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 National Library Bibliothèque nationale of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, nie Wellington Ottawa ON K1A ON4 OttawaON KtAON4 Canada Canada Your lJe Votre retermw
Our iUe Noire fefêfenfe
The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous paper or e1ectron.c formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique.
The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or othedse de celle-ci ne doivent être imprimes reproduced without the author7s ou autrement reproduits sans son permission. autorisation. 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 1would like to thank rny friends: Susan, Valerie, Martine, Simin,
Emily, Mark, Michael and Has for their heart warming words and being always there for me. They ail share this degree with me. 1 especiaily thank Edyfor moral support and her "emo tional intelligence". TABLE OF CONTENTS
I . INTRODUCTION ...... *...... *...... 1 I .I PROTEIN-LINKED OLIGOSACCHARIDES ...... L i. 1.1 Biosynthesis of N-giycans ...... 3- 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 ...... 1.1 .3.2 Endoglycosidases ...... -37 - 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 ERYTHROCYTEMEMBRANE GLYCOPROTEINS ...... 40 I.3 ...... 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- 2A 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 Lech Biots ...... 68 11.2.5 Western Biots ...... 69 11.2.6 Staining procedures ...... 69 11.2.7 Enzyme. assqs...... -69 11.2.8 Giycol~p~danalysis ...... 70 11.29 Glucose transport assay ...... -7L 11.2.10 Preparation of g lycopeptide from HEMPAS erythrocytes...... 72 11.2.1 1 Atialytical procedures ...... 7 3 III. RESULTS ...... ~...... 74 III .I Polyacrylamide gel electrophoresis of etythrocyte membrane proteins from normal. heter ozygotes and HEMPAS patien fs ...... -74 III .2 Enzymatic deglycosylation of normal and HEMPAS eryrhrocyte membrane proteins74 111.3 Lectin bittding of erythroid membrane gfycoproteins...... 77 111.4 Diagnosis of HEMPAS heterorygotes by iectin binding atiaiysis...... 82 III. 5 Staining of erythrocyte membrane sialoglycoproteins...... 89 III .6 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 11 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-meth~l-[~~~~~lucoseuptake 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
p 1-3 N-acety lglucosaminy1 tram ferase p 1-4 Galactosyltransferase
P6GlcNAcT CDA Congenitai dyserydiropoietic anernia CDGS II Carbohydrate-deficient glycoprotein syndrome type II CHO Chinese hamster ovary ce11 Con-A Concanavalin A EBV Epstein-Barr virus ECA Erythrina ciestagalli agautinin EmA Ethy lenediaminetetraacetic acid Endo H Endo-P-N-acety lglucosaminidase H ER Endoplasmic reticulum Fuc Fucose Gal Galactose GaiNAc N-acetylgaiactosamine Glc Glucose GlcNAc N-acetylglucosamine GnT Wace ty lglucosaminyltransferae GP Glycophorin Lac-Cer Lac tosy lceramide LAMF' Lysosomal associated membrane protein Man Mannose Neu Neuraminic acid PBS Phosphate buffered saline PNH Paroxysmal nocturnal hemoglubinuîa PP-Do1 Dolicholpyrophosphate SA (NeuNAc) Sialic acid (N-acety lneuraminic acid) SDS-PAGE Sodium dodecyl sulphate-polyacrylamide plelectrophoresis TBS Tris buffered saline TM Trans-membrane I. INTRODUCTION
Glycoproteins exist in al1 eukaryotic cells and indeed mos t integral membrane proteins are glycosylated or are associated w i th 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 or 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 undan t type of O-glycan in animal cells is the mucin-type gIycan in which the oligosaccharide chah starts with an N-acetylgalactosamine linked to serine or threonine. The biosynthesis of O-glycans does not 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 and 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 cm 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 the 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 alL 3 6 Man al+î-Man al 0'' Man p t j4GlcNAc 3- (Man a1 +2) Man al I
High mannose type
Hybrid type
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
P~lucosyl phosphodolichol, which provide the sugars for the luminal 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 1p comp lex, translocating chain associated membrane protein (TRAM), signal peptidase and oligosaccharyl transferase complex (Gilmore, 1993). 1t 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 and 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 are retained in the ER until proper folding has been attained. Release of fully folded glycoproteins from calnexin requires the removal of the 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 and cornplex N-glycans. In the absence of this enzyme none of the modifications by a 1-316 mannosidase (a-mannosidase II), GnT II, GnT III, GnT IV and core al-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 are 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 type 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 and 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 are 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 nuc leo tide sugars (Gahmberg and Tolvanen. 1996). In addition, the accessibility of t h e glycoprotein to the modifying enzymes determines the extent of the 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 1I 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 O Membrane 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 and consensus N-glycosylation sites. The catalytic domain cm 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 transmembrane domains play a central role in the Golgi retention by providing a site 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 be t ween different enzymes residing in the sarne compartment. Addition of an ER retention signal to GnT I cm 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 are 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 the transrnembrane segment, being 25 arnino acids long, does not mee 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 as two forms which are different in the length of their amino-terminal cytoplasmic domains. The two foms 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 been established, the longer form may be preferentially targeted to the 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 chah may be branched, as in the 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 branched lactosaminoglycan, which involves a novel P6GlcNAcT that can add
GlcNAc branch on the penultimate Gd in GalP 1-4GlcNAcB 1-3Galp-R.
Repeating N-acetyllactosamine units are present more often on tri and te traantennary N-linked oligosaccharides cornpared to biantennary chains (Komfeld and Kornfeld, 1985) and there is a preferential attachment of poly N-acetyllactosamine chains to th 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 tramte n nary poly N-acetyllactosarnine chain a2-3 sialyltransferase pre fers terminal galactose residues on the antennae of the al-6 am, 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(Fuca1-3)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 1y N-acetyllactosarninyl chains of LAMPs purified Rom human chronic myelogenous leukemia cells contain only two N-acetyllactosamine uni ts (Carlsson and Fukuda, 1WO), 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 and higher stability of lysosomes. Secretory proteins usually contain none or a small amount of this sugar. Interestingly, the fusion of the transmembrane and cytoplasmic domains of vesicular stornatitis virus G protein w i t h human chorionic gonadotropin (hCG) a chah was able to alter the 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 the 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 must 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 reglate 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 antigens Protein-linked poly N-acetyllactosamine carries AB0 blood group antigens. In O-type blood group individuals expressing H antigen, al-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 al-3 galactosyltransferase (B-enzyme) respectively. The structures of these antigens are shown in Figure 1.6.
Fuc al 42ml PL 413GlcNAc pl +R
f12 FUC a 1c2 Fuc al
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 separa ted by SDS gel electrophoresis and transferred to nitrocellulose cm b e probed by lectins to provide information about the type of glycans present. Sorne lectins interact with the core portion of 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 chah 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 pl - 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.
Lecfin Specificity LEA (Tornato) OIigomers of P 1 -3-Iinked N- (Lycopersicon 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 the 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 and 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 1-3Mana 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 do not have the outer Mana 1-3, they are resistant to endo H even af ter cheir outer chain moieties are removed. This enzyme is widely used to follow the synthesis of glycoproteins in the endomembrane system because after leaving the media1 Golgi, complex glycoproteins become resistant to endo H.
Enzyme Specificity 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 the 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 the 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 th 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 that 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 are affected by glycans, these effects cannot be generalized and must be 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 the 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
(eg. 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 the oligosaccharide "appendages" keeps glycoproteins fixed in place w i th 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- acetylglucosaminylphosphotransferase catalyzes addition of N- acetylglucosamine 1-phosphate to selected mannose residues on the 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 are 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 the 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 any 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 the of serurn glycoproteins can be regulated by alterations in their carbo hydrates. Non-reducing terminal sialic acid seems essential for continued viability of the glycoproteins in the circulation. Removal of the sialic acid b y neuraminidase exposes galactose residues of the oligosacch~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, the presence of sialic acid on hepatic receptors is required for the binding activity leading to internalization and catabolism of seru 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) and 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 the circulation via the lymphatic system. This journey provides the opportunity for exposure of lymphocytes to foreign antigens. Homing of lymphocytes to peripheral lymph nodes in part is mediated by carbohydrate-dependent mechanisms. L- (1y 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 see 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 4R 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 or eliminate the normal glycosylation pattern. These include: 1) site- 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. Inhibitor Enzyme Tunicamycin N-acetylglucosaminylphosphotransferase Castanospermine Glucosidase I and II Deoxy nojirimycin Deoxymannojirimycin Cis-Golgi a-mannosidase (a-Man 1) Swainsonine 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 the protein. For example, nonglycosylated NdK 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 against - -- ATPase Cell line Method of Properties Role modification NdK ATPase (P) Tunicaniycin Reduced: Required for Oiiabain binding to 60% çompleie surface Alboint et al., 1992 Rb upiake io 60% expression Ern, Ep (no1 due IO reduçed protein synihesis or iniraccllular Na+)
Tunicaniycin Normal: -In siabilizing ihe protein synihesis subunits Reduced: Trypsin resisiance (a, p) -A non coniplex Ncwly synihcsizcd P & u chain riiay hc ATPasr: activity sufficient; however, coniplexiiy incrcases Normal: the irypsin p & a synihesis rcsistance Trypsin sensiiiviiy ATPase aciiviiy
Xenopris ooc y ic Normal: No1 required for ihe Ia &B) ATPase üciiviiy transpori Microsoriiül nieinhrüne Ouahain binding Rh iranspori
2ulturcd chick scnsory Normal: Nol in intraccllular icuran Asscnibly ~ransloçütion or Cell surfücc çxprcssioii ratc üssenibly bgrüdalio~l Reduced: 60% pproiin syiithesis
Table 1.4 Suii~iaüry OC ralcs of oligiisüccliarides (iii soiiic transporters and clianiicls Tuble 4.a AïPases
Antiportcr Ce11 linc hlethod af Role modification rransfccicd PS 120 hariisier N75D Normal: Noi in cc11 surface N~'/H' ~ihrahlüsi wiih IINHE-1 Aniiloridç sensiiivity expression or antiporter Yield of Ht suicide-surviving cells function Cnirnillori et rd., 1994
Ylrsirfi et nl., 1988 Rot rcnai hrush horder Swüinsoiiine ia vivo Reduced: Complex iype N- Amiloride sensitive Na uptükc linked sugars on the Vm, membrane direcily Normal: or indirectly Km influence transport activiiy Rcduccd: Rüic of Nat/Hi cxchünge Vm*, No cffect
Band 3 Reduced ln correct folding of Band 3 wiih the (Ery throcytc anion Band 3 rnediatcd Cl- influx highest activiiy cxchangcr) Groves & Trmaer, 1994
Normal Not in ihe transport 2' siruciurc funcîian Proicrise sensiiivity Inliihiior hinding Incrcascd Rüic of aggregüiion
Tablc 1.4 (Coniiiiucd) l'ahlc 4,d Aniiporicrs
Transporter Cell line Method of 1 Properties Role
Na/Glucose LLC-pKI Normal: Not required for Km lransport funciion symport Glucose uptake or membrane Wir & Lever 1994 Proieiii synthesis inserlion Ce11 surface expression Lack of: Contributes io ihe NaIGlucose syniport üciivity functional integrity
-- -- - BBMV PNGF Normal: No! in iransport Transport aciiuiiy aciivity
I N248Q Reduced: Not required for the Na/glucose syriiport (30%) func tional expression
. - Tunicatnycin Reduced: Methy lglucose upiake
N at/Pi syniport Functional transporter is cxpressed
Rat kidney cortex Normal Surface delivery (NaY,-2) N32SQ Apparcrit üffiniiy for P, or Na' H[J~~?Sel cri., 1994 Double pH depciidencc Rcduced Trünspori raie Incrcüsed Intracellulür localization
Table 1.4 (Continucd) Tuhle 4,f Na-dcpciidcnt sy iiipori p. . -- -- - Transporter Ccll line Mcthod of Properties IRole modification ~r&fccied rai adipose N57Q Rcduccd: Imporlani in cclls Expression biosynthcsis of iransportcr in Norriiül expression physiologically relevant tissues GLUT 1 Endoiheliuin of the hrnin Diffcrential N- Subcellular localization (apical A'rrnrtc~cti et al., 1991 capillaries glycosy lation vs büsolateral) Epithcliurii of the clioroid plexus
Astrrro et (il., 1993 CHO cclls Reduced: -1ntraccllular Astr~io et tri,, 1991 Cell surface expression targcting Hülf lire Protein stabiliiy Affiniiy for glucose Increased: -Maintaining a high Km affinicy binding site
Ferr~easet al., 1991 Nornial: A minimum chah is Zero-tram influx required for Rediiced: irünsport aciivity Viiiiix (25%)
Exoglycosidüscs
PNGF Rcduccd: Requircd for Vniüx (50%) transport Upiüke activiiy Sninll incrcasc in Kiii
1Yhecler d Hirikle, Rcconstiiuicd in liposome Normal: The whole complcx 1981 üfter trcütrricnl Nci upirikc of glucose chah is not required 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 the 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 the 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 site- 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 transporters mimics their general function in the biosynthesis of other glycoproteins. They are involved in protein foldinp and are 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, the 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 plasma membrane, providing the flexibility and stability required for the erythrocyte's function in the circulation. Protein elements of erythrocyte membrane are of two types, integral (intrinsic) and 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 th 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 the 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 the cytoplasmic domain of Band 3, the anion exchanger. Band 3 and glycophorin are two major erythrocyte integral me 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 the MM 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 same 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 th 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 the 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 and 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 transporter is mainly a-helical with 12 predicted transrnembrane segments (Figure 1.8-b). The carboxy- and amino-termini are located in the 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). TwO 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, in 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) and Jmd (Williams et ai., 1990). Blood ce11 formation in rnammals begins in the fetal yok 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 in 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 and gradually falls over the first year after birth, maintaining a low but 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 and 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 are committed to ultimately develop into the mature cells of a single lineage. Progenitors are assayed by their capacity to form colonies in 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 matu 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) and 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 the 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 the erythropoietin biologic functions (Shivdasani and Orkin, 1996). Pluripotential stem cek 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 erythrocyte I.S. HEMPAS Crookston was the first to propose (Crookston et al., 1969) the te r m HEMPAS for the congenital dyserythropoietic anemia type II, characterized in five patients by erythroblastic rnultinuclearity i n bone marrow, ineffective erythropoiesis and positive acidified seru 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 III based on the hematological and serological findings (Heimpel and Wendt. 1968). Table 1.5 shows a surnmary of these features.
Characreristics Type 1 Type 11 Type 111 Bone marrow Megaloblastic Mu1 tinuclearity Multinuclearity Chromatin Karyorrhexis Gigantoblasts bridging Erythrocytes Macrocytosis Normocytosis Macrocytosis EM Nuclear pore Double membrane CIefts in nuclei Acidified- serum lysis test Anti-I agglutination
agglutination lnheri tance Autosomal Autosornai Autosomal recessive recessive dominant
Table 1.5 Classical features of the three types of congenitd dyserythropoietic anemia (CDA) 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 and 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) and ery thropolidiscariosis hemolitico esplenornegalica (Grignaschi, f 970) were the sme as HEMPAS. CDA II (HEMPAS) is the most common type of CDA (Marks and
Mitus, 1996). Morphological abnormali ties of the bone m arr 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 from 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 in 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 and 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 the 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 an d hemosiderin granules (Crookston et al.. 1969). Marrow iron stores were 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 structure 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 an 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 the additional membrane derives from the ER, since immunogold electron microscopy on red blood cells has shown localization of an 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 in the patient's own serum. This implies that KEMPAS red cells carry an antigen not detectable on normal cells and that some normal sera contain the corresponding antibody (Verwilghen et al., 1973). This antibody cm 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 be induced by marrow stress caused by repeated phlebotomies (Hihan 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 as 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 pattern 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 th 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 oxidase/~a~[3~]4showed 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 the 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.
HEMPAS Enzymatic Cell type Reference patient defect 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 was 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 chah on the al-3 am, which supported the presence of a lesion in GnT II. However, Band 3 purified frorn patient B. D. carried bo th 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 the al-6 arm and a subsequent reduction of lactosaminoglycans, while in 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 arm 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 the 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 am 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 five 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 am 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 een 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 stem 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. cannot make "HEMPAS glycan". In addition, a lesion in GalT blocks synthesis of poly N-acetyllactosamine on all antennae; consequently it affects tri- and tetra-antennary structures as much as biantennary. Probabfy this is the reason for the observation of membrane abnormality in G. K.'s granulocytes, which normally present tri and tetra-antennary poly N-acetyllactosamine chains. In typical cases of KEMPAS, ce11 lines other than erythroid are not usually affected, probably because they contain sugars with different structures compared to erythrocytes (Fukuda, 1990). However, analysis of carbohydrates from HEMPAS (G. K.) serum transferrin shows altered structures which may play a role in the 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 are 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 has 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 MCCorp.; UDP-[~~CIG~CNACa n d 3-0-rneth~l-['~~]~lucosefrorn NEN Research Products; UDP-[~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. Schachter' 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 1640 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 158 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), stem cell factor (50 nglml), hemin (100 PM)and al1 trans-retinoic acid (ATRA) (30 mM). The Petri dishes containing the wells were 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. II 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 mM sodium phosphate, pH 8.0 (5P8) in the presence of 1 mM 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 and filtered in the SpinBind DNA extraction unit to remove DNA. The resulting ce11 extracts were used for the enzymatic deglycosy 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 and 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 mM EDTA, 50 mM 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 an
' One unit is defined as the amount of enzyme required to remove >95% of the carbohydrate from 10 pg of denatured RNase B in a 10 pl reaction at 37°C in I hou. ' 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 dansyl- Asn(GLcNac),(Man), within 1 min nt 37T 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 mM 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 mol, 18 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 UDP-(~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 Cl8 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 as 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 WP 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~]glucosetransport 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 or 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 with 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~]~lucoseuptake in 30 s was expressed as 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 (21) 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 mM calcium chloride and 20 mg pronase. The digest was incubated at 37 OC 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 the 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 mM CaCI,, 1 mM 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 15 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 which 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 fom 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 are probably the result of proteolysis, to which HEMPAS membranes are 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 ry throcyte membrane proîeins The enzyme peptide N-glycosidase F hydrolyzes the N- bdycosidic linkage between peptides and all three types of N-linked 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 and 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 the linkage between the first two N-acetyglucosamine residues in the Figure 2.2.2 Enzymatic deglycosylation of e r y th 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 16 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 hybrid or a high mannose oligosaccharide in HEMPAS patients. ECA, a lectin specific for N-acetyllactosamine disaccharide, do es not bind to HEMPAS Band 3 and Band 4.5 (Figure 2.2.5, lanes 3 & 4), while it 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 may 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 the 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 of 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 as 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 and 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 erythrocyte membrane proteins Erythrocyte membrane proteins were resolved by SDS-PAGE on a 10% polyacrylarnide gel. ECA lectin blot was performed and 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 CO 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 II) (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 the 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 that 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 + +- PAS I (GPAdimer)
Actin + + 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 the HEMPAS condition and that the defect may be restricted to the 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 the 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 in 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 added 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 were measured as described in Methods. Panel A, GnT II assay (in duplicates); panel B, GalT assay (error bars represent standard deviations). Cnt: normal control; L.F., C.L., M.D. and M.K.: four 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 cek (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 the role of oligosaccharide in the transport function of Band 4.5, since the 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 of extracts from cultured human erythroid cells Normal cultured erythroid cells (= 3 x IO6) 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. PanelA, 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 arrow probably represents endogenous biotinylated proteins.
LOO
Figure 2.2.11 3-0-methyl-[~~~]glucoseuptake by control and HEMPAS erythrocytes The glucose uptake was measured as described in Methods and 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 goup 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 antibody 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 in 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 by glycosidase contamination in pronase or poor digestion of the precipitated glycoprotein by pronase. The purification proceeded by 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 and Figure 2.2.13-A Thin-layer chromatogram of erythrocyte oligoglycosyl ceramides €rom normal control and HEMPAS patient 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) and 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 densitometry 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 small peaks of sugar, eluted prior to the large peak of methymannoside (total volume), were prepared for NMR studies. The NMR spectra 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 short 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 chah. 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 cm 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 at 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) and 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+) re 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-
Dulycosylation of HEMPAS Band 3. Under-sialylated GPA may be ab le to interact with Band 3 more readily than normal GPA and subsequently increase the rate of Band 3 translocation to the ce11 surface. This ultirnately reduces the chance of the protein to be 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 or 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 the 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 arte ry diseasr frequently occurring before LO years of age. poly N-acetyllactosamine content of Band 3 is 50% of normal in this patient (Chmk 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 the 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 CU 1tured 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 the 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 al-3 am 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 be made in dl 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 the 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 further 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 did 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 in normal cells, a more sensitive technique has been developed, Le., the 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 are 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 red 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 the 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 ha 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 canot b e eliminated. Studies on rabbit bone marrow erythroid cells have shown a progressive increase in ce11 surface expression of Band 3, up to the reticulocyte stage (Foxwell and Tanner, 198 1). In future studies O n HEMPAS erythroid cultures, it is important to be aware that the incorporation of Band 3 into the membrane may be delayed to the 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 their 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 the 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 car 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 argeme 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 the 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 in 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 are 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 the lysosomes of the liver cells, may cause the liver cirrhosis and 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, an 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 three other siblings suffering from CDA II the increase in the glycosphingolipid content was accompanied by an increase in the 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 (Gockem et ai., 1975). Based on our 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 quantitative 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 1ts 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 in 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 the 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 the 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 cm conclude that the specific poly N-acetyllactosarnine chah is not essential for the 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 the 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 that 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 and 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 transport 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 that contribute to the understanding of the clinical and etiological aspects of EMPAS. 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 on 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 be rnetaboIically Labeled by specific radioactive sugars. Im m u no - isolated Band 3 can be analyzed for incorporation of the radiolabelled sugar. My studies on EBV-transformed lymphoblasts suggest that the glycosylation defect associated with HEMPAS is restricted to the erythroid cell Iine. Therefore, it will be of great value to maintain an erythroid ceIl culture from the HEMPAS patients. However, it is essential to first assure that the glycosylation rnachinery is active and Band 3 is expressed in these cultured cells. This work has 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 the synthesis of N-linked sugars towards the bisected hybrid structure. The activity of this enzyme can be assessed in the cultured erythroid cells by a fluorescent assay using a biantennary sugar chah and 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 trave l from the ER to the Golgi, where the oligosaccharide is processed, and between its compartments by means of non-clathrin coated vesicles
0 et ai. 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 structural 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 cm 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 the vesicles and subsequent blockage between the ER and Golgi cm be 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 two 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 or immunofluorescent microscopy using an antibody to human a- mannosidase II. If rnislocalization of the enzyme is the case, the 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. If 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 that 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. REFERENCES
Achord, D. T., Brot. F. E., Bell, C. E.. and Sly, W. S. (1978). Human beta- glucuronidase: in vivo clearance and in vitro uptake by a glycoprotein recognition system on reticuloendothelial cells. Ce11 15,269-278.
Alboim, S. V., Bak. A., and Sampson, S. R. (1992). Tunicamycin reduces Na+-Kf- pump expression in cultured skeletal muscle. J. Cell. Physiol. 150, 640-646.
Alloisio, N., Texier, P., Denoroy, L., Berger, C., Miragha del Giudice. E., Perrotta, S., Iolascon, A., Gilsanz, F., Berger, G., and Guichard, 1. (1996). The cisternae decorating the red blood ce11 membrane in congenitd dyserythropoietic anernia (type II) originate from the endoplasrnic reticulum. Blood 87,4433-4439.
Ando, K., Kikugawa, K., and Beppu, M. (1996). Binding of anti-Band 3 antibody to sialylated poly N-acetyllactosaminyl sugar chains of Band 3 glycoprotein on polyvinylidene difluoride membrane and Sepharose gel: funher evidence for anti-Band 3 autoantibody binding to the sugar chains of oxidized and senescent erythrocytes. I. Biochem. 119, 639- 647.
Anselstetter, V.. Horstmann, H. J., and Heirnpel, 8. (1977). Congenital dyserythropoietic anaemia, types 1and II: aberrant pattern of erythrocyte membrane proteins in CDA II, as revealed by two-dimensional polyacrylarnide gel electrophoresis. Br. J. Hematol. 35, 209- 2 15.
Asano, T., Katagiri, H., Takata. K., Lin, LL.. Ishihara, H., Inukai, K., Tsukuda, K., Kikuchi, M., HVano, H.. Yazaki. Y., and Oka, Y. (199 1). The role of N-glycosylation of GLUTl for glucose transport activity. l. Biol. Chem. 266,24632-24636.
Asano, T., Takata, K., Katagiri, H., Ishihara, H., Inukai, K., Anai, M., Hirano, K., Yazaki. Y.. and Oka, Y. (1993). The role of N-glycosylation in the targeting and stability of GLUT 1 glucose transporter. FEBS Lett. 324. 258-26 1. Ashwell, G., and Morell, A. G. (1974). The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv. Enzymol. Relat. Areas Mol. Biol. 41, 99- 128.
Bai, L., Fushimi, K., Sasaki, S., and Marumo, F. (1996). Stnicnire of aquaporin-2 vasopressin water channel. .J. Biol. Chem. 271, 5 17 1-5 176.
Baines, A. J., Banga, J. P., Gratzer, W. B., Linch, D. C.. and Huehns. E. R. (1982). Red celi membrane protein anomalies in congenital dyserythropoietic anaemia, type II (HEMPAS). Br. J. Hematol. 50, 563-574.
Baldwin, S. A. (1993). Mii~~lltlalianpassive glucose transporters: members of an ubiquitous family of active and passive transport proteins. Biochim. B iophys. Acta 1154, 17-49.
Barosi, G., and Cazzola, M. ( 1979). Snidies of ineffective erythropoiesis and periphenl haemolysis in congenitd dyserythropoietic anaemia type II. Br. J. Hernatol. 43,243-250.
Bergeron, J. I., Brenner, M. B., Thomas, D. Y., and Williams, D. B. (1994). Calnexin: a membrane-bound chaperone of the endoplasmic reticulum. Trends Biochem. Sci. 19, 124- 128.
Birnir, B., Lee, H.-S.. Hediger, M. A., and M., W. E. (1990). Expression and characterization of the intestinal Ndglucose cotransporter in COS-7 cells. Biochim. Biophys. Acta 1048, 100-104.
Boogaerts, M. A., and Verwilghen, R. L. (1982). Variants of congenital dyserythropoietic amernia: an update. Haematologia 15,21 1-2 19.
Bosman, G. J., and Kay, M. M. (1990). Alterations of band 3 transport protein by cellular aging and disease: erythrocyte Band 3 and glucose transporter share a fünctional relationship. Biochem. CeU Biol. 68, 14 19- 1427.
Bouhours, J. F., Bouhours, D., and Delaunary, J. (1985). Abnormal fatty acid composition of er ythrocyte gl y cosphingoiipids in congenitd dy serythropoie tic anemia type II. J. Lipid Res. 26, 435-441. Bretscher, M. S., and Munro, S. (1993). Cholesterol and the Golgi apparatus. Science 261, 1280-1281.
Bruce, L. J., Groves, J. D., Okubo, Y., Thilaganathan, B., and Tanner, M. J. A. (1994). Altered band 3 structure and function in glycophorin A- and Bdeficient (MkMk) red blood cells. BIood 84, 9 16-922.
Burke, J., Pettitt, .J. M., Humphris, D., and Gleeson, P. A. (1994). Mediai-Golgi retention of N-acetylglucosaminyl~sferase 1. Contribution from di domains of the enzyme. .J. Biol. Chem. 269, 12049-12059.
Cabantchik, 2. I., and Rothstein, A. (1974). Membrane proteins related to anion permeability of hurnan red blood cells. 1. Localization of disulfonic stilbene binding sites in proteins involved in permeation. J. Membr. Biol. 15,207-226.
Carlsson, S. R., and Fukuda, M. (1990). The polylactosaminoglycans of human lysosomal membrane glycoproteins lamp-1 and lamp-2. Localization on the peptide backbones. J. Biol. Chern. 265, 20488-20495.
Carruthers, A. (1990). Facilitated diffusion of glucose. Physiol. Rev. 70, 1 135-1 176.
Casey, J. R., Lieberman, D. M., and Reithmeier, R. A. F. (1989). M~cationand characterization of Band 3 protein. Methods Enzymol. 173,494-5 12.
Casey, J. R., Pirraglia, C. A., and Reithmeier, R. A. (1992). Enzymatic deglycosylation of human Band 3, the anion transport protein of the erythrocyte membrane. Effect on protein structure and transport properties. l. B iol. Chem. 267, 11940- 11948.
Charuk, J. H. M., Tan, J., Reithmeier, R. A. F.. laeken, J., and Schachter, H. (1995). Carbohydrate-deficient glycoprotein syndrome type II. An autosomal recessive N- acetylglucosaminyl~fenseII deficiency different from typical hereditary erythroblastic rnultinuclearity, with a positive acidified-serum lysis test (HEMPAS). Eur. J. Biochem. 230, 797-805. Chen, W., Helenius, I., Braakman, I., and Helenius, A. (1995). Cotranslational folding and calnexin binding during glycoprotein synthesis. Proc. Natl. Acad. Sci. USA 92, 6229- 6233.
Childs, R. A., Dalchau, R., and Feizi, T. (1983). Evidence for the occurrence of O- glycosidically Linked oligosaccharides of poly-N-acetyllactosarnine type on the human leucocyte cornrnon antigen. B iochem. Biophys. Res. Commun. 110,424431.
Colley, K. J., Lee, E. U., and Paulson, J. C. (1992). The signal anchor and stem regions of the beta-gaiactoside alpha 2,6-sialyltrmsferase may each act to locaiize the enzyme to the Golgi apparatus. J. Biol. Chem. 267, 7784-7793.
Counillon, L., Pouyssegur, J., and Reithrneier, R. A. F. (1994). The Na/H exchanger MIE-1 possesses N- and O-linked glycosylation restricted to the fint N-terminal extracellular domain. B iochernisüy 33, 1O463- 10469.
Crookston, J. H., Crookston, M. C., Burnie, K. L., Francombe, W., H., Dacie, J. V., Davis, J. A., and Lewis, S. M. (1969). Hereditary erythroblastic multinuclearîty associated with a positive acidified-semm test: a type of congenital dyserydiropoietic anaemia. Br. I. Hematol. 17, 1 1-26.
D'Agostaro, G., Bendiak, B., and Tropak, M. (1989). Cloning of cDNA encoding the membrane-bound form of bovine beta 1,4-galactosyltransferase. Eur. J. B iochem. 183, 2 1 1-217.
De LozWo, C. B., Valencia, J. I., md Acame, E. A. (1962). Chrornosomd study in erythroblastic endopolyploidy. Lancet 1, 1004.
Edge, C. J., loao, H. C., Woods, R. J., and Wormald, M. R. (1993). The conformational rffects of KLinked glycosylation. Biochem. Soc. Tram. 21,45245.
Evans, S. C., Lopez, L. C., and Shur, B. D. (1993). Dominant negative mutation in ceil surface beta 1.4-galactosyltransfense inhibits cell-cell and cell-matrix interactions. J. Cell. Biol. 120, 1045- 1057. Feugeas, J.-P., Neel, D., Goussault, Y., and Derappe, C. (199 1). Glycosylation of the human erythrocyte glucose transporter: a minimum structure is required for glucose transport activity. Biochim. Biophys. Acta 1066,59-62.
Feugeas, J.-P., Neel, D., Pavia, A. A., Laham. A., Goussault, Y., and Derappe, C. ( 1990). Glycosylation of the hurnan erythrocyte glucose transporter is essential for glucose transport activity. Biochim. Biophys. Acta 1030,60-64.
FIorensa, L., Woesoner, S ., Besses, C., and Sans-Sabrafen, J. ( 1994). Congenital dyserythropoietic anernia type II: rnorphological chamterkation of the erythroid colonies (BFU-E)from the bone marrow and peripheral blood of two patients. Ann. Hematol. 69, 57-59.
Foxwell, B. M. I., and Tanner, M. 1. ( 198 1). Synthesis of the erythrocyte anion-transport protein. [mmunochemical study of its incorporation into the plasma membrane of erythroid cells. Biochem. J. 195, 129-137.
Fucentese, M., Winterhalter, K., Murer. K., and Biber, 1. ( 1993). Functionai expression of rat rend Nei-cotransport (NaPi-2) in Sf9 cells by the baculovims system. J. Membr. Biol. 144. 43-48.
Fukuda, M. N., Watanabe, K., and Hakomori, S. 1. (1978). Release of oligosaccharides from various glycosphingolipids by endo-beta-galactosidase. J. Biol. Chem. 253, 68 14- 68 19.
Fukuda, M., Fukuda, M. N., and Hakomori, S. 1. (1979). Developmental change and genetic defect in the carbohydnte structure of Band 3 glycoprotein of human erythrocyte membrane. J. Biol. Chem. 254,3700-3703.
Fukuda, M., Deii, A., and Fukuda. M. N. (1984a). Structures of glycosphingolipids isolated from human granulocytes. The presence of a senes of ünear poly-N- acetyUactosaminylceramide and its sigrilncance in glycolipids of whole blood ceils. J. Biol. Chem. 259,4782479 1. Fukuda, M., Spooncer, E., Oates, J. E., Dell, A., and Klock, J. C. ( 1984b). Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes. J. Biol. Chem. 259, 10925- 10935.
Fukuda, M. N., Papayannopoulou, T., and Testa, U. (1984~).Defect in giycosylation of erythrocyte membrane proteins in congenital dyserythropoietic anaemia type II (HEMPAS). Br. J. Hematol. 56, 55-68.
Fukuda, M., Dell, A., Oates, J. E. and Fukuda, M. N. (1984d). Structure of branched lactosaminoglycan, the carbohydrate moiety of Band 3 isolated from adult human erythrocytes. I. Biol. Chem. 259, 8260-8273.
Fukuda, M. N., Bothner, B., Scartezzini, P., and Dell, A. (1986a). Isolation and characterization of poly-N-acetyllactosaminylceramides accumulated in the erythrocytes of congenital dyserythropoietic anemia type II patients. Chem. Phys. Lipids 42, 185- 197.
Fukuda, M. N., Klier, G., Yu, J., and Scartezzini, P. (1986b). Anomalous clustenng of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II. Blood 68,571-529.
Fukuda, M. N., Deil, A., and Scarteuini, P. (l987a). Primary defect of congenitd dyserythropoietic anemia type II. Failure in glycosylation of exythrocyte lactosaminoglycan proteins caused by lowered N-acetylglucosarninyltransferase II. J. Biol. Chem. 262, 7 195- 7206.
Fukuda, M. N., Klier. G., and Scartezzini, P. ( l987b). Congenital dyserythropoietic anaemia type II (HEMPAS): characterization of aberrant intracellulu organelles by immunogold electron microscopy. Br. J. Hematol. 67,95-10 1.
Fukuda, M., Guan, J.-L., and Rose, 1. K. (1988). A membrane-anchored form but not the secretory form of human chorionic gonadotropin-alpha chah acquires polylactosaminoglycan. I. Biol. Chem. 263,53 14-53 18.
Fukuda, M. N., Masri, K. A., Dell, A., Thonar, E. I., mer, G., and Lowenthal, R. M. (1989). Defective glycosylation of eIythrocyte membrane glycoconjugates in a variant of congenital dyserythropoietic anemia type II: association of low level of membrane-bound form of galactosyluansferase. Blood 73, 133 L- 1339.
Fukuda, M. N. ( 1990). HEMPAS disease: genetic defect of glycosylation. Glycobiology 1, 9-15.
Fukuda, N., Masri. K. A., Dell, A., Lunatto, L., and Moremen, K. W. (1990). Incomplete synthesis of N-glycans in congenital dyserythropoietic anemia type II caused by a defect in the pne encoding alpha-mannosidase II. Proc. Nad. Acad. Sci. USA 87, 7443- 7447.
Fukuda, M. N., Gaetani, G. F., Iuo, P., Scarteuini, P., and Dell, A. (1992). Incompletely processed N-glycans of serum glycoproteins in congenitd dyserythropoietic anaemia type U (HEMPAS). Br. J. Hematol. 82,745-752.
Fukuda, M., and Kobata, A. ( 1993). Glycobiology: A Practicai Approach, Volume 125, D. Rickwood and B. D. Hmes, eds.: IRL Press).
Fukuda, M., and Hindsgaul, 0. (1994). Molecular Glycobiology, B. D. Harnes and D. M. Glover, eds.: EU. Press).
Gahrnberg, C. G., Myllylû, G.. Leikola, J., Pirkola, A., and Nordling, S. (1976). Absence of the major sialoglycoprotein in the membrane of human En@-) erythrocytes and increased glycosylation of Band 3. J. Biol. Chem. 251.6108-6 116.
Gahmberg, C. G., and Tolvanen, M. ( 1996). Why m;unmalian ce1 surface proteins are glycoproteins? Trends Biochem. Sci. 21,308-3 11.
Gibson, R., Kornfeld, S., and Schlesinger, S. (1981). The effect of oligosaccharide chas of different sizes on the maturation and physical properties of the G protein of vesicular stomatitis virus. J. Biol. Chem. 256,456462-
Gilmore, R. ( 1993). Protein translocation across the endoplasmic reticulum: a tunnel with toli booths at entry and exit. Cell75,589-592. Gockerman, J. P., Durocher, J. R., and Conrad, M. E. ( 1975). The abnormal surface characteristics of the red blood ce11 membrane in congenital dyserythropoietic anaemia type II (HEMPAS). Br. J. Hematol. 30, 383-394.
Gooi, H. C., Veyrieres, A., Alais, J., Scudder, P., Hounsell, E. F., and Feizi, T. (1984). Further studies of the specificities of monoclonai anti-i and anti-I antibodies using chemically synthesized, linear oligosaccharides of the poly-N-acetyllactos~ne series. Mol. Immunol. 21, 1099- 1 104.
Greiner, T. C., Burns, C. P., Dick, F. R., Henry, K. M., and Mahmood, 1. (1992). Congenitd dyserythropoietic anemia type iI diagnosed in a 69-year-old patient with iron overload. Am. J. Clin. Pathol. 98, 522-525.
Grignaschi, V. 1. (1970). Eritropolidiscariosis hemolitico-esplenomegalica. Sangre 15, 57- 72.
Groves, J. D., and Tanner, M. 1. (1992). Glycophonn A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes. I. Biol. Chem. 267. 22 163- 32170.
Groves, 1. D., and Tanner, M. 1. (1994). Role of N-glycosylation in the expression of human band 3-rnediated anion transport. Mol. Membr. B iol. 11,3 1-38.
Gu, I., Nishikawa, A., and Taniguchi, N. (1992). Biosynthesis of blood group I and i antipens in nt tissues. Identification of a novel beta 1-6-N-acetylgiucosaminyltransferase. J. Biol. Chem. 267, 2994-3999.
Hakomori, S. (1981). Blood group ABH and Ii antigens of human erythrocytes: chemistry, polymorphism, and their developmental change. Semin. Hematol. 18.39-62.
Handin, R. L, Lux, S. E., and Stossel, T. P. (1995). Blood: Principles and Practice of Hematology : 1. B. Lippincott Company.
Harik, S. 1. (1992). Changes in the glucose transporter of brain capillaries. Cm. .J. Physiol. Pharmacol.70 Suppl, S 113-1 17. Hartow, R. W., and Lowenthal, R. M. (1982). Erythrocyte membrane proteins in an unusual case of congenital dyserythropoietic anaernia type II (CDA II). Br. J. Hematol. 50, 35-4 1.
Hart, G. W., Haltiwanger, R. S., Holt, G. D., and Kelly, W. G. (1989). Glycosylation in the nucleus and cytoplasm. Annu. Rev. Biochem. 58, 841-874.
Hayes, G., Busch, A., Lotscher, M., Waldegger, S., Lang, F., Verrey, F., Biber, J., and Murer, H. (1994). Role of N-linked glycosylation in nt rend NdPi cotranspon. J. Biol. Chem. 269, 34 143-24 149.
Hebert, D. N., FoeIlmer, B., and Helenius, A. (1995). Glucose uimrning and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulurn. Ce11 81,425-433.
Hediger, M. A., Mendlein, J., Lee, H.-S., and Wright, E. M. (1991). Biosynthesis of the cloned intestinal Ndglucose cotransporter. Biochim. Biophys. Acta 1064,360-364.
Heimpel, H., and Wendt, F. (1968). Congenital dyserythropoietic anemia with karyorrhexis and multinucle~tyof erythroblasts. Helv. Med. Acta 34, 103-1 15.
Helenius, A. (1994). How N-linked oiigosaccharides affect glycoprotein folding in the endoplasmic reticulum. iMol. Biol. Ce11 5, 253-265.
Hillman, R. S., and Giblett, E. R. (1965). Red cell membrane alteration associated with "marrow stress". J. Clin. Invest. 44, 1730-1734.
Hirayama, B. A., and Wright, E. M. (1992). Glycosylation of the nbbit intestinai bmsh border Na/glucose coüansporter. Biochim. Biophys. Acta liO3,37-44.
Hogue, D. L., Hodgson, K. C., and Cass, C. E. (1990). Effects of inhibition of N-linked glycosylation by ninicarnycin on nucleoside transport polypeptides of L 1210 leukernia cells. Biochem. Cell. Biol. 68, 199-209.
Hozumi, L, Nishizawa, M., Ariga, T., and Miyatake, T. (1990). Biochemical and ciinical analysis of accumuiated glycoiipids in syrnptomatic heterozygotes of angiokeratoma corpons difisum (Fabry's disease) in cornparison with hernizygotes. J. Lipid Res. 3 1, 335-340.
Hubbard, S. C., and Ivatt, R. J. (1981). Synthesis and processing of asparagine-linked oligosacchuides. Annu. Rev. Biochem. 50,555-83.
Ing, B. L., Robinson, K. A., Buse, M. G., and Quon, M. J. (1996). Characterization of a mutant GLUT4 lacking the N-glycosylation site: studies in transfected rat adipose cells. Biochem. Biophys. Res. Comm. 218,7642.
Ioffe, E., and Stanley, P. (L994). Mice lacking N-acetylglucosaminyltnnsferase 1 activiv die at mid-gestation, revealing an essential role for complex or hybnd N-linked carbohydrates. Proc. Natl. Acad. Sci. USA 91, 728-732.
Jaeken, J., Schachter, H., Carchon, H., De cock, P., Coddeville, B., and Spik, G. ( 1994). Carbohydrate deficient glycoprotein syndrome type II: a deficiency in Golgi localised N-acetyl-glucosaminyltransferaseII. Arch. Dis. Childhood71, 123- 127.
Joseph, K. C., Gockerman, J. P., and Alving, C. R. (1975). Abnormal lipid composition of the red celi membrane in congenital dyserythropoietic anexnia type II (HEMPAS). J. Lab. Clin. Med. 85, 34-40.
Karim, A. R., Virk, P. K., Rossi, M. L., Williams, A. C., and Sturman, S. G. (1993). Interaction of motor neurone disease plasma with glucose transport. Funct. Neurol. 8, 89- 94.
Kelleher, D. J., Kreibich, G., and Gilmore, R. (1992). OLigosaccharyl~ansferaseactivity is associated with a protein complex composed of ribophorins 1 and II and a 48 kd protein. Ceil 69,5565.
Kornfeld, R., and Komfeld, S. ( 1985). Assembly of asparagine-linked oligosaccharides. Annu, Rev. Biochem. 54,63 1-664.
Kornfeld, S. (1982). OLigosaccharides processing during glycoprotein biosynthesis. In The Glycoconjugates, M. 1. Horowitz, ed. (New York: Academic Press), pp. 3-23. Kornfeld, S., Reitman, M. L., Varki, A., Goldberg, D., and Gabel, C. (1982). Steps in the phosphorylation of the high mannose oligosaccharides of lysosomal enzymes. Ciba Foundation Symposium 92, 138- 156.
Koscielak. J. (1995). Diseases of aberrant glycosylation. Acta Biochimi. Pol. 43, 1-10.
Kmsius, T., Finne, 3.. and Rauvala, H. (L978). The poly(g1ycosyi) chahs of glycoproteins. Characterisation of a novel type of glycoprotein saccharide from human erythrocyte membrane. Eur. J. Biochem. 92,289-300.
Kumagai, A. K., Dwyer, K. J., and Pardndge, W. M. (1994). Differentiai glycosylation of the GLUTl glucose transporter in brain capillaries and choroid plexus. Biochim. Biophys. Acta 1193, 24-30.
Kumar, R., Yang, I., Larsen, R. D., and Stanley, P. (1990). Cloning and expression of N-acetylglucosaminyltransferase 1, the medial Golgi tramferase that initiates complex N- linked carbohydrate formation. Proc. Natl. Acad. Sci. USA 87,9948-9952.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bactenophage T4. Nature 227,680-685.
Lee, N., Wang, W. C., and Fukuda, M. (L990). Granulocytic differentiation of HL-60 ceiis is associated with increase of poly-N-acetyllactosaminein Asn-tinked oligosaccharides attached to human lysosomal membrane glycoproteins. l. Biol. Chem. 265,20476-20487.
Lew, R., Patel, A., Vaughan, R. A., Wilson, A., and Kuhar, M. J. (1992). Microheterogeneity of dopamine transportes in rat saiatum and nucleus accumbens. Brain Res. 584, 266-27 1.
Low, P. S., Waugh, S. M., Zlnke, K., and Drenckhahn, D. (1985). The role of hemogiobin denahiration and band 3 cluste~gin red blood ceil aging. Science 227, 53 1- 533.
Lowenthal, R. M., Marsden, K. A., Dewar, C. L., and Thompson, G. R. ( 1980). Congenital dyserythropoietic anaemia (CDA) with severe gout, rare Kell phenotype and erythrocyte, granulocyte and platelet membrane reduplication: a new variant of CDA type II. Br. 1. Hematol. 44, 2 1 1-320.
Lowry, O. H., Rosebrough, N. J., Fm, A. L., and Randdl, R. 1. (1% 1). Protein measurement with the folin phen01 reagent. I. Biol. Chem. 193,265-275.
Maley, F., Trimble, R. B., Tarentino, A. L., and Plumrner, J., H. (1989). Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal. Biochem. 180, 195-204.
Marks, D. L., Larkin, J. M., and McNiven, M. A. (1994). Association of kinesin with the Golgi apparatus in rat hepatocytes. J. Cell. Sci. 107,2417-2426.
Marks, P. W., and Mitus, A. J. (1996). Congenitd dyserythropoietic anernias. Am. I. Hematol. 51, 55-63.
Mawby, W. J., Tanner, M. 1. A., Anstee, D. I., and Clamo, J. R. (1983). Incomplete glycosylation of erythrocyte membrane proteins in congenital dyserythropoietic anaemia type II. Br. J. Hematol. 55, 357-368.
May, I. M. (1988). Inhibition of hexose transport and labelhg of the hexose carrier in human erythrocytes by an irnpermeant maleimide dcnvative of maltose. Biochem. J. 254, 329-336.
Melikian, H. E., McDonald, J. K., Gu, H., Rudnick, G., Moore, K. R., and Blakely, R. D. ( 1994). Human norepinephnne transporter. Biosynthetic studies using a site-directed polyclonal antibody. 1. Biol. Chem. 269, 1229042297.
Merkle, R. K., and Cummings, R. D. (1987). Relationship of the terminal sequences to the leno@ of poly-N-acetyuactosamine chahs in asparagine-linked oligosaccharides from the mouse lymphoma ceU iine BW5 147. Immobilized tornato lectin interacts with high affinity with giycopeptides containing long poly-N-acetyllactosamine chahs. I. Biol. Chem. 262, 8179-8189. Metzler, M., Gertz, A., Sarkar, M., Schachter, H., Schrader, J., and Marth, J. ( 1994). Complex asparagine-linked oligosaccharides are required for rnorphogenic events during post-implantation development. EMBO I. 13.2056-2065.
Montreuil, J., Wegenthart, J. F. G., and Schachter, H. ( 1995). Glycoproteins, Volume 29a, A. Neuberger and L. L. M. Van Deenen, eds.: Elsevier).
Nabi, 1. R., and Rodriguez-Boulan, E. (1993). Increased LAMP- polylactosamlne glycosylation is associated with its slower Golgi transit during establishment of a polarized MDCK epithelial monolayer. Mol. Biol. Cell 4,627-635.
Nilsson, T., Slusarewicz, P., Hoe, M. H., and Warren, G. (1993). Kin recognition. A mode1 for the retention of Golgi enzymes. FEBS Lett. 330, 1-4.
Nunez, E., and Aragon, C. (1994). Stmctural analysis and hinctional role of the carbohydrate component of glycine transporter. J. Biol. Chem. 269, 16920- 16924.
Olivares, L., Aragon, C., Girnenea, C., and Zafra, F. (1995). The role of N-glycosylation in the targeting and activi~of the GLYTl giycine transporter. J. Biol. Chem. 270, 9437- 943.
Orci. L., Glick, B. S., and Rothman, J. E. (1986). A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack. Ce11 46, 17 1-184.
Ott, R. J., Hui, A. C., and Giacomini, K. M. (1992). Inhibition of N-Linked glycosylation affects organic cation transport across the bmsh border membrane of opossum kidney (OK) ceils. J. Biol. Chem. 267, 133-139.
Otter, M., Zockova, P., Kuiper, J., van Berkel, T. J., Barrett-Bergshoeff, M. M., and Rijken, D. C. ( 1992). Isolation and characterization of the maonose receptor from human liver potentially involved in the plasma clearance of tissue-type plasminogen activator. Hepatol. 16, 54-59.
Parekh, R. B. (199 1). Effects of glycosylation on protein function. Curr. Opin. Structural Biol. 1, 750-754. Pelham, H. R. B ., and Munro, S. (1993). Sorting of membrane proteins in the secretory pathway. Ce11 75, 603-605.
Petry, K., Greinix, H. T., Nudelman, E., Eisen, H., hakomori, S., Levy, H. L., and Reichard, J. K. (199 1). Characterization of a novel biochemical abnormality in galactosemia: deficiency of glycoiipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes. Biochem. Med. Metab. Biol. 46,93- 104.
Piller, F., and Cartron, I.-P. ( 1983). UDP-G1cNAc:Gal beta 1-4Glc(NAc) beta 1-3N- acetylglucosaminyltransferase Identification and characterization in human serum. J. Bioi. Chem.258, 12293- 12299.
Piller, F., Cartron, J.-P., Maranduba, A., Veyrieres, A., Leroy, Y., and Foumet, B. (1984). Biosynthesis of blood group 1 antigens. Identification of a UDP-GlcNAc:GlcNAc beta 1-3Gal(-R) beta 1-6(GlcNAc to Gd) N-acetylglucosaminy1transferase in bog gastric mucosa. J. Biol. Chem. 259, 13385- 13390.
Reithrneier, R. A. F., Chan, S. L., and Popov, M. (1996). Structure of the erythrocyte Band 3 anion exchanger. In Handbook of Biological Physics, W. N. Konings, H. R. Kaback and J. S. Lolkema, eds.: Elsevier Science B. V.), pp. 28 1-309.
Roberts, P. D.. Wallis, P. G., and Jackson, A. D. M. (1962). Haemolytic mernia with multinucleated normoblasts in the marrow. Lancet 1, 1186.
Rosse, W. F. ( 1990). Phosphatidyhositol-linked proteins and paroxysmal nocnimal hemoglobinuria. Blood 75, 1595- 160 1.
Rothman. J. E., and Orci, L. (1992). Molecular dissection of the secretory pathway. Nature 355,40945.
Santacniz-Toloza, L., Huang, Y., John, S. A., and Papazian, D. M. (1994). Glycosylation of Shaker potassium channel protein in insect cell culture and in Xenopus oocytes. Biochem. 33, 5607-56 13. Sarabia, V. E., Casey, I. R., and Reithmeier, R. A. F. (1993). Molecuiar chmcterization of the band 3 protein from Southeast Asian ovalocytes. I. Biol. Chem. 268, L0676-10680.
Scartezzini, P., Forni, G. L., and Sanaone, G. (1 982). Decreased glycosylation of band 3 and band 4.5 glycoproteins of erythrocyte membrane in congenital dyserythropoietic anaernia type II. Br. J. Hematol. SI, 569-576.
Scheiffele, P., Peranen, i., and Simons, K. (1995). N-glycans as apical sorting signals in epithelial cells. Nature 378,96-98.
Schwalbe, R. A., Wang, Z., Wible, B. A., and Brown, A. M. (1995). Potassium channel structure and function as reported by single glycosylation sequon. I. Biol. Chem. 270, 15336- 15340.
Shaper, I. H., Hollis, G. F., and Shaper, N. L. (1988). Evidence for two foms of murine beta- 1,J-galactosyluansferse based on cloning smdies. Biochimie 70, 1683- 1988.
Shivdasani, R. A., and Orkin, S. H. (1996). The transcriptionai control of hematopoiesis. Blood 87, 4025-4039.
Shur, B. D. (1982). Evidence that galactosyltransferase is a surface receptor for poly(N)- acetyllactosamine glycoconjugates on embryonal carcinoma cells. I. Biol. Chem. 257, 687 1-6878.
Shur, B. D. (199 1). Cell surface beta 1,4 galactosyltransferase: twenty years later. Glycobiology 1,563-575.
Stanley, P., and Ioffe, E. (1995). Glycosylaansfer~emutants: key to new insights in glycobiology. FASEB 1. 9, 1436-1444.
Sm,Y., Hirabayashi, Y., Suniki, T., and Matsumoto, M. (1985). Occurrence of O- glycosidicdy peptide-linked oligosaccharïdes of poly-N-acetyUactosamine type (erythroglycan II) in the 1-antigenically active Sendai virus receptor sialoglycoprotein GP- 2. JI Biochem. 98, 1653-1659. Takeda, k., Noguchi, S., Sugino, A., and Kawamura, M. (1988). Functional activity of oligosaccharide-deficient (Na&) ATPase expressed in Xenopus oocytes. FEBS Lett. 238, 20 1-304.
Tamkun, M. M., and Fambrough, D. M. (1986). The (Na+ + Kf)-~TPaseof chick sensory neurons. J. Biol. Chem. 261, 100% 10 19.
Tan, I., Dunn, J., Jaeken, J., and Schachter, H. ( 1996). Mutations in the MGAT:! gene controlling complex N-glycan synthesis cause carbohydrate-deficient glycoprotein syndrome type II, an autosomal recessive disease with defective brain development. Am. J. Hum. Genet, 59, 8 10-8 17.
Tartakoff, A. M., and Vassalli, P. (1983). Lectin-binding sites as markers of Golgi subcompartments: proximal-to-distd maturation of oligosacchaides. I. Cell B iol. 9 7, 1243- 1248.
Tate, C. G., and Blakely, R. D. (1994). The effect of N-linked glycosylation on activity of the Na+- and Ckdependent serotonin transporter expressed using recombinant baculovirus in insect cells. J. Biol. Chem. 269, 26303-263 10.
Tatu, U., Hammond, C.. and Helenius, A. (1995). Foiding and oligomerization of influenza hemagglutinin in the ER and the intermediate cornpartment. EMBO J. 14, 1340- 1348.
Thomhill, W. B., Wu, M. B.. Jiang, X., Wu, X., and Morgan, P. T. (1996). Expression of Kvl. 1 delayed rectifier potassium channels in Lec mutant Chinese hamster ovary ceii lines reveals a role for sialidation in channel function. J. Biol. Chem. 271, 19093-19098.
Tornita, A., and Parker, C. 1. (1994). Aberrant regdation of cornpiement by the erythrocytes of hereditary erythroblastic multinuclearity with a positive acidified senun lysis test (HEMPAS). B lood 83,250-259.
Tsuji. T., Irimura, T., and Osawa, T. (1980). The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes. Biochem. 1.187,677-686. Vainchenker, W., Guichard, I., and Brenton-Gprius, 1. ( 1979). Morphological abnormalities in cultured erythroid colonies (BN-E)from the blood of two patients with HEMPAS. Br. J. Hernatol. 42, 363-369. van den Eijnden, D. H., Koenderman, A. H. L., and Schiphorst, W. E. C. M. (1988). Biosynthesis of blood group i-active polylactosaminoglycans. Partial purification and properties of an UDP-G1cNAc:N-acety llactosarninide P 1-3-N- acety lglucosaminy ltnnsferase from Novikoff tumor cell ascites fluid. J. B iol. Chem. 263, 1246 l-LW7 1. van Hoek, A. N., Wiener, M. C., Verbavatz, LM., Brown, D., Lipniunas, P. H., Townsend, R. R., and Verkrnan, A. S. (1995). purification and structure-function analysis of native, PNGase F-ueated, and Endo-P-galactosidase-ueated CHIP28 water channels. Biochernistry 34,2212-2219.
Verwilghen, R. L., Lewis, S. M., Dacie, J. V., Crookston, J. H., and Crookston, M. C. (1973). Hernpas: congenitd dyserythropoietic anaemia (type m.Q. J. Med. 166,257-3-78.
Wang, C., Eufemi, M., Turano, C., and Girtosio, A. (1996). influence of the carbohydrate rnoiety on the stability of glycoproteins. Biochemistry 35,7-799-7307.
Wang, W.-C., Lee, N., Aoki, D.. Fukuda, M. N., and Fukuda, M. (199 1). The poly-N- acetyllactosamines attached to lysosomal membrane glycoproteins are increased by the prolonged association with the Golgi complex. J. Biol. Chem. 266,23 185-23 190.
Ware, F. E., Vassilakos, A., Peterson, P. A., Jackson, M. R., Lehrman, M. A., and Williams, D . B. ( 1995). The molecular chaperone calnexin binds Glc lMan9GlcNAc2 oligosaccharide as an initial step in recogaizing unfolded glycoproteins. 1. Biol. Chem. 2 70,4697-7404.
Weiner, A. S., Unger, L. J., Cohen, L., and Feldman, J. (1956). Type-specific cold auto- antibodies as a cause of acquired hemolytic anemia and hemolytic transfusion reactions: biological test with bovine red cells. Am. Intem. Med. JJ, 22 1-240. Wheeler, T. J., and Hinkle, P. C. (1981). Kinetic properties of the reconstituted glucose transporter from human erythocytes. l. Biol. Chem. 256,8907-89 14.
Williams, W. J., Beutler, E. B., Erslev, A. I., Lichtman, M. A. (1990). Hematology, R. P. McGraw and M. Lemer, eds.: McGraw-Hill, Inc.
Wilson, 1. B. H., Platt, F. M., Isenberg, D. A., and Rademacher, T. W. (1993). Aberrant control of gaiactosyltransferase in penpherd B lymphocytes and Epstein-Barr virus ~ansformedB lymphob[asts from patients with rheurnatoid arthritis. I. Rheumatol. 20, 1282-1387.
Wong, K. Y., Hug, G., and Lampkin, B. C. (1972). Congenital dyserythropoietic anernia type II: ultrastructural and radioautographic snidies of blood and bone marrow. Blood 39, 23-30.
Wu, 1.-S. R., and Lever, J. E. (1994). N-linked glycosylation is not required for Ndglucose sympon activity in LLC-PKl cells. Biochim. Biophys. Acta il92,289-292.
Yamamoto, F. I., Clausen, K., White, T., Marken, S., and Hakornori, S. 1. (1990). Molecular genetic basis of the histo-blood group AB0 system. Nature 345,229-233.
Yoshimura, M., nian, Y., Ohnishi, A., Ijuhin, N., Nishiura, T., Kanakura, Y., Matsuzawa, Y., and Taniguchi, N. (1996). Bisechg N-acetylglucosamine on K562 ceils suppresses naturai killer cytotoxicity and promotes spleen colonization. Cancer Res. 56, 412-418.
Yusufi, A. N. K., Szczepanska-Konkel, M., and Dousa, T. P. (1988). Role of N-Linked oligosaccharides in the transport activity of the Na/H antiporter in rat rend brush-border membrane. J. Biol. Chem. 263, 1368343691.
Zamofing, D., Rossier, B. C., and Geering, K. (1988). Role of the Na,K-ATPase p- subunit in the cellular accumulation and maturation of the enzyme as assessed by glycosylation inhibitors. J. Membr. Biol. 104,69-79. Zuber, C., Roth, S., Misteli, T., Nakano, A., and K., Moremen. (1991). DS28-6, a temperature-sensitive mutant of Chinese hamster ovary cells, expresses key phenotypic changes associated with brefeldin A treatment. Proc. Natl. Acad. Sci. USA 88, 98 18- 9822. I ivcr7w L L v ~\LwT\I w @ v TEST TARGET (QA-3)
APPLIED IMAGE.lnc 1653 E8ç1 Main Street .-G .-G - Rochester, NY 14609 USA .==-- Phane: 71 6i482-0300 --= Fax: 7161288-5989
0 1993. Appiied Image. 11% Alt R@WS Aeserved