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

Pauline M. Rudd3, Mark R. Wormald, David J. Harvey, Mercy Devasahayam, Mark S.B. McAlister1, Marion H. Brown1, Simon J. Davis2, A. Neil Barclay1 and Raymond A. Dwek3

Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK, 1MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3QU, UK and 2Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK

Received on June 9, 1998; revised on September 4, 1998; accepted on September 8, 1998

Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specificglycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached.

Key words: CD59/Chinese hamster ovary cells/Ig superfamily/leukocyte antigens/oligosaccharides

Introduction

The synthesis of the polypeptide chain of a glycoprotein is under genetic control. In contrast, the carbohydrate chains are attached and processed by a series of enzyme reactions without the rigid direction of nucleic acid templates. Consequently, a single polypeptide which is glycosylated generally emerges from the biosynthetic pathway as a mixture of glycoforms. It is now well established that these glycoform populations are both species (Parekh et al., 1987) and cell (Parekh et al., 1989) specific. Thus, for cells in stasis, each glycoprotein has a reproducible and characteristic glycosylation profile, suggesting that each protein exerts some control over the processing of the glycans attached to it. Moreover, the processing of oligosaccharides is also site specific (Parekh et al., 1987, 1989; Shogren et al., 1989; Wyss et al., 1995a; Bloom et al., 1996), further indicating that key elements of the primary, secondary, and/or tertiary structure of the individual protein are important factors in determining the extent and nature of glycan processing.

Many recombinant glycoproteins have been expressed in Chinese hamster ovary (CHO) cells and analysis of their carbohydrates has shown that, in common with human cells, CHO cells contain a large repertoire of glycosylating enzymes and are therefore able to process a wide variety of glycans. We have shown previously (Ashford et al., 1993) that the glycan processing of individual glycoproteins expressed in independently derived CHO cell lines is broadly constant, with the exception of small variations in sialylation and fucosylation. Indeed, we have found that CHO cell lines expressing different levels of recombinant protein or grown under different conditions nevertheless generate the same set of glycoforms. The observation that the glycosylation processes are reproducible in these cell lines has allowed the CHO cell to be used as a model system in which to investigate the role of primary sequence and secondary and tertiary structure in controlling glycan processing.

While it is very clear that local protein structure has a major influence on glycan processing, it seems unlikely that this fully accounts for the mixtures of glycoforms which represent most glycoprotein populations, particularly when the cells are no longer in stasis. Additional factors likely to have a bearing on processing are expected to include: rates of protein folding, trafficking pathways within the Golgi, precursor-substrate concentration, compartmentalization, transferase regulation, and metabolic channeling. In principle, the effects of such additional factors might be expected to vary in a superfamily-dependent manner, perhaps reflecting the distinct evolutionary histories or functions implied by such classifications. To address this possibility, we have analyzed the glycosylation by CHO cells of soluble recombinant forms of six glycoproteins belonging to three protein superfamilies prominent at the leukocyte cell surface (Figure 1a): the immunoglobulin superfamily (IgSF), the Ly-6 superfamily, and the scavenger receptor cysteine-rich (SRCR; human CD5) superfamily (Figure 1a; Barclay et al., 1997).

   a, b
   c

Figure 1. (a) Schematic diagrams of the leukocyte cell surface antigens forming the basis of this study. Membrane attached rat Thy-1, human CD2, rat CD48, rat CD4, human CD5, and human CD59 are depicted with shaded or unshaded ovals, squares, and triangles representing IgSF domains, SCRC domains and Ly-6 (L6) superfamily domains, respectively. N-Glycans are represented by solid circles joined to the domains by thin lines, and O-glycans by single thick lines. The IgSF domains are designated as V-set (V) or C2-set (C2) on the basis of sequence analysis (Williams and Barclay, 1988). Sites of truncation are indicated with horizontal arrows and GPI anchors with vertical arrows. (b) The soluble derivatives of these glycoproteins characterized in this study. These are represented diagrammatically with the abbreviations used in the text to refer to the constructs, shown underneath. (c) Schematic diagrams illustrating the topology of the leukocyte antigens, where this is known, and the location of the glycosylation sites on these molecules. The classes of the major oligosaccharide populations at each site is given where this information is available. The positions of the [beta]-strands are indicated by the parallel lines with arrows showing the direction of each strand. The loops between the strands are indicated by the connecting lines. The strands are labeled according to the convention established for immunoglobulin domains (see Williams and Barclay, 1988). Data from CD54 is from Bloom et al., 1996.

Immunoglobulin superfamily (IgSF) domains are the largest group of domains found at leukocyte cell surfaces and are often involved in cell-cell recognition (Williams, 1987; Williams and Barclay, 1988). Human CD2 is expressed on most human T-lymphocytes, NK cells and thymocytes where it functions as a cell adhesion molecule binding to CD58 in humans and CD48 in rats and mice. CD48 contains a glycosylphosphatidylinositol (GPI) anchor while CD58 exists in both GPI- and transmembrane-anchored forms. The extracellular region of human CD2 is made up of two IgSF domains, each of which contains one N-glycosylation sequon, and a linker region which contains a single N-glycosylation site (Bodian et al., 1994). CD4, an accessory molecule in the recognition of foreign antigens in association with MHC Class II antigens by T cells, contains four tandem Ig-like domains. Thy-1 (CD90) is a GPI anchored molecule consisting of a single V-set IgSF domain that is implicated in the regulation of TCR-mediated signaling (Hueber et al., 1997) and in thymocyte apoptosis (Page et al., 1997). Brain and thymocyte forms of Thy-1 exhibit tissue- and site-specific glycosylation (Williams et al., 1993). The Ly-6 superfamily includes human CD59, an inhibitor of homologous lysis, which consists of a single, disk-like Ly-6 domain of 70 amino acids including 10 cysteine residues attached to a GPI anchored stalk (Sugita et al., 1993; Kieffer et al., 1994). The third protein superfamily included in this study, the scavenger receptor cysteine-rich (SRCR; human CD5) superfamily, for which there is, as yet, no detailed structural paradigm, is represented by human CD5 which consists of three SRCR domains (Freeman et al., 1990). CD5 is expressed on all mature T cells and on a subset of mature B cells (Kantor and Herzenberg, 1993), and it appears to have a regulatory effect on T-cell receptor (TCR)-mediated signal transduction (Tarakhovsky et al., 1995; Bikah et al., 1996; Aruffo et al., 1997). CD5 is related to CD6 (Bodian et al., 1997a), both in structure and distribution. CD6 binds to CD166 (Aruffo et al., 1997), suggesting that human CD5 may also have a role in cell adhesion.

The soluble forms of the glycoproteins characterized in this study (Figure 1b) contain different numbers of domains and N-glycosylation sites located on strands, loops, and helices (Figure 1c). We have analyzed the glycosylation of soluble (s) CD5 domain 1 (d1), sThy-1, sCD2, sCD4, sCD48, and sCD59, which are all single polypeptide chains, as well as that of two chimeric molecules, and we discuss the implications of the findings for the roles of glycosylation of cell surface molecules in general. The glycosylation of the leukocyte antigens has not been analyzed in detail previously, with the exception of recombinant rat and human sCD4 (Ashford et al., 1993), one of the sites on human sCD2 (Recny et al., 1992), human CD54 (Bloom et al., 1996), and human erythrocyte and platelet CD59 (Rudd et al., 1997).

Results

Preparation of glycoproteins

The glycoproteins characterized in this study consisted of some or all of the extracellular regions of the leukocyte cell surface antigens illustrated in Figure 1a. The soluble derivatives of these antigens, shown in Figure 1b, were expressed in CHO cells using the glutamine synthetase expression system as described in detail for sCD4 (Davis et al., 1990). All of the proteins were purified by affinity chromatography followed by gel filtration. The preparation of sCD59 is described in detail by Kieffer et al., 1994; sCD48 by McAlister et al., 1996; and sCD2 by Bodian et al., 1994. Figure 2.

Overall scheme of analysis of glycans released by hydrazinolysis (Figure 2)

Glycans were released from each protein by automated hydra-zinolysis at 85°C, and one-third was fluorescently labeled with 2-aminobenzamide (2AB). A second aliquot was retained unlabeled for MALDI TOF MS. The remainder was labeled with 3H for P4 GPC analysis. Each 2AB labeled glycan pool was resolved on the basis of charge using WAX chromatography (Guile et al., 1994; Figure 3, Table I). The desialylated glycan pools were resolved by normal phase (NP) HPLC (Figure 4a, Table II), Biogel P4 gel permeation chromatography (GPC; e.g., Figure 6), and MALDI TOF MS (Table II). From these data, structural assignments were made on the basis of diagnostic NP HPLC retention times (Guile et al., 1996), a mass measurement by MALDI TOF MS leading to an isobaric monosaccharide composition and by exoglycosidase digestions applied either sequentially or in arrays to the total glycan pools. The products of the exoglycosidase digestions (Table II) were analyzed by MALDI TOF MS and NP HPLC (e.g., Figure 4b).

Table I. WAX charge analysis of N-glycans
Glycoprotein %N %A1 %A2
sCD2 90 10 0
sCD48 68 28 4
sCD4 98 2 0
sThy-1 42 30 28
sCD4d3+4 92 8 0
sThy-1/CD4d3+4 57 41 2
sCD5d1 17 45 38
sCD5/CD4d3+4 0 35 65
sCD59 35 35 30
In some cases the neutral peak may have contained noncarbohydrate material.

Determination of charge state

The distribution of charge between neutral, mono-, and di-acidic structures for the glycans is shown in Table I and Figure 3. All glycans were digested to neutral oligosaccharides by both Arthrobacter ureafaciens and Newcastle disease virus sialidases (data not shown) indicating that all the charge was due to [alpha]2,3-linked sialic acid. This is consistent with previous analyses of CHO cell derived glycoproteins which indicate that, although CHO cells contain a gene for [alpha]2-6 sialyl transferase, in general this appears to be 'silent."




Table II. Structures of the glycans found in the six glycoproteins expressed in CHO cells
1Composition measured as percentage of total glycans. When the percentage of each of possible isomers is known, this is shown. Otherwise the figure is the mean value. Where the figures in the columns do not total 100%, the mixture contained some unassigned peaks.
2Composition: H, hexose; N, GlcNAc; F, deoxyhexose (fucose).
3Successive exoglycosidase digests. First cycle of galactosidase and hexosaminidase digests only are shown.
4Bovine testis [beta]-galactosidase.
5Streptococcus pneumonia [beta]-hexosaminidase.
6Charonia lampas fucosidase.
7Jack bean [alpha]-mannosidase.
8Glucose unit.
9Monoisotopic mass of the MNa+ ion. Measured masses differed by no more than 0.5 Da from the calculated masses.
10The triantennary structures shown are the major isomers.
11Abbreviated structure (see text).
12Thy-1.
13Combined peak.
14Peak detected by mass spectrometry only.

N-Glycan analysis of the soluble forms of the leukocyte antigens

The asialo sugar profiles of the soluble leukocyte antigens are compared in Figure 4a, and the data are summarized in Table II. The key conclusions from these data, combined with that from WAX analyses, MALDI TOF MS data and enzyme digests, is summarized below.


Figure 2. Overall scheme for analysis of oligosaccharides.

sThy-1. Fifty-seven percent of the glycans were neutral. More than 80% were of the bi-antennary complex type, with and without core fucose.

sCD2. The glycans were predominantly neutral and consisted of an oligomannose series (GlcNAc)2(Man)5-8 and core fucosylated bi-, tri-, and tetra-antennary complex glycans with and without lactosamine extensions.

sCD48. Sixty-eight percent of the glycans were neutral and most of the remainder were monosialylated. The pool contained a range of glycans in which the major structures were fucosylated bi-, tri-, and tetra-antennary sugars. The composition of the larger structures was determined by MALDI TOF MS (Table II), and differentiation from bisected glycans was made on the basis of their HPLC retention times and the simultaneous analysis of the glycans from sCD48 using enzyme arrays (Figure 4a,b, Table III). Mono galactosylated bi-antennary fucosylated structures were identified; arm specificity was assigned from the elution positions of known standards (Guile et al., 1996).

sCD4. Ninety-eight percent of the sugars were neutral. A series of oligomannose glycans (GlcNAc)2(Man)5-7 was identified which was susceptible to jack bean [alpha]-mannosidase. The pool of complex glycans contained the unfucosylated, biantennary glycan A2G2, its mono- and agalactosyl analogs and the fucosylated biantennary glycan A2G2F. In contrast to other glycoproteins in this study, sCD4 contained hybrid structures.

sCD5d1. The glycosylation was restricted almost entirely to the neutral (17%), mono- (38%), and di- (45%) sialylated fucosylated bi-antennary sugar, A2G2F (Figure 5a). The structure was confirmed by NP HPLC of the desialylated glycans (Figure 5b) and P4 GPC (Figure 6).sCD59. The glycans were distributed evenly among the three charge states. The pool contained glycans which were almost entirely fucosylated bi-antennary structures with and without lactosamine extensions.


O-Glycosylation of sCD5d1

sCD5d1, expressed with eight amino acids of the linker region, contained an O-linked glycan identified by its elution position on WAX chromatography (Figure 3), NP HPLC (Figure 5a), and Biogel P4 (Figure 6). The desialylated pool eluting at 3.5gu on BioGel P4 was digested with bovine testes [beta]-galactosidase to a glycan eluting at 2.5gu, consistent with the removal of a single galactose residue from Gal[beta]1-3+4-GalNAc (GGN). The reducing terminal glycan was identified by its retention time on a GLC column equipped with a radioactive detection system. The glycan was present mainly as the mono-sialylated form (Figure 5a).

Analysis of the sCD5/CD4d3+4 and sThy-1/CD4d3+4 chimeras

A comparison of the HPLC profiles of the sialylated and desialylated glycans released from sCD4d3+4 and sCD5/CD4d3+4 (Figure 5a,b, respectively) indicate that, except for an increase in sialylation, the N-glycan processing of sCD4d3+4 was not markedly altered by the attachment of the three SRCR domains of CD5. Figure 5b also implies that there was no difference between the asialo N-glycans of sCD5d1 (1 glycosylation site) and those of the sCD5/CD4d3+4 chimera (2 glycosylation sites), suggesting that the local protein structure at the two sites is similar. However, there was a significant increase in O-glycosylation of the chimeric molecule, and this presumably reflects the additional proline-rich domain derived from the intact CD5 stalk region present in the chimera.

Figure 7 shows the NP HPLC profiles of desialylated sThy-1, sCD4d3+4, and the sThy-1/CD4d3+4 chimera. There was a decrease in the population of core fucosylated glycans in the chimera, suggesting that the attachment of additional domains restricts the access of the core fucosyltransferase to one or more of the glycosylation sites.

Molecular modeling of the glycoproteins

Molecular models (Figure 8a-e) were constructed, based on the known structures of the proteins, the glycan analysis data obtained here, data from the literature and the Glycobiology Institute oligosaccharide structural data base. These models were used to probe the relationship between glycan processing and protein structure and also to examine potential roles for the sugars in the context of both the behavior and the functions of the proteins to which the glycans are attached. The results are discussed below.

Discussion

Protein- versus superfamily-specific glycan processing.

The current analysis revealed substantial differences in the extent and nature of processing of the glycans present on each of the different leukocyte antigens (Figure 4a). This is consistent with numerous other studies which show that the various glycoproteins present on the surface of individual cells display a diverse repertoire of glycans in spite of having been exposed to the same glycosylation machinery. However, there was no evidence for superfamily-specific processing of the glycans attached to the proteins. For example, the asialo glycan pools from the IgSF members, sCD48 (Figures 4a, 8b) and sCD2 (Figure 8a), contained tri- and tetra- antennary structures. In contrast, the IgSF molecule, sCD4 (Figure 8c), contained only bi-antennary complex type glycans as did the Ly-6 and SCRC superfamily proteins, sCD59 (Figure 8d) and sCD5d1, respectively. This result clearly implies that local protein structure, either directly or indirectly, is the dominant factor affecting the extent of glycan processing when cells are in stasis.

Table III. HPLC analysis of the exoglycosidase digestions of the hydrazine released, 2AB labeled pool of sCD48 desiaylated N-glycans
Peak % Assigned undigested peaks Assignment Digests (gu)
1 (panel a) 2 ( panel b) 3 (panel c) 4 (panel d)
1 2.4 M3 4.42 4.41 4.41 4.39
2 8.7 M3F 4.90 4.90    
3   M3(GN)     4.98  
4 3.9 M3(GN)F 5.43 5.43    
5 3.9 A2G0 5.43 5.43 5.48  
6 6.3 A2G0F 5.90 5.90    
7   A3G0   6.17 6.15  
8 14.5 Man 5 6.18 6.17 6.15  
9 4.5 A3G0F 6.53      
10   A4G0   6.53 6.52  
11 3.8 A2G1(1,6)F 6.67      
12 3.3 A2G1(1,3)F 6.78      
13 1.6 A4G0F 6.88 6.89    
14 23.9 A2G2F 7.55      
15   A4G1(GN)     7.37  
16   A4G1(GN)F   7.72    
17 3.2 A3G2F 8.16      
18 8.8 A3G3F 8.88      
19 2.3 A4G3F 9.23      
20 5.5 A4G4F 10.00      
21 1.4 na 10.14      
22 1.6 A4G4LF 11.11      
23 0.5 na 11.98      
The table refers to the data in Figure 4b in which enzyme arrays were used to digest five aliquots of the sCD48 glycan pool simultaneously. Peaks were initially assigned by comparison with known standards and the pre-determined incremental values for the addition of monosaccharides to glycan cores. Structures were confirmed by incubations with enzyme arrays: panel a, Arthrobacter ureafaciens [alpha]2,3-/[alpha]2,6-sialidase (ABS); panel b, Arthrobacter ureafaciens [alpha]2,3-/[alpha]2,6-sialidase + bovine testes [beta]-galactosidase (BTG); panel c, Arthrobacter ureafaciens [alpha]2,3-/[alpha]2,6-sialidase + bovine testes [beta]-galactosidase + bovine epididymis fucosidase (BEF); panel d, Arthrobacter ureafaciens [alpha]2,3-/[alpha]2,6-sialidase + bovine testes [beta]-galactosidase + bovine epididymis [alpha]1,6-fucosidase + Streptococcus pneumoniae [beta]-hexosaminidase (SPH).

Effects of local secondary structure, and quaternary structure, on glycan processing

In general, glycans attached to loops in the immunoglobulin fold were processed more extensively than those on [beta]-strands (Figure 1c). Seven of the eight glycosylation sites of sCD2 (Figure 8a) and sCD48 (Figure 8b) are located in loops and, for both of these molecules, complex-type glycans dominate. sCD2 contains a series of oligomannose structures attached to Asn-65 at the apex of the DE loop in domain 1 (Recny et al., 1992), and the modeling suggests that the glycan at this site projects into a cleft between domains 1 and 2 of the protein where it is expected to be shielded from mannosidases and GlcNAc transferase II, the key enzyme in the processing of complex type sugars. In addition, the GlcNAc-1 residue, which links the nitrogen in the side chain of Asn-65 to the remainder of the oligosaccharide, is relatively inflexible with respect to the protein (Wyss et al., 1995b). By inhibiting the movement of the glycan out of the cleft between domains 1 and 2 of the protein, inflexibility in this linkage would be expected to enhance the shielding of this glycan, thereby restricting processing at this site.


Figure 3. Separation of 2AB labeled oligosaccharides of soluble leukocyte antigens by WAX HPLC. 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: N, neutral glycans; NA1 and NA2, mono- and di-sialylated N-linked glycans, respectively. OA1 and OA2, mono- and di-sialylated O-linked glycans, respectively. Note that the neutral peaks may contain noncarbohydrate material.

   a
   b

Figure 4. (a) Separation of 2AB labeled oligosaccharides of soluble leukocyte antigens by NP HPLC. Structures were assigned to the major peaks based on (1) their elution positions, which were converted to gu by comparison with the elution positions of an external standard dextran ladder run immediately before each sample and (2) on the incremental values for the addition of monosaccharides to standard glycan cores (Table II; see Guile et al., 1996, for full details of this strategy). Structures were confirmed by MALDI TOF MS analysis of the total desialylated glycan pools and by exoglycosidase digestions using enzyme arrays monitored by HPLC (as shown for sCD48 in Figure 4b and Table III) and MALDI TOF MS. The HPLC analysis of the glycans from sThy-1 is shown in Figure 7. M, Mannose; G, galactose. Polylactosamine structures are reported by composition: H, hexose; N, N-acetyl hexosamine; F, fucose; H1 and H2 are hybrid structures. A2G0, A2G1, and A2G2 are abbreviations for complex bi-antennary glycans containing 0, 1, and 2 galactose residues, respectively. (b) Simultaneous analysis of the asialo N-glycans released from rat sCD48 using enzyme arrays. The figure shows the HPLC analysis of the desialylated glycan pool and the products resulting from the digestion of four aliquots of the pool with a series of enzyme arrays. The particular enzyme array which produced each profile is shown on the appropriate panel. The shaded areas define the peaks which contain glycans which were subsequently digested by the additional enzyme present in the next array. The gu value of each peak was calculated by comparison with the dextran hydrolysate ladder shown at the top of the figure. Structures were assigned from the gu values, previously determined incremental values for monosaccharide residues (Guile et al., 1996) and the known specificity of the exoglycosidase enzymes. The structures of the most abundant glycan populations (numbered 1-23) are shown in Figure 4a and Table III. ABS, Arthrobacter ureafaciens sialidase; BTG, bovine testes [beta]-galactosidase; BEF, bovine epididymis [alpha]-fucosidase; SPH, streptococcus pneumoniae [beta]-N-acetylhexosaminidase.

In contrast to sCD2 and sCD48, all three glycan sites in sThy-1 are predicted to be located either at the very ends (Asn-32 and -74) or the beginnings (Asn-98) of [beta]-strands rather than in the middle of loops (Figure 8e). Accordingly, restricted processing is apparent in the mixture of complex, hybrid, and oligomannose sugars present on this molecule. Similarly, the processing of the glycans of sCD4 (Figure 8c), which are also located on [beta]-strands rather than loops, is also restricted in so far as oligomannose but not lactosaminoglycans or multiantennary glycans were observed, as noted previously (Ashford et al., 1993). The simplest interpretation of these data is that the exposure to the glycosylation machinery of glycans attached to loops is significantly enhanced over that of glycans attached to [beta]-strands.

The presence of domains 1 and 2 in the four-domain form of sCD4 reduces the fucosylation of the single biantennary complex glycan attached to domain 3 by a factor of approximately 2 (compared with that of sCD4d3+4), consistent with the previous work of Ashford et al. (1993), and implying an effect of quaternary structure on the degree of processing. Interestingly, the presence of the Thy-1 domain in the sThy-1/CD4d3+4 chimera reduces the degree of fucosylation of this site even further, suggesting that the decrease in fucosylation is the result of steric effects rather than sequence-dependent phenomena. Consistent with this, the uniform modification of sCD4d3+4, sCD5d1 and the sCD5/CD4d3+4 chimera with the A2G2F glycan (Figure 5b), implies that the flexible linker of CD5 may overcome this steric limitation, thereby allowing full processing.

N-Glycosylation of sCD5d1 is restricted in CHO cells

Currently there are no three-dimensional structural data for any member of the SRCR superfamily. The glycans attached to both sCD5d1 and sCD5/CD4d3+4 were mainly restricted to the sialylated forms of the fucosylated bi-antennary sugar, A2G2F (Figure 5a). NMR studies of endoH treated sCD5d1 from a yeast expression system (McAlister et al., 1998), indicate that the GlcNAc-1 residue (which links the side chain of Asn-92 to the remainder of the oligosaccharide) is more conformationally flexible than the bulk of the domain. This is consistent with core fucosylation which requires the GlcNAc-1 residue to be accessible to the fucosyl transferase. In contrast, GlcNAc1 of the glycan attached to Asn-65 sCD2 domain 1 is nonfucosylated and relatively less flexible and the site contains only oligomannose structures (Wyss et al., 1995a).

Roles for glycosylation based on the molecular modeling

In many instances, the length of a glycan and the diameter of the protein domain to which it is attached are of the same order of magnitude. For example, the longest dimension of an immunoglobulin V-set domain is about 3 nm long and the length of a complex biantennary glycan is 3-4 nm. These steric properties are likely to affect profoundly the behavior of the proteins to which the glycans are attached.

Effect on glycoprotein presentation. Glycosylation is a prominent feature of the structure of the extracellular domain of CD2 (Figure 8a). The four CD2 homologs to date share a total of 18 glycosylation sequons predicted to occupy 12 different sites on these very similar structures (Tavernor et al., 1994). Only one site, occupying a membrane proximal position at the base of domain 2, is present in the sequences of all four homologs which, assuming full occupancy, implies that the glycan close to the membrane has an important role in the function of CD2. It is noteworthy that CD48 and CD4 also have similarly located glycans (Figure 1a; the membrane proximal site on CD4 was deleted when the CD4 gene was mutated to generate sCD4). For each of these glycoproteins, the membrane-proximal glycans could be expected to orient the ligand binding sites by limiting the conformational space available to each molecule. The presence of the glycan may thus provide a physical explanation for the 'confinement cone" of 52° described by CD2 and LFA-3 (Dustin et al., 1996) both of which are predicted to interact in a head-to-head fashion (Jones et al., 1992; Bodian et al., 1994). This restriction in the conformational space available to the protein is likely to promote trans-interactions with CD2 ligands present on other cells and to discourage cis-interactions with ligands on the same cell. It has been proposed that the sugars at Asn-65 of human CD2 domain 1 may stabilize the structure of the ligand binding domain by counterbalancing the clustering of positive charges created by five lysine residues situated on the surface of that domain (Wyss et al., 1995b), but this is controversial and in any case does not occur in rat CD2 (Davis et al., 1995; Davis and van der Merwe, 1996).

   a
   b

Figure 5. The NP-HPLC glycan profiles of (a) the total and (b) the desialylated sugars released from sCD5d1, sCD4d3+4 and the sCD5/CD4d3+4 chimera. All contain a single N-glycan, A2G2F, at each glycosylation site. The chimera contains a higher proportion of O-glycans than sCD5d1. sCD4 is not O-glycosylated.

Maximizing protein extension and ligand-binding domain exposure. No O-glycans were detected on sCD5d1 residues 1-110 by electrospray mass spectrometry (McAlister et al., 1998), therefore it is proposed that the O-glycans detected in this study are located at the C-terminal end of CD5d1 between residues 111-118 in a region rich in Thr and Pro residues, a common feature of the sites of clustered O-glycosylation. O-Glycosylation of peptides is known to produce extended and rigid structures. Electron microscopic studies indicate that the extension contributed per residue in an O-glycosylated peptide varies from 0.2-0.25 nm in CD43 (Cyster et al., 1991) and mucins (Shogren et al., 1989; Jentoft et al., 1990). The O-glycosylated peptide of CD5 may therefore separate domains 1 and 2 by up to 5 nm. It has been proposed that O-glycosylated peptides have a structural function which forces globular domains away from the membrane, perhaps for interaction with ligands (Kuwano et al., 1991; Leahy et al., 1992; Kuttner-Kondo et al., 1996). Electron microscopy studies suggest that CD5 has a linear structure (McAlister et al., 1998) consistent with the view that the function of the O-glycosylation of the linker between domains 1 and 2 of CD5 is to extend the molecule and maximize the exposure of the N-terminal domain to potential ligand(s). For other molecules, such as CD4, a similar function appears to be served by duplicated globular protein domains.


Figure 6. Biogel P4 profile of sCD5d1 asialo oligosaccharides. sCD5 contains an O-glycan, Gal[beta]1,4GalNAcitol eluting at 3.5gu on P4 GPC. The N-glycosylation of sCD5d1 is restricted almost entirely toA2G2F ± sialic acid. A2G2F elutes at 14.8gu.

Protection from proteolysis and prevention of aggregation. The sugars attached to CD59 project away from the disc-like protein domain in the plane of the active face, adjacent to the membrane surface (Figure 8d). Mutational studies by Bodian et al. (1997b) indicate that the active site residues are located on the membrane distal surface of the extracellular domain. The glycans do not appear directly to block the active site sterically, but may be expected to restrict the rotational freedom of the extracellular domain around axes parallel to the membrane. In turn this would be expected to stabilize an exposed location for the active site of CD59 (Bodian et al., 1997b; Rudd et al., 1997). In addition, the bulky, hydrophilic glycans would limit interactions with the lipid bilayer and this, together with the GPI anchor, may facilitate the diffusion of the protein in the membrane. The heterogeneity of the sugars suggests that the glycans would also influence the geometry of the packing by preventing the aggregation of CD59 on the cell surface and the formation of regular arrays. By limiting protein-protein interactions the glycans may influence the distribution of CD59 molecules at the cell surface since GPI-anchored proteins are believed to associate in microdomains in dynamic equilibrium with isolated individual molecules (Simons and Ikonen, 1997). The large N-glycans may also be important in preventing proteolysis of the extracellular domain since glycosylation has been shown to increase the dynamic stability of a protein and increase its resistance to protease digestion compared with unglycosylated forms (Rudd et al., 1994).


Materials and methods

Preparation of glycoproteins

All recombinant glycoproteins consisted of some or all of the extracellular regions of leukocyte cell surface antigens and were expressed in CHO cells using the glutamine synthetase-based gene expression system as described in detail for sCD4 (Davis et al., 1990). The preparation of sCD59 is further described by Kieffer et al., 1994; sCD48 by McAlister et al., 1996; and sCD2 by Bodian et al., 1994. All proteins were purified by affinity chromatography and gel filtration. sCD5d1 residues 1-118, including the first 8 of the 24 amino acids from the linker region between domains 1 and 2, and chimeric sCD5/CD4d3+4 constructs were prepared by polymerase chain reaction (PCR) amplification (Brown and Barclay, 1994; McAlister et al., 1998). sThy-1/CD4d3+4 was also prepared by PCR. The sThy-1 specific DNA for the PCR contained three extra amino acids (GGS) at its C-terminus. The sequence at the junction of the two molecules was VKCSGTST. The occupancy of the potential N-glycan site at Asn-217 in sCD5 was not demonstrated in this study. However, the occupied sequon in domain 2 is at an almost identical position (2 residues toward the N-terminus) within the SRCR consensus sequence (Resnick et al., 1994) as the sequon in domain 1, consistent with the assumption that Asn-217 is occupied.

Release, re-N-acetylation, and labeling of the reducing terminus of the glycans

Approximately 100 µg of each protein was dialyzed against 0.1%TFA and lyophilized. Glycans were released by hydrazine at 85°C and re-N-acetylated using a GlycoPrep 1000 (Oxford GlycoSciences Ltd.) optimized for maximum recovery (~85%) of both N- and O-linked sugars. These hydrazinolysis conditions represent a compromise between achieving nonselective release, maximization of the yield and minimization of degradation of released sugars. The recovery procedures may result in the loss of some sialic acid residues.


Figure 7. The NP-HPLC profiles of the desialylated glycan pools released from sThy-1, sCD4d3+4, and sThy-1/CD4d3+4. The addition of the extracellular domain of Thy-1 to sCD4 decreases the amount of fucosylated glycans.

3H Labeling of the reducing terminus of the glycans was carried out by reduction with sodium borohydride according to the method described by Ashford et al., 1987.

Fluorescent labeling of the reducing terminus with 2-aminobenzamide (2AB)

One-third (~8.85 nmol) of the free glycan solutions were evaporated to dryness using a vacuum centrifuge. 2AB labeling was carried out by reductive amination using the Oxford GlycoSciences (OGS, Abingdon, Oxon., UK) Signal Labeling Kit (Bigge et al., 1995).

Exoglycosidase enzyme digestions

Glycan solutions were evaporated to dryness in a vacuum centrifuge; 10 ml of standardized enzyme solutions were added as follows and the mixtures incubated for 16 h at 37°C:

(1) Arthrobacter ureafaciens neuraminidase (ABS; OGS): 1-2 U/ml in 100 mM sodium actetate buffer pH 5, substrate concentration 5-30 µM; (2) bovine testes [beta]-galactosidase (BTG; OGS): 1-2 U/ml in 100 mM citrate/phosphate buffer pH4, substrate concentration 20 µM; (3) Charonia lampas [alpha]-fucosidase (CLF; Glycobiology Institute), 9 mU/ml in 50 mM sodium actetate buffer pH 4.5 containing 0.15 M NaCl, substrate concentration 20 µM; (4) Streptococcus pneumonia hexosaminidase (SPH; OGS) 2 U/ml in 100 mM sodium citrate/phosphate buffer pH 5, substrate concentration 20 mM; (5) Newcastle disease virus (NDV; OGS): 0.2 U/ml in 50 mM sodium actetate buffer pH 5.5, substrate concentration 5-30 µM.

Simultaneous oligosaccharide sequencing on the released glycan pool

Enzyme digests were performed at 37°C for 16-24 h in 100 mM citrate/phosphate buffer pH 5 containing 0.2 mM zinc acetate and 0.15 M sodium chloride. Conditions for the individual enzymes in the arrays were as follows. ABS: 1-2 U/ml; substrate concentration 5-30 µM; almond meal fucosidase (AMF): 3 mU/ml; substrate concentration 20 µM: CLF (Oxford Glycobiology Institute):10 U/ml/1 mg/ml BSA (20µM); BTG: 1-2 U/ml; substrate concentration 20 µM; SPH: 2 U/ml; substrate concentration: 20 µM. NDV: 0.2 U/ml in 50mM sodium acetate; substrate concentration 20 µM. Samples were purified from protein and salts prior to MALDI TOF MS by passingthrough mixed bed resins of Chelex100(Na+)/Dowex AG50X12(H+)/Ag3X4A (OH-)/QAE Sephadex A-25. Samples were purified from the exoglycosidases before HPLC analysis by passing through a microcentrifuge tube inset with a protein binding filter (Microspin 45 mm CN, Pro-Mem, suppliers R. B. Radley and Co. Ltd., Shire Hill, Saffron Walden, Essex, UK). The filter was washed with 15 µl of 5% acetonitrile.


   sCD2 (a)

   sCD48 (b)

   sCD4 (c)

   sCD59 (d)

   sThy-1 (e)

Figure 8. Molecular models of selected glycoforms of the extracellular domains of the leukocyte antigens CD2 (a), CD48 (b), CD4 (c), CD59 (d), and Thy-1 (e), expressed in CHO cells. The protein structures are based on the available x-ray or NMR structures for similar or homologous proteins (see text for details). The oligosaccharides attached are the most common structures identified in the glycan analysis of each glycoprotein. Oligosaccharide structures are assigned arbitrarily to the different glycosylation sites, except where site specific information is available (see below). The numbers refer to the glycosylated residues. The arrows indicate the direction of the membrane for the membrane bound proteins. (a) sCD2-Man 6 at Asn-65, A3G3FSA3 at Asn-117, and A2G2FSA2 at Asn-126. The glycan analysis of Asn-65 is based on Wyss et al., 1995a. (b) sCD48-Man 5 at Asn-16, A4G4F at Asn-75, A2G2FSA2 at Asn-164 and Asn-181. (c) sCD4-Man 5 at Asn-159 and A2G2F at Asn-270. The site analysis is based on Ashford et al., 1993. (d) sCD59-A2G2FSA2 at Asn-18. (e) sThy-1-Man 5 at Asn-23, A2G2F at Asn-74 and A2G2SA2 at Asn-98. The site analysis is based on Parekh et al., 1987.

Separation of glycans by charge and normal phase separations of neutral and acidic oligosaccharides

Weak Anion Exchange (WAX) Chromatography was carried out using a GlycoSepC column (OGS Ltd.) according to Guile et al. (1994). Normal phase separations were performed on a Glycosep-N chromatography column (OGS Ltd.) and structures assigned according to Guile et al. (1996).

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS)

Data were acquired on a Micromass AutoSpec-QFPD magnetic sector instrument fitted with a pulsed nitrogen laser (337 nm) and an array detector (Bordoli et al., 1994; Micromass Ltd., Wythenshaw, Manchester, UK). 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 (2,5-DHB) 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 (Harvey, 1993). 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.

Gas chromatography (GC)

The GC was a Varian 3600 instrument fitted with flame ionization detection (FID) and a flow-through radioactivity detector (Lablogic, St. John's, Sheffield, UK) RAGA, 10 ml proportional counter); Supelco (Supelco UK, Dorset, UK) SP2380 column 15 m × 0.32 mm. The sample was coinjected onto the GC column with radiolabeled myo-(2-3H(N)-inositol plus unlabeled reduced sugars, used as an internal marker. The carrier gas was He. Flow rates: He 2.5 ml/min, H2 (to reactor) 3.5 ml/min Ar/CH4 (to counter) 70 ml/min. Structures were assigned by comparing the elution times of the unknown sample (detected using a radioactive detector) with standard sugars (detected by FID). The GC column is fitted with an outlet splitter; 20% of the effluent goes to the FID where the cold standards are detected. This allows direct comparison of the labeled unknowns with the cold standards in the same GC run.

Molecular modeling

This was performed on a Silicon Graphics Indigo 2 workstation using InsightII and Discover software (MSI Inc.). Figures were produced using the program Molscript (Kraulis, 1991). In all cases except for sCD2 and sCD59, structures of the exact proteins used in this study are not available, thus structures were modeled on the basis of sequence alignment with proteins of known structure (either the same protein from different species or proteins with a high degree of sequence homology). The structures of the protein components of sCD2 and sCD59 are based on the crystal structure (Bodian et al., 1994) and the solution NMR structure (Fletcher et al., 1994), respectively. The structure of the protein component of rat sCD4 is based on the crystal structure of human sCD4 (Wu et al., 1997). The structure of the protein component of rat sCD48 is modeled on the crystal structure of rat sCD2 (Jones et al., 1992). The structure of the protein component of sThy-1 is modeled on a V-set IgSF domain (Rademacher et al.,1991). Oligosaccharide structures were built using a database of disaccharide linkage conformations based on crystallographic data from saccharides, glycoproteins, and lectins (unpublished observations) to give average structures. The resulting structures were energy minimized to eliminate unfavorable steric interactions. The structures of the peptide-glycan linkages were based on the NMR results from glycopeptide studies (Wormald et al., 1991). The torsion angles around the Asn C[alpha]-C[beta] and C[beta]-C[gamma] bonds were adjusted to eliminate unfavorable steric interactions between the oligosaccharide and the protein surface.

Acknowledgments

We thank Dr. M.Tomlinson for preparation of sThy-1/CD4d3+4, Professor Henrik Clausen for discussions relating to GalNAc transferase activity in CHO cells and erythrocytes, and Cristina Colominas for excellent technical assistance with the analysis of the sCD48. S.J.D. is supported by the Wellcome Trust. The Glycobiology Institute acknowledges funding from the DTI/BBSRC LINK scheme and from the European Commission Grant BIO4-CT95-0138.

Abbreviations

2AB, 2aminobenzamide; CHO, Chinese hamster ovary; sCD4d3+4, domains 3 and 4 of native CD4; sCD5d1, domain 1 of CD5; TCR, T-cell receptor; sCD2, sCD48, etc., the prefix 's" is used to denote the soluble form of the antigen comprising the extracellular domains, distinguishing it from the intact cell surface antigen; GC, gas chromatography; GPC, gel permeation chromatography; GPI, glycosylphosphatidylinositol; gu, glucose unit; HPLC, highperformance liquid chromatography; IgSF, immunoglobulin superfamily; FID, flame ionization detection; NP, normal phase; PCR polymerase chain reaction; SRCR, scavenger receptor cysteine-rich; MALDI TOF MS, matrix assisted laser desorption ionization time of flight mass spectrometry; WAX, weak anion exchange chromato-graphy. Abbreviations used for describing oligosaccharide structures: A(1-4), the number of antennae linked to the trimannosyl core; G(0-4), the number of terminal galactose residues in the structure; F, fucose; GlcNAc, N-acetyl glucosamine; GalNAc, N-acetylgalactosamine; GGNAc, Gal[beta]1-3GalNAc; S, N-acetyl neuraminic acid; G, Gal, galactose; M, Man, mannose; H, hexose; N, N-acetylhexosamine. N, NA1, and NA2, represent neutral, mono-, and disialylated glycans, respectively. ABS, Arthrobacter ureafaciens [alpha]2,3-/[alpha]2,6-sialidase; BTG, bovine testes [beta]-galacto-sidase; BEF, bovine epididymis [alpha]1,6-fucosidase; SPH, Streptococcus pneumoniae [beta]-hexosaminidase.

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