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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