Clostridium botulinum type C hemagglutinin affects the morphology and viability of cultured mammalian cells via binding to the ganglioside GM3.
Clostridium botulinum type C hemagglutinin affects the morphology and viability of cultured mammalian cells via binding to the ganglioside GM3.
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C 型肉毒梭菌血凝素通过与神经节苷脂 GM3 结合影响培养的哺乳动物细胞的形态和活力。
DOI：
10.1111/febs.13346
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
藤永 由佳子
中科院分区：
文献类型：
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作者：
菅原 庸;岩森 正男;松村 拓大;油谷 雅広;阿松 翔;藤永 由佳子
肉毒神经毒素通常分为七个血清型，命名为A-G，通过与无毒成分如血凝素(HA)和无毒的非HA结合而产生大的蛋白质复合体。这些无毒蛋白质极大地增强了毒素复合体的口服毒性。透明质酸被认为通过碳水化合物结合和上皮屏障破坏活性在毒素通过肠上皮的运输中发挥作用。A型和B型破坏了E-钙粘附素介导的细胞黏附，进而破坏了细胞间上皮屏障。C型HA(HA/C)通过影响细胞形态和活性来破坏屏障功能，其机制尚不清楚。在本研究中，我们确定GM3为HA/C的靶分子，我们发现HA的唾液酸结合是其活性所必需的。当细胞被神经节苷脂合成抑制剂预先处理时，它被取消。与此相一致的是，HA/C与a系列神经节苷脂以多聚糖阵列结合。同时，我们从一个敏感的小鼠成纤维细胞株中分离出了对HA/C活性具有抗性的克隆。这些细胞缺乏ST-I的表达，ST-I是一种将唾液酸转化为乳糖基神经酰胺以产生GM3的酶。通过ST-I基因表达GM3后，这些克隆对HA/C活性变得敏感。通过表达神经节苷脂合成基因，成纤维细胞对HA/C的敏感性降低，其产物以GM3为底物，从而产生其他a系列神经节苷脂，提示GM3特异性机制。我们的结果表明，HA/C以GM3依赖的方式影响细胞。
Botulinum neurotoxin is conventionally divided into seven serotypes, designated A–G, and is produced as large protein complexes through associations with non‐toxic components, such as hemagglutinin (HA) and non‐toxic non‐HA. These non‐toxic proteins dramatically enhance the oral toxicity of the toxin complex. HA is considered to have a role in toxin transport through the intestinal epithelium by carbohydrate binding and epithelial barrier‐disrupting activity. Type A and B HAs disrupt E‐cadherin‐mediated cell adhesion, and, in turn, the intercellular epithelial barrier. Type C HA (HA/C) disrupts the barrier function by affecting cell morphology and viability, the mechanism of which remains unknown. In this study, we identified GM3 as the target molecule of HA/C. We found that sialic acid binding of HA is essential for the activity. It was abolished when cells were pre‐treated with an inhibitor of ganglioside synthesis. Consistent with this, HA/C bound to a‐series gangliosides in a glycan array. In parallel, we isolated clones resistant to HA/C activity from a susceptible mouse fibroblast strain. These cells lacked expression of ST‐I, the enzyme that transfers sialic acid to lactosylceramide to yield GM3. These clones became sensitive to HA/C activity when GM3 was expressed by transfection with the ST‐I gene. The sensitivity of fibroblasts to HA/C was reduced by expressing ganglioside synthesis genes whose products utilize GM3 as a substrate and consequently generate other a‐series gangliosides, suggesting a GM3‐specific mechanism. Our results demonstrate that HA/C affects cells in a GM3‐dependent manner.