Research

In Situ trans Ligands of CD22 Identified by Glycan- 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 (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 were enriched from human 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- (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. Lectin Staining and Flow Cytometry—Cells (2 ϫ 105 cells in 100 ␮l apposing B-cells using our protein-glycan cross-linking strat- of PBS, 0.5% BSA) were stained with 2 ␮g of FITC-S. nigra agglutinin egy (15). By cross-linking CD22-Fc to intact B-cells, we iden- (SNA) (Vector Laboratories) for 60 min on ice; washed twice with PBS, tified 27 candidate trans ligands of CD22 by quantitative mass 0.5% BSA; and analyzed by flow cytometry. spectrometry-based proteomics. We then looked at the in situ Photoaffinity Cross-linking and Cell Lysis—9AAzNeuAc-K20 B-cells 7 trans interactions of CD22 in the physiologically relevant cel- or human lymphocytes (1 ϫ 10 cells/ml) were incubated with 20 ␮ lular context by cross-linking CD22 expressed on one cell to g/ml CD22-Fc in PBS on ice for 30 min and then exposed to a hand-held 254 nm UV source for 30 min. Human IgG (Fc fragment) the trans ligands with photoreactive sialic acids on the appos- was used as a control. In the case of photocross-linking to CD22 ing cell. Our results indicate that only a subset of cell surface expressed on CHO cells, 9AAzNeuAc-K20 cells resuspended in PBS glycoproteins, including IgM and, to a lesser extent, CD45 and were overlaid onto CHO cell monolayers in 6-well plates at 1 ϫ 107 Basigin, are selectively recognized in trans by CD22. Indeed, cells/ml and cross-linked as described above. Unbound CD22-Fc or IgM in particular is a preferred trans ligand that is selectively IgG (Fc fragment) and K20 cells, respectively, were washed away with PBS and/or 0.2 mM glycine in PBS, pH 2.2. Cells were lysed at 1 ϫ 107 redistributed to the sites of cell contact on apposing B-cells in cells/ml in 50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Nonidet a CD22- and sialic acid-dependent manner despite a vast P-40 with a protease mixture (Calbiochem). excess of cell surface glycoproteins that carry a glycan rec- Immunoprecipitation and Western Analysis—Lysates were incu- ognized by CD22. The results support the view that factors bated overnight with Protein G-agarose (Invitrogen) or anti-V5-aga- other than glycan sequence are critical for the in situ engage- rose (Sigma) at 4 °C with end-over-end rotation. The immunoprecipi- tates were washed four times with lysis buffer and eluted with 4ϫ ment of glycan-binding proteins with glycan ligand bearing lithium dodecyl sulfate buffer (Invitrogen). Immunoprecipitates were counter-receptors on the same cell (in cis) or apposing cell (in resolved on 10 or 4–12% NuPAGE gels (Invitrogen), transferred to trans). nitrocellulose, and blocked for 1 h with 5% milk in Tris-buffered saline

1340 Molecular & Cellular Proteomics 9.6 trans Ligands of CD22

with 0.05% Tween 20 (TTBS). Blots were incubated with antibodies (across bands 1–3) in the ϩUV sample and the ϪUV sample was diluted in 5% milk, TTBS; washed with TTBS; and visualized with calculated. Next, the spectral counts (across bands 1–3) of both the chemiluminescence. ϩUV and ϪUV samples were summed to obtain the total spectral Fluorescence Microscopy—9AAzNeuAc-K20 cells stained with counts. The normalized trans ligand score was then calculated as the 5-chloromethylfluorescein diacetate (Molecular Probes) were overlaid ratio of the difference to the total spectral counts. Following analysis onto CHO cell monolayers in 6-well plates and photocross-linked. of spectral count values at the peptide level, the normalized trans Unbound K20 cells were washed off with PBS and 0.2 mM glycine in ligand scores of various peptides corresponding to a protein ID were PBS, pH 2.2, and the remaining cells were visualized with an inverted averaged to obtain a normalized trans ligand score for that protein. Zeiss fluorescence microscope. For immunofluorescence micros- The mean and S.D. of the trans ligand scores of the two replicate data copy, freshly isolated murine B-cells were stained with FITC-labeled sets were also calculated. Data were filtered to remove protein IDs anti-mouse CD22 (Southern Biotech), FITC-labeled anti-IgM (whole that were not represented in both replicate data sets and proteins IgG)/DyLight 549-labeled anti-IgM (Fab fragment) (Jackson Immu- that did not have Ͼ4 spectral counts in at least one replicate. noResearch Laboratories), PE-labeled anti-CD45, or FITC-labeled Environmental contaminants were detected by high spectral counts Sambucus nigra agglutinin (Vector Laboratories). Freshly isolated hu- across not only gel bands 1–3 but also band 4 (ϳ95–70 kDa) and man B-cells were fixed with 4% p-formaldehyde and stained with control bands corresponding to lower molecular masses and were FITC-labeled anti-human CD22 (BD Pharmingen) or anti-human IgM removed. Mean trans ligand scores of the remaining protein hits (Jackson ImmunoResearch Laboratories). Coverslips were mounted varied from 1 to Ϫ1. Proteins with mean trans ligand scores Ͼ0.33 with 4Ј,6-diamidino-2-phenylindole ProLong Gold antifade reagent were considered CD22 ligand candidates. Standard deviations be- (Invitrogen). Images were acquired on the Bio-Rad (Zeiss) Radiance tween the normalized trans ligand scores of replicate data sets were 2100 Rainbow laser scanning confocal microscope, collected on a used to assess the statistical significance of the spectral count anal- Windows 2000 PC work station running the latest Bio-Rad Laser- ysis. In addition, the standard deviation of the normalized trans ligand Sharp 2000 software, and processed with ImageJ analysis software. scores obtained for the various peptides corresponding to a protein One-dimensional Gel Electrophoresis and Mass Spectrometry (MS/ using spectral count values at the peptide level was used to measure MS)—Protein G immunoprecipitates from lysates of 9AAzNeuAc-K20 the statistical significance of the trans ligand score at the protein level. cells incubated with CD22-Fc with or without photocross-linking were Several of the candidate ligands identified by spectral count analysis resolved by gel electrophoresis and stained with EZ-Blue (Sigma). were independently verified by Western analysis. The high molecular mass region was excised into the following slices: Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)- band 1, greater than ϳ385 kDa; band 2, ϳ385–165 kDa; band 3, MuDPIT—Stable, heavy isotopes of lysine ([13C]Lys) and arginine ϳ165–95; and band 4, ϳ95–70 kDa. Gel slices were subjected to ([13C,15N]Arg) differing from their amino acid counterparts by 6 in-gel digestion as reported previously (27). Peptides from in-gel and 10 Da, respectively, were used to enable extensive coverage of trypsin digests were pressure-loaded onto a 100-␮m (inner diameter) peptides following tryptic digestion. K20 cells were maintained in fused silica capillary column containing 10 cm of C18 resin. Peptides RPMI 1640 medium supplemented with dialyzed serum and light or were eluted from the column using a 2-h gradient with a flow rate of heavy lysine and arginine (Invitrogen) for 2 weeks prior to performing 0.25 ␮l/min directly into an LTQ ion trap mass spectrometer (Thermo the SILAC experiment. Four replicates (5 ϫ 108 cells) each of light and Fisher Scientific). The LTQ was operated in data-dependent scanning heavy K20 cells were desialylated by treatment with 50 milliunits/ml A. mode with one full MS scan followed by seven MS/MS scans of the ureafaciens sialidase and sialylated with 9AAzNeuAc by enzymatic most abundant ions with dynamic exclusion enabled. Raw MS/MS engineering. Cells were incubated with 37.5 ␮g/ml CD22-Fc on ice for data were searched using the SEQUESTTM algorithm (28) using a 30 min, and two replicates each of light and heavy K20 cells were concatenated target/decoy variant of the human IPI database (version exposed to UV irradiation for photoaffinity cross-linking. All samples 3.33, released on September 13, 2007). No enzyme specificity was were washed extensively and lysed. Lysates from samples of light stated, and a static modification of ϩ57 Da was specified on cysteine K20 cells exposed to UV irradiation were mixed 1:1 with those of to account for iodoacetamide alkylation. Mass tolerance for precursor heavy K20 cells not exposed to UV irradiation, and lysates of heavy ions and fragment ions was set to 1.00 Da. SEQUEST data from each K20 cells exposed to UV irradiation were mixed 1:1 with those of light band were filtered and sorted with DTASelect version 1.9 requiring a K20 cells not exposed to UV irradiation to yield two pairs of swapped minimum ⌬CN of 0.8 and XCorr values of 1.8, 2.5, and 3.5 for 1ϩ,2ϩ, replicates. The combined lysates were subjected to immunoprecipi- and 3ϩ charge states. The false positive rates estimated by the tation with rProtein G-agarose. Reduction, alkylation of cysteines, and program from the number and quality of spectral matches to the protein digestion with trypsin were performed on-bead, and tryptic decoy database were less than 1.0%. When combining peptides into peptides were resolved by MuDPIT as described previously (29) using “proteins identified,” all isoforms/individual members of a protein an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific). Tan- family supported by the data were considered, and the entries with dem mass spectra were analyzed using the following software anal- the most annotation are reported here. ysis protocol. MS/MS spectra were searched with the ProLuCID Spectral Count Analysis of One-dimensional Gel Electrophoresis- algorithm (30) against the human IPI database (version 3.44, released MS/MS Data for Identification of trans Ligand Candidates—Two rep- on May 20, 2008 with 71,884 protein entries) concatenated to a decoy licate data sets, each containing a ϩUV sample and a ϪUV sample, database in which the sequence for each entry in the original data- were subjected to spectral count analysis. Spectral count values of base was reversed (31). The search parameters were as follows: peptides derived from proteins from gel slices corresponding to mo- precursor ion mass tolerance was 4.5 Da, fragmentation ion mass lecular mass Ͼ95 kDa (gel band 1, Ͼ385 kDa; gel band 2, ϳ385–165 tolerance was 0.6 Da, maximum number of missed cleavages was 2, kDa; and gel band 3, ϳ165–95 kDa) were analyzed in both ϪUV and and static cysteine modification was set to 57.02146. For the heavy ϩUV samples. A protein hit was considered to be a potential CD22 search, arginine and lysine were set to a static modification of trans ligand if its peptides were represented by higher spectral count 10.00827 and 6.020127, respectively. No enzyme specificity was values in the gel bands from the ϩUV sample as compared with those considered for the searches. All searches were parallelized and per- from the ϪUV sample. To identify potential CD22 trans ligands, a formed on a Beowulf computer cluster consisting of 100 1.2-GHz “normalized trans ligand score” was calculated for every protein hit as Athlon CPUs (32). The results were assembled and filtered using the follows. First, the difference between the summed spectral counts DTASelect (version 2.0) program (33, 34). DTASelect 2.0 uses a linear

Molecular & Cellular Proteomics 9.6 1341 trans Ligands of CD22

9AAz FIG.1.Photoaffinity cross-linking of CD22-Fc to glycoproteins on NeuAc-K20 B-cells. A, scheme for introducing 9AAzNeuAc onto glycoproteins of K20 cells by enzymatic engineering or metabolic incorporation. B, flow cytometry plot of native, asialo, and enzymatically engineered 9AAzNeuAc-K20 cells stained with FITC-labeled S. nigra agglutinin. C, flow cytometry plot of native, asialo, and metabolically engineered 9AAzNeuAc-K20 cells stained with FITC-labeled S. nigra agglutinin. D, scheme for photocross-linking CD22-Fc to glycoproteins on 9AAzNeuAc-K20 B-cells. E, photoaffinity cross-linking of CD22-Fc to glycoproteins on 9AAzNeuAc-K20 B-cells. Anti-CD22 and anti-Fc Western analyses of rProtein G immunoprecipitates of 9AAzNeuAc-K20 cells incubated with or without CD22-Fc and with or without UV cross-linking is shown. The band corresponding to CD22 is marked with a blue arrow, and cross-linked bands are indicated in purple. Also seen in the anti-Fc blot is the Ig heavy chain indicated with a green arrow. IP, immunoprecipitation. discriminant analysis to dynamically set XCorr and ⌬CN thresholds the statistical significance of the measurements. Several of the can- for the entire data set to achieve a user-specified false positive rate didate ligands identified were independently verified by Western (less than 1% at the peptide level in this analysis). The false positive analysis. rates are estimated by the program from the number and quality of spectral matches to the decoy database. When combining peptides RESULTS into proteins identified, all isoforms/individual members of a protein 9AAz family supported by the data were considered, and the entries with Incorporation of the Photocross-linker NeuAc onto B- the most annotation are reported here. cell Surface Glycoproteins—With the goal of identifying B-cell Analysis of SILAC-MuDPIT Data for trans Ligand Candidates—Four glycoproteins that could serve as trans ligands of CD22, we data sets were analyzed. Two replicate data sets contained proteins opted to use our approach of labeling total B-cell glycopro- from a sample containing light cells photocross-linked to CD22-Fc 9AAz (LϩUV) together with control heavy cells (HϪUV), and the remaining teins with NeuAc, which does not impair CD22 binding to two replicate data sets contained swapped samples wherein heavy its ligands and allows efficient in situ photoaffinity cross- cells were photocross-linked to CD22-Fc (HϩUV) and mixed with linking to CD22 (15). Then, following immunoprecipitation of control light cells (LϪUV). SILAC ratios were computed by the pro- the complex using CD22 as a tag, the ligands could be gram Census using a linear least squares correlation algorithm (35). identified by proteomics and immunochemical techniques. The ratio between the light versus heavy version of each peptide was calculated as the slope of the correlation line, whereas the closeness Accordingly, to label cell surface glycoproteins of B-cells, 9AAz of fit was indicated as the correlation coefficient (r). Only peptide NeuAc was installed by either metabolic labeling (15, 36) ratios with r2 Ͼ 0.5 were accepted to remove poor quality data. The or enzymatic engineering with sialyltransferase, ST6GalI (25) quantified proteins were further filtered to remove proteins that did (Fig. 1A). For optimal incorporation of 9AAzNeuAc, we used not appear in more than one replicate data set. Depending on the K20 cells cultured in serum-free medium because BJA-B K20 sample (e.g. LϪUV mixed with HϩUV), reciprocals of SILAC (light/ heavy) ratios were calculated if required to obtain ϩUV/ϪUV ratios. is a hyposialylated cell line that cannot synthesize its own Environmental contaminants were detected by high standard devia- sialic acids due to a deficiency in a key enzyme, UDP- tions between ϩUV/ϪUV ratios in swapped replicates and removed. GlcNAc-2Ј-epimerase, but will readily incorporate sialic acids Average ϩUV/ϪUV ratios of the remaining protein IDs ranged from added to the culture medium (37). Both metabolic incorporation 0.46 to 37.06. A higher cutoff of 2.17 (reciprocal of 0.46) was chosen and enzymatic engineering yielded sialylation profiles similar to to factor in the distribution of SILAC ratios. Protein IDs with ϩUV/ϪUV ratios Ͼ2.17 were considered CD22 trans ligand candidates. Stand- that of “fully sialylated” cells cultured in medium supplemented ard deviations between ϩUV/ϪUV ratios at the peptide level and with serum as detected with the sialic acid-specific lectin SNA between replicate data sets at the protein level were used to assess (Fig. 1, B and C, and supplemental Fig. S1).

1342 Molecular & Cellular Proteomics 9.6 trans Ligands of CD22

trans Ligand Candidates of CD22-Fc on B-cells Identified light or heavy amino acids were either exposed to UV irradi- by Proteomics—The ideal experiment for identifying in situ ation to induce photocross-linking or left untreated, subjected ligands of CD22 is to perform the UV cross-linking on a cell to extensive washing, and lysed. Lysates were then combined containing membrane-bound CD22 engaged in cell contact and subjected to immunoaffinity enrichment and on-bead with 9AAzNeuAc-labeled B-cells. However, in preliminary ex- trypsin digestion, peptides were analyzed by MuDPIT-MS/ periments with this approach, although trans ligand cross- MS, and the ratios of peak intensities of light and heavy linking could be readily demonstrated by immunochemical peptides in the mass spectra were used to identify proteins techniques (see below), the amount of cross-linked complex more abundantly co-immunoprecipitated with CD22-Fc upon that could be obtained was insufficient to identify the captured photocross-linking. A total of 19 proteins with SILAC-derived ligands by proteomics techniques. Therefore, we opted to first ϩUV/ϪUV ratios greater than 2.2 were identified as candidate identify a subset of the glycoproteins as candidate ligands trans ligands, all of which were annotated as membrane glyco- using intact 9AAzNeuAc-labeled B-cells as a source of in situ proteins in the UniProtKB/Swiss-Prot database (Fig. 2B, Table ligands and CD22-Fc chimera as the trans interacting recep- II, supplemental Table S1 and annexure to Table S1) (39). tor (Fig. 1D). In principle, the yield of cross-linked protein In all, the two proteomics approaches yielded 27 glycopro- could be increased 10–100-fold because B-cells could be teins as CD22 trans ligand candidates, 10 of which were prepared in large quantities in a single cell suspension, and identified in both approaches (supplemental Fig. S3A CD22-Fc had access to the entire surface of the cell, not just and Table S2). Functional analyses using the Swiss-Prot da- the point of cell contact. To test this approach, CD22-Fc was tabase (39) and the on-line DAVID knowledgebase (40) and UV cross-linked to 9AAzNeuAc-K20 cells. Uncross-linked Fatigo tools (41, 42) indicated that a majority of the CD22 CD22-Fc was then washed away by low pH, and cell lysates ligand candidates had a role in immune response, and all 27 were subjected to immunoaffinity capture with Protein G- hits were clustered by their functions in activa- agarose to remove non-cross-linked proteins and high mo- tion, signal transduction, cell adhesion, and transport lecular weight cis cross-linked CD22 from 9AAzNeuAc-K20 (supplemental Fig. S3, B and C, and Table S3). cells. Western analysis yielded anti-CD22- and anti-Fc-reac- The candidate ligands identified by the proteomics ap- tive protein species of higher molecular mass than CD22-Fc proaches above were validated by immunochemical analy- that were not seen in the absence of UV exposure (Fig. 1E)or sis of CD22-Fc photocross-linked to glycoproteins on 9AAzNeuAc labeling (supplemental Fig. S2A). Similar results 9AAzNeuAc-K20 cells. Antibodies suitable for Western anal- were obtained with 9AAzNeuAc-resialylated lymphocytes from ysis were obtained for eight of the 10 candidate glycopro- human blood (supplemental Fig. S2B). teins identified by both approaches and 11 of the 19 iden- To identify the cross-linked ligands resolved as high mo- tified by SILAC analysis (supplemental Table S2). Western lecular weight species, we used a mass spectrometry-based analysis revealed that all were photocross-linked to proteomics approach. Gel sections containing the high mo- CD22-Fc with the appearance of higher molecular mass lecular weight region of the gel were subjected to in-gel form(s) corresponding to the sum of the molecular mass of trypsin digestion and nano-LC-MS/MS. Analysis of spectral one or more molecules of CD22-Fc and the glycoprotein count values of peptides identified a total of 24 proteins that (Fig. 3 and supplemental Table S2). That cross-linking was were found in the high molecular weight region of the UV CD22-specific was verified by performing control experi- irradiation-exposed samples relative to the non-UV irradia- ments in which 9AAzNeuAc-K20 cells were incubated with tion-exposed samples. Database analysis of these revealed IgG (Fc fragment) protein instead of CD22-Fc and exposed that 18 were known glycoproteins, and six were annotated as to UV irradiation. No background was introduced in the predicted proteins or nuclear proteins. Only the known glyco- Western analysis, confirming that cross-linking was CD22- proteins were considered candidate trans ligands in the sub- specfiic. Representative Western blots with antibodies to sequent analyses (Table I). Among them, CD22 was estab- CD45, IgM, Basigin, 4F2, HLA I, HLA II, and MCAM are lished as a trans ligand candidate based on spectral hits for illustrated in Fig. 3. C-terminal CD22 peptides (amino acid 327 and above) that In Situ CD22 trans Ligands on B-cells in Cell Contacts— are not found in the recombinant CD22-Fc protein. Armed with the identities of the membrane-bound B-cell gly- As an alternative approach, we exploited SILAC to identify coproteins that are photocross-linked to CD22-Fc, we next candidate trans ligand candidates cross-linked to the sought to determine whether they would also be detected as CD22-Fc chimera (Fig. 2A) (38). SILAC experiments were in situ trans ligands in the context of cell to cell contact. To performed by culturing the BJA-B K20 B-cell line in growth examine this, we used a CHO cell line expressing CD22 with medium with light ([12C]lysine and [12C,14N]arginine) or heavy a C-terminal V5 tag for capture of glycoprotein ligands on the ([13C]lysine and [13C,15N]arginine) stable isotopes of lysine B-cell line BJA-B (subclone K20) labeled with 9AAzNeuAc (Fig. and arginine. Cell surface glycoproteins were then modified 4A). The 9AAzNeuAc-labeled or asialo K20 cells were overlaid with 9AAzNeuAc by enzymatic engineering and allowed to onto CHO cells expressing CD22-V5, subjected to UV expo- interact with CD22-Fc chimera. Cells differentially labeled with sure, and washed with low pH to remove non-covalently

Molecular & Cellular Proteomics 9.6 1343 trans Ligands of CD22

TABLE I Identification of CD22 trans ligand candidate proteins by spectral count analysis Spectral count values of peptides released by trypsin digestion from gel slices corresponding to the molecular mass of photocross-linked CD22-Fc were analyzed in CD22-Fc-cross-linked (ϩUV) replicate samples as well as control (ϪUV) replicate samples. Normalized trans ligand scores (see “Experimental Procedures”) were calculated from the spectral count values of the “ϩUV” and “ϪUV” samples for every peptide and averaged across all peptides corresponding to a protein ID to obtain the “trans ligand score” for that protein. The average of the “trans ligand scores” of replicates is reported as the “mean score.” Proteins with mean scores Ͼ0.33 were considered candidate ligands. Listed in the table are details of candidate CD22 ligands identified by this procedure. CD22 itself was established as a trans ligand candidate based on analysis of spectral count values of C-terminal CD22 peptides (amino acids 327–847) that are not found in the recombinant CD22-Fc protein. PEPTIDE COUNT, number of unique peptides; % SEQ COVERAGE, percent sequence coverage; STDEV, standard deviation.

1344 Molecular & Cellular Proteomics 9.6 trans Ligands of CD22

FIG.2.CD22 trans ligand candidates identified by SILAC analysis. A, scheme for identifying CD22 trans ligand candidates by SILAC analysis of samples subjected to photocross-linking, immunoaffinity isolation, on-bead trypsin digestion, and MuDPIT. B, spectral counts of protein hits plotted against average SILAC ratio upon UV cross-linking. Red and blue data points indicate protein hits considered as candidate trans ligands and non-ligands, respectively. IP, immunoprecipitation. bound cells. Extensive cross-linking of cells was observed a trans ligand of CD22 in cell to cell contacts, we investigated only for the 9AAzNeuAc-engineered K20 cells overlaid on the localization of CD22 and IgM on interacting primary mu- CD22-V5-CHO cells, whereas control experiments that were rine and human B-cells by immunofluorescence and micro- not subjected to UV treatment or that used K20 cells lacking scopic analysis. As observed previously, CD22 was redistrib- 9AAzNeuAc or CHO cells lacking CD22-V5 expression showed uted to sites of cell contact of interacting murine B-cells (Fig. few residual K20 cells bound to the CHO monolayer (Fig. 4B). 5A) and also was redistributed to the site of cell contact on Western analysis of anti-V5 immunoprecipitates confirmed primary human B-cells (supplemental Fig. S4). Interestingly, that UV irradiation resulted in formation of anti-CD22-reactive IgM was also localized preferentially at the sites of cell contact protein species of higher molecular mass than CD22-V5 (Fig. of interacting murine and human B-cells (Fig. 5A and 4C). To identify which glycoproteins accounted for the cross- supplemental Fig. S4). This localization of IgM was also seen linked species, representing in situ trans ligands of CD22, in samples stained with the Fab fragment of anti-IgM antibody anti-V5 immunoprecipitates were subjected to Western anal- and hence was not caused by antibody-mediated cross-link- ysis with antibodies to 11 of the 27 glycoproteins identified as ing of IgM on apposing cells. In contrast, CD45 was not trans ligand candidates in the spectral count and SILAC anal- significantly enriched at sites of cell contact (Fig. 5A). Simi- yses of the CD22-Fc-ligand complexes. Of these, only three, larly, there was diffuse staining with FITC-labeled S. nigra IgM, CD45, and Basigin, showed cross-linking to CD22 (Fig. agglutinin, which detects all glycoproteins bearing the 4D). Although we did not use CHO cells expressing other NeuAc␣2–6Gal sequence recognized by CD22 (Fig. 5A). V5-tagged proteins as controls, no significant background To assess the possibility that the IgM localization was a was observed upon Western analysis of immunoprecipitates result of trans interactions with CD22, we compared local- from controls comprising 9AAzNeuAc-K20 cells overlaid on ization of IgM on 300 pairs of contacting B-cells from wild CHO cells (not expressing CD22; Fig. 4D). Notably, cross- type and CD22 knock-out mice (representative micrographs linking of CD22 to IgM was particularly robust with little cross- are shown in Fig. 5, A and B). Remarkably, IgM was re- linking to CD45 and Basigin (Fig. 4D and supple- cruited to sites of cell contact in 35 Ϯ 5% (average Ϯ S.E. mental Table S2). Moreover, no cross-linking of CD22 was of four independent experiments) of the interacting pairs of detected to HLA I, HLA II, 4F2, Plexin-B2, Siglec-10, CD33, B-cells from wild type mice but in only 9 Ϯ 3% (average Ϯ SLC1A5, or MCAM (supplemental Table S2) despite their robust S.E. of four independent experiments) of interacting B-cell cross-linking to CD22-Fc (Fig. 3) even though several of these pairs from CD22 KO mice (Fig. 5D)(p ϭ 0.0047 in a two- glycoproteins are known to be as or more abundant than IgM on tailed, unpaired Student’s t test). CD45 and SNA localization B-cells (43, 44). The results show that the in situ recognition of remained unaltered (Fig. 5, compare A and B). The results trans ligands at the site of cell to cell contact is highly selective strongly suggest that trans interactions of CD22 are respon- with IgM as the preferred trans ligand of CD22. sible for IgM redistribution to the site of cell contact of CD22- and Sialic Acid-dependent Redistribution of IgM to interacting B-cells. Sites of Cell Contact of Apposing B-cells—We previously To investigate whether these trans interactions were me- demonstrated that CD22 is redistributed to sites of cell con- diated by CD22 binding to sialic acids on IgM, we also tact between B-cells as a result of trans ligand interactions on assessed CD22 and IgM localization on 300 pairs of con- the apposing cell (9). Given the robust cross-linking of IgM as tacting B-cells from ST6GalI knock-out mice that lack the

Molecular & Cellular Proteomics 9.6 1345 trans Ligands of CD22

TABLE II Identification of CD22 trans ligand candidate proteins by SILAC analysis SILAC (light/heavy) ratios were computed for all peptides identified after on-bead trypsin digestion and MuDPIT analysis of samples containing either light cells photocross-linked to CD22-Fc (LϩUV) and control heavy cells (HϪUV) or heavy cells photocross-linked to CD22-Fc (HϩUV) and control light cells (LϪUV). SILAC ratios of proteins, obtained by averaging the ratios of the constituent peptides, were converted to ϩUV/ϪUV ratios. Proteins with ϩUV/ϪUV ratios Ͼ2.17 were considered candidate ligands of CD22. Listed in the table are details of candidate CD22 ligands identified by this procedure. Standard deviations of the ϩUV/ϪUV ratios are not listed for single peptide spectrum-based protein identifications. PEPTIDE COUNT, number of unique peptides; %SEQ COVERAGE, percent sequence coverage; STDEV, standard deviation.

1346 Molecular & Cellular Proteomics 9.6 trans Ligands of CD22

FIG.3.Immunochemical validation of CD22 trans ligand candidates. Western analysis was conducted on rProtein G immunoprecipitates from 9AAzNeuAc-K20 cells incubated with CD22-Fc protein or IgG (Fc fragment) (abbreviated IgG-Fc in the figure) with or without subsequent UV cross-linking. Red and purple arrows indicate uncross-linked and CD22-cross-linked glycoproteins, respectively.

FIG.4. trans ligands of CD22 on apposing B-cells. A, scheme for identifying CD22 trans ligands on apposing B-cells using an immunochemical screen. B, phase and fluorescence microscopy of 5-chloromethylfluorescein diacetate-stained asialo or 9AAzNeuAc-K20 cells (green) overlaid onto CHO or CD22-CHO cell monolayers with or without UV cross-linking and subjected to a low pH wash. C, anti-CD22 Western analysis of anti-V5 immunoprecipitates conducted on lysates from 9AAzNeuAc-K20 cells overlaid onto CHO or CD22-(V5)-CHO cells with or without UV cross-linking. The band corresponding to CD22 is marked with a blue arrow, and cross-linked bands are indicated in purple. An asterisk denotes the position of CD22 dimer. D, Western analysis of anti-V5 immunoprecipitates of 9AAzNeuAc-K20 cells overlaid onto CHO or CD22-(V5)-CHO cells with or without UV cross-linking. Red and purple arrows indicate uncross-linked and CD22-V5-cross-linked glycoproteins, respectively. Also seen in the anti-Basigin Western blot is the Ig heavy chain marked with a green arrow. Uncross-linked Basigin glycoforms range from ϳ33 to 50 kDa and are not appreciably seen in anti-V5 immunoprecipitates. IP, immunoprecipitation. sialyltransferase required for synthesis of the sialylated independent experiments) of the interacting B-cell pairs CD22 ligands (representative micrographs are shown in Fig. from ST6GalI knock-out mice, 5-fold lower than contacting 5C). As expected, B-cells from ST6GalI knock-out mice B-cell pairs from wild type mice (Fig. 5D)(p ϭ 0.0026 in a were not stained by SNA, and CD22 was not redistributed to two-tailed, unpaired Student’s t test). The results demon- the sites of cell contact in interacting B-cells from ST6GalI strate that sialic acid-mediated trans interactions of CD22 knock-out mice (Fig. 5C). Notably, IgM was concentrated at are responsible for IgM redistribution to the site of contact sites of cell contact in only ϳ7 Ϯ 2% (average Ϯ S.E. of four of interacting B-cells.

Molecular & Cellular Proteomics 9.6 1347 trans Ligands of CD22

FIG.5.Microscopic analysis of trans ligand interactions on apposing B-cells. A, immunofluorescence microscopy of murine B-cells from wild type mice stained with FITC-labeled anti-CD22, FITC-labeled anti-IgM, FITC-labeled S. nigra agglutinin, or PE-labeled anti-CD45. The white scale bar represents 5 ␮m. B, immunofluorescence microscopy of murine B-cells from CD22 KO mice stained with FITC-labeled anti-CD22, DyLight 549-labeled anti-IgM (pseudocolored green), FITC-labeled S. nigra agglutinin, or PE-labeled anti-CD45. The white scale bar represents 5 ␮m. C, immunofluorescence microscopy of murine B-cells from ST6GalI KO mice stained with FITC-labeled anti-CD22, DyLight 549-labeled anti-IgM (pseudocolored green), FITC-labeled S. nigra agglutinin, or PE-labeled anti-CD45. The white scale bar represents 5 ␮m. D, histogram showing the percentage of contacting B-cells from wild type, CD22 KO, and ST6GalI KO mice in which IgM was localized at the site of contact. Values plotted are the averages of values acquired in four independent experiments, each obtained by analyzing 100 cells each in three different regions of a slide preparation. Error bars shown are the S.E. p ϭ 0.0047 (CD22 KO) and p ϭ 0.0026 (ST6GalI KO) in two-tailed, unpaired Student’s t tests. Wt, wild type.

DISCUSSION and trafficking to bone marrow that involve interactions with A major function ascribed to the glycan ligands of CD22 is trans ligands on other cell types (5, 13, 52, 53). Recent reports to modulate the activity of CD22 in BCR signaling by influ- demonstrate that high affinity cis ligands of CD22 are down- encing its proximity to the BCR complex (2, 5, 6, 45). As a regulated on B-cells during differentiation in germinal centers co-receptor of the BCR, CD22 regulates signaling by recruit- (54, 55), favoring binding of CD22 on activated B-cells to trans ment of the phosphatase SHP-1 and other Src homology 2 ligands on other contacting cells. trans ligands of CD22 have domain regulatory proteins via tyrosine motifs in its cytoplas- been documented to exist on B-cells, T-cells, monocytes, mic domain where proximity to the BCR is required for max- endothelial cells, and dendritic cells (9, 10, 13, 18, 56). Previ- imal effect. Early support of this idea came from the obser- ously, we demonstrated that CD22 is redistributed to sites of vation that sequestration of CD22 with antibody-coated B/B-cell contact mediated by sialic acid-dependent interac- beads enhances activation through BCR ligation (46). Al- tions with trans ligands on the apposing B-cell (8). Homotypic though IgM and CD22 are predominantly in distinct microdo- B-cell interactions occur in lymphoid organs and bone mar- mains in resting murine B-cells, ablation of cis ligands causes row whenever B-cells are in close contact and have been increased co-localization of CD22 and the BCR in raft-clathrin demonstrated to be important in the regulation of IgE and IgG domains, resulting in suppression of BCR signaling (47–49). synthesis (57, 58), polyclonal B-cell activation (59), mainte- Inclusion of CD22 ligands in a polymeric T-independent anti- nance of peripheral tolerance (60), differentiation of pre-B- gen also suppresses BCR activation by physically ligating cells into mature B-cells in the bone marrow (61, 62), and CD22 to the BCR complex (50, 51). Similarly, co-expression of bystander transfer of antigens from one B-cell to another (63). CD22 trans ligands on antigen-presenting cells dampens B- Because CD22 plays a role in establishing the threshold for cell activation, presumably by recruitment of CD22 to sites B-cell receptor signaling, identifying the ligands in B/B-cell of cell contact in close proximity to the BCR synapse (8, 14). contacts that regulate CD22 redistribution to sites of cell Such observations suggest that trans ligands of CD22 could contact is of significant interest. participate in recognition of self and establish a threshold for In this study, we investigated the nature of the trans ligands B-cell activation and maintenance of peripheral tolerance (14, of CD22 on B-cells that mediate redistribution of CD22 to 50, 51). sites of cell contact in homotypic B-cell interactions. Because In addition to the activation of B-cells, CD22-ligand inter- CD22 is known to recognize the majority of B-cell glycopro- actions have been proposed to mediate other aspects of teins that carry the NeuAc␣2–6Gal sequence in cell lysates B-cell biology including differentiation, antigen presentation, (15, 17, 18), a major objective was to establish whether CD22

1348 Molecular & Cellular Proteomics 9.6 trans Ligands of CD22 recognizes all B-cell glycoproteins as trans ligands in situ or trans ligand of CD22. Such factors may include the geometric preferentially recognizes a subset of them. We first identified distribution and projection of glycans from the surface of the 27 trans glycoprotein ligand candidates by proteomics anal- cell as well as microdomain localization and/or interaction ysis of complexes formed by UV cross-linking CD22-Fc chi- with the cytoskeleton of the cell. It is remarkable that IgM is mera to B-cells labeled with 9-aryl azide-NeuAc. This ex- selectively recognized by CD22 in trans but is not significantly pands the number of identified sialic acid-dependent binding recognized as a cis ligand of CD22 on B-cells (15). Thus, the partners of CD22-Fc chimera from four (CD45, IgM, CD19, factors that make IgM a preferred in situ trans ligand do not and CD22) in previous reports (15, 16, 18, 20) to 27 (see make IgM a suitable ligand for CD22 in cis. Tables I and II). The validity of the “hits” was assessed by We anticipate that the approach used here can also be confirming that the protein hits formed UV irradiation-induced used to investigate trans ligands of CD22 for other cells (e.g. cross-linked complexes with CD22 by Western blot analysis T-cells, epithelial cells, dendritic cells, etc.) known to contact using antibodies available to 11 of these glycoproteins, in- B-cells in physiologically relevant contexts (5, 6, 9–14, 53, 66, cluding eight of 10 glycoproteins identified by both the spec- 68). Stamenkovic et al. (22) provided evidence that CD45RO is tral count and SILAC analyses. The ability of CD22 to use a physiologically relevant in situ trans ligand of CD22 on these glycoproteins as trans ligands at the site of cell contact T-cells by virtue of blocking binding of T-cells to CD22 with was then assessed by cross-linking B-cells labeled with 9-aryl anti-CD45 but concluded that other ligands are also likely azide-NeuAc to CD22-expressing CHO cells followed by anal- involved (11, 69). In principle, this approach can also be used ysis of the cross-linked products by Western blot. Remark- to examine the cis and trans ligands of other siglecs, provided ably, only three of these 11 glycoproteins, IgM, Basigin, and that they can accommodate a substituent on sialic acid that CD45, were detected as trans ligands in the context of B-cells allows covalent cross-linking. In this regard, Kohler and co- bound to CD22-CHO cells. We do not suggest that they are worker (70) have demonstrated that N-acyl diazirine-modified the only in situ trans ligands of CD22, only that of the 11 sialic acids are an effective alternative for photocross-linking analyzed, these three are the only three detected with IgM of glycoprotein ligands to CD22. Although this report has appearing to be selectively recognized based on the robust focused on sialic acid-dependent ligands of CD22, it should detection of CD22-IgM complexes relative to those of CD45 be noted that some siglecs, including CD22, also selectively and Basigin (e.g. compare Figs. 3 and 4D). bind trans ligands that are not sialic acid-dependent, presum- The inference that IgM is preferentially recognized by CD22 ably via an alternative binding site(s) (56, 71, 72). Such obser- as an in situ trans ligand was further supported by the dem- vations emphasize the need to conduct in situ analyses to onstration that IgM and CD22 were co-distributed to the site distinguish physiologically relevant receptor-ligand interac- of contact of interacting primary murine and human B-cells tions from in vitro interactions that are missing the cellular and that redistribution of IgM to the site of contact was both context. CD22- and sialic acid-dependent (Fig. 5 and supple- Finally, the results presented here have broader signifi- mental Fig. S4). The conclusion that this interaction is selec- cance to the general problem of identifying the in situ ligands tive is underscored by the fact that surface IgM (43) and CD22 of glycan-binding proteins. Most glycan-binding proteins in (64) are expressed at only ϳ13,000 and ϳ25,000 molecules the major glycan-binding protein families (e.g. siglecs, C-type per cell, respectively, whereas CD45, a major cell surface lectins, and galectins) interact primarily with the glycan moiety glycoprotein, is expressed at ϳ150,000 molecules per cell of glycoproteins, which is sufficient for driving ligand interac- (65, 66). More remarkably, the total sialic acid content of a tions in vitro (e.g. from cell lysates). However, it is clear that lymphocyte is estimated to be in excess of 109 molecules per the cellular context can significantly restrict the utilization of a cell (67) of which a major fraction (Ͼ25%) would be found in glycoprotein as an in situ ligand even if it carries the glycan sequences recognized by CD22. Thus, the interaction of epitope recognized as a ligand in vitro. Thus, glycoproteins CD22 with IgM at the site of cell contact is highly selective, observed to be in vitro binding partners are best viewed as occurring in the presence of a large excess of other glyco- ligand candidates until verified as physiologically relevant li- proteins that carry the glycan sequence recognized by CD22. gands in situ. Although the functional significance of the redistribution of IgM and CD22 to the site of cell contact in instances of Acknowledgments—We thank Prof. M. Pawlita for the BJA-B K88 and K20 cell lines, Dr. Norihito Kawasaki for recloning CD22-CHO- homotypic B-cell interactions is not clear (57–63), the result is FlpIn cells, Drs. Norihito Kawasaki and Christoph Rademacher for that CD22 and IgM are brought together in close proximity on murine B-cells, Dr. Shoufa Han for 9AAz-sialic acid, Prof. Varki for both cells, which would have the expected consequence of CD22-Fc-pCDM8 plasmid, Anna Tran-Crie for expert assistance in down-regulation of signaling from BCRs at the site of cell manuscript preparation, and members of the Paulson laboratory for contact. scientific input. The preferential recognition of IgM as a trans ligand strongly * This work was supported, in whole or in part, by National Insti- suggests that factors other than glycan sequence determine tutes of Health Grants GM60938 and AI50143 (to J. C. P.) and P41 the ability of a glycoprotein on an apposing cell to serve as a RR011823 and BIMR P30NS057096 (to J. R. Y.).

Molecular & Cellular Proteomics 9.6 1349 trans Ligands of CD22

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