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Analysis of tertial structure and function of non-toxic components associating with Clostridium botulinum neurotoxins.

与肉毒杆菌神经毒素相关的无毒成分的立体结构和功能分析。

基本信息

批准号：
14370093
负责人：
OGUMA Keiji
金额：
$ 8.58万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370093/
关键词：
Botulinum Toxin Neurotoxin Progenitor toxin Hemagglutinin Binding Receptor β-trefoil domain Siglec family Neurotoxin Hemaggultinin Lactose Sialic acid Hemagglutinin

项目摘要

Clostridium botulinum type A to D hemagglutinin positive progenitor toxins (PTX) consist of three distinct components : neurotoxin (NTX), hemagglutinin (HA), and non-toxic non-HA (NTNH). The HA consists of four subcomponents designated HA1 (〜33kDa), 2 (〜17kDa), 3a (〜22kDa), and 3b (〜53kDa).We have established an easy procedure for purifying type B HA-positive PTX and NTX by employing an affinity gel column-linked lactose. By employing the purified type A to D toxins and their each HA subcomponents prepared as GST-fusion proteins, we have shown that HA, especially HA1 and HA3b, works as an adhesin in the attachment of HA-positive PTXs to the-microvilli of the upper small intestine as well as erythrocytes. Type A (and B) toxin bound to the galactose mainly via HA1, whereas type C (and D) bound to the sialic acid mainly via HA3b. By analyses of primary and tertiary structure of HA1 and HA3b, It became clear that HA1 has two β-trefoil domains like as lectin B-chain of the ricin, whereas HA3b has carbohydrate recognition domain (CRD) of sialo-adhesin family (or Siglec family). In the binding and internalization tests with C HA-positive toxin and human colon carcinoma HT-29 cells, the toxin bound to glycoproteins with high molecular mass, and internalized within 4 min, but not if the cells were pretreated with neuraminidase, indicating that sialic acid of glycoprotein is the receptor for type C toxin and the toxin can internalize quickly into the cells. It was also found that both HA1 and HA3b have adjuvant activity.

肉毒梭菌A型至D型血凝素阳性祖毒素（PTX）由三种不同的组分组成：神经毒素（NTX）、血凝素（HA）和无毒非HA（NTNH）。HA由4个亚组分组成，分别命名为HA 1（约33 kDa）、HA 2（约17 kDa）、HA 3a（约22 kDa）和HA 3b（约53 kDa）。通过采用纯化的A型至D型毒素和它们各自的HA亚组分制备成GST融合蛋白，我们已经表明HA，特别是HA 1和HA 3b，在HA阳性PTX附着于小肠上部微绒毛以及红细胞中作为粘附素起作用。A型（和B）毒素主要通过HA 1与半乳糖结合，而C型（和D型）毒素主要通过HA 3 B与唾液酸结合。通过对HA_1和HA_3b的一级和三级结构的分析，发现HA_1具有两个类似于蓖麻凝集素B链的β-三叶结构域，而HA_3b具有唾液酸粘附素家族（或Siglec家族）的碳水化合物识别结构域（CRD）。在C HA阳性毒素与人结肠癌HT-29细胞的结合和内化试验中，毒素与高分子量的糖蛋白结合，并在4 min内内化，而经神经氨酸酶预处理的细胞则不能，表明糖蛋白的唾液酸是C型毒素的受体，毒素能迅速内化入细胞。还发现HA 1和HA 3b都具有佐剂活性。