Identification of Siglec Ligands Using a Proximity Labeling Method

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Identification of Siglec Ligands Using a Proximity Labeling Method Lanyi Chang,†,⊥ Yi-Ju Chen,‡,⊥ Chan-Yo Fan,§ Chin-Ju Tang,† Yi-Hsiu Chen,† Penk-Yeir Low,† Albert Ventura,† Chun-Cheng Lin,§ Yu-Ju Chen,‡ and Takashi Angata*,†,∥ †

Institute of Biological Chemistry and ‡Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan § Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan ∥ Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan

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ABSTRACT: Siglecs are a family of receptor-type glycan recognition proteins (lectins) involved in self−nonself discrimination by the immune system. Identification of Siglec ligands is necessary to understand how Siglec−ligand interaction translates into biological outcomes. However, this is challenging because the interaction is weak. To facilitate identification of Siglec ligands, we adopted a proximity labeling method based on the tyramide radicalization principle. Cells that express Siglec ligands were labeled with Siglec−peroxidase complexes and incubated with biotin tyramide and hydrogen peroxide to generate short-lived tyramide radicals that covalently label the proteins near the Siglec−peroxidase complex. A proof-of-principle experiment using CD22 (Siglec-2) probe identified its known ligands on B cells, including CD22 itself, CD45, and IgM, among others, demonstrating the validity of this method. The specificity of labeling was confirmed by sialidase treatment of target cells and using glycan recognition-deficient mutant CD22 probes. Moreover, possible interactions between biotin-labeled proteins were revealed by literature-based protein−protein interaction network analysis, implying the presence of a molecular cluster comprising CD22 ligands. Further application of this method identified CD44 as a hitherto unknown Siglec-15 ligand on RAW264.7-derived osteoclasts. These results demonstrated the utility of proximity labeling for the identification of Siglec ligands, which may extend to other lectins. KEYWORDS: Siglec, sialic acid, lectin, ligand, tyramide, peroxidase, proximity labeling, proteomics

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INTRODUCTION Specific recognition of glycans by endogenous glycanrecognition proteins (lectins) is essential for proper regulation of cell-to-cell communication, such as leukocyte recruitment to the site of inflammation mediated by selectins and their ligands.1 Siglecs are a family of vertebrate lectins involved in immune cell recognition and cell-to-cell interaction.2−4 Siglec− ligand interactions also promote tolerance toward selfantigens.5,6 Recent studies have shown that some Siglecs recognize tumor-associated glycans, which generally favor survival or spread of tumor cells.7−10 Identification of Siglec ligands would allow us to understand the molecular mechanisms of Siglec−ligand interaction and its biological consequences in greater detail, which may eventually lead to a strategy to target such interactions for therapeutic purposes. However, identification of Siglec ligands remains technically © 2017 American Chemical Society

challenging because of the inherently weak interactions between Siglecs and their ligands. Several studies have identified Siglec ligands by conventional affinity purification,11−14 by chemical functionalization of ligands by metabolic engineering,15,16 or by a candidate-based approach.17 However, each of these approaches has shortcomings. For example, conventional affinity purification requires a relatively large amount of recombinant Siglec for the preparation of affinity media and the starting material (cells/tissues) from which the ligand shall be purified. Also, identification of membrane-integral glycoproteins such as Siglec ligands requires detergent-mediated cell/tissue lysis, which destroys higher order protein assembly that may be essential for Received: September 2, 2017 Published: September 13, 2017 3929

DOI: 10.1021/acs.jproteome.7b00625 J. Proteome Res. 2017, 16, 3929−3941

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Journal of Proteome Research

#1−330) followed by a FLAG tag and Fc fragment of human IgG1, was prepared as previously described,29 with modifications. In brief, the protein expression construct (Siglec-2-AvTEK-Fc/pcDNA3.1) was transfected into Expi293F cells (ThermoFisher Scientific, Waltham, MA) using an ExpiFectamine 293 Transfection Kit (ThermoFisher Scientific) in accordance with the manufacturer’s instructions. The cells were cultured for 7 days, with medium changed 4 days after transfection. Culture supernatant was cleared of cell debris by centrifugation at 2000g for 10 min. Cleared culture supernatant was mixed with rProtein A-Sepharose Fast Flow (GE Healthcare, Piscataway, NJ) and incubated overnight at 4 °C. The medium was packed into a disposable chromatography column (Poly-Prep Chromatography Columns, Bio-Rad, Hercules, CA), washed with Dulbecco’s phosphate-buffered saline (D-PBS), treated in column with Arthrobacter ureafaciens sialidase (Nacalai Tesque, Kyoto, Japan) to remove sialic acids, and washed again with D-PBS. Recombinant Siglec-2-Fc protein was then eluted with 0.1 M sodium citrate buffer, pH 3.0, and neutralized with 1/10 volume of 1 M sodium carbonate. The neutralized eluate was concentrated with an UltraFree ultrafiltration device (Millipore, Billerica, MA) and buffer-exchanged to D-PBS. The plasmid construct for the expression of a mutant Siglec-2[R120A]-Fc fusion protein, which is deficient in sialic acid binding (with the Arg120 residue required for sialic acid binding replaced by Ala), was prepared by site-directed mutagenesis of the Siglec-2-Fc fusion protein expression construct mentioned above using the Q5 SiteDirected Mutagenesis Kit (New England Biolabs, Ipswich, MA). The mutated protein was produced as described above. Mouse Siglec-15-Fc and mutant mouse Siglec-15[R143A]-Fc deficient in sialic acid binding were prepared in a similar manner, as previously described.30

Siglec−ligand interaction. Chemical functionalization of sialic acids (for example, by the addition of a photoreactive group) by metabolic labeling overcomes this limitation15,16,18 but requires detailed knowledge of the binding specificity of the Siglec of interest so that the functional group does not interfere with Siglec binding activity. In addition, the metabolic precursor of such chemically functionalized sialic acid must compete against endogenous precursors of sialic acid to be displayed on the cell surface, potentially limiting the efficiency of cell-surface expression.19 Hypothesis-based ligand identification, by definition, requires a short list of ligand candidates, which is obviously not available a priori. A bridging method to facilitate the identification of Siglec ligand candidates in a hypothesisfree manner will be of great help to identify biologically meaningful ligands by a candidate-based approach. Proximity labeling methods,20−23 utilizing a bait protein fused (or in complex) with an enzyme that generates shortlived radical species to label proteins in its vicinity (typically with biotin), have gained popularity as ways to identify constituents of protein complexes. In particular, tyramide radicalization,24 which has been utilized for decades in immunohistochemistry to amplify antibody binding or nucleotide hybridization signals,25−27 is now gaining popularity as a chemical principle of proximity labeling for its relatively high resolution (i.e., small diffusion radius of radicals generated), ease of use, and versatility. In this study, we adopted a proximity labeling method based on the tyramide radicalization principle to identify Siglec ligand candidates in a hypothesis-free manner to facilitate subsequent hypothesis-driven discovery of biologically relevant Siglec ligands. As a proof of principle, we targeted CD22 (Siglec-2) ligands on a B lymphoma cell line. The validity of our method was verified by the significant overlap between the CD22/ Siglec-2 ligand candidates identified by our method and those reported by a previous study that employed a chemical biology approach to systematically identify such candidates.16 Our method was further applied to identify biologically relevant ligand candidates for Siglec-15, a Siglec involved in the fusion of osteoclast precursors by interacting with a hitherto unidentified ligand, demonstrating the utility of the method to discover Siglec ligands in an exploratory setting.

Biotin Labeling of Siglec Ligand Candidates by Biotin Tyramide Radicalization

BJAB (a human B lymphoid cell line) was kindly provided by Dr. Kuo-I Lin (Genomics Research Center, Academia Sinica) and maintained in RPMI1640 supplemented with 20% fetal bovine serum (FBS) and penicillin−streptomycin. RAW264.7 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with 10% FBS and penicillin−streptomycin. Osteoclast differentiation of RAW264.7 cells was induced by culturing the cells in the presence of mouse receptor activator of nuclear factor kappa B ligand (RANKL; 40 ng/mL) for 3 to 5 days.28

Recombinant Siglec-Fc fusion protein (10 μg) was incubated with M2-peroxidase antibody (5 μg; an anti-FLAG monoclonal antibody directly conjugated to horseradish peroxidase (HRP), Sigma-Aldrich, St. Louis, MO) on ice for 30 to 60 min to facilitate complex formation. Cells (20 × 106 cells/reaction) were incubated on ice for 1 h with the Siglec−HRP complex prepared above. The cells were washed twice with 10 mL of 140 mM NaCl in 20 mM Tris-HCl buffer, pH 8.0 (TBS), then incubated with 10 μM compound 7 in 10 mM H2O2 in TBS at room temperature for 10 min. The cells were washed three times with 10 mL of TBS and lysed in 200 μL of lysis buffer (50 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxicholate, 0.1% sodium dodecyl sulfate (SDS), supplemented with protease inhibitor cocktail (cOmplete ULTRA Tablets Mini, EDTA-free; Roche, Basel, Switzerland)) on ice for 30 min. The lysate was cleared by centrifugation (15 000g, 30 min) and immediately used for purification of biotinylated proteins (as described below) or stored at −30 °C until use. For comparison, cell-surface proteins on BJAB cells were labeled with sulfo-NHS-biotin (2.3 mM in D-PBS, on ice for 1 h), which covalently introduces biotin into primary amine groups, in accordance with the protocol provided by the manufacturer (ThermoFisher Scientific).

Preparation of Siglec-Fc Fusion Proteins

Purification of Biotinylated Proteins

Recombinant fusion protein, consisting of the three N-terminal immunoglobulin-like domains of CD22/Siglec-2 (amino acid

To purify biotinylated proteins, we used either streptavidinfunctionalized paramagnetic beads or agarose resin.

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EXPERIMENTAL PROCEDURES

Synthesis of Photocleavable Biotin Tyramide

The synthesis of photocleavable biotin-tyramide (compound 7) is described in detail in the Supplementary Experimental Procedures. Cell Culture

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Journal of Proteome Research Purification with Paramagnetic beads. One mg of streptavidin-functionalized paramagnetic beads (Dynabeads Streptavidin-MyOne C1; ThermoFisher Scientific) was mixed with 100 μL of cleared cell lysate and incubated at room temperature for 1 h with constant shaking (600 rpm, on a horizontal shaker). The beads were collected by magnet (DynaMag-2, ThermoFisher Scientific) and washed extensively with D-PBS containing 0.1% SDS. Captured proteins were eluted from the beads by boiling at 100 °C for 5 min in 50 μL of 1× SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer (Bio-Rad) containing 100 mM dithiothreitol (DTT). Alternatively, the proteins were eluted by photocleavage of the linker in biotin-tyramide by exposure to ultraviolet light (2 × 1 min exposure to 365 nm light; model LC-L1 V3 equipped with collimator-type LED head unit, output ∼1000 mW/cm2; Hamamatsu Photonics, Hamamatsu, Japan) in 50 mM ammonium bicarbonate containing 0.1% RapiGest SF (Waters, Milford, MA), and lyophilized to remove salt. The eluted proteins were subjected to protein identification by mass spectrometry (MS) or SDS-PAGE/Western blot analysis, as described below. Purification with Agarose Beads. 250 μL of streptavidin sepharose high performance (GE Healthcare Life Sciences) was mixed with 250 μL of cleared lysate and incubated at room temperature with gentle agitation (40 rpm, on a rocking shaker). The mixture was centrifuged (1000g, 30 s) to remove the supernatant, and the beads were washed three times with 1 mL of D-PBS containing 0.1% SDS. The beads were aliquoted into two tubes (125 μL each). One tube received 25 μL of 6× SDS-PAGE sample buffer containing 600 mM DTT, was boiled at 100 °C for 5 min, and then was centrifuged to obtain the supernatant (∼25 μL). The other tube received 100 μL of 50 mM ammonium bicarbonate containing 0.1% RapiGest SF, was irradiated with a 365 nm UV lamp (trans-illuminator placed upside-down on the tube with the open cap; 365 nm, 8 W capacity, model LM-26, UVP) for 20 min to cleave the linker in biotin-tyramide, and was centrifuged to obtain the supernatant (∼100 μL). The supernatant was lyophilized and resuspended in 20 μL of 1× SDS-PAGE sample buffer containing 100 mM DTT and boiled at 100 °C for 5 min. The eluted proteins were subjected to protein identification by MS, as described below.

Peptides were loaded onto an analytical C18 column (Acclaim PepMap RSLC, 75 μm i.d. × 25 cm length; ThermoFisher Scientific) packed with 2 μm particles with a pore size of 100 Å and were separated using a segmented gradient for 120 min with the following mobile phases: water with 0.1% FA (buffer A) and 2 to 85% ACN with 0.1% FA (buffer B) at 500 nL/min flow rate. Survey scans of peptide precursors from 400 to 1650 m/z with charge states 2−5 were performed at 60K resolution, and the AGC target was set to 3 × 105 by Orbitrap. Tandem MS was performed by isolation window at 1.6 Da with the quadrupole and AGC target set to 1 × 105 and was used for higher-energy collisional dissociation (HCD) fragmentation detected in Orbitrap at a resolution setting of 15K with a normalized collision energy (NCE) of 27%. The top 15 given precursors were selectively fragmented and scanned out in mass spectra. Protein Identification and Quantitation

The raw data were processed using Proteome Discoverer 2.1 (PD2.1; ThermoFisher Scientific), and peptide identification was performed by Mascot search engine (version 2.3.2) and SEQUEST search engine against the Swiss-Prot database (v2015_12, total 20 193 sequences from human) with a percolator (strict false discovery rate (FDR) of 0.01 and a relaxed FDR of 0.05). The protease was specified as trypsin with two maximum missing cleavage sites. Mass tolerance for precursor ion mass was 10 ppm with the fragment ion tolerance as 0.1 Da. Methionine oxidation, cysteine alkylation by iodoacetamide (carbamidomethyl), and deamidation at asparagine or glutamine were selected as variable modifications. Peptides were considered identified if their individual ion score was higher than the identity score (p < 0.05). To evaluate the FDR ( 5× RA mutant) was 0.83, implying those proteins that were robustly identified in independent runs included a high proportion of specifically labeled proteins and thus are more likely to be ligand candidates. We also compared the eluates from streptavidin-functionalized paramagnetic beads (replicate 2) by linker photocleavage versus heat denaturation in SDS (Figure 3C). Similar numbers of proteins (153 by linker photocleavage, 209 by heat denaturation) were identified by both methods. Again, the proportions of membrane proteins in these samples were not high (0.41 for linker photocleavage, 0.52 for heat denaturation). By applying the same filter based on relative protein abundance in Siglec-2-HRP versus Siglec-2[R120A]-HRP labeled samples, the index improved to 0.68 and 0.60, respectively (Figure 3D). Furthermore, the index for the proteins detected in common in the samples eluted by the two different methods and fulfilling the filtering criterion was 0.85, implying that those proteins identified by different methods are more likely ligands.

Figure 3. Comparison of affinity media and protein elution methods. The numbers in the Venn diagrams represent the number of membrane proteins/the number of all identified proteins and (the proportion of membrane proteins) for each category. The proportion of membrane proteins among all identified proteins was calculated as a surrogate index of specifically biotin-labeled proteins because cellsurface membrane proteins are most likely biotin-labeled by our proximity labeling protocol. (A,B) Number of proteins identified in the two independent replicates of proximity labeling of BJAB cells and affinity purification. Biotinylated proteins were eluted from streptavidin beads by denaturation of streptavidin (replicate 1: streptavidinagarose; replicate 2: streptavidin-paramagnetic beads). (A) Total number of proteins identified by LC−MS. (B) Number of proteins five times or more abundant in the samples labeled with functional Siglec2-HRP than in those labeled with mutant Siglec-2[R120A]-HRP complex. (C, D) Number of proteins identified in the eluates from streptavidin-paramagnetic beads with either streptavidin denaturation or linker photocleavage elution. (C) Total number of proteins identified by LC−MS. (D) Number of proteins five times or more abundant in the samples labeled with functional Siglec-2-HRP than in those labeled with mutant Siglec-2[R120A]-HRP complexes. The full lists of identified proteins are provided in Supplementary Table 1.

Inclusion of additional screening criterion (e.g., peak area >107 or protein identification with three or more unique peptides) further improves the index (not shown). Overall, these results demonstrated that the proximity labeling and affinity purification protocol yielded sufficient amounts of proteins to enable identification by MS, and alternative elution methods (streptavidin denaturation and linker photocleavage) were both effective. Application of additional filtering criterion based on the relative protein abundance in functional versus mutant Siglec-labeled samples is effective to screen out proteins incidentally identified from the list of ligand candidates. Verification of the Enrichment of Siglec-2 Ligand Candidates by Western Blotting

To verify the ligand candidates identified by MS, we performed Western blot analysis. By applying the following criteria ([1] the estimated amount of the protein in the Siglec-2-HRP labeled sample was at least five times more than that in the Siglec-2[R120A]-HRP labeled sample; [2] the protein reproducibly fulfilled the criterion [1] in two independent 3934

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Journal of Proteome Research Table 1. Proteins Identified as CD22/Siglec-2 Ligand Candidates on BJAB Cellsa gene

protein

coverage

emPAI

Mascot score

unique peptides

P20036

HLA-DPA1

11.2

1.4

286.3

P01903b Q95IE3b

HLA-DRA HLA-DRB1

37.4 46.6

15.4 22.7

P08575b P01616c

30.3 11.6

P08195b P20273b P79483

PTPRC IGKV2D28 SLC3A2 CD22 HLA-DRB3

HLA class II histocompatibility antigen, DP alpha 1 chain HLA class II histocompatibility antigen, DR alpha chain HLA class II histocompatibility antigen, DRB1−12 beta chain receptor-type tyrosine-protein phosphatase C Ig kappa chain V−II region MIL

P04440

HLA-DPB1

P11049 P02786b P11836c Q13740 P01909

Q15375 Q10589 P32942b O15427c P01871b Q01650b Q99808 Q8N0W4 Q9H2H9 Q96S97c P30443b Q31612b

CD37 TFRC MS4A1 ALCAM HLADQA1 OR5AC2 HLADQB1 EPHA7 BST2 ICAM3 SLC16A3 IGHM SLC7A5 SLC29A1 NLGN4X SLC38A1 MYADM HLA-A HLA-B

P05107b Q14392 O15031b P20138b O95297 P08581 Q13332 P21854

ITGB2 LRRC32 PLXNB2 CD33 MPZL1 MET PTPRS CD72

accession

Q9NZP5 P01920

4F2 cell-surface antigen heavy chain B-cell receptor CD22 HLA class II histocompatibility antigen, DR beta 3 chain HLA class II histocompatibility antigen, DP beta 1 chain leukocyte antigen CD37 transferrin receptor protein 1 B-lymphocyte antigen CD20 CD166 antigen HLA class II histocompatibility antigen, DQ alpha 1 chain olfactory receptor 5AC2 HLA class II histocompatibility antigen, DQ beta 1 chain ephrin type-A receptor 7 bone marrow stromal antigen 2 intercellular adhesion molecule 3 monocarboxylate transporter 4 Ig mu chain C region large neutral amino acids transporter small subunit 1 equilibrative nucleoside transporter 1 neuroligin-4, X-linked sodium-coupled neutral amino acid transporter 1 myeloid-associated differentiation marker HLA class I histocompatibility antigen, A-1 alpha chain HLA class I histocompatibility antigen, B-73 alpha chain integrin beta-2 leucine-rich repeat-containing protein 32 plexin-B2 myeloid cell surface antigen CD33 myelin protein zero-like protein 1 hepatocyte growth factor receptor receptor-type tyrosine-protein phosphatase S B-cell differentiation antigen CD72

ratio (WT/ RA)

peak area

2

11.1

2.0 × 109

1892.1 2193.3

8 2

20.5 5.1

3.9 × 108 2.3 × 108

5.8 0.6

2237.8 98.6

36 2

24.4 WT only

1.1 × 108 1.0 × 108

29.2 24.3 43.2

2.6 2.6 10.5

1180.8 701.7 1160.0

15 16 3

WT only 44.5 17.5

9.6 × 107 8.9 × 107 8.4 × 107

22.5

2.2

456.5

4

WT only

5.1 × 107

10.7 38.3 29.0 17.2 16.1

0.6 3.4 10.8 0.8 1.8

112.1 1240.1 608.0 336.8 232.3

2 24 7 7 3

WT only 47.0 WT only WT only WT only

5.0 4.7 3.1 2.3 2.1

2.3 28.0

0.2 3.4

74.6 409.5

1 7

WT only WT only

1.7 × 107 1.4 × 107

25.6 18.3 4.8 12.7 6.0 10.8 9.0 15.0 3.9 3.1 17.5 18.5

1.2 0.9 0.3 1.2 0.3 0.8 0.6 0.7 0.3 0.2 0.8 0.9

713.8 171.9 126.3 245.9 105.6 210.9 93.0 252.4 115.2 69.5 254.1 208.0

16 3 3 5 3 3 4 8 2 1 3 2

WT WT WT WT WT WT WT WT WT WT WT WT

only only only only only only only only only only only only

1.1 1.1 1.0 1.0 8.5 8.1 7.5 6.7 5.3 4.2 3.6 2.9

× × × × × × × × × × × ×

107 107 107 107 106 106 106 106 106 106 106 106

6.4 10.7 13.5 11.3 11.9 4.2 0.6 8.6

0.2 0.5 0.7 0.5 0.7 0.2 0.0 0.2

105.8 173.3 817.3 75.1 78.6 164.6 19.3 160.7

4 5 21 3 3 6 1 2

WT WT WT WT WT WT WT WT

only only only only only only only only

2.5 2.4 2.2 2.0 2.0 1.5 4.3 1.4

× × × × × × × ×

106 106 106 106 106 106 105 105

× × × × ×

107 107 107 107 107

a Proteins that fulfilled the screening criteria ([1] the estimated amount of the protein in the Siglec-2-HRP labeled sample was at least five times more than that in the Siglec-2[R120A]-HRP labeled sample; [2] the protein fulfilled criterion [1] in two independent replicates (by heat denaturation in SDS); [3] the protein is known or predicted to be an integral plasma membrane protein) are listed in the descending order of peak area (in the Siglec-2-HRP-labeled sample in Replicate 1), which was calculated as described in the Experimental Procedures. bProteins that were identified as ligand candidates of CD22/Siglec-2 by photoactivatable sialic acid metabolic labeling/proteomics.16 cProteins that are not reported or predicted to be glycosylated.

replicates (by heat denaturation in SDS); [3] the protein is known or predicted to be an integral plasma membrane protein) and counting the HLA-DRB1 variants (individually counted in Figure 3) as one protein, we obtained a list of 36 membrane proteins identified as CD22/Siglec-2 ligand candidates (Table 1). Of these 36 proteins, 14 were reported as CD22/Siglec-2 ligand candidates on BJAB K20 (a variant of the BJAB cell line that is deficient in sialic acid biosynthesis) in a previous study by Paulson’s group, which employed a synthetic sialic acid with photoactivatable functional group.16

We obtained antibodies for 10 ligand candidates, of which seven (CD45, HLA-DRA, HLA-DRB3, CD71/transferrin receptor, plexin-B2, CD22, CD20) could be used for Western blotting (Supplementary Table 2). We used these antibodies to verify that the CD22/Siglec-2 ligand candidates were enriched by the biotin tyramide labeling and affinity purification with streptavidin beads. We found that all seven proteins were indeed enriched by this protocol (Figure 4), verifying the findings based on MS. 3935

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Journal of Proteome Research

Figure 4. Verification of the enrichment of Siglec-2 ligand candidates from BJAB cells by proximity labeling-affinity purification. Enrichment of Siglec-2 ligand candidates by proximity labeling-affinity purification was verified by Western blotting of the eluate from streptavidin beads. An equivalent amount of each ligand candidate protein is present in the cell lysates labeled with Siglec-2-HRP or Siglec-2[R120A]-HRP (lanes 1 and 2 in each panel, respectively), whereas the amount of biotinylated proteins recovered from these cell lysates (lanes 4 and 5 in each panel, respectively) showed a clear difference. The protein being probed is shown on the top of each panel. Solid arrows indicate the band corresponding to the protein of interest. Open arrow in panel F corresponds to reagent carry-over (Siglec-2-Fc).

Protein−Protein Interaction Networks among the Ligand Candidates

To reveal possible spatial relationships among the Siglec-2 ligand candidate proteins, we analyzed protein−protein interaction networks among the 36 proteins (Table 1) using IPA. The analysis revealed direct or indirect interactions, reported or implied, among these proteins (Figure 5; in which proteins are represented by their gene symbols). Interaction of CD22/Siglec-2 with CD45 (PTPRC) 17 and with IgM (IGHM)32 on B cells is well documented. Some other ligand candidates are directly (CD20/MS4A1) or indirectly (MHC class II molecules, by way of the CD20/MS4A1 node) connected to these three nodes. This result implies that some of the CD22/Siglec-2 ligand candidate proteins identified in our study (including CD22 itself) may form multiprotein clusters, although experimental verification is required to demonstrate this.

Figure 5. Protein−protein interaction network among the CD22/ Siglec-2 ligand candidates. CD22/Siglec-2 ligand candidates (Table 1) were subjected to Ingenuity Pathway Analysis to reveal protein− protein interactions reported or implied among these proteins. Unconnected nodes were removed. Proteins are represented by their gene symbols. Oval: transmembrane receptor; inverted triangle: kinase; triangle: phosphatase; trapezoid: transporter; solid line: direct interaction; hatched line: indirect interaction; circle: interaction or action on itself; arrow: action from one protein to another; arrow with an orthogonal bar: inhibition.

Identification of Siglec-15 Ligand Candidates on RAW264.7 Cells

To test whether this approach can be used to discover ligand candidates for other Siglecs, we applied the same proximity labeling method to identify Siglec-15 ligands on the RAW264.7 cell line, which has been used as an osteoclast precursor model.28,33 Previous studies have demonstrated that Siglec-15 is essential for the fusion of osteoclast precursors and RAW264.7 cells,34−36 although its ligand has not been identified. We identified over 300 proteins (Supplementary Table 3) by LC− MS/MS in the sample labeled using the Siglec-15-HRP complex. Applying the following criteria ([1] the protein is 10 times or more abundant in the sample labeled with wild-type

functional Siglec-15-HRP than in the sample labeled with mutant Siglec-15[R143A]-HRP complex; [2] identified with 3 or more unique peptides; [3] known or predicted to be glycoproteins), we narrowed down the ligand candidates to 45 3936

DOI: 10.1021/acs.jproteome.7b00625 J. Proteome Res. 2017, 16, 3929−3941

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Journal of Proteome Research Table 2. Proteins Identified as Siglec-15 Ligand Candidates on RAW264.7 Cellsa accession

gene

protein

coverage

emPAI

Mascot score

unique peptides

P97370b Q62351 Q00651b O35375 P06800b P09055b Q9CY27 P15379b Q61490b Q8R5M8 P97449b B2RXS4b P21956 P10810 Q64735 Q08857b Q91ZX7 Q8R422b Q8R366 P31651 P10852 P26011 Q9QUN7 P09581b P11688 Q8VD58 Q8C129 P97797b Q8K078 Q01965 P11835b Q504P2 Q8CFE6 P21855 Q8K4Q8 P05555 Q62192 Q6P9J9 Q6DFX2 P13597 P01900 Q64277 Q8BTY2 Q99P91b Q8K2P7

Atp1b3 Tf rc Itga4 Nrp2 Ptprc Itgb1 Tecr Cd44 Alcam Cadm1 Anpep Plxnb2 Mfge8 Cd14 Cr1l Cd36 Lrp1 Cd109 Igsf 8 Slc6a12 Slc3a2 Itgb7 Tlr2 Csf1r Itga5 Evi2b Lnpep Sirpa Slco4a1 Ly9 Itgb2 Clec12a Slc38a2 Cd72 Colec12 Itgam Cd180 Ano6 Antxr2 Icam1 H2-D1 Bst1 Slc4a7 Gpnmb Slc38a1

sodium/potassium-transporting ATPase subunit beta-3 transferrin receptor protein 1 integrin alpha-4 neuropilin-2 receptor-type tyrosine-protein phosphatase C integrin beta-1 very-long-chain enoyl-CoA reductase CD44 antigen CD166 antigen cell adhesion molecule 1 aminopeptidase N plexin-B2 lactadherin monocyte differentiation antigen CD14 complement component receptor 1-like protein platelet glycoprotein 4 prolow-density lipoprotein receptor-related protein 1 CD109 antigen immunoglobulin superfamily member 8 sodium- and chloride-dependent betaine transporter 4F2 cell-surface antigen heavy chain integrin beta-7 toll-like receptor 2 macrophage colony-stimulating factor 1 receptor integrin alpha-5 protein EVI2B leucyl-cystinyl aminopeptidase tyrosine-protein phosphatase nonreceptor type substrate 1 solute carrier organic anion transporter family member 4A1 T-lymphocyte surface antigen Ly-9 integrin beta-2 C-type lectin domain family 12 member A sodium-coupled neutral amino acid transporter 2 B-cell differentiation antigen CD72 collectin-12 integrin alpha-M CD180 antigen anoctamin-6 anthrax toxin receptor 2 intercellular adhesion molecule 1 H-2 class I histocompatibility antigen, D-D alpha chain ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2 sodium bicarbonate cotransporter 3 transmembrane glycoprotein NMB sodium-coupled neutral amino acid transporter 1

43.2 50.7 26.9 38.0 12.6 19.3 11.4 5.8 23.5 22.6 24.2 22.0 32.8 36.3 11.0 15.3 5.4 10.8 11.5 8.0 18.4 7.6 15.6 7.8 4.8 11.9 8.9 8.2 9.5 8.1 21.1 18.0 8.9 12.7 6.2 4.0 7.6 4.2 5.1 5.8 28.5 11.3 6.5 6.8 5.4

18.3 7.7 1.6 3.2 0.7 0.9 0.6 0.5 2.2 1.8 2.0 1.3 2.3 3.1 0.5 1.4 0.3 0.5 0.7 0.4 0.8 0.4 1.1 0.4 0.3 0.9 0.4 0.3 0.5 0.5 1.0 1.0 0.7 0.6 0.4 0.2 0.6 0.2 0.3 0.3 1.8 0.5 0.3 0.3 0.4

996.1 2476.7 859.5 1706.3 620.6 617.3 79.5 275.6 483.7 334.1 1261.9 1365.1 475.6 694.9 135.3 200.2 1130.0 509.4 365.2 110.7 379.2 282.8 388.8 343.5 223.8 129.8 311.6 235.3 261.2 170.1 713.8 301.4 201.6 288.4 210.2 89.0 204.4 163.5 96.5 63.4 290.8 99.9 192.4 158.4 59.1

10 33 21 26 14 12 4 4 11 8 19 32 11 10 4 5 21 13 5 3 8 5 11 7 5 4 8 3 5 4 12 5 3 4 5 4 4 4 3 3 7 3 5 3 3

peak area 3.5 2.2 1.1 8.2 7.6 6.0 5.7 5.4 5.3 4.8 4.7 4.4 4.0 3.3 3.3 2.7 2.6 2.5 2.3 2.3 2.2 2.0 1.9 1.9 1.9 1.7 1.7 1.3 1.2 1.1 1.0 1.0 9.8 9.4 9.1 8.9 7.9 7.6 7.3 7.0 6.6 6.4 5.3 3.9 2.4

× × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × ×

108 108 108 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 107 106 106 106 106 106 106 106 106 106 106 106 106 106

a Proteins that fulfilled the screening criteria ([1] the protein is 10 times or more abundant in the sample labeled with functional Siglec-15-HRP than in the sample labeled with mutant Siglec-15[R143A]-HRP complex; [2] identified with three or more unique peptides; [3] known or predicted to be glycoproteins) are listed in the descending order of peak area, which was calculated as described in the Experimental Procedures. All proteins in the list were identified only in functional Siglec-15-HRP labeled samples but not in mutant Siglec-15[R143A]-HRP labeled samples. bTested for direct interaction with Siglec-15 by coimmunoprecipitation.

chemically cross-linked, and Siglec-15 was immunoprecipitated from the cell lysate. The coprecipitated proteins were resolved by SDS-PAGE and analyzed for the presence of Siglec-15 ligand candidates by Western blotting. As shown in Figure 6A, CD44 was coimmunoprecipitated with Siglec-15 by this method, suggesting that CD44 is physically associated with Siglec-15 in this context. To the best of our knowledge, this is the first time that a glycoprotein ligand for Siglec-15 has been identified. Several other ligand candidates were also tested in a similar

proteins (Table 2). Notably, some of these candidates are reported to be involved in osteoclastogenesis.37−43 To demonstrate that at least some of these Siglec-15 ligand candidates directly interact with Siglec-15 on live cells, a coimmunoprecipitation experiment was performed for selected proteins (marked with b in Table 2). The proteins with implied involvement in osteoclast development or differentiation in the literature were selected. RAW264.7 cells were differentiated in the presence of RANKL, the cell-surface proteins were 3937

DOI: 10.1021/acs.jproteome.7b00625 J. Proteome Res. 2017, 16, 3929−3941

Article

Journal of Proteome Research

Figure 6. Evidence of direct interaction between Siglec-15 and CD44 on RAW264.7 cells. (A) RAW264.7 cells were differentiated into osteoclastlike multinucleated cells in the presence of RANKL for 5 days, and cell surface proteins were chemically cross-linked with DTSSP. Cell lysates were prepared, and Siglec-15 was immunoprecipitated with monoclonal antibody or control antibody. Coprecipitated proteins were separated by SDSPAGE and probed with rabbit antimouse CD44 antibody. Solid arrow indicates CD44. Open arrows likely correspond to reagent carry-over (mouse IgG heavy and light chains and protein G leaching from protein G-paramagnetic beads). (B) Expression of CD44 protein was efficiently suppressed by shRNA (clone TRCN0000262945). Solid arrow indicates CD44. (C) Binding of Siglec-15-Fc to CD44 knockdown RAW264.7 cells (blue) was weaker than that to control cells (red). Solid black line represents negative control staining with TREM1-Fc. (D) CD44 knockdown RAW264.7 cells formed less multinucleated (fused) cells than control RAW264.7 cells in the presence of RANKL. Cells were TRAP activity-stained.

actions in a relatively unbiased manner, which has been technically challenging thus far. Our pilot experiment, exploring the CD22/Siglec-2 ligands on a human B-cell line using a Siglec-2-HRP complex as a probe, yielded a list of proteins including those previously reported to be functional ligands of CD22/Siglec-2 (Table 1). For example, CD22/Siglec-2 on B cells forms homomultimeric clusters and functions as a cis-ligand for CD22/Siglec-2 itself.15 IgM is a cis-ligand of CD22/Siglec-232 as well as a trans-ligand involved in B-cell−B-cell interaction.16 CD45/PTPRC was also implied to be a trans-ligand of CD22/Siglec-2,17,44 although its functionality in B-cell−B-cell interaction was brought into question by another study.45 Regardless, the presence of these proteins in the list of ligand candidates identified by the protocol confirms that the method is sensitive enough to identify these important ligand candidates from a relatively small number of cells. Many proteins identified as potential ligands of CD22/Siglec-2 in our study have never been recognized as such. For example, MHC class II heterodimer was reported to functionally interact with CD22/Siglec-2 in calcium signaling,46 whereas another paper suggested that

manner, but we have not obtained convincing results demonstrating their direct interaction with Siglec-15 (data not shown). To verify that CD44 is a Siglec-15 ligand, we knocked-down CD44 in RAW264.7 cells (Figure 6B) and probed with Siglec15-Fc protein by flow cytometry (Figure 6C). CD44 was efficiently knocked down with shRNA (clone TRCN0000262945), and this resulted in the reduced binding (∼40% reduction) of Siglec-15 to the cells. Moreover, CD44 knockdown RAW264.7 cells showed reduced cell fusion in the presence of RANKL (Figure 6D), implying that the interaction between CD44 and Siglec-15 is involved in RAW264.7 cell fusion.

■

DISCUSSION We demonstrated that proximity labeling using peroxidase and biotin tyramide can be adopted to introduce a biotin label into Siglec ligand candidates on live cells and that the quantity of biotinylated proteins recovered by affinity purification is sufficient for their identification by tandem MS. This approach expands available strategies to identify Siglec−ligand inter3938

DOI: 10.1021/acs.jproteome.7b00625 J. Proteome Res. 2017, 16, 3929−3941

Article

Journal of Proteome Research CD22/Siglec-2 does not directly interact with MHC class II.47 Verification of the functional importance of the interaction between CD22/Siglec-2 and new ligand candidates will require further mechanistic investigation, which is beyond the scope of this study. Interaction between recombinant soluble Siglec−HRP complex and cell-surface glycoproteins is expected to mimic trans-interaction between native Siglec on a cell and its ligands on another cell rather than cis-interaction between Siglec and its ligands on the same cell. Therefore, our method may be more suitable for the identification of trans-ligand. However, the interaction between Siglec−HRP complex and cell-surface glycoproteins is an imperfect proxy of the natural Siglec−ligand interaction in cell−cell contact zone (e.g., immunological synapse), and the ligand candidates identified by the protocol described in this paper likely encompass broad range of glycoproteins including functional ligands. Thus an obvious caveat of our method is that not all of the proteins identified are biologically relevant. Rather, this method provides a list of likely ligand candidates, which includes biologically relevant ligands. Identification of biologically relevant ligands still requires an additional approach (such as chemical cross-linking and coimmunoprecipitation, proximity ligation, or RNAi-mediated functional evaluation) to demonstrate the interaction of the lectin and its ligand candidate in a biological context. In addition, from a technical point of view, we acknowledge the possible problem of diffusion of the biotin-tyramide radical intermediate, as attested to by the presence of nonglycosylated proteins in the list of ligand candidates (e.g., CD20/MS4A1; Table 1 and Figure 4G). Although the diffusion radius of radicalized biotin tyramide is estimated to be small (