B-Cell Maturation Antigen Is Modified by a Single N-Glycan Chain That
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B-cell maturation antigen is modified by a single N-glycan chain that modulates ligand binding and surface retention Han-Wen Huanga,b, Chein-Hung Chenb, Chun-Hung Linb,c, Chi-Huey Wonga,b,1, and Kuo-I Lina,b,1 aInstitute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan; bGenomics Research Center, Academia Sinica, Taipei 115, Taiwan; and cInstitute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan Contributed by Chi-Huey Wong, May 22, 2013 (sent for review April 11, 2013) Glycosylation, an important posttranslational modification process, which has potential implications in regulating cellular response can modulate the structure and function of proteins, but its effect to ligand-mediated stimulation (25). In this study, BCMA was on the properties of plasma cells is largely unknown. In this study, identified as a glycoprotein with a single N-glycosylation site in we identified a panel of glycoproteins by click reaction with alkynyl plasma cell lines, and its glycosylation, especially the sialylation, sugar analogs in plasma cells coupled with mass spectrometry anal- was shown to modulate the function of BCMA. ysis. The B-cell maturation antigen (BCMA), an essential membrane protein for maintaining the survival of plasma cells, was identified Results as a glycoprotein exhibiting complex-type N-glycans at a single N- Use of Sugar Probes in Plasma Cell Lines for Identification of glycosylation site, asparagine 42. We then investigated the effect Glycoproteins. We first used our designed alkynyl sugar probes as of N-glycosylation on the function of BCMA and found that the substrates for fucosylation and sialylation in cell cultures followed dexamethasone-induced apoptosis in malignant plasma cells can by the triazole-forming click reaction using an azido-biotin analog be rescued by treatment with BCMA ligands, such as a proliferation- (26) to identify the labeled glycoproteins of plasma cells (Fig. 1). inducing ligand (APRIL) and B-cell–activating factor (BAFF), whereas We first investigated if the MM cell lines, including H929, removal of terminal sialic acid on plasma cells further potentiated the RPMI8226, and U266, the malignant counterpart of plasma cells, BIOCHEMISTRY ligand-mediated protection. This effect is associated with the in- were incorporated with the sugar analogs. Indeed, we were able to creased surface retention of BCMA, leading to its elevated level on detect the fluorescent signal in cells fed with alkyne sugars, but cell surface. In addition, the α1–3,-4 fucosylation, but not the termi- not the control, after performing the click reaction with azido- nal sialylation, assists the binding of BCMA with ligands in an in biotin, followed by staining with alexa-488–conjugated streptavi- vitro binding assay. Together, our results highlight the importance din (Fig. S1A). Furthermore, immunoblot analysis showed that, of N-glycosylation on BCMA in the regulation of ligand binding and after azido-biotin labeling and streptavidin-HRP blotting, the functions of plasma cells. proteins extracted from the control had only some nonspecific background luminescence, but those extracted from cells cultured -cell maturation antigen (BCMA), classified as tumor necrosis with the probes showed strong luminescence, indicating that the fi Bfactor receptor superfamily 17 (TNFRSF 17), is primarily ex- alkyne sugar analogs were ef ciently incorporated into proteins pressed on the surface of plasma cells, but is absent on naive B (Fig. S1B). The cell surface glycoproteins labeled by the alkyne fi fl cells and on most memory B cells (1, 2). BCMA can be induced by sugar probes were also con rmed by ow cytometry analysis (Fig. stimulation with cytokines in the peripheral blood B cells (1, 2) and S1C). Because the sugar analogs were incorporated into proteins by fi is important for maintaining the survival of long-lived plasma cells plasma cell lines, we identi ed the glycoproteins that labeled by the in bone marrow (3). Upon stimulation with a proliferation- sugar analogs with mass spectrometry analysis. The list of proteins inducing ligand (APRIL) or B-cell–activating factor (BAFF) (4, labeled by the alkynyl fucose and alkynyl N-acetylmannosamine 5), BCMA becomes trimerized, subsequently eliciting a signaling (ManNAc) are shown in Table S1. However, the functions of most fi cascade involved in the activation of MAP kinases and the in- of the identi ed proteins in plasma cells were unclear. Among these duction of anti-apoptotic proteins, such as Bcl-2 and Bcl-XL (4–7). proteins, we were particularly interested in TNFRSF 17 (Inter- BCMA is also implicated in the regulation of antigen presentation national Protein Index: IPI00293877), also known as BCMA, be- cause it is essential for normal and malignant plasma cell survival activity of activated B cells (2) and in the survival, proliferation, – and differentiation of adipocytes (8, 9). The expression of BCMA is (8, 10 15). not restricted to normal tissues. Some cancer cells, such as glio- BCMA Is a Glycoprotein with a Single N-Glycosylation Site. Given that blastoma (10), multiple myeloma (MM) (11), chronic lymphocytic a previous study reported that BCMA was not subjected to N- leukemia (12, 13), and Hodgkin lymphoma (14), express BCMA, glycosylation in the MM cell lines (27) and that, according to the and blocking its interaction with BCMA ligands is known to re- protein database, UniProt (www.uniprot.org), BCMA is a non- duce cellular survival (10–12, 14, 15). In cells of autoimmune glycosylated protein, we sought to further investigate our finding. diseases, such as the fibroblast-like synoviocytes of rheumatoid Purified BCMA from H929 cells was subjected to mass spec- arthritis patients (16) and the plasmablasts from systemic lupus trometry analysis to confirm the presence of glycans. A trypsin- erythematosus (SLE) patients (17), the expression of BCMA is digested fragment, YCNASVTNSVK (amino acid 40–50), was elevated. Particularly, the expression of BCMA on the autoanti- body-producing cells is selectively increased in SLE patients (18). Therefore, disruption of the interaction between BCMA and Author contributions: H.-W.H., C.-H.L., C.-H.W., and K.-I.L. designed research; H.-W.H. its ligands by either receptor-immunoglobilin fragment crystalliz- performed research; H.-W.H., C.-H.C., C.-H.W., and K.-I.L. analyzed data; and H.-W.H., able region (Fc) chimera or antibody was proposed as a thera- C.-H.W., and K.-I.L. wrote the paper. – peutic strategy for the management of malignancies (10, 13, 19 The authors declare no conflict of interest. 21) and autoimmune diseases (22, 23). 1To whom correspondence may be addressed. E-mail: [email protected] or Glycosylation is a common modification process to modulate [email protected]. cell membrane proteins and lipids (24). In addition, the retention This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of proteins on the cell surface is also regulated by glycosylation, 1073/pnas.1309417110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1309417110 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Fig. 1. Flowchart of identification of glycoproteins. Cell extracts from sugar probe-fed cells were sub- jected to click reaction with an azido-biotin analog. Products of the click reaction were purified by strep- tavidin, followed by trypsin digestion. Sugar probe- labeled glycopeptides were released after PNGase F treatment and then analyzed by mass spectrometry. identified as the sole glycopeptide of BCMA. Consistent with the Because the trypsin-digested glycopeptide of BMCA contains result of incorporation of sugar probes, both fucose and sialic acid the N-glycosylation consensus sequence Asn-Ala-Ser (NAS) (Fig. were present in the identified complex-type N-glycans that contain 2C), we suspected that the asparagine (N) residue at amino acid abundant biantennary structure (Fig. 2A). In addition, high-man- 42 is likely the N-glycan site. The cDNA encoding the mutated nose type N-glycans were also found in BCMA (Fig. 2A). We then BCMA with the 42nd amino acid residue changed to Ala (N42A investigated if BCMA contains N-glycans by peptide-N-glycosidase BCMA) was generated for expression. Immunoblot results with anti-FLAG antibody show that only one band could be detected F (PNGase F) treatment to remove the N-glycans from proteins in the extract from 293T cells transfected with the N42A BCMA- and found that, before PNGase F treatment, two or multiple expressing vector, that its molecular weight was identical to the bands were immunoreactive to the anti-BCMA antibody in cell lower band of wild type (WT) BCMA (Fig. 2C), and that this lysates from H929, U266, and RPMI8226 cells (Fig. 2B). After single protein band was resistant to PNGase F treatment (Fig. PNGase F treatment, only one band with the predicted mo- 2C). These data demonstrated that BCMA is an N-glycan mod- lecular weight (20.165 kDa) was detected in these three cell lines ified protein and N42 is the sole N-glycosylation site. (Fig. 2B). To further confirm this result, we transfected 293T cells with a cDNA encoding the FLAG-tagged BCMA. Con- Sialylation at the Terminal N-Glycans of BCMA. Given that the cell sistently, the protein bands immunoreactive to the anti-FLAG surface of murine plasma cells displayed increased sialylation antibody in the cell lysates from the transfected cells were sensi- compared with the stimulated murine mature B cells (28) and that BCMA could be labeled by ManNAcyne, we next examined the tive to PNGase F treatment (Fig. 2C). These findings suggested sialosides linkage of BCMA on MM cells. Sambucus nigra lectin that BCMA contains N-glycans. (SNA), which preferentially binds to the α2–6-linked sialosides, and Maackia amurensis lectin (MAL), which selectively binds to the α2–3-linked sialosides, can bind to H929 and RPMI8226 cells as determined by flow cytometry analysis, but the binding was sig- nificantly reduced on cells pretreated with sialidase, which hydro- lyzes the α2–3- and α2–6-linked sialosides (Fig.