Supporting Information

Huang et al. 10.1073/pnas.1309417110 SI Materials and Methods pH 7.5) supplied with protease inhibitors and then the lysates were Plasmid. Full-length human B-cell maturation antigen (BCMA) subjected to glycoprotein identification and glycopeptide map cDNA amplified by PCR from the cDNA library of H929 cells analysis as previously described (Fig. 1) (4). was cloned into pFLAG-CMV-5a vector (Sigma-Aldrich) at fi − HindIII/BamHI sites to generate FLAG-tagged BCMA at the C Glycopeptide Profiling. Puri ed BCMA was applied to 4 16% terminus. FLAG-tagged BCMA was further amplified by PCR gradient SDS/PAGE and then transferred onto PVDF membrane. and subcloned into pGC-YFP retroviral vector (1) at the NotI Glycosylated BCMA on membrane was cut followed by trypsin site. QuikChange Site-Directed Mutagenesis Kit (Stratagene) digestion. The tryptic peptides were subjected to glycopeptide fi was used to construct N-glycosylation site-mutated BCMA, ac- pro ling as previously described (3). cording to the manufacturer’s instructions. The cDNA of the ELISA. For lectin binding, purified full-length BCMA was prebound extracellular domain of the cleaved form of human a pro- μ liferation-inducing ligand (APRIL) and B-cell–activating factor in 96-well plates coated with anti-FLAG antibody (1 g/mL). μ (BAFF) was amplified by PCR from the cDNA library of Lectins (1 g/mL) were then added to each well, followed by in- RPMI8226 and then cloned into the pSegTag2-human im- cubation with HRP-conjugated streptavidin (1:200 dilution; R&D munoglobilin G fragment crystallizable region (hIgG-Fc) vector (2) Systems). For BCMA binding, lectins were prebound in 96-well fi at BamHI/EcoRI sites to generate human IgG-Fc–tagged APRIL plates coated with streptavidin (Thermo Scienti c), followed by fi (Fc-APRIL) or BAFF (Fc-BAFF). addition of puri ed full-length BCMA (20 ng/mL) and HRP- conjugated anti-FLAG antibody (1:1,000 dilution; Sigma-Aldrich). Western Blot. Cells were lysed by RIPA lysis buffer (50 mM For ligand binding, cell extract from transfectants was prebound Tris·HCl at pH 7.4, 0.15 M NaCl, 0.25% deoxycholic acid, 1% in 96-well plates coated with anti-FLAG antibody (1 μg/mL). Fc- Nonidet P-40, and 1 mM EDTA) supplied with protease in- APRIL or Fc-BAFF (0.5 μg/mL) was then added into each well, hibitors (Roche) and then denatured in sample buffer at 95 °C for followed by incubation with HRP-conjugated anti-human IgG-Fc 5 min before being applied to 4−16% gradient SDS/PAGE. Pro- antibody (1:5,000 dilution; Bethyl). In between each step, wells teins separated in gel were then transferred onto ethanol-rinsed were washed with ELISA washing buffer (50 mM Tris, pH 8.0, PVDF membranes. Membrane was preincubated with 5% nonfat 0.14 M NaCl, and 0.05% Tween 20) five times. The activity of milk-supplied Tris-buffered saline with 0.1% (vol/vol) Tween 20 HRP was detected by 3,3′,5,5′-tetramethylbenzidine substrate. (TTBS) for 30 min before incubation with anti-BCMA antibody After addition of 2N H2SO4 to stop the reaction, absorbance at (1:1,000 dilution; Abcam) or HRP-conjugated anti-FLAG anti- OD450 was measured by SpectraMax M5 microplate reader body (1:5,000 dilution; Sigma-Aldrich) for 90 min. For click re- (Molecular Devices). action performed on membrane, membrane was preincubated with 5% (vol/vol) BSA-supplied TTBS for 30 min, followed by Production of Retroviral Vector. Retroviral vector used for the chemical click reaction with azido-biotin as previously described expression of exogenous wild type or mutated BCMA was gen- (3). In some cases, HRP-conjugated secondary antibody or erated as previously described (5). Multiple myeloma cell lines streptavidin was used to detect sugar analog-incorporated mole- were transduced with retroviral vectors at a multiplicity of in- cules for another 30 min. fection of 5, and the transduction rate was >95% in all cell lines.

Production and Purification of Fc-APRIL and -BAFF Fusion Proteins. Apoptosis Assay. Cells were treated with or without dexamethasone Fc-APRIL and -BAFF were expressed by the FreeStyle 293 ex- (DEX) (1 nM; Sigma-Aldrich), APRIL (200 ng/mL; R&D Sys- pression system according to the manufacturer’s instruction. tems), or BAFF (200 ng/mL; Peprotech) at 105 cells/mL as 293F was transfected with plasmid encoding Fc-APRIL or previously described (6). Three days later, cells were washed with -BAFF by 293fectin (Invitrogen). At 72 and 120 h later, culture PBS and incubated with allophycocyanin (APC)-conjugated an- supernatant from transfectants was collected and subjected to nexin V (1:40 dilution; BD Biosciences) in annexin V–binding protein A-Sepharose column (GE) for purification. The column buffer for 15 min on ice and then subjected to flow cytometry was washed by TBS buffer, followed by elution with 0.1 M glycine analysis. buffer at pH 3.0. The eluted protein solution was then neu- tralized by 1 M Tris buffer at pH 8.0. Circular Dichroism Analysis. The secondary structure of proteins in 20 mM sodium phosphate (pH 7.4) buffer was detected by cir- Identification of Glycoproteins. Three days after application with cular dichroism (CD) spectroscopy (JASCO) in the far-UV spec- sugar analog, cells were lyzed by sodium phosphate lysis buffer (1% tral region (190–250 nm), and obtained signal was normalized Nonidet P-40, 150 mM NaCl, and 100 mM sodium phosphate, with buffer only control.

1. Costa GL, et al. (2000) Targeting rare populations of murine antigen-specificT 4. Hanson SR, et al. (2007) Tailored glycoproteomics and glycan site mapping using lymphocytes by retroviral transduction for potential application in gene therapy for saccharide-selective bioorthogonal probes. J Am Chem Soc 129(23):7266–7267. autoimmune disease. J Immunol 164(7):3581–3590. 5. Lin KI, Calame K (2004) Introduction of genes into primary murine splenic B cells using 2. Ettinger R, Browning JL, Michie SA, van Ewijk W, McDevitt HO (1996) Disrupted splenic retrovirus vectors. Methods Mol Biol 271:139–148. architecture, but normal lymph node development in mice expressing a soluble 6. Moreaux J, et al. (2004) BAFF and APRIL protect myeloma cells from apoptosis induced lymphotoxin-beta receptor-IgG1 fusion protein. Proc Natl Acad Sci USA 93(23):13102–13107. by interleukin 6 deprivation and dexamethasone. Blood 103(8):3148–3157. 3. Liu YC, et al. (2011) Sialylation and fucosylation of epidermal growth factor receptor suppress its dimerization and activation in lung cancer cells. Proc Natl Acad Sci USA 108(28):11332–11337.

Huang et al. www.pnas.org/cgi/content/short/1309417110 1of7 Fig. S1. Plasma cell lines can incorporate sugar analog as the building block for protein glycosylation. (A) Sugar analog can be taken up by the H929 plasma cell line. Control sugar-fed or sugar analog-fed H929 cells were subjected to click reaction with azido-biotin after fixation on the glass slide by acetone. Sugar was detected by alexa488-conjugated streptavidin by fluorescence microscope. (B) Sugar analogs can be incorporated into proteins. Cell extracts from control sugar-fed or sugar analog-fed H929 cells were subjected to immunoblotting. (C) Sugar analog is present on the cell surface. H929, U266, or RPMI8226 cells were fed with control sugar or sugar analogs as indicated and then subjected to click reactions with azido-biotin at indicated time points, followed by incubation with alexa488- or APC-conjugated streptavidin to detect sugar analogs on the surface.

Huang et al. www.pnas.org/cgi/content/short/1309417110 2of7 Fig. S2. Sialylation affects ligand-mediated cell survival and death. (A–C) α2–3 sialylation affects ligand-mediated cell survival. RPMI8226 cells were pretreated with α2–3 sialidase before adding DEX together with APRIL or BAFF. Three days later, cells were stained with APC-conjugated annexin V to detect the per- centage of apoptotic cells by flow cytometry. (A) One representative result of five independent experiments is shown; the number in the dot plot indicates the percentage of annexin V–positive cells. (B) Statistic analysis of five independent experiments. (C) Percentage of rescue of apoptosis was determined by (% of apoptosis induced by DEX–% of apoptosis induced by DEX with ligand)/(% of apoptosis induced by DEX). *P < 0.05 and **P < 0.01. (D) Removal of sialylation increases tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. RPMI8226 cells were pretreated with sialidase before the addition of TRAIL (50 ng/mL). Two days later, cells were stained with APC-conjugated annexin V to detect the percentage of apoptotic cells by flow cytometry. One representative result of two independent experiments is shown; the number in the dot plot indicates the percentage of annexin V–positive cells.

Huang et al. www.pnas.org/cgi/content/short/1309417110 3of7 Fig. S3. Effect of sialylation on surface retention of BCMA. (A) α2–3 sialidase increases surface expression of BCMA. H929 or RPMI8226 cells were treated with α2–3 sialidase, and the level of surface BCMA was detected by APC-conjugated anti-BCMA antibody by FACS. (B and C) Effects of sialidase on the expression of surface BCMA. H929 or RPMI8226 cells were treated with sialidase (B)orα2–3 sialidase (C) for 4 h and then transferred to FBS-supplied RPMI1640 culture medium for 24 h. Surface level of BCMA was detected by APC-conjugated anti-BCMA antibody. (D) α2–3 sialylation is involved in the surface retention of BCMA. H929 or RPMI8226 cells were treated with cycloheximide (CHX) in the presence of α2–3 sialidase. Residual BCMA on cell surface was then detected by APC-conjugated anti-BCMA antibody. Percentage of retention of BCMA on the cell surface shown in bar graph was calculated by comparing the levels of surface BCMA of cells treated with or without sialidase divided by the levels of BCMA on mock-treated cells. (A–D) One representative result of three in- dependent experiments is shown in each histogram, and the number in the histogram indicates the mean of fluorescence. Statistical analysis of three in- dependent experiments is shown in bar graph. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. S4. Efficiency of PNGase F digestion. RPMI8226 cells were pretreated with or without PNGase F before the incubation with biotinylated SNA (A) or L-PHA (B). The binding of lectins was detected by APC-conjugated streptavidin with FACS. One representative result of two independent experiments is shown.

Huang et al. www.pnas.org/cgi/content/short/1309417110 4of7 Fig. S5. N42A BCMA is unable to accumulate on the H929 cell surface following sialidase treatment. H929 cells transfected with empty control or N42A-BCMA expression vector were treated with sialidase in the presence of CHX. Residual surface BCMA was detected by APC-conjugated anti-BCMA antibody with FACS. One representative result of three independent experiments is shown in each histogram; the number in the histogram indicates the mean of fluorescence. (Right) Statistical analysis of three independent experiments (white, mock-treated; gray, CHX-treated; black, CHX + sialidase-treated). *P < 0.05 and **P < 0.01.

Fig. S6. N-acetyl-D-lactosamine (LacNAc) fails to prevent the increased surface retention of BCMA caused by sialidase. Histograms of FACS analysis show the surface expression of BCMA on RPMI8226 cells detected by APC-conjugated anti-BCMA antibody after sialidase treatment with or without LacNAc. One representative result of two independent experiments is shown in each histogram.

Huang et al. www.pnas.org/cgi/content/short/1309417110 5of7 Fig. S7. Comparison of secondary structure of different forms of BCMA. (A) Secondary structure of wild type (WT) and N42A BCMA are similar. The secondary structure of WT and N42A BCMA purified from 293T cells was detected by CD analysis. One representative result of three independent experiments is shown in graph. (B) Glycosidase treatment did not affect secondary structure of WT BCMA. WT BCMA purified from 293F cells was treated with PNGase F to remove whole glycans. The secondary structure of mock-treated and glycosidase-treated WT BCMA was detected by CD analysis. One representative result of two independent experiments is shown in graph.

Huang et al. www.pnas.org/cgi/content/short/1309417110 6of7 Table S1. Protein ID identified by sugar analog IPI number Name

ManNAcyne IPI00000735 Tetraspanin-13 IPI00002541 CD44 antigen isoform 5 precursor IPI00003648 Isoform delta of poliovirus receptor-related protein 1 precursor IPI00003807 Lysosomal acid phosphatase precursor IPI00003813 Nectin-like protein 2 IPI00008494 Intercellular adhesion molecule 1 precursor IPI00009030 Isoform lysosome-associated membrane glycoprotein 2A of lysosome-associated membrane glycoprotein 2 precursor IPI00011578 Isoform 1 of neuroplastin precursor IPI00012877 IFNα/β receptor α-chain precursor IPI00013831 CD48 antigen precursor IPI00014055 Napsin-A precursor IPI00015476 Neutral amino acid transporter A IPI00017529 Isoform 1 of lymphocyte function-associated antigen 3 precursor IPI00018307 Leukocyte surface antigen CD53 IPI00019472 Neutral amino acid transporter B IPI00019906 Isoform 2 of basigin precursor IPI00020470 Glycosyltransferase 8 domain containing 1 IPI00022558 Isoform 1 of myelin protein zero-like protein 1 precursor IPI00023814 Isoform 1 of neogenin precursor IPI00025049 Cation-dependent mannose-6-phosphate receptor precursor IPI00027078 Carboxypeptidase D precursor IPI00030847 Transmembrane 9 superfamily protein member 3 precursor IPI00030941 Tetraspanin-3 IPI00031620 Intercellular adhesion molecule 3 precursor IPI00043883 Isoform 1 of heme carrier protein 1 IPI00072743 Isoform 1 of claudin domain-containing protein 1 IPI00106689 Isoform 2 of transmembrane protein 87A precursor IPI00107380 MHC class I chain-related protein A IPI00149097 Semaphorin-4A precursor IPI00183782 Lymphocyte antigen-9 (Ly9) IPI00215998 CD63 antigen IPI00216514 Isoform OA3-293 of leukocyte surface antigen CD47 precursor IPI00217561 Isoform β-1C of integrin β-1 precursor IPI00217766 Lysosome membrane protein 2 IPI00220737 Isoform neural cell adhesion molecule 120 of neural cell adhesion molecule 1, 120-kDa isoform precursor IPI00293074 Isoform 2 of choline transporter-like protein 2 IPI00293327 P2X purinoceptor 4 IPI00293877 Tumor necrosis factor receptor superfamily member 17/BCMA IPI00296869 Proteinase-activated receptor 1 precursor IPI00297880 Isoform Long of Integrin β-7 precursor IPI00298702 Isoform 1 of zinc transporter SLC39A6 precursor IPI00329054 Osteopetrosis-associated transmembrane protein 1 precursor IPI00375879 Hypothetical protein LOC57613/uncharacterized protein KIAA1467 IPI00394808 Embigin precursor IPI00514585 Lysosomal protein NCU-G1 IPI00556655 LAMP1 protein variant (fragment) IPI00759659 Isoform 2 of Golgi phosphoprotein 2 IPI00783380 Transmembrane protein 87B Fucyne IPI00000877 150-kDa oxygen-regulated protein precursor IPI00004670 Isoform 1 of polypeptide N-acetylgalactosaminyltransferase 3 IPI00013831 CD48 antigen precursor IPI00016150 Neuroserpin precursor IPI00020470 Glycosyltransferase 8 domain containing 1 IPI00023542 Transmembrane emp24 protein transport domain containing 9 IPI00030847 Transmembrane 9 superfamily protein member 3 precursor IPI00032825 Transmembrane emp24 domain-containing protein 7 precursor IPI00064848 β-1,3-Galactosyltransferase 6 IPI00220530 Isoform 3 of signal peptide peptidase-like 2B IPI00293088 Lysosomal alpha-glucosidase IPI00293877 Tumor necrosis factor receptor superfamily member 17/BCMA

Huang et al. www.pnas.org/cgi/content/short/1309417110 7of7