Immunoglobulin superfamily (IgSF) ()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 (CD22 (16) and CD22(17) ), one of which (CD22) lacks the sequence encoding IgSF domains 3 and 4. Human CD22 (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 NeuAc2-6Gal1-4Glc(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 2-6-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" -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

Surface Plasmon Resonance Spectroscopy

Analysis and Modification of Sialic Acids on CD45

Expression of Mutant Proteins

RESULTS AND DISCUSSION

Mouse CD22 Binds to NeuGc on CD45 N-Glycans

Figure 1: Mouse CD22Fc binds NeuGc2-6Gal1-4GlcNAc 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 Gal1-4GlcNAc 2-6-sialyltransferase (C), or sialidase-treated CD45 resialylated with NeuGc using Gal1-4GlcNAc 2-6-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, Gal1-4GlcNAc 2-6-sialyltransferase; ST3N, Gal1-3(4)GlcNAc 2-3-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 2-3- and 2-6-linked sialic acids, respectively) were also bound (Fig. 1A), indicating that CD45-thy carries both 2-3- and 2-6-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 2-6-resialylated with Gal1-4GlcNAc 2-6-sialyltransferase using NeuAc as substrate, SNA binding increased substantially, indicating successful 2-6-resialylation, but CD22Fc was still unable to bind (Fig. 1C). In contrast, CD22Fc binding was fully restored when CD45-thy was 2-6-resialylated using NeuGc as substrate (Fig. 1D). The specificity of the 2-6-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 2-6-linked NeuGc carried on CD45-thy N-glycans. Further evidence that the sequence NeuGc2-6Gal1-4GlcNAc 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 2-6-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 2-6-resialylation with NeuGc (Fig. 1E). In contrast, neither 2-6-resialylation with NeuAc nor 2-3-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 NeuGc2-6Gal1-4GlcNAc 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

Sequence Alignments and Mutagenesis Strategy

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 -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 -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 -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) ()and obtained results that agree well with structural studies(58, 64) .

Identification of the Sialic Acid-binding Site on CD22

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 -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 -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" -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" -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" -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) . ()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 -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

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

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 -sheets, which are known to display considerable structural diversity. In contrast, the -sheets show far less structural diversity. Indeed, the central portion of each -sheet (comprising the B, C, E, and F -strands) forms the structurally conserved core of the IgSF fold(60, 61) . The observation that GFC(C`C") -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.

()

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.

()

S. J. Davis, E. A. Davies, and P. A. van der Merwe, unpublished data.

()

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

ACKNOWLEDGEMENTS

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