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B-cell maturation is modified by a single N- chain that modulates 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 , 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) , an important posttranslational modification process, which has potential implications in regulating cellular response can modulate the structure and function of , 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 anal- was shown to modulate the function of BCMA. ysis. The B-cell maturation antigen (BCMA), an essential membrane for maintaining the survival of plasma cells, was identified Results as a glycoprotein exhibiting complex-type N- at a single N- Use of Sugar Probes in Plasma Cell Lines for Identification of glycosylation site, 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 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 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 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 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 in the peripheral 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 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 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), (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..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 trometry analysis to confirm the presence of glycans. A trypsin- erythematosus (SLE) patients (17), the expression of BCMA is digested fragment, YCNASVTNSVK ( 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 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]. 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 were released after PNGase F treatment and then analyzed by mass spectrometry.

identified as the sole 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 -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 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. 3 A and B). We next examined if these sialoside linkages occur on BCMA. A solution of purified FLAG-tagged BCMA isolated from RPMI8226 or H929 transfectants was added into anti-FLAG–coated plates, followed by incubation with SNA or MAL. The results in Fig. 3 C and D indicated that both recognized intact BCMA better than the sialidase-treated BCMA. Reciprocally, we preadded lectins onto streptavidin-coated plates before incubation with BCMA, and the result showed that intact BCMA bound better to SNA or MAL than the sialidase-treated BCMA did (Fig. 3 E and F). These data suggest that the N-glycans of BCMA are composed of both α2–3- and α2–6-linked sialosides at the glycan terminal.

Sialylation Suppresses the Prosurvival Activity of BCMA Ligands. Because glycosylation can be involved in the regulation of pro- tein function (24), and the major function of BCMA is to promote cell survival (3), we sought to study whether sialylation participates in BCMA-mediated cell survival in the protection of apoptosis induced by dexamethasone (DEX). DEX, a glucocorticoid analog, is a therapeutic drug used to treat patients suffering from MM. MM cells undergo apoptosis following DEX treatment, which can Fig. 2. BCMA is a glycoprotein. (A) Glycans identified on BCMA from H929 be protected by the treatment with the ligands of BCMA (29). In cells by glycopeptide profiling. Percentages represent the glycan type the basal state, RPMI8226 cells treated with or without sialidase identified from the analysis. (triangle, Fuc; diamond, NeuAc; square, Hex- have a similar number of apoptotic cells (Fig. 4A). Although DEX NAc; circle, Hex) (B) PNGase F treatment caused mobility shift of BCMA caused more apoptosis in sialidase-treated cells than in sialidase- detected by immunoblotting. Cell extracts from H929, RPMI8226, and U266 untreated cells, the difference is not statistically significant (Fig. cells were incubated with PNGase F, followed by immunoblotting with anti- BCMA antibody. (C) N42 of BCMA is the N-glycosylation site. Cell extracts 4B). Indeed, the BCMA ligand APRIL can partially protect sia- from 293T cells transfected with WT or N42A BCMA-expressing vector were lidase-untreated and -treated cells from DEX-induced apoptosis treated without or with PNGase F and then subjected to immunoblotting as previously reported (29) (Fig. 4A), but the extent of rescue is not with the indicated . significant between these two groups (Fig. 4C). It is noted that

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1309417110 Huang et al. Downloaded by guest on September 29, 2021 treatment with sialidase resulted in an elevated amount of BCMA on the plasma cell surface (Fig. 5A). Likewise, H929 or RPMI8226 cells treated with α2–3 sialidase displayed an increased level of surface BCMA (Fig. S3A). This finding implied that both α2–3and α2–6 sialylation may hamper the presence of BCMA on the cell surface. This elevated BCMA on the cell surface caused by treat- ment with sialidase was sustained for at least 24 h (Fig. S3 B and C). In addition, by treating the cells with cycloheximide (CHX) to inhibit the synthesis of new proteins, we were able to calculate the rate of BCMA accumulated on the cell surface after removing the terminal sialic acid. We found that treatment with CHX did not affect the accumulation of BCMA on RPMI8226 and H929 cells, but after sialidase treatment, the accumulation ofBCMAwasenhancedupto160–180% (Fig. 5B). Consis- tently, treatment with α2–3 sialidase also promoted the re- tention of BCMA on the surface of RPMI8226 and H929 cells (Fig. S3D). Furthermore, we examined if removal of N-glycans by PNGase F can abrogate the effect of sialidase on promoting the surface retention of BCMA. Although PNGase F only partially removed surface N-glycans (Fig. S4), we were still able to observe that treatment with PNGase F abolished the enhanced retention of BCMA resulted from sialidase treatment (Fig. 5C). In addi- tion, in the presence of CHX, treatment with sialidase cannot promote the surface retention of N42A BCMA (Fig. 5D and Fig. S5), indicating that sialylation on the N-glycans of BCMA at N42 reduces the retention of BCMA on the cell surface.

N-Glycan of BCMA Is Required for Ligand Binding. Although the in- BIOCHEMISTRY Fig. 3. Glycans on BCMA are terminally modified by sialic acid. (A and B) creased binding of ligand may result from the elevated level of FACS shows the binding with SNA (A) or MAL (B) on H929 and RPMI8226 cells receptors on the cell surface, it remains possible that the binding pretreated with or without sialidase. (C and D) ELISA shows the binding of between BCMA and the ligand per se was enhanced in the presence lectins with BCMA. Full-length BCMA purified from transfectants was pre- or absence of certain types of glycans. Therefore, we examined if treated with or without sialidase and added to anti-FLAG antibody coated sialylation and other types of N-glycans modulate ligand–receptor plates, followed by the biotinylated SNA (C) or MAL (D). (E and F) ELISA interaction by sequential enzymatic digestion with glycosidases and shows the binding of BCMA to lectins. Biotinylated SNA (E) or MAL (F) was an in vitro binding assay (Fig. 6A). We found that removal of sialic bound to streptavidin-coated plates before mixing with sialidase-treated or acid on the purified BCMA had no effect on the binding with APRIL untreated full-length BCMA. Results in A and B are representative of three and BAFF (Fig. 6 B and C, respectively). Given that the N42 residue or four independent experiments, and the number in the histogram indi- cates the mean of fluorescence. Results in C−F represent mean ± SEM of of BCMA is close to the domain required for ligand binding (30), three or four independent experiments. *P < 0.05 and **P < 0.01. we sought to examine whether the N-glycans participate in ligand binding. Indeed, of the whole N-glycan by PNGase F re- duced the binding of BCMA with exogenously added APRIL or another BCMA ligand, BAFF, can protect sialidase-treated cells BAFF compared with mock-treated BCMA (Fig. 6 D and E). from DEX-induced apoptosis more effectively (Fig. 4 A and B) Compared with wild-type BCMA,theN42ABCMAmutantalso and that the extent of rescue increased about 15% compared with consistently exhibited decreased binding with APRIL and BAFF sialidase-untreated cells (Fig. 4C), suggesting that sialylation on (Fig. 6 F and G, respectively). Following the consequence of se- BCMA may reduce the survivaleffectoftheBAFF-BCMA quential enzymatic digestion with various glycosidases, we found that pathway. We next examined which sialylation—α2–3orα2–6 digestion with sialidase and β-galactosidase to remove the sialylation—was important for this regulation. The results in Fig. residue decreased the binding of APRIL and BAFF with BCMA S2 show that removal of α2–3 sialylation can further enhance the (Fig. 6 B and C, respectively). Likewise, sequentially treating BCMA protective effect of BAFF on DEX-induced apoptosis, although with N-acetylglucosaminidase and further reduced its the extent of rescue was only marginal (Fig. S2C). These results binding with ligands (Fig. 6 B and C). Fucosylation, normally suggest that both α2–3andα2–6 sialylation on BCMA may inhibit attaching to the N-acetylglucosamine residue, may also affect ligand BAFF-mediated cell survival. binding. Indeed, BCMA treated with L- showed reduced abilitytobindwithAPRILandBAFF(Fig.6H and I, respectively). Removal of Sialylation Increases the Binding of Ligands to Plasma Cell Together, these data indicate that the N-glycans, but not terminal Surface. We next examined if sialylation modulates the binding sialylation, aid in the binding of BCMA with ligands in vitro. between BCMA and its ligands. Compared with control cells, the binding of APRIL with RPM8226 and H929 cells was slightly in- Discussion creased after removal of sialic acid (Fig. 4D). It is noted that the Here, we used sugar analogs coupled with mass spectrometry binding of BAFF to RPMI8226 or H929 cell surface was enhanced analysis (26) to identify the critical glycoproteins involved in plasma by sialidase treatment and the enhancement was more significant cell function and revealed that BCMA is an N-glycosylated protein. than APRIL (Fig. 4E). However, a previous study by Gras et al. (27) suggested that BCMA is not a glycoprotein in human MM cells. This discrepancy could be Sialylation Reduces the Retention of BCMA on the Cell Surface. The due to the use of different antibodies and protein extraction pro- enhancement of ligand binding to the cell surface after sialidase tocols. Another possibility is that the amount of glycan-modified treatment may result from the increased levels of receptor on the BCMA is not sufficient to be noted in the previous report. The cell surface. To test this possibility, we measured the receptor notion that BCMA is a glyocoprotein is further confirmed level of BCMA. Interestingly, compared with untreated control, by our analysis of the glycan profiles and identification of the

Huang et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 Fig. 4. Sialylation affects BCMA ligand-mediated protection from apoptosis induced by DEX. RPMI8226 cells pretreated with or without sialidase were treated with DEX in the absence or presence of APRIL or BAFF. Three days later, cells were subjected to annexin V staining by FACS analysis. (A) One representative result of three independent experiments is shown; the number in the dot plot indicates the percentage of annexin V–positive cells. (B) The mean value ± SEM of three independent experiments from A.(C) Percentage of rescue of apoptosis as determined by (% of apoptosis caused by DEX–% of apoptosis induced by DEX in the presence of BCMA ligand)/(% of apoptosis induced by DEX). (D and E) Histograms of FACS analysis show that removal of sialylation increases the binding of ligands for BCMA with cell surface. H929 or RPMI8226 cells were pretreated with sialidase and then incubated with Fc-APRIL (D) or Fc-BAFF (E), followed by detection with FACS analysis. The number in the histogram indicates the mean of fluorescence. Bar graphs below D and E show statistical analysis of ligand binding after treatment of cells with sialidase in three independent experiments. Results are mean ± SEM. *P < 0.05 and **P < 0.01.

N-glycosylstion site of BCMA at the N42 residue. Moreover, counteracts the constitutive endocytosis of membrane proteins disruption of the N-glycosylation site by site-directed mutagensis (39). Because BCMA has only one N-glycan chain with a low abolished the presence of the glycosylated form of BCMA. percentage of tri- and tetra-antennary glycans, BCMA may not be De-sialylation by knockdown of sialylatransferase or treatment targeted by . Another possibility is that the sialic acid- with sialidase enhanced the downstream signaling of epidermal binding Ig-like lectin (), another type of animal lectin with the receptor (EGFR) by promoting dimerization and ability to recognize sialic acids (40), may be involved. Recent studies internalization of EGFR, leading to the augmentation of cell indicated that may act as endocytic receptors, such that ov- proliferation or migration (31, 32). Apoptosis induced by the ac- albumin or cytotoxin conjugated with antibody/sugar ligands tivation of the Fas death receptor is also repressed by sialylation specific for siglecs can be delivered into B cells, , or through the reduction of receptor internalization that is required mouse embryonic fibroblasts (41–43). Especially in the case of for transduction of apoptotic signals (33). Additionally, sialylation CD22, which is expressed on B cells (44), the siglec level on the cell prevented the binding of , a type of animal lectin, with surface can be regulated by clathrin-mediated endocytosis and fi CD45 or integrins modi ed with N-acetyl-D-lactosamine (Lac- recycles between the cell surface and endosomal compartments NAc), thereby inducing apoptosis (34, 35). We also found that (42). Hence, we assume that removal of sialylation on BCMA may sialylation suppresses ligand-mediated cell survival by modulating disrupt the binding with siglecs, thereby preventing the endocytosis surface retention of BCMA and that sialylation diminished the of BCMA along with siglecs and resulting in the increased re- apoptosis of MM cells triggered by administration of the death tention of BCMA on the cell surface. However, further experi- ligand of the TNF-related apoptosis-inducing ligand () ments are required to prove our hypothesis. receptor (Fig. S2D). All these results support the notion that sia- Our finding that the N-glycans on BCMA affect its binding to lylation negatively regulates the function of protein substrates. ligands is reflected by the fact that the N-glycosylation site is The glycans on the plasma cell surface could be involved in binding with lectins. Depletion of galectin-3, for example, reduced proximal to the ligand-binding domain of BCMA. The reduction the surface expression of EGFR (36), and the retention of the of ligand binding is not due to the alternation of renal epithelial channel transient receptor potential cat- when the glycosylation site is disrupted because we have com- ion channel subfamily V member 5 was increased by binding with pared the secondary structure of N42A BCMA with WT BCMA galectin-1, leading to the decreased dynamin-dependent inter- by circular dichroism (CD) and have found that their overall nalization (37). In our case, because a previous study showed that structures were indistinguishable (Fig. S7A). We also compared removal of sialic acids on the plasma cell surface enhanced the the CD spectrum of PNGase F-treated BCMA with intact binding of galectins, such as galectin-1 and galectin-8, with the cell BCMA and found that the PNGase F treatment did not generally surface (28), we have thus hypothesized that the increased re- affect the secondary structure of BCMA (Fig. S7B). The possi- tention of BCMA on the cell surface following sialidase treatment bility that the reduced binding to ligand is due to protein stability may be due to the effect of galectins. However, we found that was excluded because BCMA with inner-core trisaccharides addition of LacNAc, the pan-inhibitor of galectins (38), did not capable of maintaining protein stability (45) still showed reduced influence the surface retention of BCMA on plasma cells regard- ligand binding. We also showed that fucosylation, but not sia- less of the sialidase treatment (Fig. S6). Previous study also showed lylation, directly affects ligand binding and that different motifs of that the number of N-glycans and their degree of branching on glycans have distinct effects. Nevertheless, we suspect that the membrane proteins affect the binding of galectin, which overall N-glycan structure of BCMA may facilitate ligand binding.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1309417110 Huang et al. Downloaded by guest on September 29, 2021 Fig. 5. Sialylation influences surface level of BCMA. (A) Histograms show that removal of sialic acid results in the increase of surface BCMA. H929 or

RPMI8226 cells treated with or without sialidase were subjected to FACS analysis with APC-conjugated anti-BCMA antibody. (B) Removal of sialic acid results in BIOCHEMISTRY the accumulation of preexisting BCMA on the cell surface. H929 or RPMI8226 cells were treated with CHX in the absence or presence of sialidase, followedby FACS analysis with APC-conjugated anti-BCMA antibody. (Right) Relative level of BCMA on H929 and RPMI8226 cell surface following treatment with sialidase (C) Removal of N-glycans by PNGase F abolishes the effect of sialidase on surface BCMA. CHX-treated H929 or RPMI8226 cells were treated with or without PNGase F in the presence of sialidase. The level of preexisting BCMA on the cell surface was detected by APC-conjugated anti-BCMA antibody by FACS analysis. (D) The level of N42A BCMA expressed on RPMI8226 cells did not change after sialidase treatment. RPMI8226 cells transfected with mock control and N42A-BCMA were treated with sialidase in the presence of CHX, followed by FACS analysis with APC-conjugated anti-BCMA antibody. (A–D) Each histogram is based on data from three independent experiments; the number in the histogram indicates the mean of fluorescence. (Right) Statistical analysis of three independent experiments. Results are mean ± SEM. *P < 0.05 and **P < 0.01.

In summary, here we used sugar analog probes to identify BCMA the life span of plasma cells or the treatment outcome of diseases. as a glycoprotein with a single N-glycan site that is involved in the On the other hand, the N-glycan and α1–3,-4 fucosylation enhance regulation of surface retention and ligand binding. Thus, manipu- the binding of ligand to BCMA, shedding on the exploitation lation of sialylation on normal or malignant plasma cells may affect of strategies to improve the efficacy of receptor-based therapeutics.

Fig. 6. N-glycans modulate the binding of BCMA with ligands. (A) Illustration of glycosidase treatment. (B and C) Sequential digestion of glycosidases reveals the glycan motifs required for binding with BCMA. Extracts from H929 and RPMI8226 transfectants expressing FLAG-tagged BCMA were prebound in 96-well plates coated with anti-FLAG antibody and then treated with glycosidases (as indicated in A) followed by adding Fc-APRIL (B) or Fc-BAFF (C). (D and E) PNGase F treatment impairs ligand binding to BCMA. Extracts from FLAG-tagged BCMA-expressed RPMI8226 transfectants were prebound by anti-FLAG antibody- coated plates and then treated with PNGase F, followed by addition of Fc-APRIL (D) or Fc-BAFF (E) to detect the binding of ligands by HRP-conjugated anti- human IgG-Fc antibody. (F and G) Binding of ligands with N42A BCMA was less than with wild-type BCMA. Extracts from 293T transfectants expressing either WT or N42A FLAG-tagged BCMA were prebound by anti-FLAG antibody in 96-well plates, followed by addition of Fc-APRIL (F) or Fc-BAFF (G). (H and I) Fucosylation is involved in ligand binding of BCMA. Extracts from H929 or RPMI8226 transfectants expressing exogenous FLAG-tagged BCMA were prebound in anti-FLAG antibody-coated 96-well plates and then treated with L-fucosidase followed by addition of Fc-APRIL (H) or Fc-BAFF (I). Statistical analysis of three or four independent experiments is shown by mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Huang et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 Materials and Methods CHX (10 μM; Sigma-Aldrich) was added to culture. The fluorescent intensity Cell Culture and Reagents. Human multiple myeloma cells H929, U266, and of stained samples was detected by FACS Canto (BD Biosciences), and results RPMI8226 were maintained in RPMI-1640 medium (Invitrogen) supplied with were analyzed by FlowJo (TreeStar). 10% (vol/vol) FBS (Biological Industries). Human 293T cells were cultured in DMEM (Invitrogen) supplied with 10% (vol/vol) FBS. 293F cells were cultured Glycosidase Treatment. Cells were washed with fasting medium [RPMI1640 in freestyle medium (Invitrogen). β-Mercaptoethanol (50 μM; Invitrogen) was with 0.5% (vol/vol) BSA] and then incubated with PNGase F (50,000 U/mL; New added to the medium for H929 cells. For sugar analog incorporation, cells England Biolabs), α2–3 sialidase (1,000 U/mL; New England Biolabs), or sia- were fed with alkyne fucose (200 μM), alkyne ManNAc (25 μM), or un- lidase (1,000 U/mL; New England Biolabs) for 4 h at 37 °C. Heparinase I (10 U/

modified sugar. All cells were cultured at 37 °C with 5% (vol/vol) CO2, except mL; Sigma-Aldrich) was added in APRIL ligand-binding assay. For the in vitro 293F cells that were cultured at 37 °C with 8% (vol/vol) CO2. ligand-binding assay or lectin binding, sialidase (50 mU/mL; QA-Bio), galac- tosidase (30 mU/mL; QA-Bio), glucosaminidase (400 mU/mL; QA-Bio), man- Flow Cytometry. For sugar analog incorporation, click reaction was performed nosidase (100 mU/mL; QA-Bio), or L-fucosidase (5 mU/mL; QA-Bio) was by incubation with azido-biotin, followed by incubation with alexa488- incubated with cell extracts or purified BCMA in supplied buffer at 37 °C conjugated streptavidin (1:100 dilution; BD Biosciences) as previously de- for overnight. scribed (46). For ligand binding, cells were incubated with Fc-APRIL or Fc-BAFF (1 μg/mL) for 30 min on ice, followed by incubation with biotin-conjugated Other Methods. Please see SI Materials and Methods. anti-human IgG-Fc antibody (1:200 dilution; eBioscience) for 15 min on ice and streptavidin-allophycocyanin (APC) (1:200 dilution; BD Biosciences) for 15 min Statistics. Two-tailed Student t test was used for all experiments. P < 0.05 was on ice sequentially. For lectin binding, cells were incubated with biotin- considered significant. conjugated SNA (1 μg/mL; Vector Laboratories), MAL (1 μg/mL; Vector Labo- μ ratories), or Phaseolus vulgaris leucoagglutinin (L-PHA) (1 g/mL; Vector Lab- ACKNOWLEDGMENTS. We thank Genomics Research Center Mass Spec- oratories) for 30 min, followed by incubation with streptavidin-APC for 15 min. trometry Core Facility in Academia Sinica. The work was supported by For detection of surface BCMA, cells were incubated with APC-conjugated Academia Sinica and by the National Science Council, Taiwan (101-2325- anti-BCMA antibody (1:20 dilution; R&D Systems) for 30 min. In some cases, B-001-009 to K.-I.L.).

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