Lack of lacto/neolacto-glycolipids enhances the formation of glycolipid-enriched microdomains, facilitating B cell activation
Lack of lacto/neolacto-glycolipids enhances the formation of glycolipid-enriched microdomains, facilitating B cell activation
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DOI：
10.1073/pnas.0914298107
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发表时间：
2010-06-29
影响因子：
11.1
通讯作者：
Narimatsu, Hisashi
中科院分区：
文献类型：
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作者：
Togayachi, Akira;Kozono, Yuko;Narimatsu, Hisashi
在先前的研究中，我们证明了β 1，3-N-乙酰葡糖胺转移酶5（B3 gnt 5）是一种乳三己糖神经酰胺（Lc（3）Cer）合酶，在体外实验中合成乳糖/新乳糖系列鞘糖脂（GSL）的前体结构。在这里，我们产生了B3 gnt 5-缺陷（B3 gnt 5（-/-））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，证实在这些小鼠的组织中不存在乳糖/新乳糖系列GSL，并且未检测到Lc（3）Cer合酶活性。这些结果表明β 3GnT 5是体内合成Lc（3）Cer的唯一酶。通过流式细胞术和荧光显微镜观察发现，神经节苷脂GM 1（称为鞘糖脂富集微结构域（GEM）标记物）在B3 gnt 5（-/-）B细胞中上调。然而，通过TLC-免疫印迹分析没有观察到GM 1的量的差异。B3 gnt 5（-/-）静息B细胞表面的GEM染色斑点比WT细胞更亮、更大。这些结果表明GEM的结构改变发生在B3 gnt 5（-/-）B细胞中。我们接下来检查了BCR信号相关蛋白，如BCR、CD 19和信号分子林恩，是否移入或移出GEM组分。在B3 gnt 5（-/-）B细胞中，这些分子富集在GEM组分或相邻组分中。此外，B3 gnt 5（-/-）B细胞对BCR刺激的细胞内磷酸化信号的诱导更敏感，并且比WT B细胞增殖更旺盛。总之，这些结果表明，乳糖/新乳糖系列GSL在GEM和系链特异性蛋白（如BCR、CD 19和GEM相关信号分子）的聚集中起重要作用。
In a previous study, we demonstrated that beta 1,3-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 beta 3GnT5 is the sole enzyme synthesizing Lc(3)Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be upregulated 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.