In Situ trans Ligands of CD22 Identified by Glycan-Protein Photocross-linking-enabled Proteomics

CD22, a regulator of B-cell signaling, is a siglec that recognizes the sequence NeuAcα2–6Gal on glycoprotein glycans as ligands. CD22 interactions with glycoproteins on the same cell (in cis) and apposing cells (in trans) modulate its activity in B-cell receptor signaling. Although CD22 predominantly recognizes neighboring CD22 molecules as cis ligands on B-cells, little is known about the trans ligands on apposing cells. We conducted a proteomics scale study to identify candidate trans ligands of CD22 on B-cells by UV photocross-linking CD22-Fc chimera bound to B-cell glycoproteins engineered to carry sialic acids with a 9-aryl azide moiety. Using mass spectrometry-based quantitative proteomics to analyze the cross-linked products, 27 glycoproteins were identified as candidate trans ligands. Next, CD22 expressed on the surface of one cell was photocross-linked to glycoproteins on apposing B-cells followed by immunochemical analysis of the products with antibodies to the candidate ligands. Of the many candidate ligands, only the B-cell receptor IgM was found to be a major in situ trans ligand of CD22 that is selectively redistributed to the site of cell contact upon interaction with CD22 on the apposing cell.Glycan-binding proteins (GBPs)¹ mediate diverse aspects of cell communication through their interactions with their counter-receptors comprising glycan ligands carried on cell surface glycoproteins and glycolipids. Identification of the in situ counter-receptors of glycan-binding proteins is problematic due to the fact that the vast majority of the glycoproteins of a cell will carry highly related glycan structures because they share the same secretory pathway that elaborates their glycans post-translationally en route to the cell surface. Thus, although many glycoproteins will carry the glycan structure recognized by a GBP, the challenge is to determine whether one, several, or all of these cell surface glycoproteins (and glycolipids) are recognized in situ as physiologically relevant counter-receptors (1–4). Standard in vitro methods, such as co-precipitation from cell lysates or Western blotting using binding protein probes, are useful for identifying glycoproteins that contain the glycan structure recognized by the GBP. However, these may not be relevant ligands in situ due to constraints imposed by their microdomain localization and the geometric arrangement of their glycans relative to the GBP presented on the apposing cell.In this report, we examine the in situ ligands of CD22 (Siglec-2), a member of the siglec family and a regulator of B-cell receptor (BCR) signaling that recognizes glycans containing the sequence NeuAcα2–6Gal as ligands (2, 5, 6). Regulation of BCR signaling by CD22 is effected by its proximity to the BCR through recruitment of a tyrosine phosphatase, SHP-1, which is in turn influenced by CD22 binding to its glycan ligands (6). Glycoproteins bearing CD22 ligands are abundantly expressed on B-cells and bind to CD22 in cis (on the same cell) (7), regulating BCR signaling (2, 5, 6). Although binding to cis ligands has been shown to “mask” CD22 from binding low avidity synthetic sialoside probes (2, 7), CD22 can also interact with ligands on apposing immune cells in trans (8–10). Interactions of CD22 with trans ligands influence T-cell signaling in vitro (11, 12), mediate B-cell homing via binding to sinusoidal endothelial cells in the bone marrow (13), and aid in “self”-recognition (14). Thus, interactions with both cis and trans ligands modulate CD22 function in immune homeostasis.Several groups have demonstrated that recombinant CD22-Fc chimera is capable of binding and precipitating the majority of glycoproteins from B- and T-cell lysates whose glycans contain the sequence NeuAcα2–6Gal (15–18). Among them, CD45, IgM, and CD22 itself were identified as specific B-cell binding partners and were postulated to have functional significance as in situ cis ligands of CD22 in regulation of BCR signaling (11, 16, 18–20). Several reports have also documented in situ interactions of CD22 with IgM and CD45, but these interactions were found to be of low stoichiometry and sialic acid-independent (19–21), leaving open the question of which glycoproteins served as in situ cis ligands of CD22 on B-cells that masked the glycan ligand binding site of CD22 (7). Subsequently, using metabolically labeled B-cells with sialic acids containing a photoactivatable 9-aryl azide moiety, we demonstrated that CD22 could be photocross-linked to its cis ligands, effectively tagging the in situ cis ligands with CD22 (15). Notably, there was no cross-linking observed to IgM or CD45, demonstrating that they are not significant in situ cis ligands of CD22 (15). Instead, only glycans of neighboring CD22 molecules interacted significantly with CD22, resulting in photocross-linking of homomultimeric complexes of CD22. Thus, despite the fact that most B-cell glycoproteins are recognized in vitro, CD22 selectively recognizes glycans of neighboring CD22 molecules as cis ligands in situ.With the perspective gained from analysis of cis ligands, we wished to determine whether CD22 was also selective in recognition of trans ligands upon cell contact. We have previously demonstrated that CD22 is redistributed to sites of cell contact of interacting B-cells and T-cells and that redistribution is mediated by the interaction of CD22 with sialic acid-containing trans ligands on the apposing cell (8). Stamenkovic et al. (22) had previously demonstrated that binding of T-cells to CD22-expressing COS cells was blocked by an anti-CD45RO antibody, suggesting that CD45 was a functional trans ligand of CD22 on T-cells. However, we found that redistribution of CD22 to sites of cell contact was also observed with CD45-deficient B-cells (8), indicating that, at a minimum, other glycoproteins must also serve as trans ligands of CD22 on B-cells.To assess whether CD22 recognizes all or a subset of glycoproteins as trans ligands on an apposing cell, we initiated an unbiased analysis of the trans ligands of CD22 on apposing B-cells using our protein-glycan cross-linking strategy (15). By cross-linking CD22-Fc to intact B-cells, we identified 27 candidate trans ligands of CD22 by quantitative mass spectrometry-based proteomics. We then looked at the in situ trans interactions of CD22 in the physiologically relevant cellular context by cross-linking CD22 expressed on one cell to the trans ligands with photoreactive sialic acids on the apposing cell. Our results indicate that only a subset of cell surface glycoproteins, including IgM and, to a lesser extent, CD45 and Basigin, are selectively recognized in trans by CD22. Indeed, IgM in particular is a preferred trans ligand that is selectively redistributed to the sites of cell contact on apposing B-cells in a CD22- and sialic acid-dependent manner despite a vast excess of cell surface glycoproteins that carry a glycan recognized by CD22. The results support the view that factors other than glycan sequence are critical for the in situ engagement of glycan-binding proteins with glycan ligand bearing counter-receptors on the same cell (in cis) or apposing cell (in trans).