans and rats and a r e
known to consist of a lectin-like basic subunit (Otter et ai., 1992).
Fukuda proposed (Fukuda et al., 1992) that accumulation of this
aberrantly glycosylated transferrin, which has to be digested in t h e
lysosomes of the liver cells, may cause the liver cirrhosis a n d
secondary tissue siderosis seen in some HEMPAS patients. The
presence of abnormally glycosylated transferrin in Our patients has
yet to be investigated.
To study the effect of HEMPAS on the composition of
erythrocyte glycolipids, lipids were extracted by c hloroform-
methanol from ghost membranes. Glycolipids were isolated by
applying a Folch partition and analyzed by thin layer
chromatography. The lower-phase glycolipid extract of h u man
erythrocyte membrane contains glycosylceramides carrying up t O
four monosaccharides, as marked by neutral glycolipid standards
(Figure 2.2.12). The results indicate an elevation of lactosyl ceramide
(Lac-Cer: GalP 1-4Glc-Cer), tri- and tetra-glycosyl ceramides i n
HEMPAS erythrocytes. Interes tingly, the increase in the lac tos y1
ceramide is limited to the species with a fatty acid of smaller chain
length. Although these results may suggest an alteration in the
metabolism of glycolipids in HEMPAS (L. F.) erythrocytes, a n
inconsistency in the lipid extraction or loading cannot be ruled out.
The ratios of Lac-Cer and tri-glycosyl ceramide to te tra-gl ycos y1
ceramide are also higher in the HEMPAS patient cornpared to the
control. It is important to mention that the method used here is only
semiquantitative, because the intensity of orcinol staining may not
be directly proportional to the amount of glycolipids. The above
oligoglycosylceramides probably include precursors for the s ynthe sis
of poly N-ace tyllactosaminyl ceramide (HEMPAS glycan) which is
known to accumulate in the erythrocytes of typical HEMPAS patients.
The elevation of these precursors probably reflects the increase in
the biosynthesis of "HEMPAS glycan" to compensate for the absence
of poly N-acetyllactosarnine on the proteins. An abnormal lipid
composition including increased concentration of glycosphingolipids,
di-, tri-, and tetrahexosyl ceramides has been shown in erythrocytes
from two siblings with clinical CDA II (Joseph et al., 1975). In th ree
other siblings suffering from CDA II the increase in t h e
glycosphingolipid content was accompanied by an increase in t h e
free cerarnide concentration (Bouhours et al., 1985). Bouhours (Bouhours et
al., 1985) reported an altered fatty acid composition of lactosyl
ceramide (increased long chain fatty acids) and the lack of increase
in glucosyl ceramide. In symptomatic heterozygotes of Fabry's
disease, accumulation of ceramide trihexoside in the heart correlates
with low activity of a-galactosidase, implying a defect of
glycosphingolipid catabolism (Honimi et al., 1990). The only O the r
disease that is reported to cause alteration of glycolipids in blood
cells (lymphocytes) is classical galactosemia (Petry et al., 199 1).
Serological studies on families of HEMPAS patients indicate a n
autosomal recessive mode of inheritance for this disease (Crookston et
al., 1969). Erythrocytes of clinically-unaffected farnily members of
HEMPAS patients show some degree of reactivity with anti-i
antibody while affected patients strongIy react with this antibody.
Polyacrylamide gel electrophoresis of red cell membrane pro teins
revealed a slightly abnormal pattern in the clinically unaffected
sibling of two HEMPAS patients ( G o c k e m et ai., 1975). Based on o u r
result (Figure 2.2.1) HEMPAS heterozygotes demonstrate a distinct
intermediary pattern in respect to the electrophoretic mobility of
Band 3. To assess this situation in a sensitive and quanti tat ive
manner, the binding of tomato lectin to Band 3 was compared in a
normal conuol, a HEMPAS patient (M. D.) and two offspring of M. D
(Figure 2.2.7). The results indicate an approximately 50% reduction
in the binding for the heterozygotes, which supports a classical
Mendelian recessive inheritance. This is the first report to Our
knowledge that quantitatively correlates the degree of glycosylation
abnormality to the mode of inheritance of HEMPAS. However this
study needs to be refined by using a higher number of controls to
establish the interpersonal variations that may exist and interfere
with the interpretation of the above results.
It is known that HEMPAS is associated with under -
glycosylation of not only Band 3 but also Band 4.5 (Fukuda et al., 1984~).
Since Band 4.5 is a diffuse band and its abundance is low, it cannot
be visualized readily on Coomassie Blue-stained gels. Our re s u 1 ts
Frorn lectin binding analysis confirm the absence of poly N-
acetyllactosamine sugar on Band 4.5 in the HEMPAS patient (Figure
2.2.3). That both Band 3 and 4.5 normally contain poly N-
acetyllactosamine and both are under-glycosylated in the HEMPAS
condition is further evidence of an abnormality of the biosynthesis of
poly N-acetyllactosamine oligosaccharide. The immunoblot analysis
of ghost membranes treated with peptide N-glycosidase F (Figure
2.2.1 1) dernonstrated that both normal and HEMPAS Band 4.5
contain N-glycans. Similarly to Band 3, removal of the N-linked
sugar on normal and HEMPAS Band 4.5 elhinates the difference
between them and results in bands with the sarne electrophoretic
mobility.
Abnormal glucose metabolism is implicated in motor neuro n
disease. Elevated glucose uptake occurs with normal erythrocytes i n
the presence of patients' plasma (Karim et al., 1993). A novel clinical
entity characterized by defective glucose transport at the blood-
brain barrier is associated with decreased density of GLUTl in
erythrocyte membranes (Harik, 1992). To investigate the possibility of
abnormal glucose transport in HEMPAS as a result of oligosaccharide
alteration in GLUTI, the cytochalasin B-sensitive uptake of t h e
radioactive non-metabolisable analogue of glucose, 3 - 0 -
methylglucose, was compared in intact erythrocytes of HEMPAS (L.
F.) and a normal control. The results showed similar glucose uptakes
for the patient and the control. In addition, surface expression of
GLUTI, estimated by Western blot analysis, was not perturbed b y
the under-glycosylation (Figure 2.2.1 1). Our result supports th e
study done on reconstituted GLUTl in proteoliposomes. In this
experiment net uptake of glucose did not change upon digestion of
poly N-acetyllactosamine on GLUTl by endo- P-galactosidase (Wheeler
and Hinkle, 198 1). The naturally occurring alteration in GLUT 1-linked
oligosaccharide in HEMPAS provides an ideal mode1 to study t h e
functional role of sugars without facing the problems related to
enzymatic deglycosylation or mutagenesis. A minimum sugar chain
probably, as large as the core, has been proposed as a requirernent
for the functional expression of GLUTl (Feugeas et ai., 199 1). From O u r
snidy we cannot rule out this possibility but c m conclude that t h e
specific poly N-acetyllactosarnine chah is not essential for t h e
transport func tion. Differential N-glycos ylation of GLUTl h a s
recently been implicated in the subceIlular localization (apical v s
basolateral) of GLUTl in the endothelium of brain capillaries (Kumagai
et ai., 1994). Furthemore, Asano (Asano et al., 1991) suggested t h e
involvement of GLUTl oligosaccharide in maintainhg a structure of
glucose transporter that has high affinity for glucose. In this study,
the reduced Km for 2-deoxyglucose uptake and the reduced
photoaffinity labeling with cytochalasin B were attributed to the
absence of oligosaccharide, while it can be secondary to the
conformational changes induced by the mutagenesis of the N-
glycosylation site.
Similarly, under-glycosylation of Band 3 does not disturb its
anion exchange function as assessed by phosphateKhloride exchange
(J. Charuk, unpublished results). A structural and functional
relationship between anion and glucose transporters has bee n
proposed, based on studies of the transport characteristics of Band 3
and glucose transporter in aged erythrocytes and in some diseases
involving erythrocytes (Bosman and Kay, 1990). It was concluded tha t
changes in the structure of Band 3 that do not affect anion transport
have no effect on glucose transport characteristics either.
124
V. CONCLUSIONS AND FUTURE DIRECTIONS
In summary, rny results have demonstrated that Bands 3 a n d
4.5 in erythrocytes from three different Toronto area HEMPAS
patients do not contain the normal poly N-acetyllactosamine
oligosaccharide. Instead, HEMPAS Band 3 is modified by a smaller
hybridlhigh mannose sugar. Offspring of a HEMPAS patient (M. D.)
show an intermediary level of glycosylation as expected for a
recessive disease. Another erythrocyte membrane glycoprotein,
GPA, is not affected significantly by this condition. Poly N-
acetyllactosamine is not a requirement for the glucose t ranspor t
function of Band 4.5. HEMPAS EBV-transformed lymphoblasts do no t
exhibit reduced activities of P4 galactosyl transferase (P4GalT) or N-
acetylglucosaminyl transferase II (GnT II), and are able to express
poly N-acetyllactosamine sugars. A defect in a-mannosidase II is
proposed as the grounds for the glycosylation abnormality in these
patients. Furthermore, some evidence for alteration in glycolipid
metabolism has been found,
From this project 1 have obtained several novel results t ha t
contribute to the understanding of the clinical and etiological aspects
of E M P A S . Some of these results need to be confirmed. For
instance, to establish the tornato lectin binding analysis as a tool to
diagnose clinically healthy heterozygotes of HEMPAS, the analysis
should be repeated in M. D.'s and other HEMPAS families. The
presence of a similar pattern (= 50% reduction in the binding for
heterozygotes compared to normal) in other HEMPAS families w ould
rule out the chance of this being due to a variation in the population.
It would be interesting to test if the same intermediary level of
binding can also be detected with Con-A lectin, to support the finding
that the defect in a glycosylation enzyme acts in a dose-dependent
manner. In addition, Con-A binding analysis needs to be performed
on the extracts of KEMPAS EBV-transformed lymphoblasts to
determine whether there is an increase in high mannoselhybrid
oligosaccharides in these cells.
Since my study was unable to isolate glycopeptides from
erythrocytes of HEMPAS patients, new strategies are needed to
determine the structure of the oligosaccharide. This may include
pronase treatment of the membranes rather than after Lipid
extraction, since pronase digestion seems not to be complered o n
precipitated glycoproteins following delipidation. The analysis can
be targeted to Band 3 oligosaccharide by purification of this protein
prior to pronase digestion. Alternatively, hydrazinolysis or N-
glycosidase F digestion can be used to release oligosaccharides from
glycoproteins, which then can be isolated by Con-A affinity
chromatography. If it can be shown that cultured erythroid cells
express the abnormal oligosaccharide on Band 3, these cells can b e
rnetaboIically Labeled by specific radioactive sugars. Im m u n o - isolated Band 3 can be analyzed for incorporation of the radiolabelled
sugar.
My studies on EBV-transformed lymphoblasts suggest that t h e
glycosylation defect associated with HEMPAS is restricted to t h e
erythroid cell Iine. Therefore, it will be of great value to maintain a n
erythroid ceIl culture from the HEMPAS patients. However, it is
essential to first assure that the glycosylation rnachinery is act ive
and Band 3 is expressed in these cultured cells. This work h a s
already started (Figure 2.2.10) and it appears that Band 3 is
expressed and glycosylated in the normal erythroid culture.
Based on my study, it is likely that under-glycosylation of Band
3 and 4.5 is the result of a-mannosidase II abnormality. Extracts of
the above cultured cells can be used to measure the a-mannosidase
II activity or to isolate poly (A)' RNA for Northern analysis with a-
mannosidase II probes. Alternatively, accumulation of a h y b r i d
sugar in place of the complex form seen in the HEMPAS patients can
be explained by a high activity of GnT III, which directs t h e
synthesis of N-linked sugars towards the bisected hybrid structure.
The activity of this enzyme can be assessed in the cultured ery throid
cells by a fluorescent assay using a biantennary sugar c h a h a n d
UDP-GlcNAc as substrates (Yoshimura et al., 1996).
In general, under-glycosylation of proteins can be the result of
an abnormality in the endomembrane system. Glycoproteins t rave l
from the ER to the Golgi, where the oligosaccharide is processed, a n d
between its compartments by means of non-clathrin coated vesicles
0 et a i . 1986). The formation of the carrier vesicles involves
microtubules and microtubule-dependent motor enzymes (Marks et al.,
1994). Any abnormality in the elements required for the s t ruc tura l
and functional integrity of the Golgi apparatus and carrier vesicles
may disturb the glycosylation pathway. The possibility of Gola
disintegration in the HEMPAS erythroid cells c m be investigated b y
immunofluorescent microscopy using antibodies against re s ide n t
proteins of Golgi, such as mannosidase II and ADP-nbosylation factor
(ARF, a protein coat cornponent of the vesicles). Disassembly of t h e
vesicles and subsequent blockage between the ER and Golgi c m b e
artificially induced by a fungal metabolite, brefeldin A, causing Golgi
proteins to redistribute to the ER. A similar phenotype has been
described in a temperature-sensitive CHO cell mutant (Zuber et al., 1991).
As mentioned in the Discussion, transferrin was shown to b e
modified with abnormal high mannose and hybrid chains in t w o
HEMPAS patients (Fukuda et al., 1992). It wouId be interesting to test
this feature in our HEMPAS patients. Such an abnormality is no t
expected, since my results suggest that the glycosylation defect may
be erythroid-specific. Transferrin can be purified from the plasma of
these patients by immobilized metal ion affinity chromatography and
compared for its binding to Con-A with normal control. A higher
binding to Con-A would suggest a glycosylation defect in HEMPAS.
The genetic defect in HEMPAS could be a mutation tha t
inactivates or mislocalizes a-mannosidase II. The latter can b e
investigated in cultured erythroid cells or rnononuclear cells isola ted
from peripheral blood by subcellular fractionation o r
immunofluorescent microscopy using an antibody to human a-
mannosidase II. If rnislocalization of the enzyme is the case, t h e
activity will probably be retained in the ce11 extracts. Furthermore,
the expression of the enzyme could be examined b y
immunoprecipitation followed by SDS-PAGE. Presence of a pro tei n
identical to normal cr-mannosidase II would suggest a single amino
acid substitution, while total absence of the enzyme could be due to a
promoter mutation or a frame-shift or stop codon in the coding
region and subsequent degradation of the defective enzyme. To
determine whether the defect is at the transcription level, Northern
blot analysis could be utilized to detect a-mannosidase II mRNA. I f
the mRNA is missing, it may be because it is unstable or that a
mutation is affecting the promoter region of the gene. Finally,
mutations in the coding region of the a-mannosidase II gene could be
identified by sequencing the PCR-amplified genomic DNA or cDNA
(obtained by reverse transcription of poly( A)' RNA) isolated fro m
cultured or fresh blood cells. frimers are selected based on a-
mannosidase II cDNA; however, if a mutation in the promoter region
is suspected, primers for PCR have to be selected in such a way tha t
the product covers this region. My results indicate a deficiency in a-
mannosidase II in erythroid cells as the grounds for HEMPAS. This
can be confirmed by determination of the Band 3 oligosaccharide
structure and the underlying genetic lesion.
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