©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand Binding by the Immunoglobulin Superfamily Recognition Molecule CD2 Is Glycosylation-independent (*)

(Received for publication, August 1, 1994; and in revised form, October 3, 1994)

Simon J. Davis (1)(§) Elizabeth A. Davies (1) A. Neil Barclay (1) Susan Daenke (2) Dale L. Bodian (3) E. Yvonne Jones (3) (4) David I. Stuart (3) (4) Terry D. Butters (5) Raymond A. Dwek (5) P. Anton van der Merwe (1)(¶)

From the  (1)MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, South Parks Road, University of Oxford, Oxford, OX1 3RE United Kingdom, (2)Molecular Sciences Division, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, OX3 9DU United Kingdom, (3)Laboratory of Molecular Biophysics and (4)Oxford Centre for Molecular Sciences, The Rex Richards Building, South Parks Road, University of Oxford, Oxford, OX1 3QU United Kingdom, and (5)Glycobiology Institute, Department of Biochemistry, South Parks Road, University of Oxford, Oxford, OX1 3QU United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-Å resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.


INTRODUCTION

The initial sequencing of immunoglobulins and beta(2)-microglobulin implied that these molecules were formed by individual protein structural units that evolved from a single primordial domain of about 100 amino acids (reviewed in (1) ). The immunoglobulin superfamily (IgSF) (^1)concept arose from the discovery that sequences characteristic of these domains are also present in molecules without an immunological function(1) . Structural studies of immunoglobulins(2) , major histocompatibility complex class I(3) , and class II antigens(4) , CD4(5, 6) , CD8(7) , and CD2 (8, 9, 10) have revealed that the IgSF fold consists of a sandwich of two anti-parallel beta-sheets stabilized in some instances by a conserved disulfide bond. The conserved patterns of sequence characteristic of IgSF domains are generally limited to beta-strand residues forming the hydrophobic core of the domain (1) which appears to be responsible for the strict conservation of the three-dimensional structure of these domains(10) .

A recent survey of the leucocyte surface has indictated that 36% of leucocyte antigens belong to the IgSF suggesting that the IgSF forms the largest single family of molecules present on the cell surface (11) . The ligand interactions of the cell-cell recognition molecule CD2 are among the best characterized of those involving IgSF cell surface molecules. In humans and rodents the ligands for CD2 are CD58(12, 13) and CD48(14, 15) , respectively. Along with CD2, CD48 and CD58 form a subset of molecules within the IgSF that also includes the carcinoembryonic antigens(16) , Ly-9 (17) and 2B4(18) . The clustering of the CD2, CD48, and CD58 genes in the genomes of humans and mice implies that CD2, CD48, and CD58 have all evolved from a common precursor involved in homophilic interactions(19) . The crystal structures of rat sCD2 (9) and human sCD2 (hsCD2) (8) have revealed that the extracellular region of CD2 consists of two IgSF domains: an NH(2)-terminal V-set domain and a C2-set domain. Mutational analyses of CD2 (20, 21, 22, 23) established that the ligand binding site is located on the GFCC`C" face of the beta-sheet of the V-set domain. The highly conserved linker region seen in the sCD2 structures places this relatively flat, highly charged face at the membrane-distal surface of the molecule(8, 9) . The interactions of rat and human CD2 with their respective ligands, CD48 (24) and CD58(25) , are characterized by relatively fast on-rates and very fast off-rates which, together with the structural data, suggest that the binding of CD2 with its ligands is not dependent on large conformational changes.

The role of glycosylation in stabilizing IgSF domains is controversial. While many IgSF molecules are N-glycosylated, the extent of glycosylation varies considerably and glycosylation sites are generally not conserved, even between species homologues. The ligand binding function of rat CD2, which has four N-glycosylation sites, is not glycosylation-dependent(15, 24) . In contrast, it has been suggested that ligand binding by human CD2, which has three N-glycosylation sites, is glycosylation-dependent(26) . This observation has significant implications given that the evolutionary success of the IgSF, and the high level of conservation of the IgSF fold, are both thought to reflect the ability of IgSF protein domains to form stable structural units for the presentation of receptor-ligand recognition motifs(1, 27) . In the present study a systematic analysis of the effect of glycosylation on the ligand binding properties of CD2 has been undertaken. The data indicate that the structural integrity and ligand binding function of human CD2 are not glycosylation dependent.


MATERIALS AND METHODS

Protein Expression

The preparation and expression of the constructs encoding hsCD2 and sCD58 in Chinese hamster ovary (CHO) cells are described in detail elsewhere(25) . In initial optimization experiments with N-butyldeoxynojirimycin (NB-DNJ), 2 times 500 cm^2 flasks (Nunc) were seeded with hsCD2 secreting CHO cells in 100 ml of GMEM-S (Applied Protein Products), 10% fetal calf serum containing NB-DNJ at 0, 0.5, 1, 1.5, or 2 mM. After 6 days 100 ml of GMEM-S, 10% fetal calf serum containing 4 mM sodium butyrate, and NB-DNJ at 0, 0.5, 1, 1.5, or 2 mM was added. Ten days after the addition of the butyrate, 0.5-ml samples of the supernatant were taken for comparison of secretion levels. The cultures were left for a total of 15 days before the supernatants were harvested. When large amounts of endoglycosidase H (endo H)-treated hsCD2 were required for crystallization, the cells were grown to confluence in cell factories as described above, and NB-DNJ was added with the sodium butyrate to a final concentration of 1.5 mM. These cultures were then left for a further 3-4 weeks prior to harvesting.

After preclearing the spent tissue culture medium at 10,000 times g for 30 min, the hsCD2 was purified by affinity chromatography according to published methods (28) using an antibody affinity column prepared with the anti-CD2 monoclonal antibody, (mAb) X/3. Final purification involved gel filtration on Sephacryl S-200 in 10 mM Hepes, 140 mM NaCl, pH 7.4.

Deglycosylation and Crystallization

After preparation of the hsCD2 in the presence of various concentrations of NB-DNJ, endo H sensitivity was determined by incubating 10-µg aliquots of the purified glycoproteins with 0.012, 0.06, or 0.3 International Union of Biochemistry (I.U.B.) units/mg of endo H (Boehringer Mannheim) in 30 µl of 100 mM sodium acetate, pH 5.2. After incubation overnight at 37 °C the digested samples were boiled in 1% SDS under reducing conditions prior to electrophoresis in 15% SDS-PAGE gels.

For large-scale endo H treatment of hsCD2 for crystallization experiments, 4 mg of the purified glycoprotein were concentrated to 1-2 mg/ml in 0.1 M sodium acetate, pH 5.2, and then digested with 0.1 I.U.B. units/ml endo H overnight at 37 °C. To purify the endo H-treated hsCD2 from the contaminating endo H-resistant fraction, the protein mixture was concentrated to 0.5 ml and then passed through a 5-ml Sephadex G-50 column to remove free oligosaccharides. The eluate was then passed through a 15-ml lectin affinity column consisting of equal parts of lentil lectin, concanavalin A and wheat germ agglutinin, each coupled to Sepharose 4B (Sigma). The homogeneity of the hsCD2 was then confirmed by SDS-PAGE on a 15% acrylamide gel. The lectin purified protein was concentrated to 2 ml and then applied to Sephadex G-75 in 10 mM Hepes, 140 mM NaCl, pH 7.4, to remove free lectin eluting from the lectin-affinity column. The deglycosylated hsCD2, in 10 mM Hepes, 140 mM NaCl, pH 7.4, was concentrated to 17 mg/ml. Crystals were grown by vapor diffusion in sitting drops at room temperature. Initial trials were conducted using Crystal Screen reagents (Hampton Research).

For large scale preparation of fully deglycosylated protein, hsCD2 (at 600 µg/ml) purified from untreated cultures was digested with peptide:N-glycosidase F (PNGase F; New England BioLabs) at 0.085 I.U.B. units/ml in 0.5 M Tris, pH 8. Deglycosylated protein was then purified by gel filtration on a Superdex G-75 fast protein liquid chromatography system (Pharmacia Biotech Inc.).

Carbohydrate Analyses and Mass Spectrometry

Amino sugars were quantitated by Dionex high performance anion exchange chromatography after hydrolysis in 6 N HCl at 100 °C for 4 h. 2-Deoxy glucose was added as an internal standard after hydrolysis, and the reaction products were separated using an isocratic eluant containing 4 mM NaOH. Mass spectrometric analysis of the proteins in H(2)O was performed on a Fisons VG BioQ mass spectrometer equipped with an electrospray interface operating in positive ion mode.

Binding Experiments

Antibodies were obtained as previously described(25) . Analysis of the interaction of hsCD2 with sCD58 and anti-CD2 mAbs was performed on a BIAcore biosensor (Pharmacia). Experiments were performed at 25 °C at the indicated buffer flow rates (3-20 µl/min). The buffer used was Hepes-buffered saline which contained (in mM): NaCl, 150; MgCl(2), 1; CaCl(2), 1; NaN(3), 10; 0.005% Surfactant P-20 (Pharmacia), and Hepes, 10 (pH 7.4). NaN(3) was omitted during the immobilization procedures. The various forms of hsCD2 were covalently coupled to a CM5 sensor chip via primary amine groups (Pharmacia) as described using the Amine Coupling Kit (Pharmacia)(25) . For immobilization of hsCD2, the glycosylation variants were used at 22-85 µg/ml in 10 mM sodium acetate (pH 5), and the activation times were varied from 75 to 360 s. Immobilized untreated and endo H-treated hsCD2 were regenerated after immobilization or antibody binding with 10-100 mM HCl. However, HCl treatment of immobilized PNGase F-treated hsCD2 reduced its ability to bind ligand and antibody, and so it was omitted when analyzing PNGase F-treated hsCD2 (see Fig. 6D). sCD58 loses its ligand and antibody binding activity when coupled via primary amines. Therefore sCD58 was coupled, as described previously(25) , via thiol groups on two cysteine residues present within the second IgSF domain which can be exposed by mild reduction.


Figure 6: Crystallization of hsCD2 and diffraction analysis of crystals. Crystals of purified endo H-treated hsCD2 were grown by vapour diffusion against 1.25 M sodium citrate, 0.1 M Hepes, pH 7.4. Typical crystals are shown in Panel A; the largest crystals from 2-4-µl drops grew to 0.6 mm along the longest axis. In Panel B a typical two-pass 1.5° oscillation image taken at Daresbury Synchrotron Radiation Source (line 9.5, = 0.999 Å, T = 20 °C) is shown. The crystal to film distance was 212.2 mm. The edge of the image corresponds to diffraction to Bragg spacings of 2.5 Å. The diffraction pattern was displayed with the program PSIMAGE (R. Esnouf, Oxford).



The equilibrium binding data (see Fig. 3) were analyzed by 1) nonlinear curve fitting of the Langmuir binding isotherm to the primary data and 2) linear curve fitting of the Scatchard plots. The dissociation phases (see Fig. 4) were analyzed by first normalizing them so that the response before dissociation was 100% and the base line response was 0%. Dissociation rate constants (k) for each dissociation phase were then determined by fitting mono- or bi-exponential decay functions to the data (see Fig. 4). All curve fitting was performed using the curve-fitting functions of the program Origin version 2 (MicroCal Software Inc, Northampton, MA) which was run on a Compaq PC. Linear curve fitting was by linear least squares regression. Nonlinear curve fitting employed iterative least squares curve fitting using the Marquardt-Levenberg algorithm.


Figure 3: Measurement of the affinity of sCD58 binding to untreated, endo H-treated and PNGase F-treated hsCD2. Panel A, sCD58 was injected for 6 s at the indicated concentrations over a flow cell with immobilized untreated (open circles) or endo H-treated (closed circles) hsCD2. The levels of untreated and endo H-treated hsCD2 immobilized were 3160 and 4983 response units (RU), respectively. At saturation, 39 and 34% of the immobilized untreated and endo H-treated hsCD2 had bound sCD58, respectively. Panel B, untreated or PNGase F-treated hsCD2 were injected for 6 s at the indicated concentration over a flow cell with sCD58 immobilized. The level of immobilization of sCD58 was 10,863 RU. The equilibrium binding levels shown in Panels A and B were calculated as described elsewhere (24) by subtracting the response obtained when the same sCD58 and hsCD2 samples are injected over a control flowcell with nothing immobilized. The flow rate was 20 µlbulletmin. Insets, Scatchard plots of the binding data. The K values were determined by linear-regression analysis of the Scatchard plots as well as by nonlinear curve fitting of the saturation binding curve. Both methods gave the same K values.




Figure 4: Comparison of the rates of dissociation of sCD58 from untreated hsCD2, endo H-treated hsCD2, and PNGase F-treated hsCD2. Panel A, the dissociation of sCD58 (0.22 mg/ml) from immobilized untreated (open triangles, 3160 RU) or endo H-treated (filled triangles, 4983 RU) hsCD2. The fall in response following injection of sCD58 over a flow cell with nothing immobilized is also shown (dotted line). The equilibrium responses (100%) following injection of sCD58 over nothing, untreated hsCD2 and endo H-treated hsCD2 were 92, 373, and 705 RU, respectively. Panel B, dissociation of untreated hsCD2 (open triangles, 0.9 mg/ml) or PNGase F-treated hsCD2 (closed triangles, 1 mg/ml) from immobilized sCD58 (10,863 RU immobilized). The fall in response following injection of untreated (open triangles) and PNGase F-treated (closed triangles) hsCD2 over a flow cell with nothing (dotted line) immobilized is shown. The equilibrium responses (100%) following injection of untreated and PNGase F-treated hsCD2 over immobilized sCD58 were 850 and 1193 RU, respectively. The same samples injected though a control flow cell gave responses of 161 and 317 RU. The samples were injected at flow rates of 20 µl/min. The apparent dissociation times were obtained by fitting mono-exponential decay curves to the data (see dotted and solid lines). One exception was the dissociation of PNGase F-treated hsCD2 from immmobilized sCD58 which was fitted using a bi-exponential decay function.




RESULTS

Effects of NB-DNJ and Digestion of hsCD2 with Endo H

A full-length cDNA clone encoding human CD2 (29) was truncated and mutated to encode a soluble form of CD2 with three potential glycosylation sites (Asn-65, Asn-117, and Asn-126) and terminating at Lys-182 of the fully processed product as described elsewhere(25) . The glucosidase I inhibitor, NB-DNJ, has previously been shown to prevent the maturation of N-linked oligosaccharides on recombinant human immunodeficiency virus gp120 expressed in CHO cells(30) . These sugars remain as oligomannose forms that are readily cleaved with endo H under denaturing conditions to single N-acetylglucosamine (GlcNAc) residues. To determine the effect of NB-DNJ on the endo H sensitivity of undenatured glycoprotein, hsCD2 expressing cells were cultured in the absence or in the presence of 0.5, 1, 1.5, or 2 mM NB-DNJ. Quantitative assays of the expression levels indicated that there was a 3-4-fold reduction in expression in the presence of 0.5-2 mM NB-DNJ (data not shown). After expression in the presence of increasing concentrations of NB-DNJ the hsCD2 migrated more slowly and as a narrower band on SDS-PAGE (Fig. 1), consistent with an increase in the size and uniformity of its N-linked glycosylation(30) .


Figure 1: Effect of NB-DNJ on endo H sensitivity of hsCD2 oligosaccharides. Soluble CD2 was expressed in the presence of 0, 0.5, 1.0, 1.5, or 2 mM NB-DNJ, purified to homogeneity and then digested overnight with endo H at 0.012, 0.06 or 0.3 I.U.B. units/mg of hsCD2. The digestion products (3 µg) were then electrophoresed with undigested hsCD2 on a 15% SDS-PAGE gel alongside equivalent amounts of the starting material for each digestion. The gel was then stained with Coomassie Blue.



Digestion of the 2 mM NB-DNJ-treated hsCD2 with limiting amounts of endo H produced three smaller products each differing by 2-3 kDa (Fig. 1, lane 18). These are likely to correspond to hsCD2 forms bearing zero, one, or two oligosaccharides indicating that all three glycosylation sites can be utilized. There was a concomitant increase in sensitivity to endo H with increasing NB-DNJ concentrations. However, at the two highest NB-DNJ concentrations there was little difference in the endo H sensitivity. Thus, 1.5 mM NB-DNJ appears to completely inhibit glucosidase I. Densitometric analysis of the gel revealed that at the highest NB-DNJ concentration 15% of the hsCD2 is endo H-resistant indicating that a relatively inefficient glucosidase I bypass mechanism exists in these cells (data not shown). Some of the N-linked glycosylation of hsCD2 expressed in the absence of NB-DNJ was also sensitive to endo H. The 2-3-kDa change in SDS-PAGE mobility is consistent with the presence of a single unprocessed site on hsCD2 expressed in CHO cells as observed previously(26) . This contrasts with rat sCD2 expressed in CHO cells which is completely endo H-resistant(31) .

Milligram quantities of hsCD2 were produced in the presence of NB-DNJ, purified and treated with endo H. The digested material was then purified to homogeneity by lectin affinity chromatography and gel filtration chromatography for functional studies and crystallization trials (Fig. 2, lane 2). The electrophoretic mobility of the endo H-treated hsCD2 suggested that the oligosaccharides had been removed from each glycosylation site. Using methods which quantitatively release amino sugars (incubation with 6 N HCl at 100 °C for 4 h), 3.36 ± 0.31 and 14.79 ± 0.32 mol of GlcNAc/mol of protein were detected in endo H-treated and untreated hsCD2, respectively. Mass spectrometric analysis of the endo H-treated hsCD2 gave a mass expected for the polypeptide backbone with three GlcNAc residues (21, 575 Daltons; data not shown). This data indicates that all three glycosylation sites are fully utilized in CHO cells, and that endo H truncates the oligosaccharides at each site to single GlcNAc residues.


Figure 2: Gel electrophoretic analysis of the purified endo H and PNGase F treated hsCD2. Three micrograms of untreated hsCD2 (lane 1), 3 µg of hsCD2 expressed in the presence of 1.5 mM NB-DNJ that had been endo H-treated and lectin purified (lane 2) and 3 µg of PNGase F-treated hsCD2 that had been purified by gel filtration chromatography (lane 3) were electrophoresed on a 15% SDS-PAGE gel. The gel was then stained with Coomassie Blue.



Ligand Binding Properties of Endo H-treated hsCD2

Previous studies of the interaction of rat CD2 with its ligand CD48 (24, 32) and of human CD2 with its ligand CD58 (25) have shown that the BIAcore instrument (33) offers significant advantages over conventional assays for analyzing the affinity and kinetics of weak protein interactions. The instrument uses the optical phenomenon of surface plasmon resonance to detect binding of macromolecules to ligands immobilized on a dextran matrix within a small flow cell. An important advantage of the BIAcore is that interactions are followed in real time. This provides kinetic data which enable the binding of monomers to be readily distinguished from that of aggregates, thereby reducing the likelihood that the measured affinities are artefactually high(24, 32) .

The binding affinities of sCD58, prepared as described elsewhere(25) , for untreated hsCD2 and for endo H-treated hsCD2 prepared after expression in the presence of NB-DNJ, were measured by equilibrium binding analysis on the BIAcore, as described previously (24) . Endo H-treated hsCD2 was compared with untreated hsCD2 by injecting sCD58 at increasing concentrations through flow cells in which either untreated or endo H-treated hsCD2 had been immobilized (Fig. 3A). sCD58 clearly binds endo H-treated hsCD2 and untreated hsCD2 with similar affinities (Fig. 3A). Scatchard plots of the data (Fig. 3A, inset) indicated that the affinities of sCD58 for untreated and endo H-treated hsCD2 were K(d) 7 µM and 9 µM, respectively.

While the affinity of endo H-treated hsCD2 for sCD58 was essentially unchanged, this does not rule out the possibility that the kinetics of binding are altered by glycosidase treatment. Very low affinity interactions, such as those between rat CD2 (24) and human CD2 (25) and their respective ligands, reach equilibrium very rapidly, making kinetic analysis of the association phase impossible on the BIAcore. However, the dissociation phase of such interactions can usually be analyzed, and so the dissociation rates of untreated and endo H-treated hsCD2 were compared (Fig. 4A). It has previously been shown that the apparent dissociation rate constant (k) observed on the BIAcore is slower than the actual k because 1) it takes time for the dissociated protein to wash out of the flow cell and 2) rebinding can occur during this washing phase(24, 25) . As a result, the measured k values shown in this study represent a lower limit for the actual k and are used here for comparative purposes. Under identical experimental conditions sCD58 dissociated from untreated and endo H-treated hsCD2 at about the same rate (with observed values of k 0.8 s and 0.7 s, respectively). In both cases the shape of the dissociation curve was mono-exponential, consistent with the bound sCD58 having a single k (Fig. 4A). A similar result was obtained with endo H-treated hsCD2 binding to immobilized sCD58 (not shown).

Antibody Binding Properties of Endo H-treated hsCD2

Concomitant loss of antibody binding capacity accompanied the loss of the CD58 binding function when a proteolytic fragment of human CD2 was deglycosylated(26) . To compare their antibody binding properties, untreated and endo H-treated hsCD2 were covalently immobilized in BIAcore flow-cells and then thirteen purified anti-CD2 mAbs were sequentially injected for six minutes into the flow cells and eluted with 10 mM HCl. These mAbs include seven that block ligand binding and bind to ``region 1'' on domain 1 of CD2 (9.6, 7E10, MT110, MT910, 95-5-49, T11/3PT2H9 and 9-2), five that also block ligand binding and bind to ``region 2'' on domain 1 (T11/3T4-8B5, NU-TER, CLB-T11/1, TS1/8.1.1, and F92-3A11) and one that does not block ligand binding and which binds to ``region 3'' on domain 2 (OCH.217)(20) . Changes in responses of 3,500-6,500 RU upon injection of the mAbs indicated that each of the mAbs bound strongly to the immobilized proteins and that binding to the untreated and endo H-treated molecules was essentially indistinguishable (Fig. 5A). It is noteworthy that both untreated and endo H-treated hsCD2 were unaffected by exposure to 10 mM HCl.


Figure 5: Binding of anti-CD2 mAbs to untreated, endo H-treated, and PNGase F-treated hsCD2. Panel A, ascites (1 in 20 dilution) containing the indicated human CD2 mAbs was injected for 6 min through flow cells with immobilized untreated (2678 RU) or endo H-treated (3502 RU) hsCD2. Bound mAbs were eluted with 10 mM HCl (arrow) prior to injection of the subsequent mAb. The flow rate was 5 µlbulletmin. Panel B, ascites (1 in 50 dilution) containing the indicated human CD2 mAb was injected for 10 min through flow cells in which untreated hsCD2 (4689 RU), endo H-treated hsCD2 (5955 RU), and PNGase F-treated hsCD2 (5568 RU) were immobilized. The flow rate was 3 µlbulletmin.



Crystallization and Structural Analysis of Endo H-treated hsCD2

During structural studies, it was found that the endo H-treated hsCD2 readily formed crystals which diffract to 2.5-Å resolution (Fig. 6). The crystals were of sufficient quality to yield a high resolution structure for the extracellular region of human CD2 which showed a single well defined main-chain conformation throughout(8) . The overall structure is very similar to that of endo H-treated rat sCD2 (9) with both homologues consisting of two domains with standard IgSF topology. The ligand binding GFCC`C" face of hsCD2 domain 1 is generally well defined, and the flexibility of the loops is not substantially greater than that seen in the rat sCD2 structure; the human and rat domain 1 structures differ by only 0.93-Å root mean square distance for 90 equivalent residues(8) . The similarity of the two structures confirms that the endo H-treated hsCD2 is not structurally disordered. The result also indicates that the use of NB-DNJ represents a useful alternative to utilizing glycosylation mutants (31) for producing natively folded glycoproteins with endo H-sensitive oligosaccharides.

The ligand and antibody-binding data alone do not rule out the possibility that the single GlcNAc left by endo H digestion directly stabilizes the ligand binding face of hsCD2. However, the location of the GlcNAc residue on domain 1 of the hsCD2 crystal structure indicates that the monosaccharide is unlikely to stabilize the protein structure in general and the spatially distant GFCC`C" face in particular (Fig. 7).


Figure 7: Location of the domain 1 GlcNAc residue in the crystal structure of endo H-treated hsCD2. Domain 1 is shown in space-filling format with the line of view parallel to the GFCC`C`` and DEBA faces. The side chains of residues previously shown to form part of the CD58 binding site (left) and the GlcNAc residue left by endo H treatment (right) are shaded grey (the mutagenesis of human CD2 is discussed in detail by Bodian et al.(8) ).



Properties of PNGase F-treated hsCD2

To confirm that the GlcNAc residue does not stabilize the GFCC`C" face, the ligand and antibody binding properties of PNGase F-treated hsCD2 were determined. PNGase F completely removes the oligosaccharides from glycosylation sites in contrast to endo H which leaves a single GlcNAc residue. Using relatively high concentrations of enzyme and after purification by gel filtration the molecular mass of the protein was reduced to that of the polypeptide backbone (20,966 daltons; Fig. 2, lane 3). Amino sugars were undetectable in hydrolysates of the purified protein, and mass spectrometric analyses of the PNGase F-treated protein were consistent with the expected mass of the polypeptide backbone (data not shown). The gel filtration step of the purification procedure indicated that the fully deglycosylated hsCD2 had a significant tendency to aggregate. Therefore, binding experiments were always conducted immediately following gel filtration. Nevertheless kinetic analysis of the dissociation of PNGase F-treated hsCD2 from sCD58 indicated that the PNGase F-treated hsCD2 contained multimeric aggregates even when used immediately (see below).

PNGase F-treated hsCD2 could not be coupled at high levels to the dextran matrix of the BIAcore flow cell (not shown) and so its affinity for sCD58 was determined in the opposite orientation, with sCD58 immobilized. When untreated or PNGase F-treated hsCD2 were injected over sCD58 they both bound with a similar affinity (Fig. 3B). The affinities determined from a Scatchard plot were K(d) 3 and 3.2 µM for untreated and PNGase F-treated hsCD2, respectively (Fig. 3B, inset). It should be noted that the affinities measured in this study (3-9 µM) were obtained at 25 °C for comparative purposes and are slightly higher than the affinity at 37 °C (22 µM) obtained previously for the interaction of sCD2 and sCD58(25) .

In contrast to untreated and endo H-treated hsCD2, PNGase F-treated hsCD2 dissociated from immobilized sCD58 in two phases (Fig. 4B), with fast initial dissociation (k 0.8 s) and then some slow dissociation (k 0.16 s), suggesting that a proportion of the PNGase F-treated hsCD2 has a high avidity. This high avidity binding is specific since it is not seen when PNGase F-treated hsCD2 is injected over 1) a control flow-cell with no immobilized protein (Fig. 4B) or over 2) immobilized CD58 which had been pre-saturated with the inhibitory mAb TS2/9 (data not shown). The observation that PNGase F-treated hsCD2 has a tendency to aggregate in solution during purification (see above) suggests that this high avidity binding represents the binding of multimeric aggregates of the PNGase F-treated hsCD2. Although the immobilization of PNGase F-treated hsCD2 was difficult and relatively inefficient, the binding of the regions 1-, 2-, and 3-reactive mAbs, T11/3PT2H9, TS1/8.1.1, and OCH.217 was tested and each was shown to bind to the immobilized PNGase F-treated protein (Fig. 5D).


DISCUSSION

The results of this study indicate that the structural integrity of human CD2 is glycosylation independent. First, the affinity of two-domain hsCD2 for its ligand, CD58, and the kinetics of this interaction are not significantly affected by truncation of the oligosaccharides to single GlcNAc residues with endo H. Second, the binding of ligand-blocking and other mAbs to endo H-treated hsCD2 is essentially indistinguishable from the binding of the same mAbs to untreated hsCD2. The antibody epitopes of protein antigens with significant secondary structure are usually formed by discontinuous polypeptide segments (34) and thus antibody binding can generally be considered to be good evidence for the correct folding and conformation of protein antigen derivatives if the antibodies also recognize the correctly-folded native antigen. Third, since local or global losses in structural integrity would be expected to prevent the crystallization of hsCD2 as this depends on the formation of stable, reproducible lattice contacts, the crystallization of the endo H-treated hsCD2 implies that the protein is not destabilized by oligosaccharide truncation. It is not inconceivable, however, that the endo H-treated hsCD2 adopts a new configuration that is sufficiently stable to crystallize. This possibility seems unlikely given that the crystallographic analysis has shown that the endo H-treated hsCD2 consists of two domains with conventional IgSF folds(8) . The structural analysis also indicates that the apparent stability of the ligand binding face is unlikely to be due to any stabilizing effect of the single GlcNAc residue located on the DE loop of domain 1. Finally, hsCD2 fully deglycosylated with PNGase F bound CD58 with an affinity similar to that of untreated hsCD2 and bound a series of anti-CD2 mAbs. The slightly reduced dissociation rate for the binding of PNGase F-treated hsCD2 to immobilized CD58 is more likely to be due to aggregation of a fraction of the deglycosylated hsCD2 molecules during the experiment than to disruption of the structure per se. The stability of human CD2 in the absence of glycosylation is consistent with the view that the evolutionary success of the IgSF reflects the ability of IgSF domains to form stable structural units for the presentation of protein recognition motifs.

The results of this study contrast with those of Recny et al.(26) who have proposed that the ligand binding function and stability of CD2 are glycosylation-dependent. The conclusions of that study were based on two observations. First, deglycosylated CD2 domain 1, produced by proteolysis of a two domain form of hsCD2 expressed in Chinese hamster ovary cells, fails to bind CD58 and anti-CD2 mAbs. Second, the removal of the domain 1 glycosylation site at Asn-65 by mutagenesis also prohibits ligand and mAb binding to cell-surface expressed CD2. As an explanation for these observations, Withka et al.(35) and Wyss et al.(36) have suggested that the Asn-65 oligosaccharide interacts with and stabilizes residues surrounding the GFCC`C" face involved in ligand binding by filling a cavity between the BC, C`C", and FG loops at the top of domain 1.

The present experiments do not provide an explicit explanation for the contrary data of Recny et al.(26) . The first observation, that in the absence of domain 2 deglycosylated CD2 domain 1 loses its ligand and antibody binding properties, is not consistent with studies of rat CD2 which have shown that domain 1 of the rat homologue retains its ligand and mAb binding properties when expressed in bacteria in an unglycosylated state and in the absence of domain 2(15, 37) . Inspection of the crystal structures of human and rat CD2 fails to reveal any obvious differences between rat and human CD2 domain 1 that could account for the differing stabilities of the isolated unglycosylated domains. The B factors for residues of domain 1 in the human and rat sCD2 crystal structures, which give some insight into the overall stability of the domain, are not significantly different for the two homologues(8) . The PNGase F-treated hsCD2 used in the present studies did tend to aggregate as discussed above, and it is conceivable that this is exacerbated by proteolysis. However, the interface residues located between domains 1 and 2 of human and rat CD2 are very highly conserved(8) , suggesting that exposure of this interface in human CD2 is unlikely to reduce its stability any more than exposure of the same region of rat CD2 domain 1. Finally, the side chain of Asn-65 and the GlcNAc moiety attached to this residue project toward the interface region of the molecule (data not shown); assuming that this reflects the orientation of the intact oligosaccharide, there are no conspicuous hydrophobic residues that are likely to be shielded by the intact oligosaccharide and exposed by deglycosylation. Irrespective of the underlying cause of these differences, the two domain form of sCD2 used in the present study more closely resembles the natural state of CD2 than does the proteolytic fragment studied by Recny et al. It therefore seems reasonable to conclude that the in vitro behavior of two domain sCD2 more faithfully reflects the structural properties of the molecule in vivo.

It is less difficult to explain the second observation that the ligand and antibody binding properties of human CD2 expressed at the cell surface are disrupted by the mutation of Asn-65 Gln(26) . It is now a widely held view that the folding and stability of glycoproteins is, in many cases, glycosylation dependent(38) . Much of the evidence supporting this view has been obtained from experiments in which glycosylation has been blocked with inhibitors such as tunicamycin or by mutation of glycosylation sites. In many instances it is clear that under these conditions the unglycosylated proteins do not leave the rough endoplasmic reticulum and are degraded (see, for example, (39, 40, 41, 42) ). It can also readily be envisaged that bulky, hydrophilic oligosaccharides might influence protein folding pathways by limiting the number of alternative kinetically accessible conformations or by reducing their thermodynamic stability. Work in this laboratory on T cell receptor/CD4 chimeras has also shown that incorrectly folded proteins can in some instances emerge from the endoplasmic reticulum as indicated by the tendency of the chimeras to form disulfide-bonded aggregates outside the cell(43) . Also, major histocompatibility class I and II antigens reach the surface of transfected cells in the absence of beta(2)-microglobulin and invariant chain, respectively, and these antigens both fail to bind conformationally sensitive antibodies(44, 45) . Consistent with the observations of Recny et al.(26) >90% of human CD2 domain 1 expressed in an unglycosylated state in Escherichia coli forms large aggregates and the remaining material appears to be incorrectly folded according to antibody binding assays(37) . Thus, the inability of the cell-surface expressed Asn-65 mutant of CD2 to mediate antibody and ligand binding (26) may be due to incorrect folding in the endoplasmic reticulum and its subsequent transport to the surface in an inappropriately folded, or unfolded, state.

These studies of deglycosylated CD2 emphasize the difficulty of distinguishing, on the basis of mutational and inhibitor studies, between any effects of glycosylation on protein folding and any role in maintaining protein conformation once folding is complete. The post-folding, structural role of glycosylation has not thus far been explored in great detail presumably because it is relatively difficult to deglycosylate glycoproteins under non-denaturing conditions in vitro. Structural comparisons of naturally occurring glycosylated and unglycosylated forms of bovine pancreatic RNase by crystallographic (46) and solution ^1H NMR analyses (47, 48) indicate that glycosylation has little effect on the overall conformation of the enzyme although studies of hydrogen-deuterium solvent exchange rates have indicated that the oligosaccharide enhances the global dynamic stability of the protein(47, 49) . Conversely, structural studies of glycoproteins and glycopeptides suggest that N-linked oligosaccharides are largely unaffected by the presence of the protein (48, 50, 51) . Molecular dynamic simulations of the interaction of the oligosaccharide of RNase B with the polypeptide backbone suggest that while the di-N-acetylchitobiose core is relatively rigid, flexibility in the linkages to outer arm residues of the oligosaccharide and in the asparagine-GlcNAc linkage allow the oligosaccharide potentially to contact relatively large areas of the protein surface(52) . In toto, these studies suggest that, while oligosaccharides may significantly influence the functions of glycoproteins, this is probably likely to be due in most cases to steric effects, and that oligosaccharides forming intimate contacts with the protein backbone which influence the polypeptide conformation are likely to be rare. CD2 may be unusual in that nuclear Overhauser effects involving a terminal mannose residue(s) of the Asn-65 oligosaccharide and Gly-90 of domain 1 have been tentatively identified (35, 36) . While the current study does not rule out the possibility that the domain 1 oligosaccharide interacts directly with CD2 in this way in vivo, the weight of evidence is against the view that such interactions affect the conformation of the domain or are important for ligand binding.


FOOTNOTES

*
This work was supported by the Human Frontier Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 0865-275597; Fax: 0865-275591.

P. A. van der Merwe is an Oxford Nuffield Medical Fellow.

(^1)
The abbreviations used are: IgSF, immunoglobulin superfamily; endo H, endoglycosidase H; PNGase F, peptide:N-glycosidase F; NB-DNJ, N-butyldeoxynojirimycin; GlcNAc, N-acetylglucosamine; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; RU, response unit(s); hsCD2, human soluble CD2; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We are indebted to Gunilla B. Karlsson and Frances M. Platt for helpful discussion, to Antony Willis for assistance with the amino acid analysis, to David Harvey and Robin Aplin for help with the mass spectrometric analyses, and to Paul C. Driscoll, Marion H. Brown, and Don W. Mason for critical appraisal of the manuscript.


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