Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Central-2 OSL, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):11900-5. doi: 10.1073/pnas.0914298107. Epub 2010 Jun 14.
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.
在之前的研究中，我们证明了β1,3-N-乙酰氨基葡萄糖基转移酶 5（B3gnt5）是一种乳糖三己糖苷脂（Lc（3）Cer）合酶，它在体外实验中合成乳糖/新乳糖系列糖脂（GSL）的前体结构。在这里，我们生成了 B3gnt5 缺陷（B3gnt5(-/-)）小鼠，以研究乳糖/新乳糖系列 GSL 的体内生物学功能。在生化分析中，确认这些小鼠的组织中不存在乳糖/新乳糖系列 GSL，也检测不到 Lc（3）Cer 合酶活性。这些结果表明β3GnT5 是体内合成 Lc（3）Cer 的唯一酶。神经节苷脂 GM1 是一种富含糖脂的微区（GEM）标志物，通过流式细胞术和荧光显微镜发现 B3gnt5(-/-)B 细胞中的 GM1 上调。然而，通过 TLC-免疫印迹分析未观察到 GM1 量的差异。B3gnt5(-/-)静息 B 细胞表面染色的 GEM 斑点比 WT 细胞更亮更大。这些结果表明 B3gnt5(-/-)B 细胞中的 GEM 结构发生了改变。我们接下来检查了 BCR 信号相关蛋白，如 BCR、CD19 和信号分子 Lyn 是否已经进入或离开 GEM 部分。在 B3gnt5(-/-)B 细胞中，这些分子在 GEM 部分或相邻部分富集。此外，B3gnt5(-/-)B 细胞在 BCR 刺激诱导细胞内磷酸化信号方面更为敏感，比 WT B 细胞更强烈地增殖。总之，这些结果表明乳糖/新乳糖系列 GSL 在后 GEM 簇和特定于 tether 的蛋白（如 BCR、CD19 和相关信号分子）与 GEM 的聚类中发挥重要作用。
