Lack of lacto/neolacto-glycolipids enhances the formation of glycolipid-enriched microdomains, facilitating B cell activation

Abstract

In a previous study, we demonstrated that beta1,3-N-acetylglucosaminyltransferase 5 (B3gnt5) is a lactotriaosylceramide (Lc(3)Cer) synthase that synthesizes a precursor structure for lacto/neolacto-series glycosphingolipids (GSLs) in in vitro experiments. Here, we generated B3gnt5-deficient (B3gnt5(-/-)) mice to investigate the in vivo biological functions of lacto/neolacto-series GSLs. In biochemical analyses, lacto/neolacto-series GSLs were confirmed to be absent and no Lc(3)Cer synthase activity was detected in the tissues of these mice. These results demonstrate that beta3GnT5 is the sole enzyme synthesizing Lc(3)Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be up-regulated in B3gnt5(-/-) B cells by flow cytometry and fluorescence microscopy. However, no difference in the amount of GM1 was observed by TLC-immunoblotting analysis. The GEM-stained puncta on the surface of B3gnt5(-/-) resting B cells were brighter and larger than those of WT cells. These results suggest that structural alteration of GEM occurs in B3gnt5(-/-) B cells. We next examined whether BCR signaling-related proteins, such as BCR, CD19, and the signaling molecule Lyn, had moved into or out of the GEM fraction. In B3gnt5(-/-) B cells, these molecules were enriched in the GEM fraction or adjacent fraction. Moreover, B3gnt5(-/-) B cells were more sensitive to the induction of intracellular phosphorylation signals on BCR stimulation and proliferated more vigorously than WT B cells. Together, these results suggest that lacto/neolacto-series GSLs play an important role in clustering of GEMs and tether-specific proteins, such as BCR, CD19, and related signaling molecules to the GEMs.

Fig. 1.

Confirmation of Lc₃Cer synthase activity in B3gnt5^−/− mice and MS analysis of the glycans derived from GSLs of mouse B cells. (A) Homogenates (100 μg of protein) of mouse tissues were used to assay Lc₃Cer synthase activity. The products were separated on a high-performance twin-layer chromatography (HPTLC) plate with a solvent system of chloroform/methanol/0.2% CaCl₂ (60:35:8 vol/vol/vol). The intensities of the radioactive bands were measured with a FLA3000 Image Analyzer (Fujifilm, Tokyo). HL-60, which has Lc₃Cer synthase activity, and Jurkat, which does not, were used as positive and negative controls, respectively. Lc₃Cer synthase assays were performed on two TLC plates, with positive and negative control on each plate. This figure was prepared from the results of two TLC plates to demonstrate Lc₃Cer synthase activity in cerebellum, spleen, and colon between WT and B3gnt5^−/− mice. (B) MS spectra of the glycans derived from GSLs of mouse B cells. Arrows indicate glycan signals that are absent from GSLs of B3gnt5^−/− B cells. Arrowheads indicate glycan signals that are decreased in GSLs of B3gnt5^−/− B cells. Table S1 summarizes the results of the assigned GSL-derived glycan structures of mouse B cells. Detailed methods for MS of glycans derived from GSLs are described in Fig. S2.

Fig. 2.

Up-regulation of CT-B staining as a GEM marker but no alteration of surface protein expression on B cells. FCM analysis of GEMs and surface proteins on splenic B cells (A) and T cells (B). The expression on the splenocyte cell surface was analyzed by FCM using FITC-conjugated CT-B, anti-CD3ε, anti-BCR (sIgM), anti-CD48 Abs, and phycoerythrin (PE)-conjugated anti-CD19. Results are shown as histograms of fluorescence intensity. The shaded peak in each panel represents the negative control. Max, maximum.

Fig. 3.

Fluorescence microscopy of GEMs on the cell surface. (A) Fluorescence microscopy showed a CT-B–induced punctate pattern indicating GEM structures on the B cell surface. Resting or stimulated isolated B cells were stained with FITC-conjugated CT-B. The results shown are representative of several independent experiments revealing markedly stronger staining of B3gnt5^−/− than WT B cells. (B) Fluorescence intensity of positive signals for each CT-B–stained punctate region was analyzed by means of a BZ-Analyzer (KEYENCE). The total value of the positive signals on each cell surface was measured. Statistical analysis of the difference between WT and B3gnt5^−/− B cells, with or without stimulation, was performed using one-way ANOVA and Tukey testing by means of PRISM4 software. Data are given as each cell type's mean (bold line) and SD. *1, P < 0.01; *2, P < 0.01.

Fig. 4.

Distribution of BCR, CD19, and Lyn in GEMs separated into fractions by sucrose density gradient centrifugation. B cells stimulated with anti-BCR and anti-CD19 Abs were lysed with 1% Triton X-100 solubilization buffer. After sucrose gradient centrifugation, fractions were collected from the top of gradient. Then, every second fraction was combined starting from the top fraction. GM1, a GEM marker, was detected using HRP-conjugated CT-B as shown. After fractionation, immunoprecipitation of BCR and CD19 was performed using anti-BCR and anti-CD19 Abs, respectively. BCR (sIgM) and CD19 proteins in B3gnt5^−/− B cells were up-regulated in the GEM fraction (fractions 1–4). Lyn proteins were also up-regulated in the GEM fraction of B3gnt5^−/− B cells. The results shown are representative of several independent experiments. *Nonspecific bands.

Fig. 5.

Analysis of phosphorylation status and enhanced response to BCR cross-linking in resting B cells. (A) Resting B cells from spleen were stimulated by cross-linking anti-BCR Ab for the indicated time. The same volume of lysates was added to each lane. Phosphorylated proteins were analyzed by immunoblots using anti-phospho-Tyr mAb (4G10). B3gnt5^−/− B cells exhibited significantly up-regulated phosphorylation signals. (B) Resting B cells from B3gnt5^−/− mice show enhanced responses to BCR-mediated stimulation. Resting B cells were cultured with the indicated dose of F(ab′)₂ anti-IgM. Proliferative responses are given as [³H]-thymidine (TdR) incorporation for the final 6 h of the 42-h culture period. Each assay was performed in triplicate, and all data are representative of three experiments. Open circles indicate WT mice, and closed triangles indicate B3gnt5^−/− mice. (C) Response to TI-II antigen. Mice were immunized i.p. with 25 μg of TNP-Ficoll in PBS at day 0. Sera were collected sequentially from the eye socket and tested at a 1:100 dilution. The levels of TNP-specific IgM Ab in sera were determined by ELISA. Representative data were obtained for five (WT) and six (B3gnt5^−/−) mice, respectively. Open circles indicate WT mice, and closed circles indicate B3gnt5^−/− mice. Statistical analysis of the difference between WT and B3gnt5^−/− B cells with days after stimulation was performed by two-way ANOVA using PRISM4 software. The levels of IgM in serum are presented as mean (symbols) ± SE. *P < 0.05.