Research In Situ trans Ligands of CD22 Identified by Glycan-Protein Photocross-linking-enabled Proteomics*□S T. N. C. Ramya‡, Eranthie Weerapana‡§, Lujian Liao‡, Ying Zeng‡, Hiroaki Tateno‡, Liang Liao‡, John R. Yates III‡, Benjamin F. Cravatt‡§, and James C. Paulson‡¶ CD22, a regulator of B-cell signaling, is a siglec that rec- atic due to the fact that the vast majority of the glycoproteins ognizes the sequence NeuAc␣2–6Gal on glycoprotein gly- of a cell will carry highly related glycan structures because cans as ligands. CD22 interactions with glycoproteins on they share the same secretory pathway that elaborates their the same cell (in cis) and apposing cells (in trans) modu- glycans post-translationally en route to the cell surface. Thus, late its activity in B-cell receptor signaling. Although CD22 although many glycoproteins will carry the glycan structure predominantly recognizes neighboring CD22 molecules recognized by a GBP, the challenge is to determine whether as cis ligands on B-cells, little is known about the trans ligands on apposing cells. We conducted a proteomics one, several, or all of these cell surface glycoproteins (and scale study to identify candidate trans ligands of CD22 on glycolipids) are recognized in situ as physiologically relevant B-cells by UV photocross-linking CD22-Fc chimera bound counter-receptors (1–4). Standard in vitro methods, such as to B-cell glycoproteins engineered to carry sialic acids co-precipitation from cell lysates or Western blotting using with a 9-aryl azide moiety. Using mass spectrometry- binding protein probes, are useful for identifying glycoproteins based quantitative proteomics to analyze the cross-linked that contain the glycan structure recognized by the GBP. products, 27 glycoproteins were identified as candidate However, these may not be relevant ligands in situ due to trans ligands. Next, CD22 expressed on the surface of one constraints imposed by their microdomain localization and cell was photocross-linked to glycoproteins on apposing the geometric arrangement of their glycans relative to the B-cells followed by immunochemical analysis of the prod- ucts with antibodies to the candidate ligands. Of the many GBP presented on the apposing cell. candidate ligands, only the B-cell receptor IgM was found In this report, we examine the in situ ligands of CD22 to be a major in situ trans ligand of CD22 that is selectively (Siglec-2), a member of the siglec family and a regulator of redistributed to the site of cell contact upon interaction B-cell receptor (BCR) signaling that recognizes glycans con- with CD22 on the apposing cell. Molecular & Cellular taining the sequence NeuAc␣2–6Gal as ligands (2, 5, 6). Proteomics 9:1339–1351, 2010. Regulation of BCR signaling by CD22 is effected by its prox- imity to the BCR through recruitment of a tyrosine phospha- tase, SHP-1, which is in turn influenced by CD22 binding to its 1 Glycan-binding proteins (GBPs) mediate diverse aspects glycan ligands (6). Glycoproteins bearing CD22 ligands are of cell communication through their interactions with their abundantly expressed on B-cells and bind to CD22 in cis (on counter-receptors comprising glycan ligands carried on cell the same cell) (7), regulating BCR signaling (2, 5, 6). Although surface glycoproteins and glycolipids. Identification of the in binding to cis ligands has been shown to “mask” CD22 from situ counter-receptors of glycan-binding proteins is problem- binding low avidity synthetic sialoside probes (2, 7), CD22 can also interact with ligands on apposing immune cells in trans From the ‡Department of Chemical Physiology and §The Skaggs (8–10). Interactions of CD22 with trans ligands influence T-cell Institute for Chemical Biology, The Scripps Research Institute, La signaling in vitro (11, 12), mediate B-cell homing via binding to Jolla, California 92037 Received, October 1, 2009, and in revised form, February 18, 2010 sinusoidal endothelial cells in the bone marrow (13), and aid in Published, MCP Papers in Press, February 19, 2010, DOI 10.1074/ “self”-recognition (14). Thus, interactions with both cis and trans mcp.M900461-MCP200 ligands modulate CD22 function in immune homeostasis. 1 The abbreviations used are: GBP, glycan-binding protein; SILAC, Several groups have demonstrated that recombinant stable isotope labeling by amino acids in cell culture; BCR, B-cell receptor; SNA, S. nigra agglutinin; MuDPIT, multidimensional protein CD22-Fc chimera is capable of binding and precipitating the identification technology; MCAM, melanoma cell adhesion molecule; majority of glycoproteins from B- and T-cell lysates whose HLA, human leukocyte antigen; SHP-1, Src homology 2 domain- glycans contain the sequence NeuAc␣2–6Gal (15–18). containing protein-tyrosine phosphatase 1; CHO, Chinese hamster Among them, CD45, IgM, and CD22 itself were identified as ovary; rProtein, recombinant protein; HRP, horseradish peroxidase; specific B-cell binding partners and were postulated to have 9AAz, 9-aryl azide; SFM, serum-free medium; PE, phycoerythrin; LTQ, linear trap quadrupole; IPI, International Protein Index; ID, iden- functional significance as in situ cis ligands of CD22 in regu- tifier; KO, knock-out. lation of BCR signaling (11, 16, 18–20). Several reports have © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics 9.6 1339 This paper is available on line at http://www.mcponline.org trans Ligands of CD22 also documented in situ interactions of CD22 with IgM and EXPERIMENTAL PROCEDURES CD45, but these interactions were found to be of low stoichi- Cells—CHO-FlpIn cells and stably transfected CHO-FlpIn cells ex- ometry and sialic acid-independent (19–21), leaving open the pressing human CD22 with a C-terminal V5 tag (23) were maintained question of which glycoproteins served as in situ cis ligands of in Dulbecco’s modified Eagle’s medium/F-12 medium supplemented ␮ CD22 on B-cells that masked the glycan ligand binding site of with 10% fetal bovine serum and either 100 g/ml Zeocin (Invitrogen) or 500 ␮g/ml hygromycin B (Roche Applied Science), respectively. CD22 (7). Subsequently, using metabolically labeled B-cells CD22-Fc was purified from culture supernatants of COS-1 cells by with sialic acids containing a photoactivatable 9-aryl azide Protein A affinity chromatography 3–7 days after transfection with moiety, we demonstrated that CD22 could be photocross- CD22-Fc-pCDM8 using LipofectamineTM 2000 (24). BJA-B K20 cells linked to its cis ligands, effectively tagging the in situ cis were cultured in RPMI 1640 medium supplemented with 10% fetal ligands with CD22 (15). Notably, there was no cross-linking bovine serum. Human lymphocytes were enriched from human blood with Ficoll-Hypaque density gradient centrifugation. Human and mu- observed to IgM or CD45, demonstrating that they are not rine B-cells were enriched from blood and spleen, respectively, using significant in situ cis ligands of CD22 (15). Instead, only glycans B-cell negative isolation kits (Dynal, Invitrogen). of neighboring CD22 molecules interacted significantly with Antibodies—Antibodies and dilutions for Western blots were as CD22, resulting in photocross-linking of homomultimeric com- follows: anti-CD22 (Santa Cruz Biotechnology Inc., sc-7932, 1:500), plexes of CD22. Thus, despite the fact that most B-cell glyco- anti-CD45 (Santa Cruz Biotechnology Inc., sc-25590, 1:500), anti-4F2 (Santa Cruz Biotechnology Inc., sc-9160, 1:200), anti-major histocom- proteins are recognized in vitro, CD22 selectively recognizes patibility complex I (Santa Cruz Biotechnology Inc., sc-25619, 1:200), glycans of neighboring CD22 molecules as cis ligands in situ. anti-Plexin-B2 (Santa Cruz Biotechnology Inc., sc-34504, 1:200), anti- With the perspective gained from analysis of cis ligands, we ST6GalI (Santa Cruz Biotechnology Inc., sc-20926, 1:200), anti-HLA II wished to determine whether CD22 was also selective in DR␣ (Dako, M0746, 1:200), anti-Basigin (R&D Systems, MAB972, recognition of trans ligands upon cell contact. We have pre- 1:200), anti-Siglec-10 (R&D Systems, AF2130, 1:200), anti-transferrin receptor (Invitrogen, A-11130, 1:200), anti-IgM-HRP (Bethyl Labora- viously demonstrated that CD22 is redistributed to sites of cell tories, A80–100P, 1:2000). HRP-conjugated secondary antibodies contact of interacting B-cells and T-cells and that redistribu- were obtained from Jackson ImmunoResearch Laboratories. For pri- tion is mediated by the interaction of CD22 with sialic acid- mary antibodies raised in rabbit, Reliablot (Bethyl Laboratories) milk containing trans ligands on the apposing cell (8). Stamenkovic and HRP-conjugated secondary antibody were used. et al. (22) had previously demonstrated that binding of T-cells Metabolic Incorporation and Enzymatic Engineering—BJA-B K20 cells were weaned to serum-free medium (Hyq-SFM, Clontech, to CD22-expressing COS cells was blocked by an anti- Thermo Fisher Scientific, San Jose, CA) over 2 days and acclimatized CD45RO antibody, suggesting that CD45 was a functional to serum-free medium for 4 days to reduce the sialic acid content of trans ligand of CD22 on T-cells. However, we found that K20 cells. For metabolic incorporation, medium was supplemented redistribution of CD22 to sites of cell contact was also ob- with 3 mM NeuAc (Taiyo Kagaku Co. Ltd.) or 9-aryl azide-substituted 9AAz served with CD45-deficient B-cells (8), indicating that, at a sialic acid ( NeuAc) (synthesized as described earlier (15)) for 18 h. Enzymatic engineering of cell surfaces was performed as described minimum, other glycoproteins must also serve as trans li- previously (25, 26) on K20 cells cultured in Hyq-SFM or on cells gands of CD22 on B-cells. desialylated by treatment with 200 milliunits/ml Arthrobacter ureafa- To assess whether CD22 recognizes all or a subset of ciens sialidase at 37 °C for 1 h. Briefly, 1 ϫ 108 asialo cells were glycoproteins as trans ligands on an apposing cell, we initi- incubated in 2 ml of PBS, 0.5% BSA containing 0.65 mM CMP- 9AAz ated an unbiased analysis of the trans ligands of CD22 on NeuAc and 5 milliunits/ml ST6GalI at 37 °C for 1 h.