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
a, b
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c
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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
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
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
Table I.
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 |
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
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N-Glycan analysis of the soluble forms of the leukocyte antigens
The asialo sugar profiles of the soluble leukocyte antigens are compared in Figure
Figure 2. Overall scheme for analysis of oligosaccharides.
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
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
Figure
Molecular modeling of the glycoproteins
Molecular models (Figure
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
Table III.
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 |
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
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
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
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
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
a
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b
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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
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.
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.
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.
3To whom correspondence should be addressed