In Situ trans Ligands of CD22 Identified by Glycan … · In Situ trans Ligands of CD22 Identified by Glycan-Protein Photocross-linking-enabled Proteomics* S T. N. C. Ramya‡, Eranthie

In Situ trans Ligands of CD22 Identified byGlycan-Protein Photocross-linking-enabledProteomics*□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-ognizes the sequence NeuAc�2–6Gal on glycoprotein gly-cans as ligands. CD22 interactions with glycoproteins onthe same cell (in cis) and apposing cells (in trans) modu-late its activity in B-cell receptor signaling. Although CD22predominantly recognizes neighboring CD22 moleculesas cis ligands on B-cells, little is known about the transligands on apposing cells. We conducted a proteomicsscale study to identify candidate trans ligands of CD22 onB-cells by UV photocross-linking CD22-Fc chimera boundto B-cell glycoproteins engineered to carry sialic acidswith a 9-aryl azide moiety. Using mass spectrometry-based quantitative proteomics to analyze the cross-linkedproducts, 27 glycoproteins were identified as candidatetrans ligands. Next, CD22 expressed on the surface of onecell was photocross-linked to glycoproteins on apposingB-cells followed by immunochemical analysis of the prod-ucts with antibodies to the candidate ligands. Of the manycandidate ligands, only the B-cell receptor IgM was foundto be a major in situ trans ligand of CD22 that is selectivelyredistributed to the site of cell contact upon interactionwith CD22 on the apposing cell. Molecular & CellularProteomics 9:1339–1351, 2010.

Glycan-binding proteins (GBPs)1 mediate diverse aspectsof cell communication through their interactions with theircounter-receptors comprising glycan ligands carried on cellsurface glycoproteins and glycolipids. Identification of the insitu counter-receptors of glycan-binding proteins is problem-

atic due to the fact that the vast majority of the glycoproteinsof a cell will carry highly related glycan structures becausethey share the same secretory pathway that elaborates theirglycans post-translationally en route to the cell surface. Thus,although many glycoproteins will carry the glycan structurerecognized by a GBP, the challenge is to determine whetherone, several, or all of these cell surface glycoproteins (andglycolipids) are recognized in situ as physiologically relevantcounter-receptors (1–4). Standard in vitro methods, such asco-precipitation from cell lysates or Western blotting usingbinding protein probes, are useful for identifying glycoproteinsthat contain the glycan structure recognized by the GBP.However, these may not be relevant ligands in situ due toconstraints imposed by their microdomain localization andthe geometric arrangement of their glycans relative to theGBP 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 ofB-cell receptor (BCR) signaling that recognizes glycans con-taining the sequence NeuAc�2–6Gal as ligands (2, 5, 6).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 itsglycan ligands (6). Glycoproteins bearing CD22 ligands areabundantly expressed on B-cells and bind to CD22 in cis (onthe same cell) (7), regulating BCR signaling (2, 5, 6). Althoughbinding to cis ligands has been shown to “mask” CD22 frombinding low avidity synthetic sialoside probes (2, 7), CD22 canalso interact with ligands on apposing immune cells in trans(8–10). Interactions of CD22 with trans ligands influence T-cellsignaling in vitro (11, 12), mediate B-cell homing via binding tosinusoidal endothelial cells in the bone marrow (13), and aid in“self”-recognition (14). Thus, interactions with both cis and transligands modulate CD22 function in immune homeostasis.

Several groups have demonstrated that recombinantCD22-Fc chimera is capable of binding and precipitating themajority of glycoproteins from B- and T-cell lysates whoseglycans contain the sequence NeuAc�2–6Gal (15–18).Among them, CD45, IgM, and CD22 itself were identified asspecific B-cell binding partners and were postulated to havefunctional significance as in situ cis ligands of CD22 in regu-lation of BCR signaling (11, 16, 18–20). Several reports have

From the ‡Department of Chemical Physiology and §The SkaggsInstitute for Chemical Biology, The Scripps Research Institute, LaJolla, California 92037

Received, October 1, 2009, and in revised form, February 18, 2010Published, MCP Papers in Press, February 19, 2010, DOI 10.1074/

mcp.M900461-MCP2001 The abbreviations used are: GBP, glycan-binding protein; SILAC,

stable isotope labeling by amino acids in cell culture; BCR, B-cellreceptor; SNA, S. nigra agglutinin; MuDPIT, multidimensional proteinidentification technology; MCAM, melanoma cell adhesion molecule;HLA, human leukocyte antigen; SHP-1, Src homology 2 domain-containing protein-tyrosine phosphatase 1; CHO, Chinese hamsterovary; rProtein, recombinant protein; HRP, horseradish peroxidase;9AAz, 9-aryl azide; SFM, serum-free medium; PE, phycoerythrin;LTQ, linear trap quadrupole; IPI, International Protein Index; ID, iden-tifier; KO, knock-out.

Research

© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Molecular & Cellular Proteomics 9.6 1339This paper is available on line at http://www.mcponline.org

also documented in situ interactions of CD22 with IgM andCD45, but these interactions were found to be of low stoichi-ometry and sialic acid-independent (19–21), leaving open thequestion of which glycoproteins served as in situ cis ligands ofCD22 on B-cells that masked the glycan ligand binding site ofCD22 (7). Subsequently, using metabolically labeled B-cellswith sialic acids containing a photoactivatable 9-aryl azidemoiety, we demonstrated that CD22 could be photocross-linked to its cis ligands, effectively tagging the in situ cisligands with CD22 (15). Notably, there was no cross-linkingobserved to IgM or CD45, demonstrating that they are notsignificant in situ cis ligands of CD22 (15). Instead, only glycansof neighboring CD22 molecules interacted significantly withCD22, resulting in photocross-linking of homomultimeric com-plexes of CD22. Thus, despite the fact that most B-cell glyco-proteins are recognized in vitro, CD22 selectively recognizesglycans of neighboring CD22 molecules as cis ligands in situ.

With the perspective gained from analysis of cis ligands, wewished to determine whether CD22 was also selective inrecognition of trans ligands upon cell contact. We have pre-viously demonstrated that CD22 is redistributed to sites of cellcontact of interacting B-cells and T-cells and that redistribu-tion is mediated by the interaction of CD22 with sialic acid-containing trans ligands on the apposing cell (8). Stamenkovicet al. (22) had previously demonstrated that binding of T-cellsto CD22-expressing COS cells was blocked by an anti-CD45RO antibody, suggesting that CD45 was a functionaltrans ligand of CD22 on T-cells. However, we found thatredistribution of CD22 to sites of cell contact was also ob-served with CD45-deficient B-cells (8), indicating that, at aminimum, other glycoproteins must also serve as trans li-gands of CD22 on B-cells.

To assess whether CD22 recognizes all or a subset ofglycoproteins as trans ligands on an apposing cell, we initi-ated an unbiased analysis of the trans ligands of CD22 onapposing B-cells using our protein-glycan cross-linking strat-egy (15). By cross-linking CD22-Fc to intact B-cells, we iden-tified 27 candidate trans ligands of CD22 by quantitative massspectrometry-based proteomics. We then looked at the in situtrans interactions of CD22 in the physiologically relevant cel-lular context by cross-linking CD22 expressed on one cell tothe trans ligands with photoreactive sialic acids on the appos-ing cell. Our results indicate that only a subset of cell surfaceglycoproteins, including IgM and, to a lesser extent, CD45 andBasigin, are selectively recognized in trans by CD22. Indeed,IgM in particular is a preferred trans ligand that is selectivelyredistributed to the sites of cell contact on apposing B-cells ina CD22- and sialic acid-dependent manner despite a vastexcess of cell surface glycoproteins that carry a glycan rec-ognized by CD22. The results support the view that factorsother than glycan sequence are critical for the in situ engage-ment of glycan-binding proteins with glycan ligand bearingcounter-receptors on the same cell (in cis) or apposing cell (intrans).

EXPERIMENTAL PROCEDURES

Cells—CHO-FlpIn cells and stably transfected CHO-FlpIn cells ex-pressing human CD22 with a C-terminal V5 tag (23) were maintainedin Dulbecco’s modified Eagle’s medium/F-12 medium supplementedwith 10% fetal bovine serum and either 100 �g/ml Zeocin (Invitrogen)or 500 �g/ml hygromycin B (Roche Applied Science), respectively.CD22-Fc was purified from culture supernatants of COS-1 cells byProtein A affinity chromatography 3–7 days after transfection withCD22-Fc-pCDM8 using LipofectamineTM 2000 (24). BJA-B K20 cellswere cultured in RPMI 1640 medium supplemented with 10% fetalbovine serum. Human lymphocytes were enriched from human bloodwith Ficoll-Hypaque density gradient centrifugation. Human and mu-rine B-cells were enriched from blood and spleen, respectively, usingB-cell negative isolation kits (Dynal, Invitrogen).

Antibodies—Antibodies and dilutions for Western blots were asfollows: anti-CD22 (Santa Cruz Biotechnology Inc., sc-7932, 1:500),anti-CD45 (Santa Cruz Biotechnology Inc., sc-25590, 1:500), anti-4F2(Santa Cruz Biotechnology Inc., sc-9160, 1:200), anti-major histocom-patibility complex I (Santa Cruz Biotechnology Inc., sc-25619, 1:200),anti-Plexin-B2 (Santa Cruz Biotechnology Inc., sc-34504, 1:200), anti-ST6GalI (Santa Cruz Biotechnology Inc., sc-20926, 1:200), anti-HLA IIDR� (Dako, M0746, 1:200), anti-Basigin (R&D Systems, MAB972,1:200), anti-Siglec-10 (R&D Systems, AF2130, 1:200), anti-transferrinreceptor (Invitrogen, A-11130, 1:200), anti-IgM-HRP (Bethyl Labora-tories, A80–100P, 1:2000). HRP-conjugated secondary antibodieswere obtained from Jackson ImmunoResearch Laboratories. For pri-mary antibodies raised in rabbit, Reliablot (Bethyl Laboratories) milkand HRP-conjugated secondary antibody were used.

Metabolic Incorporation and Enzymatic Engineering—BJA-B K20cells were weaned to serum-free medium (Hyq-SFM, Clontech,Thermo Fisher Scientific, San Jose, CA) over 2 days and acclimatizedto serum-free medium for 4 days to reduce the sialic acid content ofK20 cells. For metabolic incorporation, medium was supplementedwith 3 mM NeuAc (Taiyo Kagaku Co. Ltd.) or 9-aryl azide-substitutedsialic acid (9AAzNeuAc) (synthesized as described earlier (15)) for 18 h.Enzymatic engineering of cell surfaces was performed as describedpreviously (25, 26) on K20 cells cultured in Hyq-SFM or on cellsdesialylated by treatment with 200 milliunits/ml Arthrobacter ureafa-ciens sialidase at 37 °C for 1 h. Briefly, 1 � 108 asialo cells wereincubated in 2 ml of PBS, 0.5% BSA containing 0.65 mM CMP-9AAzNeuAc and 5 milliunits/ml ST6GalI at 37 °C for 1 h.

Lectin Staining and Flow Cytometry—Cells (2 � 105 cells in 100 �lof PBS, 0.5% BSA) were stained with 2 �g of FITC-S. nigra agglutinin(SNA) (Vector Laboratories) for 60 min on ice; washed twice with PBS,0.5% BSA; and analyzed by flow cytometry.

Photoaffinity Cross-linking and Cell Lysis—9AAzNeuAc-K20 B-cellsor human lymphocytes (1 � 107 cells/ml) were incubated with 20�g/ml CD22-Fc in PBS on ice for 30 min and then exposed to ahand-held 254 nm UV source for 30 min. Human IgG (Fc fragment)was used as a control. In the case of photocross-linking to CD22expressed on CHO cells, 9AAzNeuAc-K20 cells resuspended in PBSwere overlaid onto CHO cell monolayers in 6-well plates at 1 � 107

cells/ml and cross-linked as described above. Unbound CD22-Fc orIgG (Fc fragment) and K20 cells, respectively, were washed away withPBS and/or 0.2 mM glycine in PBS, pH 2.2. Cells were lysed at 1 � 107

cells/ml in 50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NonidetP-40 with a protease mixture (Calbiochem).

Immunoprecipitation and Western Analysis—Lysates were incu-bated overnight with Protein G-agarose (Invitrogen) or anti-V5-aga-rose (Sigma) at 4 °C with end-over-end rotation. The immunoprecipi-tates were washed four times with lysis buffer and eluted with 4�lithium dodecyl sulfate buffer (Invitrogen). Immunoprecipitates wereresolved on 10 or 4–12% NuPAGE gels (Invitrogen), transferred tonitrocellulose, and blocked for 1 h with 5% milk in Tris-buffered saline

trans Ligands of CD22

1340 Molecular & Cellular Proteomics 9.6

with 0.05% Tween 20 (TTBS). Blots were incubated with antibodiesdiluted in 5% milk, TTBS; washed with TTBS; and visualized withchemiluminescence.

Fluorescence Microscopy—9AAzNeuAc-K20 cells stained with5-chloromethylfluorescein diacetate (Molecular Probes) were overlaidonto CHO cell monolayers in 6-well plates and photocross-linked.Unbound K20 cells were washed off with PBS and 0.2 mM glycine inPBS, pH 2.2, and the remaining cells were visualized with an invertedZeiss fluorescence microscope. For immunofluorescence micros-copy, freshly isolated murine B-cells were stained with FITC-labeledanti-mouse CD22 (Southern Biotech), FITC-labeled anti-IgM (wholeIgG)/DyLight 549-labeled anti-IgM (Fab fragment) (Jackson Immu-noResearch Laboratories), PE-labeled anti-CD45, or FITC-labeledSambucus nigra agglutinin (Vector Laboratories). Freshly isolated hu-man B-cells were fixed with 4% p-formaldehyde and stained withFITC-labeled anti-human CD22 (BD Pharmingen) or anti-human IgM(Jackson ImmunoResearch Laboratories). Coverslips were mountedwith 4�,6-diamidino-2-phenylindole ProLong Gold antifade reagent(Invitrogen). Images were acquired on the Bio-Rad (Zeiss) Radiance2100 Rainbow laser scanning confocal microscope, collected on aWindows 2000 PC work station running the latest Bio-Rad Laser-Sharp 2000 software, and processed with ImageJ analysis software.

One-dimensional Gel Electrophoresis and Mass Spectrometry (MS/MS)—Protein G immunoprecipitates from lysates of 9AAzNeuAc-K20cells incubated with CD22-Fc with or without photocross-linking wereresolved by gel electrophoresis and stained with EZ-Blue (Sigma).The high molecular mass region was excised into the following slices:band 1, greater than �385 kDa; band 2, �385–165 kDa; band 3,�165–95; and band 4, �95–70 kDa. Gel slices were subjected toin-gel digestion as reported previously (27). Peptides from in-geltrypsin digests were pressure-loaded onto a 100-�m (inner diameter)fused silica capillary column containing 10 cm of C18 resin. Peptideswere eluted from the column using a 2-h gradient with a flow rate of0.25 �l/min directly into an LTQ ion trap mass spectrometer (ThermoFisher Scientific). The LTQ was operated in data-dependent scanningmode with one full MS scan followed by seven MS/MS scans of themost abundant ions with dynamic exclusion enabled. Raw MS/MSdata were searched using the SEQUESTTM algorithm (28) using aconcatenated target/decoy variant of the human IPI database (version3.33, released on September 13, 2007). No enzyme specificity wasstated, and a static modification of �57 Da was specified on cysteineto account for iodoacetamide alkylation. Mass tolerance for precursorions and fragment ions was set to 1.00 Da. SEQUEST data from eachband were filtered and sorted with DTASelect version 1.9 requiring aminimum �CN of 0.8 and XCorr values of 1.8, 2.5, and 3.5 for 1�, 2�,and 3� charge states. The false positive rates estimated by theprogram from the number and quality of spectral matches to thedecoy database were less than 1.0%. When combining peptides into“proteins identified,” all isoforms/individual members of a proteinfamily supported by the data were considered, and the entries withthe most annotation are reported here.

Spectral Count Analysis of One-dimensional Gel Electrophoresis-MS/MS Data for Identification of trans Ligand Candidates—Two rep-licate data sets, each containing a �UV sample and a �UV sample,were subjected to spectral count analysis. Spectral count values ofpeptides derived from proteins from gel slices corresponding to mo-lecular mass �95 kDa (gel band 1, �385 kDa; gel band 2, �385–165kDa; and gel band 3, �165–95 kDa) were analyzed in both �UV and�UV samples. A protein hit was considered to be a potential CD22trans ligand if its peptides were represented by higher spectral countvalues in the gel bands from the �UV sample as compared with thosefrom the �UV sample. To identify potential CD22 trans ligands, a“normalized trans ligand score” was calculated for every protein hit asfollows. First, the difference between the summed spectral counts

(across bands 1–3) in the �UV sample and the �UV sample wascalculated. Next, the spectral counts (across bands 1–3) of both the�UV and �UV samples were summed to obtain the total spectralcounts. The normalized trans ligand score was then calculated as theratio of the difference to the total spectral counts. Following analysisof spectral count values at the peptide level, the normalized transligand scores of various peptides corresponding to a protein ID wereaveraged to obtain a normalized trans ligand score for that protein.The mean and S.D. of the trans ligand scores of the two replicate datasets were also calculated. Data were filtered to remove protein IDsthat were not represented in both replicate data sets and proteinsthat did not have �4 spectral counts in at least one replicate.Environmental contaminants were detected by high spectral countsacross not only gel bands 1–3 but also band 4 (�95–70 kDa) andcontrol bands corresponding to lower molecular masses and wereremoved. Mean trans ligand scores of the remaining protein hitsvaried from 1 to �1. Proteins with mean trans ligand scores �0.33were considered CD22 ligand candidates. Standard deviations be-tween the normalized trans ligand scores of replicate data sets wereused to assess the statistical significance of the spectral count anal-ysis. In addition, the standard deviation of the normalized trans ligandscores obtained for the various peptides corresponding to a proteinusing spectral count values at the peptide level was used to measurethe statistical significance of the trans ligand score at the protein level.Several of the candidate ligands identified by spectral count analysiswere independently verified by Western analysis.

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-MuDPIT—Stable, heavy isotopes of lysine ([13C]Lys) and arginine([13C,15N]Arg) differing from their light amino acid counterparts by 6and 10 Da, respectively, were used to enable extensive coverage ofpeptides following tryptic digestion. K20 cells were maintained inRPMI 1640 medium supplemented with dialyzed serum and light orheavy lysine and arginine (Invitrogen) for 2 weeks prior to performingthe SILAC experiment. Four replicates (5 � 108 cells) each of light andheavy K20 cells were desialylated by treatment with 50 milliunits/ml A.ureafaciens sialidase and sialylated with 9AAzNeuAc by enzymaticengineering. Cells were incubated with 37.5 �g/ml CD22-Fc on ice for30 min, and two replicates each of light and heavy K20 cells wereexposed to UV irradiation for photoaffinity cross-linking. All sampleswere washed extensively and lysed. Lysates from samples of lightK20 cells exposed to UV irradiation were mixed 1:1 with those ofheavy K20 cells not exposed to UV irradiation, and lysates of heavyK20 cells exposed to UV irradiation were mixed 1:1 with those of lightK20 cells not exposed to UV irradiation to yield two pairs of swappedreplicates. The combined lysates were subjected to immunoprecipi-tation with rProtein G-agarose. Reduction, alkylation of cysteines, andprotein digestion with trypsin were performed on-bead, and trypticpeptides were resolved by MuDPIT as described previously (29) usingan LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific). Tan-dem mass spectra were analyzed using the following software anal-ysis protocol. MS/MS spectra were searched with the ProLuCIDalgorithm (30) against the human IPI database (version 3.44, releasedon May 20, 2008 with 71,884 protein entries) concatenated to a decoydatabase in which the sequence for each entry in the original data-base was reversed (31). The search parameters were as follows:precursor ion mass tolerance was 4.5 Da, fragmentation ion masstolerance was 0.6 Da, maximum number of missed cleavages was 2,and static cysteine modification was set to 57.02146. For the heavysearch, arginine and lysine were set to a static modification of10.00827 and 6.020127, respectively. No enzyme specificity wasconsidered for the searches. All searches were parallelized and per-formed on a Beowulf computer cluster consisting of 100 1.2-GHzAthlon CPUs (32). The results were assembled and filtered using theDTASelect (version 2.0) program (33, 34). DTASelect 2.0 uses a linear


Molecular & Cellular Proteomics 9.6 1341

discriminant analysis to dynamically set XCorr and �CN thresholdsfor the entire data set to achieve a user-specified false positive rate(less than 1% at the peptide level in this analysis). The false positiverates are estimated by the program from the number and quality ofspectral matches to the decoy database. When combining peptidesinto proteins identified, all isoforms/individual members of a proteinfamily supported by the data were considered, and the entries withthe most annotation are reported here.

Analysis of SILAC-MuDPIT Data for trans Ligand Candidates—Fourdata sets were analyzed. Two replicate data sets contained proteinsfrom a sample containing light cells photocross-linked to CD22-Fc(L�UV) together with control heavy cells (H�UV), and the remainingtwo replicate data sets contained swapped samples wherein heavycells were photocross-linked to CD22-Fc (H�UV) and mixed withcontrol light cells (L�UV). SILAC ratios were computed by the pro-gram Census using a linear least squares correlation algorithm (35).The ratio between the light versus heavy version of each peptide wascalculated as the slope of the correlation line, whereas the closenessof fit was indicated as the correlation coefficient (r). Only peptideratios with r2 � 0.5 were accepted to remove poor quality data. Thequantified proteins were further filtered to remove proteins that didnot appear in more than one replicate data set. Depending on thesample (e.g. L�UV mixed with H�UV), reciprocals of SILAC (light/heavy) ratios were calculated if required to obtain �UV/�UV ratios.Environmental contaminants were detected by high standard devia-tions between �UV/�UV ratios in swapped replicates and removed.Average �UV/�UV ratios of the remaining protein IDs ranged from0.46 to 37.06. A higher cutoff of 2.17 (reciprocal of 0.46) was chosento factor in the distribution of SILAC ratios. Protein IDs with �UV/�UVratios �2.17 were considered CD22 trans ligand candidates. Stand-ard deviations between �UV/�UV ratios at the peptide level andbetween replicate data sets at the protein level were used to assess

the statistical significance of the measurements. Several of the can-didate ligands identified were independently verified by Westernanalysis.

RESULTS

Incorporation of the Photocross-linker 9AAzNeuAc onto B-cell Surface Glycoproteins—With the goal of identifying B-cellglycoproteins that could serve as trans ligands of CD22, weopted to use our approach of labeling total B-cell glycopro-teins with 9AAzNeuAc, which does not impair CD22 binding toits ligands and allows efficient in situ photoaffinity cross-linking to CD22 (15). Then, following immunoprecipitation ofthe complex using CD22 as a tag, the ligands could beidentified by proteomics and immunochemical techniques.Accordingly, to label cell surface glycoproteins of B-cells,9AAzNeuAc was installed by either metabolic labeling (15, 36)or enzymatic engineering with sialyltransferase, ST6GalI (25)(Fig. 1A). For optimal incorporation of 9AAzNeuAc, we usedK20 cells cultured in serum-free medium because BJA-B K20is a hyposialylated cell line that cannot synthesize its ownsialic acids due to a deficiency in a key enzyme, UDP-GlcNAc-2�-epimerase, but will readily incorporate sialic acidsadded to the culture medium (37). Both metabolic incorporationand enzymatic engineering yielded sialylation profiles similar tothat of “fully sialylated” cells cultured in medium supplementedwith serum as detected with the sialic acid-specific lectin SNA(Fig. 1, B and C, and supplemental Fig. S1).

FIG. 1. Photoaffinity cross-linking of CD22-Fc to glycoproteins on 9AAzNeuAc-K20 B-cells. A, scheme for introducing 9AAzNeuAc ontoglycoproteins of K20 cells by enzymatic engineering or metabolic incorporation. B, flow cytometry plot of native, asialo, and enzymaticallyengineered 9AAzNeuAc-K20 cells stained with FITC-labeled S. nigra agglutinin. C, flow cytometry plot of native, asialo, and metabolicallyengineered 9AAzNeuAc-K20 cells stained with FITC-labeled S. nigra agglutinin. D, scheme for photocross-linking CD22-Fc to glycoproteins on9AAzNeuAc-K20 B-cells. E, photoaffinity cross-linking of CD22-Fc to glycoproteins on 9AAzNeuAc-K20 B-cells. Anti-CD22 and anti-Fc Westernanalyses of rProtein G immunoprecipitates of 9AAzNeuAc-K20 cells incubated with or without CD22-Fc and with or without UV cross-linking isshown. The band corresponding to CD22 is marked with a blue arrow, and cross-linked bands are indicated in purple. Also seen in the anti-Fcblot is the Ig heavy chain indicated with a green arrow. IP, immunoprecipitation.



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trans Ligand Candidates of CD22-Fc on B-cells Identifiedby Proteomics—The ideal experiment for identifying in situligands of CD22 is to perform the UV cross-linking on a cellcontaining membrane-bound CD22 engaged in cell contactwith 9AAzNeuAc-labeled B-cells. However, in preliminary ex-periments with this approach, although trans ligand cross-linking could be readily demonstrated by immunochemicaltechniques (see below), the amount of cross-linked complexthat could be obtained was insufficient to identify the capturedligands by proteomics techniques. Therefore, we opted to firstidentify a subset of the glycoproteins as candidate ligandsusing intact 9AAzNeuAc-labeled B-cells as a source of in situligands and CD22-Fc chimera as the trans interacting recep-tor (Fig. 1D). In principle, the yield of cross-linked proteincould be increased 10–100-fold because B-cells could beprepared in large quantities in a single cell suspension, andCD22-Fc had access to the entire surface of the cell, not justthe point of cell contact. To test this approach, CD22-Fc wasUV cross-linked to 9AAzNeuAc-K20 cells. Uncross-linkedCD22-Fc was then washed away by low pH, and cell lysateswere subjected to immunoaffinity capture with Protein G-agarose to remove non-cross-linked proteins and high mo-lecular weight cis cross-linked CD22 from 9AAzNeuAc-K20cells. Western analysis yielded anti-CD22- and anti-Fc-reac-tive protein species of higher molecular mass than CD22-Fcthat were not seen in the absence of UV exposure (Fig. 1E) or9AAzNeuAc labeling (supplemental Fig. S2A). Similar resultswere obtained with 9AAzNeuAc-resialylated lymphocytes fromhuman blood (supplemental Fig. S2B).

To identify the cross-linked ligands resolved as high mo-lecular weight species, we used a mass spectrometry-basedproteomics approach. Gel sections containing the high mo-lecular weight region of the gel were subjected to in-geltrypsin digestion and nano-LC-MS/MS. Analysis of spectralcount values of peptides identified a total of 24 proteins thatwere found in the high molecular weight region of the UVirradiation-exposed samples relative to the non-UV irradia-tion-exposed samples. Database analysis of these revealedthat 18 were known glycoproteins, and six were annotated aspredicted proteins or nuclear proteins. Only the known glyco-proteins were considered candidate trans ligands in the sub-sequent analyses (Table I). Among them, CD22 was estab-lished as a trans ligand candidate based on spectral hits forC-terminal CD22 peptides (amino acid 327 and above) thatare not found in the recombinant CD22-Fc protein.

As an alternative approach, we exploited SILAC to identifycandidate trans ligand candidates cross-linked to theCD22-Fc chimera (Fig. 2A) (38). SILAC experiments wereperformed by culturing the BJA-B K20 B-cell line in growthmedium with light ([12C]lysine and [12C,14N]arginine) or heavy([13C]lysine and [13C,15N]arginine) stable isotopes of lysineand arginine. Cell surface glycoproteins were then modifiedwith 9AAzNeuAc by enzymatic engineering and allowed tointeract with CD22-Fc chimera. Cells differentially labeled with

light or heavy amino acids were either exposed to UV irradi-ation to induce photocross-linking or left untreated, subjectedto extensive washing, and lysed. Lysates were then combinedand subjected to immunoaffinity enrichment and on-beadtrypsin digestion, peptides were analyzed by MuDPIT-MS/MS, and the ratios of peak intensities of light and heavypeptides in the mass spectra were used to identify proteinsmore abundantly co-immunoprecipitated with CD22-Fc uponphotocross-linking. A total of 19 proteins with SILAC-derived�UV/�UV ratios greater than 2.2 were identified as candidatetrans ligands, all of which were annotated as membrane glyco-proteins in the UniProtKB/Swiss-Prot database (Fig. 2B, TableII, supplemental Table S1 and annexure to Table S1) (39).

In all, the two proteomics approaches yielded 27 glycopro-teins as CD22 trans ligand candidates, 10 of which wereidentified in both approaches (supplemental Fig. S3Aand Table S2). Functional analyses using the Swiss-Prot da-tabase (39) and the on-line DAVID knowledgebase (40) andFatigo tools (41, 42) indicated that a majority of the CD22ligand candidates had a role in immune response, and all 27hits were clustered by their functions in lymphocyte activa-tion, signal transduction, cell adhesion, and transport(supplemental Fig. S3, B and C, and Table S3).

The candidate ligands identified by the proteomics ap-proaches above were validated by immunochemical analy-sis of CD22-Fc photocross-linked to glycoproteins on9AAzNeuAc-K20 cells. Antibodies suitable for Western anal-ysis were obtained for eight of the 10 candidate glycopro-teins identified by both approaches and 11 of the 19 iden-tified by SILAC analysis (supplemental Table S2). Westernanalysis revealed that all were photocross-linked toCD22-Fc with the appearance of higher molecular massform(s) corresponding to the sum of the molecular mass ofone or more molecules of CD22-Fc and the glycoprotein(Fig. 3 and supplemental Table S2). That cross-linking wasCD22-specific was verified by performing control experi-ments in which 9AAzNeuAc-K20 cells were incubated withIgG (Fc fragment) protein instead of CD22-Fc and exposedto UV irradiation. No background was introduced in theWestern analysis, confirming that cross-linking was CD22-specfiic. Representative Western blots with antibodies toCD45, IgM, Basigin, 4F2, HLA I, HLA II, and MCAM areillustrated in Fig. 3.

In Situ CD22 trans Ligands on B-cells in Cell Contacts—Armed with the identities of the membrane-bound B-cell gly-coproteins that are photocross-linked to CD22-Fc, we nextsought to determine whether they would also be detected asin situ trans ligands in the context of cell to cell contact. Toexamine this, we used a CHO cell line expressing CD22 witha C-terminal V5 tag for capture of glycoprotein ligands on theB-cell line BJA-B (subclone K20) labeled with 9AAzNeuAc (Fig.4A). The 9AAzNeuAc-labeled or asialo K20 cells were overlaidonto CHO cells expressing CD22-V5, subjected to UV expo-sure, and washed with low pH to remove non-covalently











TABLE IIdentification 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-linkedCD22-Fc were analyzed in CD22-Fc-cross-linked (�UV) replicate samples as well as control (�UV) replicate samples. Normalized trans ligandscores (see “Experimental Procedures”) were calculated from the spectral count values of the “�UV” and “�UV” samples for every peptideand averaged across all peptides corresponding to a protein ID to obtain the “trans ligand score” for that protein. The average of the “transligand scores” of replicates is reported as the “mean score.” Proteins with mean scores �0.33 were considered candidate ligands. Listed inthe table are details of candidate CD22 ligands identified by this procedure. CD22 itself was established as a trans ligand candidate based onanalysis 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.



bound cells. Extensive cross-linking of cells was observedonly for the 9AAzNeuAc-engineered K20 cells overlaid onCD22-V5-CHO cells, whereas control experiments that werenot subjected to UV treatment or that used K20 cells lacking9AAzNeuAc or CHO cells lacking CD22-V5 expression showedfew residual K20 cells bound to the CHO monolayer (Fig. 4B).

Western analysis of anti-V5 immunoprecipitates confirmedthat UV irradiation resulted in formation of anti-CD22-reactiveprotein species of higher molecular mass than CD22-V5 (Fig.4C). To identify which glycoproteins accounted for the cross-linked species, representing in situ trans ligands of CD22,anti-V5 immunoprecipitates were subjected to Western anal-ysis with antibodies to 11 of the 27 glycoproteins identified astrans ligand candidates in the spectral count and SILAC anal-yses of the CD22-Fc-ligand complexes. Of these, only three,IgM, CD45, and Basigin, showed cross-linking to CD22 (Fig.4D). Although we did not use CHO cells expressing otherV5-tagged proteins as controls, no significant backgroundwas observed upon Western analysis of immunoprecipitatesfrom controls comprising 9AAzNeuAc-K20 cells overlaid onCHO cells (not expressing CD22; Fig. 4D). Notably, cross-linking of CD22 to IgM was particularly robust with little cross-linking to CD45 and Basigin (Fig. 4D and supple-mental Table S2). Moreover, no cross-linking of CD22 wasdetected to HLA I, HLA II, 4F2, Plexin-B2, Siglec-10, CD33,SLC1A5, or MCAM (supplemental Table S2) despite their robustcross-linking to CD22-Fc (Fig. 3) even though several of theseglycoproteins are known to be as or more abundant than IgM onB-cells (43, 44). The results show that the in situ recognition oftrans ligands at the site of cell to cell contact is highly selectivewith IgM as the preferred trans ligand of CD22.

CD22- and Sialic Acid-dependent Redistribution of IgM toSites of Cell Contact of Apposing B-cells—We previouslydemonstrated that CD22 is redistributed to sites of cell con-tact between B-cells as a result of trans ligand interactions onthe apposing cell (9). Given the robust cross-linking of IgM as

a trans ligand of CD22 in cell to cell contacts, we investigatedthe localization of CD22 and IgM on interacting primary mu-rine and human B-cells by immunofluorescence and micro-scopic analysis. As observed previously, CD22 was redistrib-uted to sites of cell contact of interacting murine B-cells (Fig.5A) and also was redistributed to the site of cell contact onprimary human B-cells (supplemental Fig. S4). Interestingly,IgM was also localized preferentially at the sites of cell contactof interacting murine and human B-cells (Fig. 5A andsupplemental Fig. S4). This localization of IgM was also seenin samples stained with the Fab fragment of anti-IgM antibodyand hence was not caused by antibody-mediated cross-link-ing of IgM on apposing cells. In contrast, CD45 was notsignificantly enriched at sites of cell contact (Fig. 5A). Simi-larly, there was diffuse staining with FITC-labeled S. nigraagglutinin, which detects all glycoproteins bearing theNeuAc�2–6Gal sequence recognized by CD22 (Fig. 5A).

To assess the possibility that the IgM localization was aresult of trans interactions with CD22, we compared local-ization of IgM on 300 pairs of contacting B-cells from wildtype and CD22 knock-out mice (representative micrographsare shown in Fig. 5, A and B). Remarkably, IgM was re-cruited to sites of cell contact in 35 � 5% (average � S.E.of four independent experiments) of the interacting pairs ofB-cells from wild type mice but in only 9 � 3% (average �

S.E. of four independent experiments) of interacting B-cellpairs from CD22 KO mice (Fig. 5D) (p 0.0047 in a two-tailed, unpaired Student’s t test). CD45 and SNA localizationremained unaltered (Fig. 5, compare A and B). The resultsstrongly suggest that trans interactions of CD22 are respon-sible for IgM redistribution to the site of cell contact ofinteracting B-cells.

To investigate whether these trans interactions were me-diated by CD22 binding to sialic acids on IgM, we alsoassessed CD22 and IgM localization on 300 pairs of con-tacting B-cells from ST6GalI knock-out mice that lack the

FIG. 2. CD22 trans ligand candidates identified by SILAC analysis. A, scheme for identifying CD22 trans ligand candidates by SILACanalysis of samples subjected to photocross-linking, immunoaffinity isolation, on-bead trypsin digestion, and MuDPIT. B, spectral counts ofprotein hits plotted against average SILAC ratio upon UV cross-linking. Red and blue data points indicate protein hits considered as candidatetrans ligands and non-ligands, respectively. IP, immunoprecipitation.








TABLE IIIdentification 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 samplescontaining either light cells photocross-linked to CD22-Fc (L�UV) and control heavy cells (H�UV) or heavy cells photocross-linked to CD22-Fc(H�UV) and control light cells (L�UV). SILAC ratios of proteins, obtained by averaging the ratios of the constituent peptides, were converted to�UV/�UV ratios. Proteins with �UV/�UV ratios �2.17 were considered candidate ligands of CD22. Listed in the table are details of candidateCD22 ligands identified by this procedure. Standard deviations of the �UV/�UV ratios are not listed for single peptide spectrum-based proteinidentifications. PEPTIDE COUNT, number of unique peptides; %SEQ COVERAGE, percent sequence coverage; STDEV, standard deviation.



sialyltransferase required for synthesis of the sialylatedCD22 ligands (representative micrographs are shown in Fig.5C). As expected, B-cells from ST6GalI knock-out micewere not stained by SNA, and CD22 was not redistributed tothe sites of cell contact in interacting B-cells from ST6GalIknock-out mice (Fig. 5C). Notably, IgM was concentrated atsites of cell contact in only �7 � 2% (average � S.E. of four

independent experiments) of the interacting B-cell pairsfrom ST6GalI knock-out mice, 5-fold lower than contactingB-cell pairs from wild type mice (Fig. 5D) (p 0.0026 in atwo-tailed, unpaired Student’s t test). The results demon-strate that sialic acid-mediated trans interactions of CD22are responsible for IgM redistribution to the site of contactof interacting B-cells.

FIG. 3. Immunochemical validation of CD22 trans ligand candidates. Western analysis was conducted on rProtein G immunoprecipitatesfrom 9AAzNeuAc-K20 cells incubated with CD22-Fc protein or IgG (Fc fragment) (abbreviated IgG-Fc in the figure) with or without subsequentUV 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 animmunochemical 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-CD22Western analysis of anti-V5 immunoprecipitates conducted on lysates from 9AAzNeuAc-K20 cells overlaid onto CHO or CD22-(V5)-CHO cellswith 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 CHOor CD22-(V5)-CHO cells with or without UV cross-linking. Red and purple arrows indicate uncross-linked and CD22-V5-cross-linkedglycoproteins, respectively. Also seen in the anti-Basigin Western blot is the Ig heavy chain marked with a green arrow. Uncross-linked Basiginglycoforms range from �33 to 50 kDa and are not appreciably seen in anti-V5 immunoprecipitates. IP, immunoprecipitation.



DISCUSSION

A major function ascribed to the glycan ligands of CD22 isto modulate the activity of CD22 in BCR signaling by influ-encing its proximity to the BCR complex (2, 5, 6, 45). As aco-receptor of the BCR, CD22 regulates signaling by recruit-ment of the phosphatase SHP-1 and other Src homology 2domain regulatory proteins via tyrosine motifs in its cytoplas-mic domain where proximity to the BCR is required for max-imal effect. Early support of this idea came from the obser-vation that sequestration of CD22 with antibody-coatedbeads enhances activation through BCR ligation (46). Al-though IgM and CD22 are predominantly in distinct microdo-mains in resting murine B-cells, ablation of cis ligands causesincreased co-localization of CD22 and the BCR in raft-clathrindomains, resulting in suppression of BCR signaling (47–49).Inclusion of CD22 ligands in a polymeric T-independent anti-gen also suppresses BCR activation by physically ligatingCD22 to the BCR complex (50, 51). Similarly, co-expression ofCD22 trans ligands on antigen-presenting cells dampens B-cell activation, presumably by recruitment of CD22 to sitesof cell contact in close proximity to the BCR synapse (8, 14).Such observations suggest that trans ligands of CD22 couldparticipate in recognition of self and establish a threshold forB-cell activation and maintenance of peripheral tolerance (14,50, 51).

In addition to the activation of B-cells, CD22-ligand inter-actions have been proposed to mediate other aspects ofB-cell biology including differentiation, antigen presentation,

and trafficking to bone marrow that involve interactions withtrans ligands on other cell types (5, 13, 52, 53). Recent reportsdemonstrate that high affinity cis ligands of CD22 are down-regulated on B-cells during differentiation in germinal centers(54, 55), favoring binding of CD22 on activated B-cells to transligands on other contacting cells. trans ligands of CD22 havebeen documented to exist on B-cells, T-cells, monocytes,endothelial cells, and dendritic cells (9, 10, 13, 18, 56). Previ-ously, we demonstrated that CD22 is redistributed to sites ofB/B-cell contact mediated by sialic acid-dependent interac-tions with trans ligands on the apposing B-cell (8). HomotypicB-cell interactions occur in lymphoid organs and bone mar-row whenever B-cells are in close contact and have beendemonstrated to be important in the regulation of IgE and IgGsynthesis (57, 58), polyclonal B-cell activation (59), mainte-nance of peripheral tolerance (60), differentiation of pre-B-cells into mature B-cells in the bone marrow (61, 62), andbystander transfer of antigens from one B-cell to another (63).Because CD22 plays a role in establishing the threshold forB-cell receptor signaling, identifying the ligands in B/B-cellcontacts that regulate CD22 redistribution to sites of cellcontact is of significant interest.

In this study, we investigated the nature of the trans ligandsof CD22 on B-cells that mediate redistribution of CD22 tosites of cell contact in homotypic B-cell interactions. BecauseCD22 is known to recognize the majority of B-cell glycopro-teins that carry the NeuAc�2–6Gal sequence in cell lysates(15, 17, 18), a major objective was to establish whether CD22

FIG. 5. Microscopic analysis of trans ligand interactions on apposing B-cells. A, immunofluorescence microscopy of murine B-cells fromwild type mice stained with FITC-labeled anti-CD22, FITC-labeled anti-IgM, FITC-labeled S. nigra agglutinin, or PE-labeled anti-CD45. Thewhite scale bar represents 5 �m. B, immunofluorescence microscopy of murine B-cells from CD22 KO mice stained with FITC-labeledanti-CD22, DyLight 549-labeled anti-IgM (pseudocolored green), FITC-labeled S. nigra agglutinin, or PE-labeled anti-CD45. The white scale barrepresents 5 �m. C, immunofluorescence microscopy of murine B-cells from ST6GalI KO mice stained with FITC-labeled anti-CD22, DyLight549-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 thesite of contact. Values plotted are the averages of values acquired in four independent experiments, each obtained by analyzing 100 cells eachin three different regions of a slide preparation. Error bars shown are the S.E. p 0.0047 (CD22 KO) and p 0.0026 (ST6GalI KO) in two-tailed,unpaired Student’s t tests. Wt, wild type.



recognizes all B-cell glycoproteins as trans ligands in situ orpreferentially recognizes a subset of them. We first identified27 trans glycoprotein ligand candidates by proteomics anal-ysis of complexes formed by UV cross-linking CD22-Fc chi-mera to B-cells labeled with 9-aryl azide-NeuAc. This ex-pands the number of identified sialic acid-dependent bindingpartners of CD22-Fc chimera from four (CD45, IgM, CD19,and CD22) in previous reports (15, 16, 18, 20) to 27 (seeTables I and II). The validity of the “hits” was assessed byconfirming that the protein hits formed UV irradiation-inducedcross-linked complexes with CD22 by Western blot analysisusing antibodies available to 11 of these glycoproteins, in-cluding eight of 10 glycoproteins identified by both the spec-tral count and SILAC analyses. The ability of CD22 to usethese glycoproteins as trans ligands at the site of cell contactwas then assessed by cross-linking B-cells labeled with 9-arylazide-NeuAc to CD22-expressing CHO cells followed by anal-ysis of the cross-linked products by Western blot. Remark-ably, only three of these 11 glycoproteins, IgM, Basigin, andCD45, were detected as trans ligands in the context of B-cellsbound to CD22-CHO cells. We do not suggest that they arethe only in situ trans ligands of CD22, only that of the 11analyzed, these three are the only three detected with IgMappearing to be selectively recognized based on the robustdetection of CD22-IgM complexes relative to those of CD45and Basigin (e.g. compare Figs. 3 and 4D).

The inference that IgM is preferentially recognized by CD22as an in situ trans ligand was further supported by the dem-onstration that IgM and CD22 were co-distributed to the siteof contact of interacting primary murine and human B-cellsand that redistribution of IgM to the site of contact was bothCD22- and sialic acid-dependent (Fig. 5 and supple-mental Fig. S4). The conclusion that this interaction is selec-tive is underscored by the fact that surface IgM (43) and CD22(64) are expressed at only �13,000 and �25,000 moleculesper cell, respectively, whereas CD45, a major cell surfaceglycoprotein, is expressed at �150,000 molecules per cell(65, 66). More remarkably, the total sialic acid content of alymphocyte is estimated to be in excess of 109 molecules percell (67) of which a major fraction (�25%) would be found insequences recognized by CD22. Thus, the interaction ofCD22 with IgM at the site of cell contact is highly selective,occurring in the presence of a large excess of other glyco-proteins that carry the glycan sequence recognized by CD22.Although the functional significance of the redistribution ofIgM and CD22 to the site of cell contact in instances ofhomotypic B-cell interactions is not clear (57–63), the result isthat CD22 and IgM are brought together in close proximity onboth cells, which would have the expected consequence ofdown-regulation of signaling from BCRs at the site of cellcontact.

The preferential recognition of IgM as a trans ligand stronglysuggests that factors other than glycan sequence determinethe ability of a glycoprotein on an apposing cell to serve as a

trans ligand of CD22. Such factors may include the geometricdistribution and projection of glycans from the surface of thecell as well as microdomain localization and/or interactionwith the cytoskeleton of the cell. It is remarkable that IgM isselectively recognized by CD22 in trans but is not significantlyrecognized as a cis ligand of CD22 on B-cells (15). Thus, thefactors that make IgM a preferred in situ trans ligand do notmake IgM a suitable ligand for CD22 in cis.

We anticipate that the approach used here can also beused to investigate trans ligands of CD22 for other cells (e.g.T-cells, epithelial cells, dendritic cells, etc.) known to contactB-cells in physiologically relevant contexts (5, 6, 9–14, 53, 66,68). Stamenkovic et al. (22) provided evidence that CD45RO isa physiologically relevant in situ trans ligand of CD22 onT-cells by virtue of blocking binding of T-cells to CD22 withanti-CD45 but concluded that other ligands are also likelyinvolved (11, 69). In principle, this approach can also be usedto examine the cis and trans ligands of other siglecs, providedthat they can accommodate a substituent on sialic acid thatallows covalent cross-linking. In this regard, Kohler and co-worker (70) have demonstrated that N-acyl diazirine-modifiedsialic acids are an effective alternative for photocross-linkingof glycoprotein ligands to CD22. Although this report hasfocused on sialic acid-dependent ligands of CD22, it shouldbe noted that some siglecs, including CD22, also selectivelybind trans ligands that are not sialic acid-dependent, presum-ably via an alternative binding site(s) (56, 71, 72). Such obser-vations emphasize the need to conduct in situ analyses todistinguish physiologically relevant receptor-ligand interac-tions from in vitro interactions that are missing the cellularcontext.

Finally, the results presented here have broader signifi-cance to the general problem of identifying the in situ ligandsof glycan-binding proteins. Most glycan-binding proteins inthe major glycan-binding protein families (e.g. siglecs, C-typelectins, and galectins) interact primarily with the glycan moietyof glycoproteins, which is sufficient for driving ligand interac-tions in vitro (e.g. from cell lysates). However, it is clear thatthe cellular context can significantly restrict the utilization of aglycoprotein as an in situ ligand even if it carries the glycanepitope recognized as a ligand in vitro. Thus, glycoproteinsobserved to be in vitro binding partners are best viewed asligand candidates until verified as physiologically relevant li-gands in situ.

Acknowledgments—We thank Prof. M. Pawlita for the BJA-B K88and K20 cell lines, Dr. Norihito Kawasaki for recloning CD22-CHO-FlpIn cells, Drs. Norihito Kawasaki and Christoph Rademacher formurine B-cells, Dr. Shoufa Han for 9AAz-sialic acid, Prof. Varki forCD22-Fc-pCDM8 plasmid, Anna Tran-Crie for expert assistance inmanuscript preparation, and members of the Paulson laboratory forscientific input.

* This work was supported, in whole or in part, by National Insti-tutes of Health Grants GM60938 and AI50143 (to J. C. P.) and P41RR011823 and BIMR P30NS057096 (to J. R. Y.).





□S This article contains supplemental Figs. S1–S4, Tables S1–S3,and an annexure to Table S1.

¶ To whom correspondence should be addressed: The ScrippsResearch Inst., 10550 N. Torrey Pines Rd., Maildrop MEM-L71, LaJolla, CA 92037. Tel.: 858-784-9634; Fax: 858-784-9690; E-mail:[email protected].

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