Comparison of the N-linked glycans from soluble and GPI-anchored CD59 expressed in CHO cells

Susan F. Wheeler2, Pauline M. Rudd2, Simon J. Davis3, Raymond A. Dwek2 and David J. Harvey1,2

2Oxford Glycobiology Institute, Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, United Kingdom; and 3Molecular Sciences Division, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 9DU, United Kingdom

Received on July 16, 2001; revised on January 7, 2002; accepted on January 7, 2002.


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The N-linked glycosylation of recombinant human CD59, expressed in Chinese hamster ovary (CHO) cells with and without a membrane anchor, was compared to examine the effect of the anchor on glycan processing. N-Linked glycans were released with peptide-N-glycosidase F (PNGase F) within gel from SDS–PAGE-isolated soluble and glycosylphosphatidylinositol (GPI)-anchored human CD59 expressed in CHO cells. The anchored form contained core-fucosylated neutral and sialylated bi-, tri-, and tetraantennary glycans with up to four N-acetyllactosamine extensions. Exoglycosidase digestions and analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were used to define the relative amounts of the bi-, tri-, and tetraantennary glycans and to investigate the distribution of N-acetyllactosamine extensions between their antennae. Biantennary structures accounted for about 60% of the glycans, 30% of the triantennary structures, and about 10% of the tetraantennary structures. For tri- and tetraantennary glycans, those with extended antennae were found to be more abundant than those without extensions. The soluble form of CD59, expressed in CHO cells without the GPI anchor signal sequence, consisted almost entirely (97%) of biantennary glycans, of which 81% were unmodified, 17% contained one N-acetyllactosamine extension, and 2% contained two extensions. No compounds with longer extensions were found. A MALDI spectrum of the intact glycoprotein showed a distribution of glycans that matched those released with PNGase F. In addition, the protein was substituted with several small glycans, such as HexNAc, HexNAc->Fuc, and HexNAc->HexNAc, probably as the result of degradation of the mature N-linked glycans. The results show that the presence of the anchor increases the extent of glycan processing, possibly as the result of longer exposure to the glycosyltransferases or to a closer proximity of the protein to these enzymes.

Key words: CD59/CHO cells/MALDI MS/N-acetyllactosamine extensions/N-linked glycosylation


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CD59 (protectin) is a human glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that serves as the principal cellular inhibitor of the C5b-9 membrane attack complex (MAC) of human complement (Davies and Lachmann, 1993Go). The cells that are in contact with blood plasma are resistant to attack by the homologous complement system under normal conditions. Cell damage by the complement system is mediated by the MAC, which is a macromolecular complex of C5b, C6, C7, C8, and multiple C9 molecules (Muller-Eberhard, 1986Go). When CD59 inhibits MAC formation by interacting with C8 in C5b-8 and C9 in C5b-9, it prevents poly-C9 formation. CD59 is widely distributed on the surface of both hemopoietic and nonhemopoietic cells (i.e., erythrocytes, all types of leukocytes, fibroblasts, epithelial cells, and myeloma lines) (Davies et al., 1989Go) and has also been found in many extravascular tissues (Meri et al., 1991bGo; Walsh et al., 1992Go). A soluble form of CD59 (sCD59) has been reported in urine, saliva, tears, sweat, cerebrospinal fluid, seminal plasma, amniotic fluid, and breast milk (Davies et al., 1989Go; Meri et al., 1991aGo) with the presence of a lipid tail characterized in the latter three fluids (Lehto et al., 1995Go). At present, the origin or role of urinary CD59 (usCD59) is unknown, although possible explanations include (1) CD59 being shed from the cell surfaces following release from the anchor by the action of phospholipase D and phospholipase C, or (2) incomplete addition of the GPI anchor to all CD59 molecules during their biosynthesis, resulting in secretion of the soluble form. The latter explanation, however, appears unlikely because GPI-anchored proteins with signal peptides that do not receive a GPI anchor are usually degraded intracellularly and not released in a soluble form (Ali et al., 2000Go).

The amino acid sequence of CD59, predicted from its cDNA nucleotide sequence, consists of 128 amino acids (Sugita et al., 1989Go), including an amino-terminal signal peptide of 25 residues and a carboxyl-terminal of 26 residues. This C-terminal sequence is a signal that directs the attachment of a GPI membrane anchor (Sugita et al., 1993Go) The mature glycoprotein, after removal of the signal peptide, has 77 amino acids (sequence mass = 8707.8; Figure 1). The soluble form of CD59 used in this study was truncated at Asn70, giving a sequence mass of 8076.2. It was expressed in Chinese hamster ovary (CHO) cells (Rudd et al., 1999Go).



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Fig. 1. Amino acid sequence of CD59.

 
CD59 carries a single N-glycosylation site at Asn18. The glycans at this site account for approximately 30–50% of the total apparent mass (Ninomiya et al., 1992Go). The site is conserved in all known sequences except that from rat, where the site is at Asn16 (Rushmere et al., 1994Go). Several roles for this glycosylation have been suggested. Recombinant soluble CD59 isolated from bacterial cell cultures (which do not possess N-linked glycosylation machinery) shows markedly diminished MAC inhibitory activity (Nakano et al., 1993Go; Suzuki et al., 1994Go) supporting the role of glycosylation in protecting the cells from lysis. Menu et al. (1994)Go have found that the ability of CD59 to enhance CD58-dependent T cell responses is dependent on N-linked glycosylation. In contrast, more recent studies involving unglycosylated rCD59 (Bodian et al., 1997Go; Rushmere et al., 1997Go; Suzuki et al., 1996Go; Yu et al., 1997Go) or rCD59 in which the glycosylation site has been transported to another site in the polypeptide (Rother et al., 1996Go), suggest that glycosylation of CD59 is not required for normal surface expression and function. Rudd et al. (1997)Go proposed, from molecular modeling studies, that the glycans might facilitate diffusion by preventing nonspecific protein–protein interactions on the membrane and prevent proteolysis of the extracellular domain. However, no biochemical studies have confirmed this theory. Nuclear magnetic resonance studies confirmed that the N-linked glycans do not interact with any other portion of the protein except via the Asn–sugar linkage (Kieffer et al., 1994Go).

The N-linked glycosylation of soluble (urinary) human CD59 (shCD59) has been analyzed by several investigators (Meri et al., 1996Go; Nakano et al., 1994Go). Rudd et al. (1999)Go analyzed the glycosylation of the soluble form expressed in CHO cells. The glycans identified in all three studies consisted mainly of core-fucosylated biantennary compounds. Less than 1% of the total were triantennary complex structures. Small amounts of glycans with N-acetyllactosamine extensions to the antennae were also found. All complex glycans were variably sialylated. Rudd et al. (1997)Go additionally analyzed the N-glycosylation of human erythrocyte CD59 containing the GPI anchor and identified more than 100 glycans, including a wide range of biantennary complex structures with multiple lactosamine extensions and several outer arm fucose residues. In addition, two potential O-glycosylation sites at Thr51 and Thr52 (Rudd et al., 1997Go) adjacent to the active site residues, Arg53 and Glu56 (Bodian et al., 1997Go) were found to be partially occupied with the O-glycans, Neu5Ac{alpha}2->3Galß1->3GalNAc and Galß1->3[Neu5Ac{alpha}2->6]GalNAc.

This article presents a more detailed description of the N-linked glycosylation of the soluble form expressed in CHO cells than that reported earlier (Rudd et al., 1999Go) and compares this with the glycosylation of the GPI-anchored form of the glycoprotein, again expressed in CHO cells. The aim was to investigate whether the glycan processing of CD59 is affected by the presence of a GPI anchor.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
GPI-anchored CD59
Recombinant human CD59 (rhCD59) expressed in CHO cells was dispersed in 10 mM 3-[3-cholamidopropyldimethylammonio]-1-propanesulfonate (CHAPS) and phosphate-buffered saline (PBS), pH 7.4, to ensure effective solubilization because of the presence of the hydrophobic GPI anchor moiety. The glycoprotein migrated as a single broad band of apparent molecular weight 20–25 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). During the electrophoresis process, the glycoprotein remained soluble in the presence of SDS, whereas the CHAPS surfactant, which interferes with the clean-up methodology and subsequent mass spectrometry (MS), was effectively removed. Glycans were released from the glycoprotein with PNGase F using the in-gel release method described by Küster et al. (1997)Go. Glycans were also released with this enzyme in solution, and SDS–PAGE analysis of the residual protein confirmed complete glycan removal. The in-gel released sample was divided into two; half was used directly for structural studies using exoglycosidase digestions, and the other half was treated with methyl iodide to convert the constituent sialic acids into their methyl esters (Powell and Harvey, 1996Go).

Figure 2 shows the positive ion matrix-assisted laser desorption/ionization (MALDI) mass spectrum of the methyl ester fraction from a 12.5% aliquot of the total glycan pool obtained from 3 µg of rhCD59. An identical profile was obtained from the solution-released sample. Glycans released by automated hydrazinolysis (Rudd et al., 1999Go) and examined as 2-aminobenzamide (2-AB) derivatives by either high-performance liquid chromatography (HPLC) or MALDI MS showed a similar profile.



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Fig. 2. Positive ion MALDI mass spectrum of the glycans released from GPI-anchored CD59. Sialic acids have been converted into their methyl esters. Peaks are identified in Table I. The numbers in circles represent the numbers of sialic acids associated with the compounds in each peak.

 
Masses, compositions, and structures of the glycans released by PNGase F are listed in Table I. The glycans consisted of both neutral and sialylated species as shown by incubation with Arthrobacter ureafaciens sialidase, which cleaves both {alpha}2->3 and {alpha}2->6 linked sialic acid, digested the glycans to those represented by the profile in Figure 3. Because CHO cells normally do not appear to express {alpha}2->6 sialyltransferase (Sasaki et al., 1987Go), all sialic acids were assumed to be {alpha}2->3-linked. However, as there has been at least one report of {alpha}2->6-linked sialic acids on CHO cell–derived recombinant human plasminogen (Davidson and Castellino, 1991Go), the possibility of sialic acid linked in this fashion cannot be ruled out. The peaks in Figure 2 appearing 16 mass units above those of the major peaks were mainly due to [M+K]+ ions of the glycans. However, for the sialylated glycans, there was also a contribution from the [M+Na]+ ions of the corresponding N-glycolyl analogs, as demonstrated by the presence of another set of ions appearing at 32 mass units above the mass of the parent compound. These latter ions corresponded to the [M+K]+ ions of the glycolylneuraminic acids. Addition of 60 pmole of sodium acetate solution to the glycan mixture prior to MALDI MS resulted in a large reduction in the relative abundance of all the [M+K]+ ions from the neutral glycans, consistent with the presence of an extra oxygen atom in the glycolylneuraminic acids. Following desialylation, these latter "[M+32]+" ions were absent, confirming the presence of the glycolylneuraminic acid moiety. The presence of glycolylneuraminic acids was not detected in the earlier study (Rudd et al., 1999Go) in which MALDI MS was only performed on the neutral glycans. The spectra of the sialylated glycans also contained ions at 42 and 58 mass units higher than those of the [M+Na]+ ions. These higher mass ions can be attributed to the [M+Na]+ and [M+K]+ ions from the corresponding 9-O-acetylneuraminic acids. Both N-glycolyl- and 9-O-acetyl-neuraminic acids have been reported before as products of CHO cell glycosylation (Bergwerff et al., 1993Go; Hokke et al., 1995Go; Noguchi et al., 1995Go, 1996).


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Table I. Masses and compositions for the glycans released from the recombinant GPI-anchored and soluble forms of rhCD59
 


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Fig. 3. Positive ion MALDI mass spectrum of the glycans released from GPI-anchored CD59 following treatment with A. ureafaciens sialidase. Peaks are identified in Table I. Key to symbols: circle = mannose, squares = N-acetylglucosamine, diamonds with dots = fucose, open diamonds = galactose.

 
The major oligosaccharide in the spectrum of the desialylated glycans (Figure 3) corresponded to a composition of (Hex)5(HexNAc)4(Fuc)1 and was shown by further exoglycosidase digestions (removal of two hexose residues with Saccharomyces pneumoniae ß-galactosidase and two HexNAc residues with jackbean N-acetyl-hexosaminidase) to be the fucosylated biantennary glycan (I, Figure 4). All other glycans in the sample were also reduced to the same core (Man)3(GlcNAc)2(Fuc)1 structure by this enzymatic treatment, confirming that they were all complex rather than high-mannose or hybrid sugars. Further treatment with bovine epididymis {alpha}-fucosidase reduced this core glycan to (Man)3(GlcNAc)2 and, after additional incubation with jackbean {alpha}-mannosidase, the only structure detected corresponded to (Man)1(GlcNAc)2 (m/z 609.3), thus confirming the common N-linked glycan core structure for all glycans in the mixture. The major glycans in the sample formed a series differing by the equivalent of a Gal-GlcNAc group. Such groups could form tri- and tetraantennary glycans with N-acetyllactosamine extensions or they could all be present as N-acetyllactosamine extensions of the biantennary glycan. To resolve this point, a series of additional exoglycosidase digestions was performed.



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Fig. 4. Structures of some of the compounds discussed in the text. Symbols for the glycan structures are as defined in the legend to Figure 3 plus star = sialic acid.

 
Digestion of the glycans with a mixture of A. ureafaciens sialidase, endo-ß-galactosidase from B. fragilis, and ß-galactosidase from S. pneumoniae reduced all glycans to core structures whose antennae terminated in GlcNAc and showed that bi- (compound I, Figure 4), tri- (II and III), and tetraantennary (IV) structures were present. Treatment of the sample with A. ureafaciens sialidase, S. pneumoniae ß-galactosidase, and S. pneumoniae hexosaminidase at low concentrations (8 mU/ml) showed that approximately 80% of the triantennary compounds contained a branched 6-antenna (III). Under the conditions used for this digestion, the only GlcNAc residues that are removed are those that are attached to the 2-position of the core mannose residues, providing that these mannose residues are not substituted by GlcNAc in the 6-position (Chen et al., 1998Go).

A parallel sequential digestion of the total glycan pool with A. ureafaciens sialidase and S. pneumoniae ß-galactosidase removed only the terminal ß1->4-linked galactose residues; those forming integral parts of N-acetyllactosamine repeat units remained intact on their respective core structures. Thus the number of antennae in each glycan could be determined by the number of galactose residues removed. The resulting mass spectrum (Figure 5) contained varying amounts of complex glycans consisting of biantennary structures with up to three N-acetyllactosamine extensions, triantennary glycans with up to three N-acetyllactosamine units, and tetraantennary glycans with two extensions. Only a small percentage of tri- and tetraantennary sugars without N-acetyllactosamine groups was present. Thus in Figure 5, the ion at m/z 1485.5 originates from the biantennary glycan, the ion at m/z 1688.6 from a triantennary compound (addition of 203 mass units), and the ion at m/z 1891.7 from a native tetraantennary structure. Ions at m/z 1850.7, 2215.9, and 2581.0, all of which lost two galactose residues, are from compounds containing a biantennary core (I) and one, two, and three N-acetyllactosamine extensions, respectively. The ions at m/z 2053.7, 2419.0, and 2784.0 arise from similarly extended triantennary glycans. N-acetyllactosamine extensions to the tetraantennary glycan gave the ions at m/z 2256.9 and 2621.0. It would appear that the more highly branched glycans were more likely to be present with N-acetyllactosamine extensions, although the parent tri- and particularly the tetraantennary compounds were less abundant than the biantennary glycans. This observation correlates with that reported by others (Fukuda, 1994Go) who have also observed that tetraantennary structures are preferable acceptors for poly-N-acetyllactosamine extensions. The relative percentages of the bi-, tri-, and tetraantennary glycans (with and without N-acetyllactosamine extensions) measured in this experiment were 62%, 32%, and 6%, respectively.



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Fig. 5. Positive-ion MALDI mass spectrum of the glycans from GPI-anchored CD59 after sequential treatment with A. ureafaciens sialidase and S. pneumoniae ß-galactosidase. Symbols for the glycan structures are as defined in the legend to Figure 3.

 
Removal of the fucose from the products of the digestions with bovine epididymis {alpha}-fucosidase confirmed that this residue was attached to the reducing-terminal GlcNAc residue. Because of the higher signal:noise ratio of the MALDI spectrum obtained in this experiment, a further N-acetyllactosamine group was detected on each of the three parent glycans. This experiment gave a composition of 63%, 30%, and 7% for the bi-, tri-, and tetraantennary glycans, respectively.

Distribution of the N-acetyllactosamine extensions
The distribution of the N-acetyllactosamine extensions among the antennae was probed by incubation with sialidase and endo-ß-galactosidase. The latter enzyme will reduce N-acetyllactosamine-containing antennae to GlcNAc but leave unextended antennae as Galß1->4GlcNAc. Thus in Figure 6, the biantennary glycans are represented by ions at m/z 1809.6 (two terminal galactose, no extensions, compound I), 1647.6 (one antennae extended), and 1485.5 (two antennae extended) in the relative proportions of 38%, 44%, and 18%, respectively, showing that most biantennary glycans that carried N-acetyllactosamine extensions did so on only one antenna. Corresponding figures for the tri- and tetraantennary glycans are listed in Table II.



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Fig. 6. Positive-ion MALDI mass spectrum of the glycans from GPI-anchored CD59 after treatment with A. ureafaciens sialidase and B. fragilis endo-ß-galactosidase. Symbols for the glycan structures are as defined in the legend to Figure 3.

 

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Table II. Percent of antennae carrying N-acetyllactosamine extensions for the bi-, tri-, and tetraantennary glycans from GPI-anchored human CD59
 
Distribution of sialic acids
Antennae that are capped with sialic acid are not substrates for S. pneumoniae ß-galactosidase, whereas uncapped antennae will be digested. If, as described, S. pneumoniae hexosaminidase is used at low concentration (i.e., 8 mU/ml), only GlcNAc residues attached to the 2-positions of the core mannose residues will be removed and then only if no substituent is present at the 6-position of that mannose (Chen et al., 1998Go). Thus incubation of the intact glycan pool with this mixture of enzymes will provide information on the length of the antennae capped with sialic acid and, for some compounds, give information on their position of substitution. The sialylated glycans in the total glycan pool were, thus, incubated with the S. pneumoniae hexosaminidase and S. pneumoniae ß-galactosidase and the products were converted into their methyl esters for examination by MALDI MS. In a parallel experiment on the desialylated glycans, it was shown that at this concentration of ß-N-acetylhexosaminidase, the N-acetyllactosamine chains were almost completely digested. Thus, it can be concluded that the masses of the monosialylated glycans in Figure 7 indicate the length of the poly-N-acetyllactosamine chain. The profile shown in this figure revealed the following information: three glycans (m/z 1952.5, 2317.6, and 2683.1) were found with compositions that indicated triantennary structures with an unsubstituted 3-linked mannose residue as the result of removal of the GlcNAc residue by the N-acetyl-hexosaminidase (structure V, Figure 4 shows the structure of the compound of mass 2683.1). As the 3-branched triantennary structures (III, Figure 4) contributed only 20% to the total triantennary glycans, they will contribute little to the profile in Figure 7. Thus, for the major triantennary glycans (which contained branched 6-antenna), the compounds with an unsubstituted mannose residue must have originated from glycans having zero, one, two, three, or four Gal->GlcNAc groups substituted onto the mannoses of the 6-antenna (i.e., zero, one, or two N-acetyllactosamine extensions). However, as to whether these groups constituted the 2- or 6-branches of this antenna could not be determined. The other triantennary glycan peaks contained an extra GlcNAc residue and were, therefore, substituted on the 3-antenna. These ions were observed at m/z 2155.1, 2520.1, 2884.7, and 3250.1 and corresponded to the presence of one, two, three and four Galß1->GlcNAc groups terminating in sialic acid. Most of these compounds must have originated from the major 6-branched triantennary glycans, with the sialylated N-acetyllactosamine-extended antenna located at the 3-position of the core mannose (VI, Figure 4) or at the 6-position of the minor (20%) of triantennary glycans with the branched 3-antenna. Thus, the triantennary glycans appeared to contain longer N-acetyllactosamine-extended antennae on the unbranched arm, which, in the case of GPI-anchored, recombinant CD59, was the 3-antenna.



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Fig. 7. Positive-ion MALDI mass spectrum of the glycans from GPI-anchored CD59 after treatment with S. pneumoniae ß-galactosidase and S. pneumoniae N-acetyl-ß-D-glucosaminidase at low concentration. Sialic acids have been methylated. Symbols for the glycan structures are as defined in the legends to Figures 3 and 4.

 
Fukuda (1991)Go reported that poly-N-acetyllactosaminyl repeats were not uniformly distributed on different antennae of human erythrocyte–derived glycans but that they were preferentially attached to the 6-branch of the 6-antenna, unlike the compounds found in GPI-anchored recombinant CD59 expressed in CHO cells used in the present study. The relatively high abundance of the larger glycans indicated that the longer chains were more likely to terminate in sialic acid than the shorter ones, generally in agreement with the observations made by Merkle and Cummings (1987)Go in mice. Preferential association of sialic acid with the longer chains can also be seen in Figure 2, where the relative proportions of the more highly sialylated glycans increased with mass.

Soluble CD59
The soluble form of rhCD59 migrated as two broad bands on SDS–PAGE with the apparent molecular weights of 16–22 kDa (band 1) and 6–14 kDa (band 2). Each band was excised and digested in-gel with PNGase F, followed by either esterification of the sialic acid–containing sugars or by exoglycosidase digestions. Figure 8 shows the positive-ion MALDI mass spectra of the total glycan pool obtained from band 1. No N-linked glycans were recovered from band 2, indicating that, in contrast to GPI-anchored CD59, the soluble form of CD59 can be secreted in an unglycosylated form, similar to Thy-1 (Devasahayam et al., 1999Go). Exoglycosidase digestion showed that the glycans were essentially similar to those found in the GPI-anchored sample but that the extent of poly-N-acetyllactosamine extensions to the antennae was much less. The molecular weights and compositions of the glycans are compiled in Table I. Simultaneous treatment of the sugars with A. ureafaciens sialidase and S. pneumoniae ß-galactosidase showed that approximately 97% of the sample consisted of biantennary structures of which 81% contained no N-acetyllactosamine extensions, 17% contained one N-acetyllactosamine extension, and 2% had two of these units. The presence of N-acetyllactosamine extensions was confirmed by incubation with a mixture of endo-ß-galactosidase, A. ureafaciens sialidase, and S. pneumoniae galactosidase. The remaining 3% of the glycans were triantennary. Approximately 50% of these were unmodified, and the remainder carried one or two N-acetyllactosamine chains.



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Fig. 8. Positive-ion MALDI mass spectrum of N-linked glycans released from soluble CD59. Sialic acids have been methylated. Only one isomer of the triantennary glycans is shown for clarity. Symbols for the glycan structures are as defined in the legends to Figures 3 and 4. The peaks appearing 32 mass units below those of the main peaks were produced by lactones from the sialic acid groups.

 
Figure 9 shows the MALDI mass spectrum of intact soluble rhCD59 (Leu1 to Asn70) recorded from sinapinic acid. The peak at m/z 8077.1 corresponds to the unglycosylated protein (calculated average mass, assuming five Cys–Cys bonds = 8077.1). By subtracting this mass from that of the other peaks, the glycoforms were found to contain the same glycans that were identified after release (see Table III). Three glycans of lower mass corresponding to HexNAc, Fuc->HexNAc, and HexNAc->HexNAc were also found. These may be degraded N-linked glycans. The possibility that they may be O-linked glycans is unlikely because no molecules were identified that contained both these compounds and the fully processed N-linked glycans. In addition, the compositions of these mono- and disaccharides did not correspond to those of reported O-linked glycans (Rudd et al., 1997Go). Such degraded compounds have been reported before from rCD59 (Meri et al., 1996Go) and from tissue inhibitor of metalloproteinase (Sutton et al., 1994Go) expressed in CHO cells.



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Fig. 9. Positive-ion MALDI mass spectrum of soluble CD59. The letter P attached to the glycan structures represents protein. Symbols for the glycan structures are as defined in the legends to Figures 3 and 4.

 

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Table III. Masses and compositions of the glycans found in the MALDI mass spectrum of intact soluble CD59
 
Conclusions
This study has used selective exo- and endoglycosidases to provide additional information on the detailed structure of glycans from GPI-anchored and soluble rhCD59. The N-linked oligosaccharides observed for the GPI-anchored rhCD59 were similar to those found earlier on human erythrocyte CD59 (Rudd et al., 1997Go) in that they carried extensive poly-N-acetyllactosamine extensions to their antennae. Although these glycans were also similar to those found on the soluble CD59, namely complex bi-, tri-, and tetraantennary structures, the chains of the glycans from the soluble form were not extended to nearly the same extent as those from the GPI-anchored glycoprotein. This difference in glycosylation shows that soluble CD59 is glycosylated differently from the form possessing a GPI anchor. The soluble form studied in this article was expressed without the GPI-signal peptide and therefore did not contain the part of the anchor present on human soluble (urinary) CD59 (Meri et al., 1996Go). Neither of these glycoproteins contained the extended antennae present on human CD59 anchored to erythrocytes (Rudd et al., 1997Go). This dissimilarity indicates that the urinary CD59 does not derive from erythrocytes and is consistent with cell-specific glycosylation.

A possible reason for the difference between the glycosylation of the glycoprotein with and without a GPI anchor is that the glycoprotein with the GPI anchor passes through the Golgi at a slower rate than the soluble form allowing longer exposure to the glycosyltransferases (Nabi and Dennis, 1998Go). A similar result has been reported by Fukuda et al. (1988)Go for the {alpha}-chain of human chorionic gonadotropin fused to the transmembrane and cytoplasmic domains of vesicular stomatitis virus-G protein, making a chimeric membrane glycoprotein. The N-glycans attached to this protein were extensively modified by poly-N-acetyllactosamines, whereas the native secretory hCG-{alpha} protein contained typical complex-type oligosaccharides. It is also possible that GPI-anchored proteins are more efficiently processed because they are held closer to the glycan-processing enzymes, which are also membrane-bound. The results presented herein, therefore, clearly show that great care should be taken in extrapolating from the analysis of the glycans of soluble proteins to those from the anchored state.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
All standard laboratory chemicals and biochemicals were purchased from Sigma (Poole, Dorset, UK). Solvents were from Merck (Poole). Milli-Q water was freshly distilled over sodium permanganate to remove residual organic material. 2,5-Dihydroxybenzoic acid (2,5-DHB), the MALDI matrix, was obtained from Aldrich (Poole) and was recrystallized from methanol/chloroform. The 2-AB fluorescent labeling kit was obtained from Oxford GlycoSciences (Abingdon, UK). AG3-X4 (free base form) and AG 50-X12 (hydrogen form) ion exchange resins were purchased from Bio-Rad (Hemel Hampstead, UK). Sep-Pak C18 cartridge packing was from Waters (Milford, MA). All microcolumns were packed into Eppendorf GELoader pipette tips from BDH. Acrylamide and bis-acrylamide were purchased from National Diagnostics (Atlanta, GA). Recombinant PNGase F from Flavobacterium meningosepticum expressed in Escherichia coli was purchased from Boehringer Mannheim (Mannheim, Germany). A. ureafaciens sialidase, S. pneumoniae ß-galactosidase, jackbean ß-N-acetyl-hexosaminidase, S. pneumoniae N-acetyl-ß-D-glucosaminidase, bovine epididymis {alpha}-fucosidase, jackbean {alpha}-mannosidase, and Bacteriodes fragilis endo-ß-galactosidase were all from Oxford GlycoSciences. PNGase F and A. ureafaciens sialidase were both dialyzed into 20 mM NaHCO3, pH 6.8, before use. rhCD59 (GPI-anchored and soluble form, lacking the GPI anchor and signal peptide) were prepared from CHO cells as detailed by Morgan (1992)Go and Bodian et al. (1997)Go, respectively.

Gel electrophoresis and in-gel PNGase F digestion
SDS–PAGE gels (80 x 80 x 0.75 mm) were run using a Mini PROTEAN II cell (Bio-Rad) at 200 V constant voltage in 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3, as previously described (Küster et al., 1997Go). Approximately 120 pmole of the GPI-anchored form of CD-59 (3 µg) and 900 pmole of the soluble form (10 µg) were loaded onto the gel and visualized by Coomassie staining for 15 min. After destaining, the glycoprotein bands were excised from the gel, reduced, alkylated, and digested with PNGase F as described previously (Küster et al., 1997Go). The glycans were extracted from the gel pieces by three changes of 200 µl water with sonication for 30 min followed by extraction with 200 µl of freshly distilled acetonitrile. All extracts were combined, incubated with 30 µl AG50-X12 (hydrogen form) for 5 min, then filtered (Millipore syringe filter type FH, 0.45 µm) and dried in vacuo. The sample was split into two aliquots. In one aliquot, the sialic acids were then converted into their methyl esters using the procedure described by Powell and Harvey (1996)Go, and the other was passed through a microcolumn of 5 µl each of AG3-X4 (free base form), AG50-X12 (hydrogen form), and C18 resins that had been prewashed with 3 x 50 µl water. Glycans were eluted with 3 x 50 µl water, collected, and dried in vacuo.

PNGase F solution digest of CD-59 (GPI anchor form)
Approximately 3 µg of rhCD-59 (GPI anchor form), in 20 mM PBS, pH 7.4, containing 10 mM CHAPS surfactant, was denatured at 100°C with 0.1% SDS for 5 min and diluted with 24 mM phosphate buffer, pH 8.6, containing 34 mM octylglucoside (160 µl). A solution of 200 U/ml PNGase F (50 µl) was added to the sample and incubated at 37°C for 24 h. Complete digestion was confirmed by SDS–PAGE from an aliquot (70 µl) of the digest mixture. Glycans were recovered by applying to a microcolumn packed with 30 µl C18 resin that had been washed with 200 µl water. Sugars were eluted with x 60 µl of water and dried in vacuo. The sample was split into two; half was digested with 2 U/ml A. ureafaciens sialidase (5 µl), and the remainder was converted to methyl esters as already described.

Glycan release by automated hydrazinolysis
Glycans were released with hydrazine at 95°C for 5 h and re-N-acetylated using a Glycoprep 1000 (Oxford GlycoSystems). Samples were filtered and rotary-evaporated to dryness before 2-AB labeling.

Fluorescent labeling of glycans
Glycans were fluorescently labeled with 2-AB by reductive amination according to the procedure described by Bigge et al. (1995)Go.

Normal phase HPLC
2-AB-Labeled glycans were separated on a 4.6 x 250 mm GlycoSep N column (Oxford GlycoSciences) using a Waters 2690 Alliance separation module with a column heater at 30°C and a Waters 474 scanning fluorescence detector. The gradient used was as described by Guile et al. (1996)Go: solvent A, 50 mM ammonium formate, pH 4.4; solvent B, acetonitrile. Initial conditions were 20% A at a flow rate of 0.4 ml/min followed by a linear gradient of 20–58% A over 152 min followed by 58–100% A in the next 3 min. The column was washed with 100% A for 5 min at a flow rate of 1 ml/min before being re-equilibrated in 20% A for the next sample.

Exoglycosidase digestions
The unmethylated glycans obtained from in-gel PNGase F digestion were incubated with A. ureafaciens sialidase and then split into seven aliquots (1 µl) and incubated overnight at 37°C in 20 mM sodium acetate at pH 5.5, in parallel with 1 µl of each of the following exoglycosidase arrays: (1) S. pneumoniae ß-galactosidase (1 U/ml); (2) ß-galactosidase and bovine epididymis {alpha}-fucosidase (1 U/ml); (3) ß-galactosidase, {alpha}-fucosidase, and jackbean ß-N-acetylhexosaminidase (30 U/ml); (4) ß-galactosidase, {alpha}-fucosidase. and S. pneumoniae N-acetyl-ß-D-glucosaminidase (6 mU/ml); (5) ß-galactosidase, {alpha}-fucosidase, jackbean ß-N-acetylhexosaminidase, and jackbean {alpha}-mannosidase (100 U/ml); and (6) ß-galactosidase and B. fragilis endo-ß-galactosidase (0.8 U/ml). Each reaction mixture was prepared for MALDI MS by using the neutral glycan microcolumn clean-up procedure described herein.

MALDI MS
An aliquot (1–2 µl) of the soluble CD59 solution was drop-dialyzed against water for 10 min using a Spectra/Por CE 2000 molecular weight cut-off membrane (Spectrum Medical Industries, TX). The solution was then transferred to the MALDI target plate, mixed with 1 µl of a solution of sinapinic acid (10 mg/ml in 70:30 v/v acetonitrile:0.1% trifluoroacetic acid) and allowed to dry. Released glycans (50–100 pmoles) were loaded onto the mass spectrometer target in 1 µl of water, mixed with a fresh solution of 2,5-DHB (1 µl of 10 mg/ml in acetonitrile), and allowed to dry. The mixture was then recrystallized from the minimum amount of alcohol (Harvey, 1993Go).

All glycan mass spectra were acquired in positive ion mode with a PerSeptive Biosystems Voyager Elite reflectron time-of-flight mass spectrometer equipped with a delayed extraction MALDI ion source. The delay time was 75 ns. Grid wire and guide wire voltages were 65% and 0.1% of the accelerating voltage (20 kV), respectively. Between 150 and 256 scans were averaged for each spectrum. The instrument was externally calibrated with a mixture of dextran oligomers. All molecular weights are quoted as monoisotopic masses. Neutral and derivatized sialic acid–containing oligosaccharides were observed as [M+Na]+ ions accompanied by lower amounts of [M+K]+ ions.

For glycoprotein samples, the delay time was 100 ns, and the grid wire and guide wire voltages were 90% and 0.3% of the accelerating voltage (20 kV), respectively. The instrument was externally calibrated with horse heart cytochrome C. Glycoproteins were observed as [M+H]+ ions in the positive ion mode, and all molecular weights are quoted as average (chemical) masses.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Professor E.M. Southern for accesses to the PerSeptive Voyager mass spectrometer. This work was supported by the Biotechnology and Biological Sciences Research Council and Oxford GlycoSciences.


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
2-AB, 2-aminobenzamide; CHAPS, 3-[3-cholamidopropyldimethylammonio]-1-propanesulfonate; CHO, Chinese hamster ovary; DHB, 2,5-dihydroxybenzoic acid; GPI, glycosylphosphatidylinositol; HPLC, high-performance liquid chromatography; MAC, membrane attack complex; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PNGase F, peptide N-glycosidase F; SDS, sodium dodecyl sulfate.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Ali, B.R., Claxton, S., and Field, M.C. (2000) Export of a missprocessed GPI-anchored protein from the endoplasmic reticulum in vitro in an ATP- and cytosol-dependent manner. FEBS Lett., 483, 32–36.[CrossRef][ISI][Medline]

Bergwerff, A.A., van-Oostrum, J., Asselbergs, F.A., Burgi, R., Hokke, C.H., Kamerling, J.P., and Vliegenthart, J.F. (1993) Primary structure of N-linked carbohydrate chains of a human chimeric plasminogen activator K2tu-PA expressed in Chinese hamster ovary cells. Eur. J. Biochem., 212, 639–656.[Abstract]

Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., and Parekh, R.B. (1995) Nonselective and efficient fluorescent labeling of glycans using 2-aminobenzamide and anthranilic acid. Anal. Biochem., 230, 229–238.[CrossRef][ISI][Medline]

Bodian, D.L., Davis., S.J., Morgan, B.P., and Rushmere, N.K. (1997) Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59. J. Exp. Med., 185, 507–516.[Abstract/Free Full Text]

Chen, Y.-J., Wing, D.R., Guile, G.R., Dwek, R.A., Harvey, D.J., and Zamze, S. (1998) Neutral N-glycans in adult rat brain tissue: complete characterization reveals fucosylated hybrid and complex structures. Eur. J. Biochem., 251, 691–703.[Abstract]

Davidson, D.J. and Castellino, F.J. (1991) Oligosaccharide structures present on asparagine-289 of recombinant human plasminogen expressed in a Chinese hamster ovary cell line. Biochemistry, 30, 625–633.[ISI][Medline]

Davies, A. and Lachmann, P.J. (1993) Membrane defence against complement lysis: the structure and biological properties of CD59. Immunol. Res., 12, 258–275.[ISI][Medline]

Davies, A., Simmons, D.L., Hale, G., Harrison, R.A., Tighe, H., Lachmann, P.J., and Waldmann, H. (1989) Cd59, an Ly-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells. J. Exp. Med., 170, 637–653.[Abstract]

Devasahayam, M., Catalino, P.D., Rudd, P.M., Dwek, R.A., and Barclay, A.N. (1999) The glycan processing and site occupancy of recombinant Thy-1 is markedly affected by the presence of a glycosylphosphatidylinositol anchor. Glycobiology, 9, 1381–1387.[Abstract/Free Full Text]

Fukuda, M. (1991) Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking. J. Biol. Chem., 266, 21327–21330.

Fukuda, M. (1994) Cell surface carbohydrates: cell type–specific expression. In Fukuda, M. and Hindsgaul, O., eds., Molecular glycobiology. IRL Press, Oxford, pp. 1–52.

Fukuda, M., Guan, J.L., and Rose, J.K. (1988) A membrane-anchored form but not the secretory form of human chorionic gonadotropin-a chain acquires polylactosaminoglycan. J. Biol. Chem., 263, 5314–5318.[Abstract/Free Full Text]

Guile, G.R., Rudd, P.M., Wing, D.R., Prime, S.B., and Dwek, R.A. (1996) A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem., 240, 210–226.[CrossRef][ISI][Medline]

Harvey, D.J. (1993) Quantitative aspects of the matrix-assisted laser desorption mass spectrometry of complex oligosaccharides. Rapid Commun. Mass Spectrom., 7, 614–619.[ISI][Medline]

Hokke, C.H., Berwerff, A.A., Van Dedem, G.W.K., Kamerling, J.P., and Vligenthart, J.F.G. (1995) Structural analysis of the sialylated N- and O-linked carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. Eur. J. Biochem., 228, 981–1008.[Abstract]

Kieffer, B., Driscoll, P.C., Campbell, I.D., Willis, A.C., van der Merwe, P.A., and Davis, S.J. (1994) Three-dimensional structure of the extracellular region of the complement regulatory protein CD59, a new cell surface protein domain related to snake venom neurotoxins. Biochemistry, 33, 4471–4482.[ISI][Medline]

Küster, B., Wheeler, S.F., Hunter, A.P., Dwek, R.A., and Harvey, D.J. (1997) Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high performance liquid chromatography. Anal. Biochem., 250, 82–101.[CrossRef][ISI][Medline]

Lehto, T., Honkanen, E., Teppo, A.M., and Meri, S. (1995) Urinary excretion of protectin (CD59), complements C5b-9 and cytokines in membranous glomerulonephritis. Kidney Int., 47, 1403–1411.[ISI][Medline]

Menu, E., Tsai, B.C., Bothwell, A.L., Sims, P.J., and Bierer, B.E. (1994) CD59 costimulation of T cell activation. CD58 dependence and requirement for glycosylation. J. Immunol., 153, 2444–2456.[Abstract/Free Full Text]

Meri, S., Lehto, T., Sutton, C.W., Tyynelä, J., and Baumann, M. (1996) Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser-desorption mass spectrometric analysis. Biochem. J., 316, 923–935.[ISI][Medline]

Meri, S., Vakeva, A., Laari, T., and Lachmann, P.J. (1991a) Soluble forms of CD59-antigen: distribution in body fluids and functional activity. Comp. Inflam., 8, 193.

Meri, S., Waldmann, H., and Lachmann, P.J. (1991b) Distribution of protectin (CD59), a complement membrane attack inhibitor, in normal human tissues. Lab. Invest., 65, 532–537.[ISI][Medline]

Merkle, R.K. and Cummings, R.D. (1987) Relationship of the terminal sequences to the length of the poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from mouse lymphoma cell line BW5147. J. Biol. Chem., 262, 8179–8189.[Abstract/Free Full Text]

Morgan, B.P. (1992) Isolation and characterisation of the complement-inhibiting protein CD59 antigen from platelet membranes. Biochem. J., 282, 409–413.[ISI][Medline]

Muller-Eberhard, H. (1986) The membrane attack complex of complement. Annu. Rev. Immunol., 4, 503–528.[CrossRef][ISI][Medline]

Nabi, I.R. and Dennis, J.W. (1998) The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time. Glycobiology, 8, 947–953.[Abstract/Free Full Text]

Nakano, Y., Noda, K., Endo, T., Kobata, A., and Tomita, M. (1994) Structural study on the glycosyl-phosphatidylinositol anchor and the asparagine-linked sugar chains of the soluble form of CD59 in human urine. Arch. Biochem. Biophys., 311, 117–126.[CrossRef][ISI][Medline]

Nakano, Y., Sugita, T., Tobe, T., Mirura, N.H., and Tomita, M. (1993) Receptor-independent activation of GTP-binding, regulatory proteins by C5b-9. Mol. Immunol., 30, suppl. 1, 37.

Ninomiya, H., Stewart, B.H., Rollins, S.A., Zhao, J., Bothwell, A.L.M., and Sims, P.J. (1992) Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity. J. Biol. Chem., 267, 8404–8410.[Abstract/Free Full Text]

Noguchi, A., Mukuria, C.J., Suzuki, E., and Naiki, M. (1995) Immunogenicity of N-glycolylneuraminic acid–containing carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. J. Biochem. (Tokyo), 117, 59–62.[Abstract]

Noguchi, A., Mukuria, C.J., Suzuki, E., and Naiki, M. (1996) Failure of human immunoresponse to N-glycolylneuraminic acid epitope contained in recombinant human erythropoietin. Nephron, 72, 599–603.[ISI][Medline]

Powell, A.K. and Harvey, D.J. (1996) Stabilisation of sialic acids in N-linked oligosaccharides and gangliosides for analysis by positive ion matrix-assisted laser desorption-ionization mass spectrometry. Rapid Commun. Mass Spectrom., 10, 1027–1032.[CrossRef][ISI][Medline]

Rother, R.P., Zhao, J., Zhou, Q., and Sims, P.J. (1996) Elimination of potential sites of glycosylation fails to abrogate complement regulatory function of cell surface CD59. J. Biol. Chem., 271, 23842–23845.[Abstract/Free Full Text]

Rudd, P.M., Morgan, B.P., Wormald, M.R., Harvey, D.J., Van den Berg, C.W., Davis, S.J., Ferguson, M.A.J., and Dwek, R.A. (1997) The glycosylation of the complement regulatory protein, human erythrocyte CD59. J. Biol. Chem., 272, 7229–7244.[Abstract/Free Full Text]

Rudd, P.M., Wormald, M.R., Harvey, D.J., Devashayam, M., McAlister, M.S.B., Brown, M.H., Davis, S.J., Barclay, A.N., and Dwek, R.A. (1999) Oligosaccharide analysis and molecular modeling of soluble forms of glycoproteins belonging to the Ly-6, scavenger receptor, and immunoglobulin superfamilies expressed in Chinese hamster ovary cells. Glycobiology, 9, 443–458.[Abstract/Free Full Text]

Rushmere, N.K., Harrison, R.A., van den Berg, C.W., and Morgan, B.P. (1994) Molecular cloning of the rat analogue of human CD59: structural comparison with human CD59 and identification of a putative active site. Biochem. J., 304, 595–601.[ISI][Medline]

Rushmere, N.K., Tomlinson, S., and Morgan, B.P. (1997) Expression of rat CD59: functional analysis confirms lack of species selectivity and reveals that glycosylation is not required for function. Immunology, 90, 640–646.[ISI][Medline]

Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J. Biol. Chem., 262, 12059–12076.[Abstract/Free Full Text]

Sugita, Y., Nakano, Y., Oda, E., Tobe, T., Miura, N.H., and Tomita, M. (1993) Determination of carboxyl-terminal residue and disulphide bonds of MACIF (CD59), a glycosyl-phosphatidylinositol-anchored membrane protein. J. Biochem. (Tokyo), 114, 473–477.[Abstract]

Sugita, Y., Tobe, T., Oda, E., Tomita, M., Yasukawa, K., Yamaji, N., Takemoto, T., Furuichi, K., Takayama, M. and Yano, S. (1989) Molecular cloning and characterisation of MACIF, an inhibitor of membrane channel formation of complement. J. Biochem. (Tokyo), 106, 555–557.[Abstract]

Sutton, C.W., O’Neill, J.A., and Cottrell, J.S. (1994) Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry. Anal. Biochem., 218, 34–46.[CrossRef][ISI][Medline]

Suzuki, H., Ito, K., Yamaji, N., Egashira, A., Sugita, Y., and Masuho, Y. (1994) Differences in activities of human recombinant soluble CD59s and Urine CD59. Clin. Exp. Immunol., 97, suppl. 2, 46.

Suzuki, H., Yamaji, N., Egashira, A., Yasunaga, K., Sugita, Y., and Masuho, Y. (1996) Effect of the sugar chain of soluble recombinant CD59 on complement inhibitory activity. FEBS Lett., 399, 272–276.[CrossRef][ISI][Medline]

Walsh, L.A., Tone, M., Thiru, S., and Waldmann, H. (1992) The CD59 antigen—a multifuctional molecule. Tissue Antigens, 40, 213–220.[ISI][Medline]

Yu, J., Abagyan, R., Dong, S., Gilbert, A., Nussenzweig, V., and Tomlinson, S. (1997) Mapping the active site of CD59. J. Exp. Med., 185, 745–753.[Abstract/Free Full Text]