©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Localization of the Putative Sialic Acid-binding Site on the Immunoglobulin Superfamily Cell-surface Molecule CD22 (*)

(Received for publication, November 13, 1995; and in revised form, January 24, 1996)

P. Anton van der Merwe (1)(§) Paul R. Crocker (2) Mary Vinson (2) A. Neil Barclay (1) Roland Schauer (3) Sørge Kelm (3)

From the  (1)Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom, the (2)Imperial Cancer Research Fund Laboratories, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom, and the (3)Biochemisches Institut der Universität Kiel, Olshausenstraße 40, 24098 Kiel, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

B-lymphocyte antigen CD22 is a member of the recently described sialoadhesin family of immunoglobulin-like cell-surface glycoproteins that bind glycoconjugates terminating in sialic acid. One prominent ligand for CD22 is the highly glycosylated leukocyte surface protein CD45. Using surface plasmon resonance spectroscopy, we characterized the interaction of recombinant mouse CD22 with native CD45 purified from rat thymus (CD45-thy). By in situ desialylation and resialylation of immobilized CD45-thy, we show that mouse CD22 binds to the sialoglycoconjugate NeuGcalpha2-6Galbeta1-4GlcNAc carried on CD45-thy N-glycans. Previous studies have shown that the sialic acid-binding site lies within the two membrane-distal domains of CD22 (domains 1 and 2), which are V-set and C2-set immunoglobulin superfamily domains, respectively. To further localize the binding site, we have made 42 single amino acid substitutions throughout both domains. All 12 mutations that abrogated binding to CD45-thy without disrupting antibody binding were of residues within the GFCC`C" beta-sheet of domain 1. These residues are predicted to form a contiguous binding site centered around an arginine residue in the F strand that is conserved in all members of the sialoadhesin family. Our results provide further evidence that immunoglobulin superfamily cell adhesion molecules use the GFCC`C" beta-sheet of membrane-distal V-set domains to bind structurally diverse ligands, suggesting that this surface is favored for cell-cell recognition.


INTRODUCTION

Immunoglobulin superfamily (IgSF) (^1)domains are probably the commonest domain type involved in cell-surface recognition, being present in 40% of all proteins identified on the surface of leukocytes(1) . One possible reason for this is that IgSF domains provide a stable, but versatile, recognition platform, capable of binding to structurally diverse ligands(2) . Typically, IgSF cell adhesion molecules bind either to other IgSF molecules or to integrins (3, 4) , but recent reports indicate that some IgSF cell adhesion molecules bind carbohydrate ligands (reviewed in (5) ). The best characterized of these lectin-like IgSF proteins are a group of homologous proteins (termed the sialoadhesin family) that bind carbohydrate structures terminating in sialic acid(5, 6, 7) . Members of this family include the leukocyte proteins CD22, sialoadhesin, and CD33 as well as myelin-associated glycoprotein and Schwann cell myelin protein(5, 6, 7) .

CD22 is expressed on a subpopulation of mature B-cells and has been implicated in cell adhesion as well as in modulating signaling through the B-cell antigen receptor (BCR) (reviewed in (8) ). CD22 associates loosely with the BCR (9, 10) and is tyrosine-phosphorylated following BCR ligation(11) . This leads to association with and activation of the tyrosine phosphatase SHP(12) , which can inhibit signaling through the BCR(13, 14, 15) . The binding of anti-CD22 antibody-coated beads to B-cells decreases the activation threshold of the BCR, presumably by removing CD22 (and associated SHP) from the vicinity of the BCR(12) . Together, these findings suggest that physiological interactions between CD22 and natural cell-surface ligands may function to modulate signaling through the BCR(12) .

The extracellular region of mouse CD22 (18) consists of a single membrane-distal V-set IgSF domain (domain 1), followed by six C2-set IgSF domains (domains 2-7). However, two cDNA clones of human CD22 have been identified (CD22alpha (16) and CD22beta(17) ), one of which (CD22alpha) lacks the sequence encoding IgSF domains 3 and 4. Human CD22beta (which is equivalent to mouse CD22 and is henceforth called CD22) binds with a low affinity (K 30 µM at 4 °C(18) ) to the sialylated glycoconjugate NeuAcalpha2-6Galbeta1-4Glc(NAc)(19, 20) . Although this structure is very common on N-glycans, recombinant CD22 appears to bind only to a limited number of lymphocyte cell-surface (19, 21, 22) and plasma (23) glycoproteins, suggesting that some of these molecules are preferred ligands. One prominent ligand is the large, abundant, and highly glycosylated leukocyte cell-surface glycoprotein CD45(22, 24, 25) , which carries multiple N-glycans terminating in alpha2-6-linked sialic acid(26) .

With the exception of antibody-carbohydrate interactions, little is known about carbohydrate recognition by IgSF molecules(5) . As a first step toward understanding the structural basis of sialic acid recognition, we undertook to identify the sialic acid-binding site on CD22. Previous studies on human (27) and mouse (22, 28) CD22 have shown that the sialic acid-binding site lies within domains 1 and 2. In the present study, we extend this work by making single amino acid substitutions of surface residues throughout domains 1 and 2 of mouse CD22. Our results suggest that the CD22 sialic acid-binding site is situated on the GFCC`C" beta-sheet of domain 1 centered on an arginine residue in the F strand that appears to be essential for sialic acid recognition.


MATERIALS AND METHODS

Proteins, Lectins, and Monoclonal Antibodies

Native CD45 (CD45-thy) and thy-1 were purified from rat thymus as described(29, 30) . The purified proteins were precipitated in cold ethanol and dissolved in water(29, 30) . Rat alpha(1)-acid glycoprotein (orosomucoid) was purchased from Sigma. Recombinant soluble rat CD45 (including the A, B, and C exons (sCD45ABC-CHO)) was expressed in Chinese hamster ovary cells and purified as described(31) . The lectins Maackia amurensis agglutinin (MAA) (which binds alpha2-3-linked sialic acid(32) ) and Sambucus nigra agglutinin (SNA) (which binds alpha2-6-linked sialic acid(33) ) were from Boehringer (Mannheim, Germany). The purified mouse anti-human IgG monoclonal antibody (mAb) R10Z8E9 (34) was kindly provided by Professor R. Jefferis and Dr. M. Goodall and is available from Recognition Systems (University of Birmingham Science Park, Birmingham, United Kingdom). The hybridoma CY34.1.2, which produces the mouse (IgG1) anti-mouse CD22 antibody CY34(35) , was obtained from the American Type Culture Collection (Rockville, MD).

Surface Plasmon Resonance Spectroscopy

All BIAcore experiments were performed on a BIAcore biosensor (Pharmacia Biosensor, Uppsala) at 25 °C in the running buffer HBS, which contains 150 mM NaCl, 1 mM CaCl(2), 1 mM MgCl(2), 10 mM HEPES, pH 7.4, and 0.005% Surfactant P-20 (Pharmacia Biosensor). Proteins were covalently coupled via amine groups onto the carboxymethylated dextran surface of CM5 (research-grade) sensor chips (Pharmacia Biosensor) using the standard amine coupling kit (Pharmacia Biosensor) as recommended(36) , with the following modifications. During coupling, CD45-thy and sCD45ABC-CHO were injected for 7 min at 20-40 µg/ml in 10 mM sodium formate, pH 3, and in 10 mM sodium acetate, pH 4, respectively. Both proteins were regenerated by injecting 100 mM HCl for 3 min. The anti-human Fc antibody was injected at 28 µg/ml in 10 mM sodium acetate, pH 4.5, and regenerated by sequential 3-min injections of 0.1 M glycine HCl, pH 2.5, and 5 mM NaOH. All experiments were performed at a flow rate of 1 µl/min, except for the amine coupling reactions, which were performed at a flow rate of 5 µl/min.

Analysis and Modification of Sialic Acids on CD45

Sialic acids were released by hydrolysis with 0.1 N HCl, derivatized with 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride, and then analyzed by fluorescent high pressure liquid chromatography as previously described(37) . Proteins covalently coupled to the BIAcore sensor surface were desialylated by injection of 100 milliunits/ml Vibrio cholerae sialidase (Behringwerke Ag, Marburg, Germany) in 50 mM sodium acetate buffer, pH 5.5, with 2 mM CaCl(2) for 30 min at a flow rate of 1 µl/min at 25 °C. Repeated V. cholerae sialidase injections at higher concentrations (1 unit/ml) failed to abolish SNA binding to CD45, and Arthrobacter ureafaciens sialidase (Calbiochem) was no more effective (data not shown). Desialylated proteins immobilized on the sensor surface were resialylated with Galbeta1-4GlcNAc alpha2-6-sialyltransferase (beta-galactoside alpha-2,6-sialyltransferase, EC 2.4.99.1) or Galbeta1-3(4)GlcNAc alpha2-3-sialyltransferase (sialyltransferase, EC 2.4.99.6). Galbeta1-4GlcNAc alpha2-6-sialyltransferase (200 milliunits/ml in buffer A) or Galbeta1-3(4)GlcNAc alpha2-3-sialyltransferase (75 milliunits/ml in buffer A) was injected for 40 min at a flow rate of 1 µl/min at 25 °C together with 1 mM CMP-NeuAc or CMP-NeuGc. Buffer A comprised 50 mM MES, pH 6.5, 0.1% bovine serum albumin (BSA), and 12.5 units/ml calf intestinal alkaline phosphatase. Galbeta1-4GlcNAc alpha2-6-sialyltransferase was purified from rat liver (38) , whereas recombinant Galbeta1-3(4)GlcNAc alpha2-3-sialyltransferase (39) was provided by Dr. J. C. Paulson (Cytel Inc., La Jolla, CA). CMP-NeuAc was from Boehringer, and CMP-NeuGc was provided by Dr. L. Shaw (Biochemisches Institut der Universität Kiel).

Expression of Mutant Proteins

The DNA fragment encoding domains 1-3 of mouse CD22 (C57Bl allele; see ``Results and Discussion'') had previously been cloned into the EcoRI site of the expression vector pIG(28, 40) , yielding a chimeric protein comprising domains 1-3 of CD22 fused to the Fc portion of human IgG1 (CD22Fc). The entire CD22Fc fragment was excised with HindIII and NotI, blunt-ended, and subcloned by blunt-end ligation into the XbaI site of the phagemid expression vector pEF-BOS(41) . CD22 mutants were generated directly in CD22Fc/pEF-BOS as described (42) using the Muta-Gene phagemid mutagenesis kit (Version 2, Bio-Rad). All mutations were confirmed by DNA sequencing. When CD22 mutations disrupted CD45 or CY34 binding, the DNA encoding domains 1 and 2 was sequenced in order to exclude spurious mutations. Mutant CD22Fc chimeras were expressed by transient transfection of COS-7 cells as described previously(42) . Tissue culture supernatants (TCS) were concentrated 3-4-fold before analysis using Centricon-10 concentrators (Amicon, Inc.).


RESULTS AND DISCUSSION

Mouse CD22 Binds to NeuGc on CD45 N-Glycans

Previous studies have shown that only the two NH(2)-terminal domains of human (27) and mouse (22, 28) CD22 (domains 1 and 2) are required for sialic acid binding. However, we have found that a mouse CD22 construct containing domains 1 and 2, but lacking domain 3, was somewhat unstable (28) . We therefore used a construct containing domains 1-3 of mouse CD22 fused to the Fc portion of human IgG1 (CD22Fc)(6) . Using surface plasmon resonance spectroscopy, as implemented in the BIAcore instrument(43) , we have shown that CD22Fc binds to native CD45 (CD45-thy) purified from rat thymus(28) . To further characterize this interaction, we modified the sialoglycoconjugates present on CD45-thy and examined the effect on CD22Fc binding (Fig. 1).


Figure 1: Mouse CD22Fc binds NeuGcalpha2-6Galbeta1-4GlcNAc carried on CD45 N-glycans. A-D, CD45-thy was covalently coupled to the BIAcore sensor surface. CD22Fc and the sialic acid-binding lectins MAA and SNA were then injected at 0.5 mg/ml for 4 min each (bars) over unmodified thymic CD45 (A), sialidase-treated CD45 (B), sialidase-treated CD45 resialylated with NeuAc using Galbeta1-4GlcNAc alpha2-6-sialyltransferase (C), or sialidase-treated CD45 resialylated with NeuGc using Galbeta1-4GlcNAc alpha2-6-sialyltransferase (D). Following each injection, bound protein was eluted with a 4-min injection of 100 mM HCl (arrows mark the beginning of these injections). E, sCD45ABC-CHO was coupled to the sensor surface. Mouse CD22Fc, MAA, and SNA were injected (0.5 mg/ml for 4 min) first over unmodified sCD45ABC-CHO and then after the indicated desialylation and resialylation steps. The binding response during each injection was measured 20 s after the injection (to eliminate the bulk phase effect) and is expressed as a percentage of the maximal response seen for each ligand during the experiment, which was 1380, 3640, and 7300 response units for CD22Fc, MAA, and SNA, respectively. ST6N, Galbeta1-4GlcNAc alpha2-6-sialyltransferase; ST3N, Galbeta1-3(4)GlcNAc alpha2-3-sialyltransferase.



When CD22Fc was injected over a sensor surface to which CD45-thy had been covalently immobilized, there was an increase in the response (measured in response units), which indicates binding of CD22Fc to CD45-thy (Fig. 1A). Following completion of the injection, the response decreased slowly, reflecting dissociation of bound CD22Fc (Fig. 1A). The remaining CD22Fc was eluted rapidly by the injection of 100 mM HCl (Fig. 1A, arrow). The lectins MAA and SNA (which are specific for alpha2-3- and alpha2-6-linked sialic acids, respectively) were also bound (Fig. 1A), indicating that CD45-thy carries both alpha2-3- and alpha2-6-linked sialic acids. Treatment of the immobilized CD45-thy with sialidase abolished CD22Fc binding and substantially decreased both MAA and SNA binding (Fig. 1B). When desialylated CD45 was alpha2-6-resialylated with Galbeta1-4GlcNAc alpha2-6-sialyltransferase using NeuAc as substrate, SNA binding increased substantially, indicating successful alpha2-6-resialylation, but CD22Fc was still unable to bind (Fig. 1C). In contrast, CD22Fc binding was fully restored when CD45-thy was alpha2-6-resialylated using NeuGc as substrate (Fig. 1D). The specificity of the alpha2-6-resialylation is indicated by the increase in SNA (but not MAA) binding following resialylation (Fig. 1, C and D).

These results indicate that mouse CD22 binds to alpha2-6-linked NeuGc carried on CD45-thy N-glycans. Further evidence that the sequence NeuGcalpha2-6Galbeta1-4GlcNAc was both necessary and sufficient for CD22Fc binding was obtained in an experiment using sCD45ABC-CHO. The lectin MAA bound unmodified sCD45ABC-CHO, whereas SNA did not, indicating that sCD45ABC-CHO contains no detectable alpha2-6-linked sialic acid (Fig. 1E, Untreated). Therefore, it is not surprising that CD22Fc did not bind unmodified sCD45ABC-CHO (Fig. 1E, Untreated). However, CD22Fc did bind sCD45ABC-CHO following alpha2-6-resialylation with NeuGc (Fig. 1E). In contrast, neither alpha2-6-resialylation with NeuAc nor alpha2-3-resialylation with NeuGc could restore CD22Fc binding (Fig. 1E). Taken together, these results establish that mouse CD22Fc binds to CD45-thy through an interaction with the structure NeuGcalpha2-6Galbeta1-4GlcNAc carried on CD45-thy N-glycans. This is consistent with a recent analysis of the specificity of mouse CD22Fc using resialylated erythrocytes(44) .

Analysis of the Sialic Acid Composition of CD45-thy

Since mouse CD22 requires alpha2-6-linked NeuGc for binding, biologically relevant ligands for CD22 should contain this sialic acid rather than NeuAc. Normal human tissues do not contain NeuGc, but this sialic acid is common in rodents(45, 46) . However, the relative amount of NeuGc differs between cell types and is developmentally regulated(47, 48, 49, 50) . Previous studies of mouse lymphocytes found that NeuGc constituted 40-50% of the sialic acid in glycolipids(51, 52) . However, no analysis of mouse or rat lymphocyte glycoproteins has been reported. We therefore analyzed the sialic acid composition of glycoproteins isolated from rat thymus (Table 1). For comparison, we also studied a rat serum protein and rat CD45 that had been expressed in CHO cells (Table 1). This analysis revealed that most (>98.8%) of the sialic acid in the thymic proteins CD45-thy and thy-1 is NeuGc. In contrast, the serum protein alpha(1)-acid glycoprotein, which is synthesized by hepatocytes, contains mainly (>89%) NeuAc (Table 1). NeuAc constituted 98% of the sialic acid in sCD45ABC-CHO (Table 1), which is in agreement with other studies of glycoproteins expressed in CHO cells(53) . Taken together, these results demonstrate that CD45-thy is a suitable ligand for murine CD22 since it contains abundant alpha2-6-linked NeuGc. In support of a physiological role for this interaction, Law et al.(22) recently demonstrated that CD45 is prominent among the glycoproteins that are immunoprecipitated from mouse B-cell lines using mouse CD22Fc.



Sequence Alignments and Mutagenesis Strategy

Two mouse CD22 alleles have been isolated from BALB/c and DBA/2J mice, respectively (54, 55) . While sequencing the CD22 construct used in the present study (which originated from C57Bl mice(6) ), it emerged that it encodes a third allele (Fig. 2, CD22 C57Bl). This allele is identical to the BALB/c allele in the region encoding domains 1-3, with the exception of the codons for residues 79 (Val instead of Cys, numbered from the initiation codon), 247 (Arg instead of Cys), and 250 (Arg instead of His), in which the DNA sequence is identical to the DBA/2J allele (Fig. 2). These changes result in the loss of an unusually positioned pair of cysteine residues that are present in the CD22 BALB/c allele, but not in any of the other sialoadhesin family members(56) .


Figure 2: Alignment of CD22 with IgSF molecules of known structure. The predicted protein sequences of domains 1 and 2 of the mouse CD22 alleles C57Bl (this study), BALB/c (GenBank/EMBL accession number L02844), and DBA/2J (GenBank/EMBL accession number L16928) were manually aligned with human CD22 (GenBank/EMBL accession number X59350), mouse sialoadhesin (GenBank/EMBL accession number Z36293), and either rat CD2 (domain 1 (d1)) (SwissProt accession number P08921) or VCAM-1 (domain 2 (d2)) (SwissProt accession number P19320). The beta-strand assignments (solid bars) were based on the structures of CD2 (57, 64) and VCAM-1 (59) as well as on structural data from other IgSF domains(2, 60, 61) . The division between domains 1 and 2 of mouse CD22 is made at the junction of exons 4 and 5(55) . Dashed lines instead of bars are shown where there are no grounds for making precise assignments to beta-strands. Boxed mouse CD22 residues were mutated in the present study, whereas boxed sialoadhesin residues were mutated in the accompanying study(65) .



To aid in the selection of residues to mutate, domains 1 and 2 of CD22 were aligned with IgSF domain sequences for which there are structural data available (Fig. 2). Domain 1 of CD22 was aligned with the V-set domain (domain 1) of rat CD2 (57, 58) (Fig. 2), whereas domain 2 was aligned with domain 2 of VCAM-1(59) . CD22 residues in domains 1 and 2 could be assigned accurately to the structurally conserved B, C, E, and F beta-strands (Fig. 2) by aligning residues characteristic of IgSF domains(2, 60, 61) . In a similar manner, residues in CD22 domain 1 could be assigned to the beginning of the D strand and to the end of the G strand, and residues in domain 2 could be assigned to the A strand and to the end of the G strand (Fig. 2). In contrast, CD22 residues could not reliably be assigned to the C` and C" strands of domain 1 or to the C`/D strand of domain 2 (Fig. 2). The assignment of residues to the loop regions was tentative except for the E-F loop, which is structurally conserved in V-set and C2-set IgSF domains(2, 60, 61) .

The sialic acid-binding site on sialoadhesin has been definitively localized to its V-set domain (domain 1(28) ), but in the case of CD22, a contribution from domain 2 has not been ruled out(27, 28) . To further localize the sialic acid-binding site on CD22, we mutated residues predicted to lie on the surface of domain 1 or 2. We introduced drastic changes rather than mutating to alanine because our primary aim was to delineate the structural binding site. It has been shown that alanine mutagenesis may only identify a fraction (25-40%) of the residues within the binding site(62, 63) . We have previously used this approach of making drastic mutations to identify the interacting surfaces of the cell adhesion molecules CD2 and CD48 (42) (^2)and obtained results that agree well with structural studies(58, 64) .

Identification of the Sialic Acid-binding Site on CD22

Mutant CD22Fc chimeras were expressed by transient transfection of COS-7 cells and then analyzed for ligand and antibody binding by surface plasmon resonance spectroscopy using the approach outlined schematically in Fig. 3A (upper left). TCS containing wild-type or mutant CD22Fc was injected over a sensor surface to which an anti-Fc mAb had been covalently coupled (Fig. 3, A and B, long bars). The initial rapid increase is due to the high bulk refractive index of the injected TCS (``bulk phase effect''), whereas the slower, more sustained increase reflects the binding of CD22Fc to the anti-Fc mAb on the sensor surface (Fig. 3, A and B, long bars). The contribution from the bulk phase effect ends when the injection of the TCS is completed and the flow of the running buffer resumes. The response then drops rapidly to a new, elevated base line, the level of which is proportional to the mass of bound CD22Fc, with 1000 response units representing 1 ng/mm^2 of bound protein(43) . The control protein BSA and CD45-thy (both at 26 µg/ml) were injected over the sensor surface both before (to control for a bulk phase effect) and after the binding of wild-type or mutant CD22Fc to the sensor surface. A substantially increased response is seen when CD45 is injected over immobilized wild-type CD22Fc, reflecting binding, whereas the response to the injection of BSA is unchanged (Fig. 3, A and B, Wild type). The mutant CD22Fc constructs were analyzed in the same way and compared with wild-type CD22Fc (Fig. 3).


Figure 3: Analysis of CD45 binding to CD22 mutants. An outline of the approach used in these experiments is shown in A (upper left). TCS containing the indicated CD22Fc mutant was injected (long bars) for 20-40 min over a sensor surface to which an anti-Fc mAb had been covalently coupled. BSA and purified rat CD45 (both at 26 µg/ml) were injected for 3 min each (short bars) before and after CD22Fc was bound to the sensor surface. An increase in the response with the second injection of CD45 reflects binding to that particular CD22 mutant. The mutants in A and B were analyzed in different experiments and should be compared with wild-type CD22Fc in A and B, respectively. The results with R130A (solid lines) and R130K (dotted line) are superimposed (B, lower left)



Initially, nine mutations were made in each of domains 1 and 2 (Table 2). Of these, only two mutations, both in domain 1, led to a decrease in CD45 binding (R130E and E140K) (Fig. 3A and Table 2). Both mutants bound normally to mAb CY34 (Fig. 4A and Table 2). The sequence alignment (Fig. 2) places Arg-130 and Glu-140 on adjacent F and G beta-strands in domain 1 (Fig. 5). Interestingly, Arg-130 is one of only five residues in domain 1 (apart from residues characteristic of IgSF domains) that are completely conserved within the sialoadhesin family (indicated by &cjs3622; in Fig. 2)(2, 56, 60, 61) , suggesting that it may play an important role in sialic acid recognition. To provide stronger evidence for this, we made the substitutions R130A and R130K, which are less likely to abrogate binding by introducing unfavorable effects. Both mutations abolished CD45 binding (Fig. 3B) without affecting the binding of mAb CY34 (Table 2), suggesting that Arg-130 is critical for sialic acid recognition.




Figure 4: A, analysis of CY34 binding to CD22 mutants. TCS containing the indicated CD22Fc mutants was injected for 12 or 40 min over a sensor surface to which an anti-Fc mAb had been covalently coupled (see Fig. 3). The anti-CD22 mAb CY34 (in TCS) was then injected over the immobilized CD22 mutants for 4-6 min. An elevated base line following the injection of CY34 TCS indicates binding. B, CY34 and CD45 bind to different regions of CD22. In this experiment, an anti-Fc mAb was immobilized to the sensor surface. Left, BSA and CD45 (65 µg/ml each) were injected for 4 min before and after the binding of wild-type CD22Fc (30 µg/ml, 10 min) to the sensor surface. Middle, purified CY34 (30 µg/ml, 6 min) was injected before and immediately after the binding of CD22Fc to the sensor surface, followed by injection of BSA and CD45. Right, after the binding of CD22Fc, BSA and CD45 are injected, followed by CY34.




Figure 5: Approximate positions of mutations in domain 1 of mouse CD22 that disrupt CD45 and CY34 binding. This ribbon drawing is intended to represent a typical V-set IgSF domain and is drawn with MOLSCRIPT (73) using the coordinates of domain 1 of human CD2(58) . The positioning of the mutated CD22 residues is based on the alignment of CD22 with rat CD2 in Fig. 2. Because the alignment is poor over the entire C`-C" region, the precise positions of Lys-74, Thr-76, Lys-85, and Lys-88 cannot be predicted, and so they are depicted with broken circles. For clarity, the residues mutated in beta-strand B (Arg-43 and Lys-47) and the B-C loop (Lys-49 and Asp-58) are not labeled. Lys-149 is included in this figure, although it lies at the junction of domains 1 and 2.



The binding site was further defined with 22 additional mutations in and around the GFCC`C" beta-sheet of domain 1 ( Fig. 2and Table 2). Of a total of 42 mutations made (Table 2), 30 had little or no effect on CD45 binding (examples include K74E, R120D, K149D, and K185E (Fig. 3)), 10 completely abolished CD45 binding (examples include R130E, R130K, R130A, and W138R (Fig. 3)), and 2 substantially decreased, but did not abolish, CD45 binding (E140K and K73E (Fig. 3)). The partial effect of the latter mutants suggests that they lie on the periphery of the binding site. According to the alignment shown in Fig. 2, the mutations that abrogate CD45 binding fall within the GFCC`C" beta-sheet and are predicted to form a well defined contiguous region centered around Arg-130 in the F strand ( Fig. 2and Fig. 5). The positioning of the F and C strand mutations is likely to be correct because the alignment of CD22 with CD2 in both these regions is excellent (Fig. 2), and these strands form part of the structurally conserved core of IgSF domains(60, 61) . Because of a poor alignment with CD2, the positioning of the G and C` strand mutants is more tentative (Fig. 2). However, it is clear that residues in the F-G loop and/or the beginning of the G strand contribute to the CD45-binding site.

Our finding that none of the nine mutations in domain 2 affect CD45 binding ( Fig. 3and Table 2) suggests that domain 2 does not contribute directly to sialic acid recognition. This is consistent with the observation that domain 1 of sialoadhesin is sufficient for sialic acid binding(28) . Furthermore, mutagenesis of sialoadhesin (65) suggests that its sialic acid-binding site is also localized to the GFCC`C" beta-sheet of domain 1, centered around the same conserved F strand arginine ( Fig. 2and Fig. 6). Taken together, these data suggest that sialic acid recognition by CD22 and sialoadhesin involves only domain 1. Prior observations that domain 1 of CD22 binds poorly (22) or not at all (27, 28) to ligand when expressed in the absence of domain 2 may be explained by an inability of domain 1 to fold correctly in the absence of domain 2. Support for this is provided by two lines of evidence that suggest that domains 1 and 2 of CD22 are intimately associated. First, the conserved cysteines present in domains 1 (A-B loop) and 2 (B-C loop) of sialoadhesin family members appear to form an interdomain disulfide bridge (66) . (^3)Second, residues in both domains 1 and 2 contribute to the CY34 epitope (see below and Table 2).


Figure 6: Ligand-binding sites on the cell-cell recognition molecules CD22, sialoadhesin, VCAM-1, CD80, and CD2. Shown are the positions (filled circles) of mutations reported to disrupt the following interactions: CD22 (this study) and sialoadhesin (65) with sialoglycoconjugates, VCAM-1 with VLA-4(59) , CD2 with CD58(58, 69, 70, 71) , and CD80 (72) with CD28 or CTLA-4. All the mutations lie within membrane-distal V-set IgSF domains of these molecules. The ribbon drawings of human CD2 and VCAM-1 are based on their crystal structures(58, 59) . A ribbon drawing of domain 1 of CD2 is used as a template to display the CD22, sialoadhesin, and CD80 mutants. The positioning of the residues was guided by the beta-strand assignments in Fig. 2and (72) . This figure was drawn using MOLSCRIPT(73) .



A potential source of artifact in the present study is the possibility that some or all of the mutants do not lie within the sialic acid-binding site, but instead disrupt the overall folded structure of CD22. While this possibility cannot be eliminated, several considerations suggest that this is unlikely. First, all mutants that did not bind CD45 still bound mAb CY34. Our mutagenesis studies suggest that CY34 binds to a ``discontinuous'' or ``conformational'' epitope on CD22 (see below), which requires the correct folding of domain 1 and 2. This is supported by our observation that several, widely spaced mutants that were expressed only at very low levels bound neither CD45 not CY34 (see Footnote a to Table 2). Second, the 12 mutations that decrease CD45 binding (without affecting CY34 binding) lie within a single contiguous area, with the mutations that have a partial effect (K73E and E140K) situated on the edge of this area. And finally, mutations in the equivalent region of sialoadhesin also disrupt sialic acid binding without disrupting the binding of mAbs directed to this domain 1(65) .

The CY34 Epitope Includes Portions of Domains 1 and 2

CY34 is an allele-specific mouse anti-mouse CD22 mAb (35) that has been reported to bind the CD22 BALB/c allele, but not the DBA/2J allele (55) . The CD22 C57Bl allele identified in the present study also binds CY34(28) . Using truncation mutants, it has been shown that the CY34-binding site lies within domains 1 and 2 of CD22(22, 28) . Of the 42 mutants in domains 1 and 2, three (R120D, K149D, and K185E) abolished CY34 binding (Fig. 4A and Fig. 5and Table 2). None of these three mutations affected CD45 binding, suggesting that they do not disrupt the overall structure of CD22 (and Fig. 4A and Table 2). The mutated residues are widely distributed in the primary sequence, with Arg-120 in the E-F loop of domain 1, Lys-149 at the junction of domains 1 and 2, and Lys-185 in the C strand of domain 2. Although distant in the primary sequence (Fig. 2), Arg-120, Lys-149, and Lys-185 are likely to lie in close proximity in the folded structure (see Fig. 5for the predicted positions of Lys-149 and Arg-120). Thus, as with the majority of monoclonal antibodies(67, 68) , CY34 binds a discontinuous (and therefore conformationally sensitive) epitope that includes portions of domains 1 and 2 and is some distance from the putative sialic acid-binding site (Fig. 5). In agreement with the latter, CD45 binding to immobilized CD22 is not inhibited by bound CY34, nor is CY34 binding inhibited by bound CD45 (Fig. 4B), demonstrating that their binding sites on CD22 do not overlap.

IgSF Molecules Involved in Cell-Cell Recognition Bind Structurally Diverse Ligands Using the Same beta-Sheet

This analysis of CD22 and the accompanying study on sialoadhesin (65) suggest that both these proteins bind sialoglycoconjugates through the GFCC`C" beta-sheet of their membrane-distal V-set domain (Fig. 6). As discussed in the accompanying paper(65) , it seems likely that other members of the sialoadhesin family bind sialoglycoconjugates through the same site. The ligand-binding sites of several cell-surface IgSF molecules involved in cell-cell recognition have recently been characterized (Fig. 6). These include the T-cell surface molecule CD2(58, 69, 70, 71) , which binds to the closely related IgSF molecules CD48 and CD58; VCAM-1 (59) , which binds to the integrin VLA-4; and the B-lymphocyte molecule CD80(72) , which binds to the T-cell surface molecules CD28 and CTLA-4. In each instance, the binding sites have been localized to different portions of the GFCC`C" (CD2 and CD80) or GFC (VCAM-1) beta-sheet of the membrane-distal domain (Fig. 6). This beta-sheet appears to be favored for interactions mediating cell-cell recognition, presumably because of its membrane-distal location and because, as shown for CD2 (58, 64) and VCAM-1(59) , it is well exposed at the top of these molecules, making it accessible to ligands on the opposing cell surface.

The variable IgSF domains in B- and T-cell antigen receptors are capable of binding to an enormous variety of structures. However, antigen recognition involves loop regions between the beta-sheets, which are known to display considerable structural diversity. In contrast, the beta-sheets show far less structural diversity. Indeed, the central portion of each beta-sheet (comprising the B, C, E, and F beta-strands) forms the structurally conserved core of the IgSF fold(60, 61) . The observation that GFC(C`C") beta-sheets bind to structures as diverse as integrins, IgSF molecules, and sialoglycoconjugates provides impressive evidence of the versatility of IgSF domains(2) .


FOOTNOTES

*
This work was supported by the Medical Research Council, the Imperial Cancer Research Fund, the Mizutani Foundation for Glycosciences, and NATO. 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.: 44-1865-275593; Fax: 44-1865-275591; vdmerwe{at}molbiol.ox.ac.uk.

(^1)
The abbreviations used are: IgSF, immunoglobulin superfamily; BCR, B-cell antigen receptor; CD45-thy, CD45 purified from rat thymus; sCD45ABC-CHO, soluble rat CD45 (including the A, B, and C exons) expressed in CHO cells; MAA, M. amurensis agglutinin; SNA, S. nigra agglutinin; mAb, monoclonal antibody; MES, 2-(N-morpholino)ethanesulfonic acid; BSA, bovine serum albumin; CD22Fc, chimeric protein consisting of domains 1-3 of mouse CD22 fused to the hinge and Fc portion of human IgG1; TCS, tissue culture supernatant(s); CHO, Chinese hamster ovary; VCAM-1, vascular cell adhesion molecule-1.

(^2)
S. J. Davis, E. A. Davies, and P. A. van der Merwe, unpublished data.

(^3)
A. May, E. Y. Jones, A. C. Willis, A. N. Barclay, and P. R. Crocker, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Yvonne Jones for supplying the coordinates for VCAM-1, Liz Davies for assistance with the DNA sequencing, and Drs. L. Shaw and J. C. Paulson for kindly donating CMP-NeuGc and Galbeta1-3(4)GlcNAc alpha2-3-sialyltransferase, respectively.


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