B-cell maturation antigen is modified by a single N-glycan chain that modulates ligand binding and surface retention

Abstract

Glycosylation, an important posttranslational modification process, can modulate the structure and function of proteins, but its effect on the properties of plasma cells is largely unknown. In this study, we identified a panel of glycoproteins by click reaction with alkynyl sugar analogs in plasma cells coupled with mass spectrometry analysis. The B-cell maturation antigen (BCMA), an essential membrane protein for maintaining the survival of plasma cells, was identified as a glycoprotein exhibiting complex-type N-glycans at a single N-glycosylation site, asparagine 42. We then investigated the effect of N-glycosylation on the function of BCMA and found that the dexamethasone-induced apoptosis in malignant plasma cells can be rescued by treatment with BCMA ligands, such as a proliferation-inducing ligand (APRIL) and B-cell-activating factor (BAFF), whereas removal of terminal sialic acid on plasma cells further potentiated the ligand-mediated protection. This effect is associated with the increased surface retention of BCMA, leading to its elevated level on cell surface. In addition, the α1-3,-4 fucosylation, but not the terminal sialylation, assists the binding of BCMA with ligands in an in vitro binding assay. Together, our results highlight the importance of N-glycosylation on BCMA in the regulation of ligand binding and functions of plasma cells.

Fig. 1.

Flowchart of identification of glycoproteins. Cell extracts from sugar probe-fed cells were subjected to click reaction with an azido-biotin analog. Products of the click reaction were purified by streptavidin, followed by trypsin digestion. Sugar probe-labeled glycopeptides were released after PNGase F treatment and then analyzed by mass spectrometry.

Fig. 2.

BCMA is a glycoprotein. (A) Glycans identified on BCMA from H929 cells by glycopeptide profiling. Percentages represent the glycan type identified from the analysis. (triangle, Fuc; diamond, NeuAc; square, HexNAc; circle, Hex) (B) PNGase F treatment caused mobility shift of BCMA detected by immunoblotting. Cell extracts from H929, RPMI8226, and U266 cells were incubated with PNGase F, followed by immunoblotting with anti-BCMA antibody. (C) N42 of BCMA is the N-glycosylation site. Cell extracts from 293T cells transfected with WT or N42A BCMA-expressing vector were treated without or with PNGase F and then subjected to immunoblotting with the indicated antibodies.

Fig. 3.

Glycans on BCMA are terminally modified by sialic acid. (A and B) FACS shows the binding with SNA (A) or MAL (B) on H929 and RPMI8226 cells pretreated with or without sialidase. (C and D) ELISA shows the binding of lectins with BCMA. Full-length BCMA purified from transfectants was pretreated with or without sialidase and added to anti-FLAG antibody coated plates, followed by the biotinylated SNA (C) or MAL (D). (E and F) ELISA shows the binding of BCMA to lectins. Biotinylated SNA (E) or MAL (F) was bound to streptavidin-coated plates before mixing with sialidase-treated or untreated full-length BCMA. Results in A and B are representative of three or four independent experiments, and the number in the histogram indicates the mean of fluorescence. Results in C−F represent mean ± SEM of three or four independent experiments. *P < 0.05 and **P < 0.01.

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.

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 the accumulation of preexisting BCMA on the cell surface. H929 or RPMI8226 cells were treated with CHX in the absence or presence of sialidase, followed by 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 expression vector 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.

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.