The Glycosylation of the Complement Regulatory Protein, Human Erythrocyte CD59*

(Received for publication, June 14, 1996, and in revised form, December 17, 1996)

Pauline M. Rudd Dagger §, B. Paul Morgan , Mark R. Wormald Dagger , David J. Harvey Dagger , Carmen W. van den Berg , Simon J. Davis par **, Michael A. J. Ferguson Dagger Dagger §§ and Raymond A. Dwek Dagger §

From the Dagger  Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom, the  University of Wales College of Medicine, Department of Medical Biochemistry, Heath Park, Cardiff, CF4 4XN, United Kingdom, the par  Molecular Sciences Division, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom, and the Dagger Dagger  Department of Biochemistry, University of Dundee, Dundee, DD1 4HN, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Human erythrocyte CD59 contains N- and O-glycans and a glycosylphosphatidylinositol (GPI) anchor, all of which have been analyzed in this study. The anchor consists principally of the minimum core glycan sequence Manalpha 1-2Manalpha 1-6Manalpha 1-4GlcN-linked to a phosphatidylinositol moiety with the structure sn-1-O-alkyl(C18:0 and C18:1)-2-O-acyl(C20:4)glycerol-3-phospho-1-(2-O-palmitoyl(C16:0))myo-inositol. This structure is essentially identical to that of human erythrocyte cholinesterase (Deeg, M. A., Humphrey, D. R., Yang, S. H., Ferguson, T. R., Reinhold, V. N., and Rosenberry, T. L. (1992) J. Biol. Chem. 267, 18573-18580). This first comparison of GPI anchors from different proteins expressed in the same tissue suggests that human reticulocytes produce only one type of anchor structure. The N- and O-glycans were sequenced using a novel approach involving digestion of the total glycan pool with multiple enzyme arrays. The N-glycan pool contained families of bi-antennary complex-type structures with and without lactosamine extensions and outer arm fucose residues. The predominant O-glycans were NeuNAcalpha 2-3Galbeta 1-3GalNAc and Galbeta 1-3[NeuNAcalpha 2-3]GalNAc. Inspection of a molecular model of CD59, based on the NMR solution structure of the extracellular domain and the structural data from this study, suggested several roles for the glycans, including spacing and orienting CD59 on the cell surface and protecting the molecule from proteases. This work completes the initial structural analysis of CD59, providing the most complete view of any cell surface glycoprotein studied to date.


INTRODUCTION

CD59 is a cell surface glycoprotein that binds to the complement proteins C8 and/or C9 in the nascent membrane attack complex, thereby protecting host cells from lysis (1). CD59 belongs to the Ly-6 superfamily (2) and is present on a wide variety of cell types, including leukocytes, platelets, epithelial and endothelial cells, placental cells, and erythrocytes, where it is present at 2.5-5 × 104 copies/cell (3). In addition to its role in protecting host tissues from homologous complement, it has been proposed that CD59 mediates T cell adhesive interactions by synergizing with CD58 via direct interactions with CD2 (4, 5) although this is controversial (6, 7). It has also been proposed that CD59 participates in T cell activation pathways and platelet secretory responses (1).

CD59 is normally attached to the cell surface via a glycosylphosphatidylinositol (GPI)1 anchor (8) although a number of soluble forms have been found, for example in saliva, amniotic fluid, milk, and urine (1). The complementary DNA sequence of the CD59 gene (8) has shown that the translated precursor contains 128 amino acids, including a 25-amino acid N-terminal signal peptide and a C-terminal sequence that contains a cluster of 18 mainly hydrophobic amino acids preceded by eight polar amino acids. This sequence is typical of signal peptides for GPI anchored proteins (9). The cleavage site for the anchor attachment is between Asn77 and Gly78 (10). The molecule contains eight potential O-glycosylation sites at Thr10, Thr15, Thr29, Thr51, Thr52, and Thr60, and at Ser20 and Ser21. There is one fully occupied N-glycosylation site (Asn18-Cys-Ser) (8) which is completely conserved in all known CD59 sequences except rat, which is glycosylated at the adjacent residue, Asn16 (11). The role of the glycans at this site in CD59 is controversial. Removal or modification of the N-glycans has been shown to reduce the co-stimulation of proliferation by CD59 and to eliminate its complement inhibitory properties (12, 13), whereas other studies have shown that this enhances or has no effect on CD59 function (14-16).

NMR analyses of recombinant (17) and urine-derived (18) glycosylated forms of soluble human CD59 revealed that the extra-cellular region consists of a single disk-like domain, formed by single two- and three-stranded beta sheets and a short alpha helix, that is attached to the GPI anchor by a short 7-residue stalk. A recent mutational analysis2 has shown that the active site is centered on Trp40 of the exposed glycosylated face of CD59 and that this is equivalent to the "concave" face used by neurotoxins to bind to target nicotinic and muscarinic acetylcholine receptors and acetylcholinesterases.

GPI membrane anchors are used by a wide variety of cell surface glycoproteins (reviewed in Refs. 1, 19-22). All GPI anchors characterized to date contain a conserved core of ethanolamine-HPO4-6Manalpha 1-2Manalpha 1-6Manalpha 1-4GlcNalpha 1-6myo-inositol-1HPO4-lipid where the lipid can be a diacylglycerol, an alkylacylglycerol, or a ceramide (22). In some cases, the inositol is substituted with a fatty acid (palmitate), rendering the structure resistant to the action of bacterial PI-specific phospholipase C (PIPLC) (23, 24). The core glycan can be substituted with other sugars, and all GPI anchors from metazoan sources contain one or two additional ethanolamine phosphate groups attached to the conserved trimannosyl backbone (22).

In this study, the glycan component of the GPI anchor of human erythrocyte CD59 was characterized, enabling its structure to be compared with that of the glycan anchor attached to another human erythrocyte GPI-anchored glycoprotein, acetylcholinesterase (25, 26). Further, the N- and O-linked glycans covalently attached to human erythrocyte CD59 were characterized, and the full HPLC glycosylation profile compared with that of human platelet CD59 to explore the extent of cell-specific glycosylation. Molecular modeling, based on the solution structure of the protein (18) was used to explore the spatial relationships between the lipid bilayer, the protein, and the N-and O-linked glycans. Several possible roles for the heterogeneous array of glycans attached to CD59 were proposed, including orientation of the active site, protection from protease cleavage, and limiting interactions with other CD59 molecules and with the cell surface. This work provides the most complete view of any cell surface glycoprotein studied to date.


EXPERIMENTAL PROCEDURES

Purification of Human Erythrocyte and Platelet CD59

CD59 was purified from human erythrocytes and platelets by immunoaffinity chromatography of butanol-extracted erythrocyte membranes essentially as described previously (27, 28). The purity of the proteins was confirmed by analysis on SDS-PAGE (15% gels) and by N-terminal amino acid sequencing. Protein concentration was measured using the Pierce protein assay reagent.

Nitrous Acid Deamination and Sodium Borohydride Reduction

CD59 (200 µg) was dissolved in 10 µl of pH 4.0 buffer (0.1 M sodium acetate adjusted to pH 4 with acetic acid). A freshly prepared solution of NaNO2 (10 µl, 0.5 M) was added, and the mixture was incubated for 2.5 h at room temperature after which boric acid (5 µl, 0.8 M) was added. The amount of NaOH required to adjust the pH to about 10.5 (determined using buffer controls and typically 8-10 µl) was added to the reaction mixture. NaB3H4 (5 µl, 10-15 Ci/mmol, 36 mM in 0.1 M NaOH) was added immediately, followed 2 h later by freshly prepared NaBH4 (1 M aqueous) to give a final concentration of 0.2 M NaBD4. After a further 2 h, the reduction mixture was acidified with acetic acid (1 M). The reaction was carried out in the fume hood as tritium gas was evolved. The sample was dialyzed against water using a Spectra/Por membrane (Orme Scientific) with a 2,000 Da cut off and was freeze dried.

Aqueous HF Dephosphorylation and Re-N-acetylation

Deaminated and reduced CD59 (40 µg), prepared as described above, was placed in a screw top Eppendorf tube and incubated with 50 µl of 48% aqueous HF at 0 °C for 48 h. The aqueous HF was neutralized with saturated LiOH and the products re-N-acetylated (29).

Dionex HPLC

The system was fitted with a Dionex CarboPac PA-1 column. A calibration standard of mixed glucose oligomers was co-injected with the sample and detected by pulsed amperometric detection. The elution positions of the neutral glycans were defined in Dionex units (Du) by linear interpolation of their elution positions between two adjacent internal standards. These Du have no specific meaning but are a reliable chromatographic property that correlate with particular glycan structures. The radiolabeled glycans were detected on line by a Raytest Romana radioactive monitor and by scintillation counting of collected fractions.

Bio-Gel P4 Gel Filtration

Filtration was performed using the Oxford GlycoSystems GlycoMapTM Instrument, which is based on a BioGel P4 gel permeation chromatography system (GPC).

Exoglycosidase Sequencing

Aspergillus phoenicus (Saitoi) alpha 1-2mannosidase digestions were performed in 10 µl of 0.1 M sodium acetate, pH 5.0, containing 0.01 milliunit (0.04 mg) of Aspergillus phoenicus (Saitoi) alpha 1-2mannosidase (X-5009, Oxford GlycoSystems Ltd.) for 16-24 h at 37 °C under a toluene atmosphere. The products were desalted by passage through 0.2 ml of Dowex AG50X12 (H+) and eluted with water (1 ml). The samples were evaporated to dryness, and any residual acetic acid was removed by co-evaporation with toluene. Jack bean alpha -mannosidase digestions were performed in 30 µl of 0.1 M sodium acetate, pH 5, containing 25 units/µl of Jack bean alpha -mannosidase (Boehringer Mannheim) under the same conditions.

Electrospray Mass Spectrometry (ES-MS)

A sample of CD59 (200 µg) in 0.5 ml of phosphate-buffered saline, 0.05% CHAPS, 0.02% sodium azide was extracted four times with an equal volume of butan-1-ol saturated with water to remove the CHAPS detergent and any non-covalently associated lipids. The aqueous phase was freeze dried and redissolved in 100 µl of 0.3 M sodium acetate buffer, pH 4.0). The sample was deaminated by addition of 100 µl of freshly prepared 0.5 M NaNO2, sonication for 15 min, and incubation for 3 h at 37 °C. The PI fraction, released by the deamination procedure, was recovered by two extractions with 200 µl and one extraction with 100 µl of butan-1-ol saturated with water. The combined butan-1-ol phases were washed with 300 µl of water, dried under a stream of N2 and redissolved in 100 µl of chloroform/methanol (2:3) containing 1 mM NH3. Aliquots (20 µl) were introduced into the electrospray source of a Micromass (Altringham, Cheshire, UK) mass spectrometer in a stream of the same solvent at 10 µl/min. Negative ion mass spectra were recorded over the mass range m/z 700-1500 and processed using MassLynx software. The source conditions were optimized using soybean PI from Sigma. For tandem mass spectrometry, mass resolution in mass spectrometer 1 was reduced to allow the simultaneous transmission of m/z 1135 and m/z 1137 into a hexapole collision cell containing argon (2.5 × 10-3 Torr) under an accelerating voltage of 60 V. Daughter ions were collected over the mass range m/z 200-450.

GC-MS Monosaccharide Analysis

A sample of CD59 (50 µg) was analyzed for monosaccharide content by GC-MS following methanolysis, re-N-acetylation, and trimethylsilylation (29).

Release and Re-N-acetylation of Glycans

0.6 µg (31.6 nmol) of CD59 was dialyzed against 0.1% trifluoroacetic acid and lyophilized. Glycans were released from CD59 by hydrazine at 95 °C and re-N-acetylated using a GlycoPrep 1000 (Oxford GlycoSystems, Ltd.) optimized for maximum recovery (approximately 85%) of both N- and O-linked sugars. The recovery was assessed by desialylating and degalactosylating bi-antennary complex sugars and then incubating with UDP-[3H]Gal and galactosyl transferase. The free glycans associated with the anchor were also released under these conditions. However, since these glycans terminate in inositol, they cannot be reduced or 2-aminobenzamide-labeled and are therefore not detected in the oligosaccharide analyses.

Fluorescent Labeling of the Oligosaccharide-reducing Terminus with 2-aminobenzamide (2AB)

2AB labeling was carried out using the Oxford GlycoSystems Signal labeling kit (30).

Weak Anion Exchange (WAX) Chromatography

WAX was performed on a GlycoSep-C column (Oxford GlycoSystems, Ltd.) (7 × 50 mm) attached to a Waters HPLC system as described previously (31).

Normal Phase Chromatographic Separations of Neutral and Acidic Oligosaccharides

Separations were performed on a GlycoSep-N HPLC column (Oxford GlycoSystems, Ltd.) (4.6 × 250 mm) as described previously (32).

Simultaneous Oligosaccharide Sequencing of the Released Glycan Pool

Enzyme digests were performed at 37 °C for 16-24 h in 100 mM citrate/phosphate buffer, pH 4.5, containing 0.2 mM zinc acetate and 0.15 M sodium chloride. Conditions for the individual enzymes in the arrays were as follows: Arthrobacter ureafaciens sialidase (Oxford GlycoSystems), 1-2 units/ml; almond meal (AM) fucosidase (Oxford GlycoSystems), 3 milliunits/ml; Charonia lampas alpha -fucosidase 9 miliunits/ml (Oxford Glycobiology Institute); bovine testes beta -galactosidase (Oxford GlycoSystems), 1-2 units/ml; Streptococcus pneumoniae beta -N-acetylhexosaminidase (Oxford Glycobiology Institute), 2 units/ml. Bacteroides fragilis endo-beta -galactosidase (EBG) (Oxford GlycoSystems), 0.5 units/ml in 50 mM sodium actetate buffer, pH 5.8. No exogalactosidase activity was detected in this enzyme preparation (data not shown).

Matrix-assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS)

MALDI data were acquired on a Micromass (Altringham, Cheshire, UK) AutoSpec-FPDQ magnetic sector instrument fitted with a pulsed nitrogen laser (337 nm) and an array detector (33). Samples were prepared by adding the oligosaccharide sample (1 µl) in water to the matrix solution (3 µl of a saturated solution of 2,5-dihydroxybenzoic acid in acetonitrile) on the mass spectrometer target and allowing it to dry at room temperature. The mixture was then recrystallized from 1 µl of ethanol (34). The array detector was set to the high resolution position, and the mass range was set to be appropriate to the sample being examined. For data acquisition, the laser was operated at full power, and the laser beam was moved manually over the sample in order to compensate for sample depletion under the laser beam.

N-terminal Sequence Analysis

Purified CD59 (10 µg) was run under reducing conditions on a 15% Tris/glycine gel-PAGE (35) and blotted onto ProblottTM membrane (Applied Biosystems, Warrington, UK). The blot was stained with Coomassie Brilliant Blue, the CD59 band was excised, and the protein was reduced and alkylated with dithiothreitol and iodoacetamide. For enzymatic digestion 60 µg of CD59 was precipitated with ice-cold acetone, resuspended in phosphate-buffered saline, 2 mM EDTA, 2 mM dithiothreitol, and boiled for 5 min. 10 mM iodoacetamide and 3 µg of endoproteinase Lys C (Promega, Southampton, UK) were added. Digestion was carried out for 16 h at 37 °C. Fragments were separated under reducing conditions on a 15% Tris/Tricine gel (36) and blotted onto ProblottTM membrane. The blot was stained with Amido black, and bands were excised. N-terminal sequence analysis of the excised bands was performed using an Applied Biosystems 476A protein sequencer and 610A data analysis software.

N-deglycosylation of CD59 with Peptide-N-glycosidase F

CD59 was incubated for 16 h at 37 °C with Flavobacterium meningoseptum peptide-N-glycosidase F (PNGase F) (analytical grade) 7.5 units/100 µl in 20 mM sodium phosphate buffer, pH 7.5, containing 50 mM EDTA and 0.02% azide buffer to deglycosylate the protein.

Molecular Modeling

Molecular modeling was performed on a Silicon Graphics 4D/35 workstation using the program Insight II and Discover (Biosym Tech. Inc.). Coordinates for the CD59 solution structure were retrieved from the Protein Data Bank, Brookhaven National Laboratory (37).


RESULTS

CD59 was purified from butanol-extracted erythrocyte membranes by affinity chromatography and gel-filtration as described previously (27, 28). On SDS-PAGE, the purified protein migrated as a broad band in the apparent molecular mass range 20-25 kDa. No other bands were detected even on overloaded gels. N-terminal sequencing indicated that no N-terminal residues other than that of CD59 (leucine) were detectable (data not shown). Together these data indicate that the preparation was at least 95% pure.

The strategy for the subsequent analysis of the GPI anchor and the N- and O-glycans attached to CD59 is outlined in Fig. 1A, and the results of the study are correspondingly organized into sections A-H below. Fig. 1B shows a sialylated bi-antennary complex glycan with lactosamine extensions and some of the cleavage positions of the enzymes used in this study.


Fig. 1. A, strategy for the analysis of the GPI anchor and the N- and O-glycans of CD59. The letters A-H refer to the paragraph under "Results" where the data are presented. B, a schematic diagram of a sialylated bi-antennary complex glycan with lactosamine extensions, showing some of the cleavage positions of the enzymes used in this study.
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GC-MS Composition Analysis of All N- and O-linked CD59 Sugars (Fig. 1A)

The monosaccharide composition of the total CD59 oligosaccharides, following methanolysis, re-N-acetylation, and trimethylsilylation, was determined by GC-MS (29) (Table I). A number of conclusions were drawn. First, the detection of 2.3 nmol of GalNAc/nmol of CD59 suggested the presence of O-linked glycans. Second, the high proportion of N-acetyl glucosamine (10.4 nmol) and galactose (9.5 nmol) compared with mannose (1.6 nmol) suggested that CD59 was associated with a high percentage of multi-antennary- and polylactosamine-type oligosaccharides. Third, the proportion of fucose (2.5 nmol) suggested that many structures contained outer arm as well as core fucose. Finally, the detection of 3 nmol of sialic acid residues indicated that many of the glycans were charged.

Table I.

GC-MS monosaccharide composition analysis of the glycans associated with CD59


Monosaccharide residue nmol/nmol of CD59

N-acetyl galactosamine 2.3
N-acetyl glucosamine 10.4
Mannose 1.6
Galactose 9.5
Fucose 2.5
Sialic acid 3

Analysis of the Glycans Associated with the CD59 GPI Anchor (Fig. 1B)

Intact CD59 was subjected to nitrous acid deamination which converted the non-N-acetylated GlcN residue, unique to the GPI anchor, to 2,5-anhydromannose and simultaneously released the PI moiety. Subsequent reduction with NaB3H4 converted the 2,5-anhydromannose to [1-3H]2,5-anhydromannitol (AHM), and the radiolabeled GPI glycan was released from the protein and ethanolamine substituents by dephosphorylation with cold 48% aqueous HF. The resulting GPI neutral fraction was fractionated by Dionex high pH anion exchange chromatography (HPAEC) into three species that eluted at 2.51 (91%), 3.07 (8%), and 3.72 (1%) Dionex units (Du) (Fig. 2). The major species eluted at 4.2 P4 glucose units (gu) on Bio-Gel P4 and the 3.07-Du species eluted at 5.6 gu. These Du and gu values are identical to those of authentic standards of Manalpha 1-2Manalpha 1-6Manalpha 1-4AHM and Manalpha 1-2Manalpha 1-6(GalNAcbeta 1,4)Manalpha 1-4AHM, respectively (29). The major 2.51 Du/4.2 gu species was subjected to A. saitoi Manalpha 1-2Man-specific alpha -mannosidase and produced a labeled glycan that eluted at 3.2 P4 gu on Bio-Gel P4, consistent with a structure Manalpha 1-6Manalpha 1-4AHM (29). This material was further digested by Jack Bean alpha -mannosidase to a labeled component that eluted at 1.7 gu, consistent with free AHM. Taken together with the chromatographic data, these results confirm that the major GPI neutral glycan species is Manalpha 1-2Manalpha 1-6Manalpha 1-4AHM, derived from Manalpha 1-2Manalpha 1-6Manalpha 1-GlcN by deamination and reduction. The assignment of the minor 3.07 Du/5.6 P4 gu species as Manalpha 1-2Manalpha 1-6(GalNAcbeta 1,4)Manalpha 1-4AHM (derived from Manalpha 1-2Manalpha 1-6(GalNAcbeta 1,4)Manalpha 1-GlcN) is based solely on the chromatographic data since insufficient material was available for exoglycosidase digestions. However, to date no GPI neutral glycans have been found to co-chromatograph on both Dionex HPAEC and BioGel P4. The very minor 3.72 Du species was not characterized, but this Du value would be consistent with the structure Manalpha 1-2Manalpha 1-2Manalpha 1-6(GalNAcbeta 1,4)Manalpha 1-4AHM.


Fig. 2. HPAEC analysis of neutral glycans from the CD59 GPI anchor. 91% of the glycans eluted at 2.51 Dionex units, consistent with the assignment of the structure Manalpha 1-2Manalpha 1-6Manalpha 1-4GlcNH- to the glycan in the anchor.
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Characterization of the PI Moiety of the GPI Anchor (Fig. 1C)

The PI moiety released by nitrous acid deamination of CD59 was recovered by extraction into butan-1-ol and analyzed by negative ion ES-MS. Pseudomolecular [M-H]- ions were observed at m/z 1135 and m/z 1137 (Fig. 3a). Simultaneous collision induced dissociation of the two ions resulted in a daughter ion spectrum (Fig. 3b) that showed the presence of ions at m/z 331 (consistent with a C22:4 fatty acid carboxylate ion) and at m/z 403 and m/z 405. The latter fragment ions are characteristic of alkyl-PI species which produce intense [alkyl-O-CH2-CHdouble bond CH-HPO4]- fragments (38). The m/z values of 403 and 405 suggest that the alkyl chains are C18:1 and C18:0, respectively. The m/z values of the parent ions are consistent with 1-O-(C18:1)alkyl-2-O-(C22:4)acyl-PI and 1-O-(C18:0)alkyl-2-O-(22:4)acyl-PI species that are further substituted with hydroxyester-linked palmitic acid (C16:0). The absence of a detectable palmitate carboxylate ion (m/uz 255) and of an [inositol-1,2-cyclic phosphate]- ion (m/z 241) in the daughter ion spectrum is consistent with the attachment of the palmitoyl group to the 2-position of the inositol ring (24). Thus, the ES-MS data strongly suggest that the PI moiety of CD59 consists of approximately equal amounts of 1-O-(C18:1)alkyl-2-O-C(22:4)acylglycerol-3-HPO4-1-(2-O-(C16:0)acyl)myo-inositol and 1-O-(C18:0)alkyl-2-O-(C22:4)acylglycerol-3-HPO4-1-(2-O-(C16:0)acyl)myo-inositol. These so called acyl-PI structures are identical to the structures previously described for the GPI anchor of human erythrocyte acetylcholinesterase (23). The presence of the acyl (palmitoyl) group attached to the 2-position of the inositol ring explains the resistance of CD59 to the action of PIPLC enzymes that require a free 2-hydroxyl group on the inositol ring to participate in the cleavage reaction, i.e. nucleophilic attack of the phosphodiester phosphorus atom (39).


Fig. 3. Negative ion ES-MS analysis of the PI fraction of CD59. a, detail of the pseudo-molecular ion region of the ES-MS spectrum of the PI fraction of CD59 showing two ions at m/z 1135 and m/z 1137. b, daughter ion spectrum of the two parent ions shown in panel a.
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The glycan and PI data described here are summarized in Fig. 4, which is a schematic representation of the GPI anchor of CD59. The inclusion of the ethanolamine groups in this model is solely by analogy with other mammalian GPI anchors such as rat brain Thy-1 antigen (40), human erythrocyte acetylcholinesterase (26), and human leukocyte CD52 (24).


Fig. 4. Schematic drawing of the CD59 glycan anchor. The structures of the lipids and glycans are based on data derived in this paper. The inclusion of the ethanolamine groups in this model is solely by analogy with other mammalian GPI anchors such as rat brain Thy-1 antigen (40), human erythrocyte acetylcholinesterase (26), and human leukocyte CD52 (24).
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Comparison of the Glycosylation Profiles of Human Erythrocyte and Human Platelet CD59 (Fig. 1D)

The 2AB-labeled sialylated glycan pools released from human erythrocyte and human platelet CD59 were resolved by normal phase HPLC (Fig. 5a and b). The differences in these profiles, which compare the overall glycosylation of CD59 expressed in two different cell types, confirm previous findings that glycosylation is cell type-specific (41). There are more than 40 peaks in each of these profiles indicating that platelet and erythrocyte CD59 are both expressed as a population of many different glycoforms. The bar chart below the figure indicates the elution positions of standard 2AB-labeled oligosaccharides. Using this as a calibration, it can be predicted that both platelet and erythrocyte CD59 contain bi- and multi-antennary structures and glycans with lactosamine extensions. Both contain sugars, for example peaks A and B, in the region of the profile where small O-link sugars are predicted to elute. The N- and O-glycans from HuE CD59 were further characterized by MALDI-MS and HPLC.


Fig. 5. Comparison of the normal phase HPLC profiles of 2AB-labeled glycans released from CD59 isolated from HuE (a) and human platelets (b). The elution positions of the dextran ladder and of the different classes of oligosaccharides are shown in the scale above and the bar chart below the figure. A and B are O-linked glycans.
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Characterization of the N-Glycans Attached to HuE CD59 (Fig. 1E)

MALDI-MS Analysis of CD59 N-linked Asialo Glycan Pool (Fig. 1E.1)

MALDI-MS of the desialylated N-glycan pool (Fig. 6a, Table II) indicated that asialo CD59 contained at least 123 glycan structures associated with the one N-glycosylation site. These included families with compositions consistent with those of bi-, tri-, and tetra-antennary sugars with and without polylactosamine extensions and outer arm fucose. Since MALDI-MS determines only molecular masses, this type of analysis does not give information with respect to isomers which arise from differences in linkage and arm specificity. These additional variations, as well as diversity arising from the addition of sialic acid residues to many of the neutral oligosaccharides, may be expected to increase the number of structures even further. The maximum size of the sugars could not be determined because their relative abundance decreased with increasing molecular weight and eventually fell below the detection limit of the MALDI experiment. The largest structures detected contained approximately 24 monosaccharide residues.


Fig. 6. MALDI-MS analyses of glycans from HuE CD59. a, asialo glycans; b, asialo glycans following digestion with C. lampas fucosidase; c, asialo glycans following digestion with C. lampas fucosidase and endo beta -galactosidase; and d, asialo glycans following digestion with C. lampas fucosidase and endo beta -galactosidase and bovine testes galactosidase. The data are analyzed in Table II.
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Table II.

Oligosaccharides from CD59 (2-AB, [MNa]+)


Mass
Hex HNAc Fuc % Exoglycosidase incubations
Measureda Calculated Endo-beta -galactosidaseb
C. lampas fucosidasec
C. lampas fucosidase endo-beta -Gald
C. lampas fucosidase endo-beta -Gal galactosidasee
% Remf- Fmdg % Remh Fmd % Rem Fmd % Rem Fmd

1460.7 1460.4 3 4 0 1.7 1.6 No No 3.6 No Y No 7.3 No
1605.7 1606.5 3 4 1 1.8 1.9 No No 0.3 Y(0) No 0 No
1622.1 1622.5 4 4 0 0.9 1.1 No No 4.3 No Y 3.2 No Y 0 Y
1664.0 1663.6 3 5 0 0.5 1.2 No Y 1.7 No Y 4.9 No Y 56.4 No Y
1703.3 1702.6 7 2 0 0.3 0.2 No No 0 No No 1.9 3.7 No
1768.4 1768.7 4 4 1 2.8 2.2 No No 0.6 Y(0) No 0 No 3.3
1784.4 1784.7 5 4 0 2.6 1.6 No No 7.0 No Y 7.2 No 0 Y
1809.3 1809.7 3 5 1 1.0 3.7 No Y 0 Y(0) No 0 No 0
1825.5 1825.7 4 5 0 0.8 2.5 No Y 2.6 No Y 21.6 No Y 2.2 Y
1866.6 1866.8 3 6 0 0.4 0.7 No Y 0.6 No No 4.4 No Y 5.5 Y No
1930.7 1930.8 5 4 1 1.8 2.6 No No 0.3 Y(0) No 0 No 0
1947.1 1946.8 6 4 0 0.6 0.5 No No 1.2 No No 1.9
1971.9 1971.9 4 5 1 1.4 15.3 No Y 1.4 No(1) No 2.9 No Y
1987.8 1987.9 5 5 0 6.0 5.6 No No 33.8 No Y 26.1 No Y
2013.6 2012.9 3 6 1 0.9 2.3 No Y 0.3 Y(0) No 0 No
2030.1 2028.9 4 6 0 1.0 0.8 No No 4.1 No Y 4.1 No 2.2 Y No
2070.0 3 7 0 0 2.2 Y
2092.7 2092.9 6 4 1 0.5 0.6 No No 0.3 Y(0) No 0 No
2108.2 2108.9 7 4 0 0.6 0.8 No No 0.3 No Y
2117.9 2118.0 4 5 2 0.1 6.2 No Y 0.9 Y(0) No
2134.0 2134.0 5 5 1 15.2 14.6 No No 0 Y(0) No 2.6 No 3.3 No
2149.8 2150.0 6 5 0 3.1 0.6 Y No 4.1 No Y No 0 Y
2175.8 2175.1 4 6 1 2.0 2.6 No No 0.4 Y(0) No 3.4 No Y 30.3 No (No)
2191.0 2191.1 5 6 0 0.5 0.2 No 0.9 No Y 2.2 Y Y
2832.7 2832.7 5 7 3 1.1 0.9 No No 0 Y(2) No 0 No 0
2848.2 2848.5 6 7 2 1.8 0.3 No No 0 Y(1) No 0 No 0
2864.9 2864.7 7 7 1 2.0 0.4 (Y) No 0 Y(0) No 0 Y 0
2880.7 8 7 0 0.3 0
2890.3 2889.7 5 8 2 0.3 0.1 Y No 0 Y No 0 0
2905.7 2905.7 6 8 1 0.3 0.2 No 0 0
2953.3 2953.8 7 6 3 0.4 0 Y No 0 Y No 0
2969.3 2969.8 8 6 2 0.1 0 No 0.3 No No 0 0
2993.9 2994.8 6 7 3 0.5 0.3 No No 0 Y(1) No 0 0
3010.6 3010.8 7 7 2 0.8 0.5 No No 0.3 Y(0) No No 0
3026.3 3026.8 8 7 1 0.6 0 Y No 0.6 No Y 0
3035.6 3035.9 5 8 3 0.3 0 No 0 0
3051.6 3051.9 6 8 2 0.5 0.2 Y No 0 Y(0) No 0
3067.8 3067.9 7 8 1 0.3 0 Y No 0.7 Y 0
3083.9 8 8 0 0.7 No
3108.9 6 9 1 0.3 Y
3116.3 3115.9 8 6 3 0.3 0 Y No 0.3 No No 0
3124.9 7 9 0 0.4 Y
3156.2 3157.0 7 7 3 0.6 0.3 No No 0 Y(0) No No
3172.6 3173.0 8 7 2 1.0 0.3 (Y) No 0 Y(1) No
3188.2 3188.9 9 7 1 0.3 0 Y No 0 Y No
3197.3 3198.0 6 8 3 0.7 0.2 Y No 0 Y No 0
3214.4 3214.0 7 8 2 1.2 0.3 (Y) No 0 Y(1) No
3229.7 3230.0 8 8 1 0.9 0 Y No 0 Y(0) Y
3239.4 3239.1 5 9 3 0.3 0 Y No 0 Y No
3255.4 3255.1 6 9 2 0.5 0 Y No 0 Y No
3246.0 9 8 0 0.1 Y
3271.5 3271.1 7 9 1 0.1 0 Y No
3319.2 3319.1 8 7 3 0.5 0 Y No 0 Y(1) No
3333.3 3335.1 9 7 2 0.3 0 Y No 0 Y No
3360.4 3360.1 7 8 3 0.6 0.2 Y No 0 Y(1) No
3375.5 3376.2 8 8 2 0.6 0 Y No 0 Y(0) Y
3390.6 3392.2 9 8 1 0.2 No 0.3 Y
3401.9 3401.2 6 9 3 0.3 0 Y No
3416.0 3417.2 7 9 2 0.3 0.1 Y No
3433.0 3433.2 8 9 1 0.2 0.2
3449.4 3449.2 9 9 0 0.2 0.7 No Y
3459.3 3458.3 6 10 2 0.1
3474.2 7 10 1 0.1 Y
3480.4 3481.2 9 7 3 0.2 0 Y
3490.3 8 10 0 0.3 Y
3521.6 3522.3 8 8 3 0.4 Y 0 Y No
3538.2 3538.3 9 8 2 0.3 0 Y No
3562.9 3563.3 7 9 3 0.5 0 Y No
3579.1 3579.3 8 9 2 0.7 0 Y No
3594.9 3595.3 9 9 1 0.8 0 Y(0) No
3607.0 3604.4 6 10 3 0.3 Y No
3621.8 3620.4 7 10 2 0.3 Y No
3636.8 3636.4 8 10 1 0.5 Y(0) No
3725.8 3725.5 8 9 3 0.1 0
3740.6 3741.5 9 9 2 0.4 0 Y(0) No
3753.4 3750.5 6 10 4 0.1 Y No
3757.5 10 9 1 0.3 Y
3768.2 3766.5 7 10 3 0.1
3782.3 3782.5 8 10 2 0.3 0.1
3797.6 3798.5 9 10 1 0.2 0.3 Y
3814.4 10 10 0 0.4 Y
3827.0 3823.6 7 11 2 0.1 Y No
3839.6 8 11 1 0.1 Y
3844.2 3846.5 10 8 3 0.2 0
3855.6 9 11 1 0.1 Y
3873.2 3871.6 8 9 4 0.1 0
3887.2 3887.6 9 9 3 0.1 0 Y No
3903.1 3903.6 10 9 2 0.1 Y No
3945.4 3944.7 9 10 2 0.3 Y No
3960.8 3960.7 10 10 1 0.5 Y(0) No
3968.3 3969.7 7 11 3 0.1
3976.7 11 10 0 0.1 Y
3985.8 3985.7 8 11 2 0.1 Y No
4003.5 4001.7 9 11 1 0.1 Y(0) No
4032.9 4033.8 9 9 4 0.1 Y No
4048.6 4049.8 10 9 3 0.1 Y No
4064.2 4065.8 11 9 2 0.1 Y No
4075.1 4074.8 8 10 4 0.2 Y No
4090.6 4090.8 9 10 3 0.3 Y No
4107.7 4106.8 10 10 2 0.2 Y No
4163.9 10 11 1 0.1 Y
4180.0 11 11 0 0.3 Y
4204.9 8 12 1 0.1 Y
4211.4 4211.9 11 9 3 0.1 Y No
4234.4 4237.0 9 10 4 0.1 Y No
4254.6 4253.0 10 10 3 0.1 Y No
4268.9 4269.0 11 10 2 0.2 Y No
4293.9 4294.0 9 11 3 0.1 Y No
4310.3 4310.0 10 11 2 0.2 Y No
4325.2 4326.0 11 11 1 0.3 Y(0) No
4330.9 4529.2 11 12 1 0.1 0.1
4340.0 4342.0 12 11 0 0.1 No
4354.0 4351.1 9 12 2 0.1 Y No
4367.6 4367.1 10 12 1 0.1 Y(0) No
4379.3 4278.0 8 11 4 0.1 Y No
4472.3 4472.2 11 11 2 0.1 Y No
4488.7 4488.2 12 11 1 0.1 Y(0) No
4513.6 4513.2 10 12 2 0.1 Y No
4546.6 4545.2 12 12 0 0.1 0.3 No
4570.3 10 13 1 0.1
4580.4 4577.2 12 10 3 0.2 Y No
4619.2 4618.3 11 11 3 0.1 Y No
4636.8 4634.3 12 11 2 0.1 Y No

a Mass (average or chemical mass of the MNa+ ion) measured in original sample.
b Products following digestion with endo-beta -galactosidase.
c Products after incubation with C. lampas fucosidase.
d Products after successive incubations with C. lampas fucosidase and endo-beta -galactosidase.
e Products after successive incubations with C. lampas fucosidase, endo-beta -galactosidase, and bovine testis beta -galactosidase.
f Rem = oligosaccharide was removed during the incubation. No = not removed, Y = removed.
g Fmd = oligosaccharide was formed during the incubation. No = not formed, Y = formed.
h Numbers in parentheses after "Y" indicate the number of fucose residues remaining.
Blank space indicates uncertain or peak not observed. In most cases the spectra were too weak for any peaks to be visible.

Normal Phase HPLC Analysis of CD59 N-linked Glycan Pool (Fig. 1E.2)

The sialylated N-glycan pool from HuE CD59 was resolved into at least 36 peaks by normal phase HPLC (Fig. 7a). Each peak was numbered and assigned an HPLC glucose unit value by comparison with the elution positions of a standard 2AB-labeled dextran hydrolysate mixture (shown at the top of the figure). Individual peaks were then assigned structures from their glucose unit values using the elution positions of standard glycans and pre-determined incremental values for monosaccharide residues (32) (Table III). The data indicated that human erythrocyte CD59 contained families of bi-, tri-, or tetra-antennary complex glycans, which were both neutral and sialylated; neither hybrid nor oligomannnose structures were detected.


Fig. 7. a-f, HPLC profiles of the N-glycan population of HuE CD59 simultaneously digested with a series of enzyme arrays. The HPLC analysis of the total glycan pool (a) and the products resulting from the digestion of five aliquots of the total CD59 glycan pool with a series of enzyme arrays (b-f). The shaded areas define the peaks that contain glycans which were subsequently digested by the additional enzyme present in the next array. The glucose unit value of each peak was calculated (Table III) by comparison with the dextran hydrolysate ladder shown at the top of the figure. Structures were assigned from the glucose unit values, previously determined incremental values for monosaccharide residues (32) and the known specificity of the exoglycosidase enzymes (58).
[View Larger Version of this Image (38K GIF file)]


Table III.

The glucose unit values and structures assigned to the N-linked glycans released from human erythrocyte CD59 (columns 1-3) and HPLC analysis of the products of a series of five simultaneous exoglycosidase digestions of aliquots of the total pool of CD59 sugars (columns 4-8)

Colume 4-8 refer to the products of the enzyme assay digests shown in Fig. 7, b-f respectively. The numbering of the peaks is shown in Fig. 7 a, denotes a shoulder on the main peak.
1
2
3
4
5
6
7
8
Peak no. Assignment gu gu gu gu gu gu

0 M3N2 4.41
1 A2G0 5.5 5.5 5.5 5.5 5.5
2a A2G0B 5.81 5.81 5.81 5.81 5.81
2 A3G0 5.91 5.91 5.91 5.91 5.91
3 A2G0F 5.93 5.93
4a A3G0B 6.07 6.07 6.07 6.07 6.07
4 A2GOFB 6.24 6.24 6.24
5a O-link n/a 6.28
5 A2G1(1,6) 6.32 6.32 6.32 6.32
6 A2G1(1,3) 6.47 6.47 6.47 6.47
7 A2G1(1,6)B 6.47 6.47 6.47 6.47
7a A2G1(1,3)B 6.67 6.67 6.67 6.67
8 A4G0B 6.79 6.79 6.79 6.79 6.79
8a A2G1(1,6)F 6.65 6.65 6.65
9 A2G1(1,3)F 6.89 6.89 6.89
9a A2G1(1,6)FB 6.9 6.9 6.9
9b A2G1(1,3)BGNG 6.88 6.88 6.88 6.88
10b A2G1(1,6)BGNG 6.95 6.95 6.95 6.92
10 A2G1(1,3)FB 6.9 6.9 6.9
11a O-link n/a 7.05
11 A2G2 7.15 7.15 7.15 7.15
12 A4G0BGN 7.3 7.3 7.3 7.3 7.3
A2G2B 7.3 7.3 7.3 7.3
13 A2G2F 7.57 7.57 7.57
14 A2G2FB 7.67 7.67 7.67
15 O-link n/a 7.8
16 A2G2FFalpha 1,3 7.88 7.88
17 A3G2BF 8.17 8.17 8.17
18 A3G3 8.32 8.32 8.32 8.32
19 A2G2FBSA(1,6) 8.42
20a A2G2BFF 8.53 8.53
20b A4G2F 8.46 8.46 8.46
20c A3G3F 8.69 8.69 8.69
21 A2G2B(GN)2G 8.96 8.96 8.96 8.96
21a A3G3BF 8.83 8.83 8.83
22 A2G2FBS2(6) 9.19
23 A2G2FB(GN)2G 9.33 9.33 9.33
23a A3G3FF(alpha 1,3) 9.49 9.49
A4G4 9.67
24 A4G3BF 9.71 9.71 9.71
A4G4B 9.77
25 A2G2BF(GNG)2 10.04 10.04 10.04
26 n/a 10.18 10.18 10.18 10.18
26a n/a 10.29 10.29 10.29
27 n/a 10.45 10.45
28 n/a 10.6 10.6 10.6
29 A2G2FB(GN)2GS(1,6) 10.68
30 A2G2FB(GN)2GS(1,3) 10.7
31 A2G2B(GNG)3 11.02 11 11 11
32 n/a 11.19
33 n/a 11.64
34 n/a 12.01 12.09 12.09 12.09
35 n/a 12.58 12.66 12.42
36 n/a 14.9

In view of the wide heterogeneity revealed by both the MALDI-MS and HPLC analyses of the CD59 N-glycans, no attempts were made to purify and define every individual oligosaccharide. Classically, enzyme arrays and P4 GPC have been used to sequence individual sugars purified to >80% homogeneity from the glycan pool. In the case of CD59, this would have required an extremely lengthy and difficult separations procedure. Therefore, a strategy was developed which involved the simultaneous sequencing of aliquots of the total glycan pool with enzyme arrays followed by HPLC analysis (Fig. 7). The glycan structures assigned by this novel strategy were confirmed as follows: (i) the sialylation status of the sugars was confirmed by WAX chromatography followed by normal phase HPLC; (ii) further studies with exoglycosidase enzymes confirmed the structures of the bi-antennary non-extended complex glycans and the oligosaccharides containing core and/or outer arm fucose; and (iii) glycans with lactosamine extensions were further characterized using endo-beta -galactosidase.

Simultaneous Sequencing of the Total Glycan Pool Using Enzyme Arrays (Fig. 1E.3)

A preliminary assignment of structures to the N-glycans from HuE CD59 was made from the HPLC gu values (Fig. 7a and Table III). These assignments were confirmed by digesting aliquots of the total pool of CD59 glycans with five different enzyme arrays. The digestion mixtures were resolved by normal phase HPLC (Fig. 7, b-f) (Table III, columns 4-8). The assignment of each of the main peaks in the CD59 glycan pool was checked by following its predicted elution position through each of the enzyme digests. Predicted elution positions were based on predetermined incremental values for monosaccharide additions to standard glycan cores (32) and the known specificity of the enzymes (Fig. 1b).

Charge Analysis of CD59 N-Glycans (Fig. 1E.4)

The 2AB-labeled CD59 glycan pool was separated on the basis of charge by WAX chromatography (Fig. 8). Five fractions were collected that contained neutral (N, 37%), monosialylated (A1, 22%), O-linked (5%), disialylated (A2, 14%), and trisialylated (A3, 22%) sugars. All glycans eluted with the naturally neutral sugars after incubation of the glycan pool with A. ureafaciens neuraminidase, indicating that all the charge was derived from sialic acid.


Fig. 8. Analysis of CD59 glycans on the basis of charge using WAX HPLC. a, WAX analysis of HuE CD59. Five pools (N, A1, O, A2, A3) were collected. The charges of the N-glycan pools were assigned by comparison with the elution positions of standard fetuin N-linked sugars on the same system (b). N, neutral glycans; A1, A2, and A3, mono-, di- and tri-sialylated N-links respectively. The peak A4 contains tetra-sialylated N-linked sugars not present in CD59. Peak O in CD59 contains O-glycans. The conductivity of the gradient is shown as a line in Fig. 8b.
[View Larger Version of this Image (19K GIF file)]


The naturally neutral N-glycans (pool N) and de-sialylated N-glycans prepared from pools A1, A2, and A3 were analyzed by normal phase HPLC (Fig. 9). The monosialylated (A1) N-glycan pool contained bi-, tri-, and tetra-antennary sugars; the disialylated (A2) N-glycans were mainly of the bi-antennary type, while the trisialylated (A3) N-glycan fraction contained tri- and tetra- antennary glycans with and without lactosamine extensions. Table IV indicates the charge states which were associated with each glycan.


Fig. 9. Normal phase HPLC analysis of the neutral, mono-, di-, and trisialylated N-glycan populations of HuE CD59 after desialylation with A. ureafaciens sialidase. a, total glycan pool; b, desialylated total glycan pool; c, neutral fraction; d, desialylated A1 (monosialylated) WAX fraction; e, desialylated A2 (disialylated) WAX fraction; and f, desialylated A3 (trisialylated) WAX fraction.
[View Larger Version of this Image (14K GIF file)]


Table IV.

Analysis of CD59 N-linked glycans by charge


1
2
3
4
5
6
7
8
Peak gu Structure Composition Neutral A1 A2 A3

1 5.5 A2G0 H3N4 N
2 5.81 A2G0B H3N5 N
3 5.93 A2G0F H3N4F N
4 6.24 A2G0FB H3N5F N
5 6.32 A2G1(1,6) H4N4 N
6 6.47 A2G1(1,3) H4N4 N
6a 6.47 A2G1(1,6)B H4N5 N
7 6.67 A2G1(1,3)B H4N5 N
8a 6.74 A2G1(1,6)F H4N4F N
9 6.89 A2G1(1,3)F H4N4F N A1
9a 6.9 A2G1(1,6)FB H4N5F N A1
10 6.9 A2G1(1,3)FB H4N5F N A1
11 7.15 A2G2 H5N4 N A1 A2
12 7.3 A2G2B H5N5 N A1 A2
13 7.57 A2G2F H5N4F N A1 A2
14 7.67 A2G2FB H5N5F N A1 A2
16 7.88 A2G2FFalpha 1,3 H5N4F2 N A1 A2
17 8.14 A2G2BFF H5N5F2 N A1 A2
18 8.32 A3G3 H6N5 N A1 A2 A3
19 8.42 A2G2FBS1(1,6) H5N5FS1
20 8.53 A2G2BFF H5N5F2 N A1 A2
20a 8.46 A4G2F H5N6F N A1 A2
20c 8.69 A3G3F H6N5F N A1 A2 A3
21a 8.83 A3G3BF H6N6F N A1 A3
22 9.19 A2G2FBS2 H5N5FS2
23 9.33 A2G2FB(GN)2G H6N7F A1
23a 9.49 A3G3BFFalpha (1,3) H6N6F2 A3
24 9.71 A4G3BF H6N6BF N A1 A3
25 10.04 A2G2BF(GNG)2 H5N8BF N
25a not assigned A1 A3
26 10.29 not assigned A1
27 10.45 not assigned N/D
28 10.6 not assigned A1

Analysis of the Major Glycan Population of Bi-antennary Non-extended Complex N-glycans (Fig. 1E.5)

As a result of the above analyses, peaks 1-14, 16, and 17 in Fig. 7a were assigned to a series of neutral complex bi-antennary glycans. The major neutral glycan, A2G2FB, was isolated and its structure further confirmed by the reagent array analysis method (42) (data not shown). A comparison of the native and asialo HPLC profiles (Fig. 7, a and b) indicated that A2G2FB eluted in peak 14, while the mono- and di-sialylated forms of this sugar eluted in peaks 19 and 22, respectively. This was consistent with the WAX chromatograhy data (Table IV).

Further Characterization of Oligosaccharides Containing Fucose (Fig. 1E.6)

The 2AB-labeled asialo glycan pool was incubated with C. lampas fucosidase, which removes both core fucose (GlcNAcbeta 1-4(Fucalpha 1,6)GlcNAc) and outer arm fucose residues linked Fucalpha 1-2Gal and Galbeta 1-3(Fucalpha 1-4 GlcNAc)right-arrowGalbeta 1-4(Fucalpha 1-3)GlcNAc (43). MALDI-MS analysis of the digestion products indicated that most fucose residues were removed by the enzyme (Fig. 6b and Table 2, footnote c), but a number of structures, in particular the mono, di, and tri-fucosylated forms of H7N6, were resistant. These glycans contained fucose residues linked alpha 1,3 to N-acetyl glucosamine in the outer arm and which were susceptible to almond meal fucosidase that is specific for this linkage (data not shown). These data were consistent with the results of the enzyme digests (Fig. 7, b and c).

Further Characterization of Multi-antennary Glycans and Glycans with Polylactosamine Extensions (Fig. 1E.7)

In the normal phase HPLC separation of the total glycan pool (Fig. 7a), peaks 18 and 20-36 were assigned to multi-antennary and polylactosamine structures. The largest structure resolved by this system was a bi-antennary extended glycan with the composition formula H7N7 (peak 36). However, MS identified 75 additional structures which were larger than H7N7. Most of these contained more than one fucose residue, and 57 were present at 0.3% or less (Table 2). Interestingly, some of the compositions were consistent with those of multi-antennary glycans with outer arm branching such as have been detected on the lactosamine sugars released from erythrocyte band 3 protein (44).

The polylactosamine-type structures were investigated by digesting the asialo glycan pool with EBG after defucosylation with C. lampas fucosidase. The digestion mixture was analyzed using both MALDI-MS (Fig. 6d and Table 2) and HPLC (Fig. 10b). In both cases, the major products of the EBG digestion were A2G0B (H3N5), A2G1B or A3G1 (H4N5), A4G2B (H5N7), A4G3B (H6N7), and A4G4B (H7N7). These structures are drawn in Fig. 10c. The glycan with the composition H7N7 has been shown with the fourth galactose attached to the bisecting GlcNAc since the alternative oligosaccharide with this composition is the fully galactosylated tetra-antennary structure, which is not a product of an EBG digest. There is no further evidence for the proposal that this structure contains galactose linked to the bisecting GlcNAc, but, interestingly, it has been noted (44) in the glycans attached to erythrocyte band 3 protein.


Fig. 10. a-c, analysis of polylactosamine structures attached to CD59. a, HPLC profile of the total pool of CD59 glycans after digestion with A. ureafaciens sialidase and C. lampas fucosidase. Glycans with lactosamine extensions elute from approximately 9-12 gu. 20% of the glycans associated with CD59 are larger than this and elute later in a broad peak which is not resolved using this HPLC gradient. b, HPLC profile of CD59 glycans after incubation with A. ureafaciens sialidase, C. lampas fucosidase, and EBG. The major products of the digestion are in peaks 1-5. c, a schematic diagram showing the structures of the major digestion products. The structure with the composition H7N7 has been assigned tentatively to a tetra-antennary structure (A4G4B*) containing a galactose linked to the bisecting GlcNAc.
[View Larger Version of this Image (16K GIF file)]


These data indicate that CD59 contains (i) bi-antennary glycans with lactosamine extensions on both arms, (ii) tetra-antennary glycans with two and three lactosamine extensions, and (iii) many larger polylactosamine structures present at low levels which were digested by EBG to A2G1(1,3)B and/or A3G1 (H4N5). These latter structures are derived from tri-antennary glycans with extensions on two arms and/or bi-antennary glycans with bisecting GlcNAc and lactosamine extensions on one arm. Since no A2G1(alpha 1,6) glycans were detected by HPLC analysis after the EBG digestion, A2G1(1,3)B was derived from bi-antennary glycans extended on the alpha 1,6 arm and containing only one (terminal) galactose on the alpha 1,3 arm. This tendency for the 1,6 arm to be extended in preference to the 1,3 arm has been noted by Fukuda et al. (44) in band 3 protein from erythrocytes.

A2G1(1,3)B and A3G1 co-elute on this HPLC system and cannot be resolved by MS since they have the same composition (H4N5). The basic core structures were therefore determined by incubating the EBG-treated glycan pool with bovine testes galactosidase and analyzing the products by MS (Fig. 6d and Table 2). The major core structures were A2G0 (30%), A2G0B/A3G0 (60%), and A3G0B (5%).

Characterization of the O-Glycans Attached to Human Erythrocyte CD59 (Fig. 1F)

The presence of O-linked sugars, not previously reported on CD59, was initially suggested by GC-MS composition analysis of the glycan pool (Table I). Consistent with this finding, the HPLC profile of the pool of sugars released from CD59 contained peaks eluting with glucose unit values corresponding to O-linked glycans (Fig. 5a). The two major peaks (A and B) were analyzed by enzyme digests coupled with normal phase HPLC (data not shown). After incubation with A. ureafaciens sialidase, both A and B were digested to one peak. This was assigned by comparison with known standards to Galbeta 1, 3GalNAc. After incubation with an enzyme array containing both sialidase and bovine testes galactosidase, both A and B were digested to GalNAc. The elution positions of A and B and the products of the digests were consistent with the presence of two species of glycans, both containing the disaccharide Galbeta 1,3GalNAc but with sialic acid-linked alpha 2,3 to the Gal in one case (A) and alpha 2,6 to the GalNAc in the other (B).

Location of Possible O-Glycosylation Sites (Fig. 1G)

Since O-glycans attached to CD59 have not been reported previously, attempts were made to locate the glycosylation sites in the protein. Edman degradation of intact CD59 was carried out for 30 cycles. This covered all the Thr (10, 13, 29) and Ser (20, 21) residues, which are potential O-glycosylation sites, in the first half of the CD59 molecule. The data that were obtained were identical to the published amino acid sequence (45, 46). The repetitive yields for the five residues at potential sites for O-glycosylation were close to the predicted value (data not included). The previously published amino acid sequence of the second half of the molecule was derived solely from the cDNA sequence. No amino acid analysis had been carried out on this section of the molecule, with the exception of the last six amino acids which contain the GPI-anchoring site (47). Here protein enzymatic digestion was carried out with endoproteinase Lys-C, which specifically digests at the C terminus of lysine. As predicted, this approach yielded a 3-kDa fragment from amino acid 42 to amino acid 64 of the mature protein. This fragment was separated from partial digests and smaller fragments on a Tris/tricine gel, blotted, and sequenced. The amino acid sequence was identical to the cDNA derived sequence. All potential O-glycosylation sites were detected, and the repetitive yields for residues Thr51 and Thr52 were reduced, although not entirely absent (data not included). The level of the reduction was consistent with the finding that approximately 30% of the glycan pool consists of O-linked sugars. These findings, while not definitive, suggest that O-linked sugars may be variably attached to residues Thr51 and Thr52. Further studies are being undertaken to confirm the presence of the O-linked glycans, and to determine the linkage positions since these structures have not been identified in earlier studies of CD59.

Molecular Modeling Studies of CD59 (Fig. 1H)

Molecular modeling studies using the x-ray crystallographic co-ordinates (48) and the enzyme binding site residues (49) indicate that PNGase F will only cleave N-glycosidic linkages when the sugar is in a conformation in which there are no interactions of the outer arm residues and the target protein. In this study, CD59 was incubated with PNGase F to probe for possible interactions of the outer arm residues with the protein. Analysis of CD59 by SDS-PAGE showed that the protein migrated as a broad band with an apparent molecular mass of 20-25 kDa (data not shown), consistent with the presence of a range of glycoforms. After incubation of native, undenatured CD59 with PNGase F, the protein migrated as a narrow band with an apparent molecular mass of approximately 15 kDa. This indicated that the glycoform population had been digested by the enzyme and that the Asn18-sugar amide linkage is fully accessible to PNGase F. These data are consistent with NMR analyses of glycosylated forms of CD59, which provided little evidence for protein-sugar interactions beyond the first N-acetylglucosamine residue of the N-linked glycan (17, 18). Moreover, incorporation of model oligosaccharides in structure calculations (18) suggested that the N-glycan projects away from the protein in the plane of the disc-like extracellular domain.


DISCUSSION

The GPI-anchored glycoprotein, CD59, consists of a heterogeneous mixture of more than 120 glycoforms. The full significance of glycosylation for the structure and function of CD59 can only be explored properly when its glycan population is viewed in the context of the complete structure of the molecule. In previous work, the three-dimensional structure of the protein component of CD59 (17, 18) and the location of the active site of the molecule2 have been determined. The aim of the present study was to determine the structure of both the GPI anchor and the N- and O-linked glycans of CD59 in order to complete the initial structural analysis and to provide a context for considering the function of the glycosylation of the molecule. With respect to its structure, CD59 is now the best defined cell surface molecule studied to date.

A Comparison of the GPI Anchors Attached to CD59 and Acetylcholinesterase Suggests That Structural Features of GPI Anchors May Be Cell-type Specific

This study has enabled the first comparison of GPI anchors expressed on two different glycoproteins in the same tissue, human erythrocytes. The majority (91 mol %) of the GPI anchors of CD59 were identical, in terms of PI and glycan structure, to that described for acetylcholinesterase (23, 26). The identical PI moieties in particular, which contain alkyl chains and C22:4 fatty acid components that are not common in mammalian PI pools (50), suggest that, in reticulocytes, the two proteins receive the same GPI precursor in exchange for their different COOH-terminal GPI signal peptides. The small proportion of CD59 anchors subsequently modified by the addition of beta -GalNAc suggests that at least some human reticulocytes possess the beta -GalNAc transferase required for this relatively common GPI modification.

Analysis of the GPI Anchor and the N- and O-Glycans of Human Erythrocyte CD59 Indicates That Soluble Urinary CD59 (U-CD59) Does Not Originate From Erythrocytes

While CD59 is normally attached to cell surfaces, a number of soluble forms have been found including urine (U-) CD59. Two studies (51, 52) have shown that soluble U-CD59 contains a GPI anchor without any lipid moieties, consistent with a PIPLD cleavage product of PIPLC-sensitive material. In addition, there was extensive modification of the tri-mannosyl core with GalNAc (45 mol %). This is in contrast to the present finding that only 9 mol % of the trimannosyl core of the anchor of membrane bound erythrocyte CD59 was modified. Analysis of the N-linked glycans of U-CD59 also revealed significant differences between urine and erythrocyte CD59 and, in addition, no O-glycans have been reported on U-CD59. Thus, an examination of the structures of the GPI anchor and the glycans suggests that little or no urinary CD59 originates from erythrocytes. An explanation for the apparent sequestration of erythrocyte CD59 from urine can be proposed on the basis of the new data. GPI-anchored proteins which appear in a soluble form in the urine may be the result of PIPLC- or PIPLD- type cleavages. The PIPLC resistance of HuE CD59 may prevent its appearance in urine even if it undergoes transfer between plasma membranes as has been suggested by several studies (53, 54).

Human Erythrocyte CD59 Consists of at Least 120 Different Glycoforms

While the structure of the GPI anchor glycan was relatively homogeneous, the N-glycan population was extremely heterogeneous. However, despite the apparent heterogeneity of the N-glycans, the major population of glycoforms consisted of a family of structures based on bisected, core-fucosylated, bi-antennary glycans carrying varying numbers of lactosamine extensions. A high proportion of the larger sugars were bisected bi-antennary glycans with polylactosamine extensions on one arm containing one or more fucose residues alpha 1-3 linked to a GlcNAc residue.

Molecular Modeling Suggests Several Roles for the Glycans Attached to CD59

The N-linked oligosaccharides (size range 3-6 nm in length) attached to the disc-like extracellular region of CD59 (diameter of approximately 3 nm) project away from the protein domain in the plane of the active face and adjacent to the membrane surface (Fig. 11). The glycans do not appear to restrict access to proposed active site residues of human CD59 (Asp24, Trp40, Arg53, and Glu56) located on the membrane distal surface of the extracellular domain2. However, the glycans would be expected to restrict the rotational freedom of the extracellular domain around axes parallel to the membrane that may, in turn, stabilize an exposed location for the active face (55). Removal of the conserved N-linked glycan might therefore reduce the affinity of CD59 for the membrane attack complex without eliminating it completely. The effects of removing the N-linked glycan might therefore be expected to depend on the density of expression of the glycoprotein at the cell surface, and this may explain the observed variation in the activities of unglycosylated CD5923 (13, 45). A discrepancy between the measured two-dimensional affinity of the cell surface recognition molecules CD2 and LFA-3 and of the two-dimensional affinity predicted on the basis of the measured three dimensional affinity (6) suggests that the mobility of the CD2 ligand binding site is restricted perpendicular to the cell surface (56). It seems likely that N-glycans attached to the highly conserved, membrane proximal glycosylation site at the base of CD2 may contribute to this effect. This suggests that restricting the conformational space available to cell surface glycoproteins may be a key function of N-glycosylation.


Fig. 11. A molecular model of CD59 based on the protein coordinates from the solution structure (18). a and b, two orthogonal views of the model drawn in space-filling format using the program RasMol (59). Active site residues D24, W40, R53, and E562 are shown in purple. The glycan anchor (G, green), is modeled with a trimannosyl core (gold), an ethanolamine bridge at Man3, and additional ethanolamine groups at Man1 and Man2. Two lipids are attached to inositol via phosphate, and the third is attached directly to the inositol ring through an ester linkage. A trisialylated, tetra-antennary complex N-glycan (N, light blue) is shown attached to Asn18. The structure of the N-glycosidic linkage is based on a study by Wormald et al. (60). The O-glycan (O, dark blue), NeuNAc2,3Galbeta 1-3GalNAc, is attached to Thr51 to indicate one of the possible linkage positions. To illustrate the probable relationship between CD59 and the erythrocyte cell surface, the plasma membrane is depicted by two parallel red lines; the distance between these lines reflects the maximum dimensions of the bilayer based on the work of Hauser et al. (61). Views c and d, which are slightly enlarged with respect to views a and b, are orthogonal to the plane of the membrane which, for clarity, is not depicted. In view c, all residues are colored as in views a and b, whereas in view d, additional potential (exposed) O-glycosylation sites, rather than active site residues, are shown in purple and are labeled.
[View Larger Version of this Image (40K GIF file)]



Fig. 12. A schematic figure showing the effect of CD59 glycosylation on the flexibility of the protein relative to the GPI anchor. Three different glycoforms are shown. Both the O- and N-linked oligosaccharides (size range 2-6 nM) attached to CD59 (diameter of approximately 3 nM) restrict the conformational space available to the protein and limit its interaction with the lipid bilayer. The sugars may therefore orient the active site of CD59 toward the C5b-9 complex, which is also inserted into the cell membrane. In addition, the heterogeneity of the sugars suggests that the glycans influence the geometry of the packing, and it is unlikely that CD59 molecules will form a regular array on the cell surface. The active site residues are highlighted in CPK.
[View Larger Version of this Image (21K GIF file)]


Molecular modeling of the GPI-anchored glycoprotein Thy-1 (57) suggested that the glycoprotein sits directly on the membrane and that there are extensive interactions between the protein and the GPI glycan. In contrast to Thy-1, where the anchor is attached to a cysteine residue involved in an intra-molecular disulfide bond (40), CD59 contains a hydrophilic C-terminal sequence linking the protein to the GPI anchor. The protein component of CD59 would therefore have considerable dynamic freedom relative to the membrane, and it seems likely that there would be very few non-covalent interactions between the protein and the anchor. This view is supported by NMR data from the analysis of the soluble form of U-CD59, which shows that the last few hydrophilic residues have a poorly defined structure. The bulky, hydrophilic glycans in the anchored form of CD59 would be expected to limit interactions with the lipid bilayer and in this way facilitate diffusion of the glycosylated protein in the membrane.

Fig. 12 shows three differently glycosylated CD59 molecules inserted into the cell membrane. The heterogeneity of the sugars suggests that the glycans influence the geometry of the packing, and it is likely that they will also prevent the aggregation of CD59 molecules on the cell surface. By limiting such protein-protein interactions, the glycans could influence the distribution of CD59 molecules at the cell surface where GPI anchored proteins may associate in micro-domains in dynamic equilibrium with isolated individual molecules. The large N-glycans may also be important in preventing proteolysis of the extracellular domain since N-glycosylation has been shown to increase the dynamic stability of a protein while different glycoforms variably increase its resistance to protease digestion (55).

Human Erythrocyte CD59 Contains Potential O-Glycosylation Sites

A population of sialylated O-glycans was recovered from human erythrocyte CD59; the major species that were identified were two mono-sialylated forms of the disaccharide Galbeta 1,3GalNAc. Interestingly, asialo human erythrocyte CD59 failed to bind peanut agglutinin (13), a lectin which is specific for Galbeta 1,3GalNAc. This suggests that the O-linked Galbeta 1, 3GalNAc glycans detected on CD59 may not be accessible to the lectin. In view of this result (13), and the fact that O-glycans have not been identified previously on CD59, further studies are being undertaken to confirm the presence of the O-linked sugars and to determine their attachment sites. In the present study, attempts to locate the O-glycosylation sites on the basis of signal supression during protein sequencing suggested that the O-glycans may partially occupy Thr51 and Thr52.

In support of this, inspection of the molecular model suggested that several potential sites are accessible. Two structures are available, both derived from NMR solution studies (17, 18). Rotation and inspection of both structures indicates that Ser20, which forms part of the N-glycosylation sequon, may not be readily accessible once the N-linked sugar is attached at Asn18. However, the side chain of the adjacent residue, Ser21, presents a feasible site for O-glycosylation. Thr15 is also close to Asn18, but is accessible, as are the side chains of Thr52 and Thr60. Thr29 is inaccessible. In the structure determined by Fletcher et al. (18), the side chain of Thr51 is also accessible (Fig. 11d). Interestingly, in the structure determined by Kieffer et al. (17), a small difference in the conformation of the helix to which Thr51 is attached causes the side chain to be differently oriented so that, in this case, it is inaccessible. Thr10 is partially hindered in both models. As a result, it may be concluded that potential sites at Ser20, Thr10, and Thr29 are unlikely to be O-glycosylated, while both Thr51 and Thr52 are situated on a helix and sufficiently well exposed to be O-glycosylated. These residues are also adjacent to active site residues (Arg53 and Glu56) identified by site-directed mutagenesis2 (Fig. 11). This raises the possibility that the O-glycans may form part of the active site of CD59. Soluble, recombinant CD59 expressed in insect cells is active in complement inhibition assays (47) but the extent of O-glycosylation of this form of the molecule has not been determined. Conversely, Chinese hamster ovary cell-derived soluble CD59 (17) is not O-glycosylated,4 but it is not clear if protein from this source is active.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed. Tel.: 44 1 865-275340; Fax: 44 1 865-275216.
**   Supported by the Welcome Trust.
§§   Supported by the Welcome Trust and is a Howard Hughes Medical Institute International Research Scholar.
1   The abbreviations and other trivial names used are: GPI, glycosylphosphatidylinositol; 2AB, 2-aminobenzamide; AHM, anhydromannitol; Du, Dionex units; ES-MS, electrospray mass spectrometry; GC-MS, gas chromatography/mass spectrometry; HPLC, high performance liquid chromatography; gu, HPLC glucose units; HuE, human erythrocyte; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry; EBG, endo-beta -galactosidase; P4 GPC, BioGel P4 gel permeation chromatography; PI, phosphatidylinositol; PIPLC/D, PI-specific phospholipase C/D, respectively; PNGase F, peptide N-glycosidase F; U-CD59, urine CD59; WAX, weak anion exchange; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis, and the abbreviations used for describing oligosaccharde structures are: A(1-4), indicates the number of antennae linked to the trimannosyl core; G(0-4) indicates the number of terminal galactose residues in the structure; Fuc, fucose; B, bisecting N-acetyl glucosamine (GlcNAc); GalNAc, N-acetyl galactosamine; S (Sia), sialic acid; G (Gal), galactose; M (Man), mannose; H (Hex), hexose; N (HexNAc), N-acetylhexosamine; (GlcN), glucosamine; HPAEC, high performance anion exchange chromatography.
2   D. L. Bodian, S. J. Davis, N. K. Rushmere, and B. P. Morgan, in press (Feb. issue) J. Exp. Med.
3   P. B. Morgan, unpublished data.
4   P. M. Rudd, K. O. McLellan, S. J. Davis, and R. A. Dwek, unpublished data.

Acknowledgments

The authors thank Dr. David Wing for critically reading the manuscript and Joshua Dwek for valuable contributions to the molecular modeling studies.


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