Exploration of the toxic site by use of genetic diversity on Clostridium botulinum neurotoxin genes.

利用肉毒杆菌神经毒素基因的遗传多样性探索毒性位点。

基本信息

批准号：
13660323
负责人：
KOZAKI Shunji
金额：
$ 2.24万
依托单位：
Osaka Prefecture University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Clostridium botulinum neurotoxin (BoNT) binds presynaptic neuronal cells and inhibits specifically neurotransmitter release. We have reported that BoNT/B recognizes a complex of synaptotagmin 2 (Stg2) and ganglioside GT1b as a type-specific receptor. We were cloning carboxyl-terminal heavy chain (Hc) gene and expressed the recombinant He in E. coli. The recombinant Hc inhibited ^<125>I-labeled BoNT/B binding to the Stg2/GT1b lipid vesicles and rat brain synaptosomes to the same extent, in comparison with unlabeled BoNT/B, suggesting that the recombinant Hc retains binding properties of holotoxin to the receptor. The blockade of neurotransmitter release by BoNT/B at the neuromuscular junction (NMJ) was rescued in the presence of recombinant Hc. We then constructed several site-directed mutants of Hc to investigate the role of individual residues in recognition of the receptor and GT1b. The binding capabilities of Hc mutants to the receptor and GTlb were examined by competition experiments with ^<125>I-labeled Hc. Three mutants, S1259A, W1261A and Y1262A, were found to drastically decrease the binding activities to the receptor and GT1b, although W1261 and Y1262 have been identified as contact residues between Hc domain and sialyllactose. Interestingly, K1260A hardly inhibited ^<125>I-labeled Hc binding to GT1b, whereas the binding of ^<125>I-labeled Hc to the receptor was effectively interfered by K1260A. From these results, it seems that the residues relating with ganglioside binding do not necessarily participate in formation of receptor recognition site. In the Hc derived from type C neurotoxins, the recombinant bound to the extract from chicken brain synaptosomes in the absence of ganglioside. We then purified further the binding substance by ion exchange and hydrophobic chromatography. Far-western analysis revealed that a acidic glycoprotein interacted with the Hc.

肉毒梭菌神经毒素 (BoNT) 结合突触前神经元细胞并特异性抑制神经递质释放。我们报道了 BoNT/B 将突触结合蛋白 2 (Stg2) 和神经节苷脂 GT1b 的复合物识别为类型特异性受体。我们克隆了羧基端重链（Hc）基因，并在大肠杆菌中表达了重组He。与未标记的BoNT/B相比，重组Hc以相同程度抑制125 I标记的BoNT/B与Stg2/GT1b脂囊泡和大鼠脑突触体的结合，这表明重组Hc保留了全毒素与Stg2/GT1b脂囊泡和大鼠脑突触体的结合特性。受体。在重组 Hc 存在下，BoNT/B 对神经肌肉接头 (NMJ) 神经递质释放的阻断得以恢复。然后，我们构建了 Hc 的几个定点突变体，以研究各个残基在识别受体和 GT1b 中的作用。通过与125 I-标记的Hc的竞争实验检查Hc突变体与受体和GT1b的结合能力。尽管 W1261 和 Y1262 已被鉴定为 Hc 结构域和唾液酸乳糖之间的接触残基，但发现三种突变体 S1259A、W1261A 和 Y1262A 显着降低了受体和 GT1b 的结合活性。有趣的是，K1260A几乎不抑制 125 I-标记的Hc与GT1b的结合，而125 I-标记的Hc与受体的结合被K1260A有效地干扰。从这些结果看来，与神经节苷脂结合有关的残基不一定参与受体识别位点的形成。在源自 C 型神经毒素的 Hc 中，重组体在不存在神经节苷脂的情况下与鸡脑突触体提取物结合。然后我们通过离子交换和疏水色谱进一步纯化结合物质。 Far-western 分析表明酸性糖蛋白与 Hc 相互作用。