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Studies on the Interaction between Subunits of the Botulinum Toxin Complex and X-Ray Crystallography

肉毒毒素复合物亚基间相互作用与X射线晶体学的研究

基本信息

批准号：
15570103
负责人：
OHYAMA Tohru
金额：
$ 2.37万
依托单位：
Tokyo University of Agriculture
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Botulinum toxin is produced as a 650 kDa toxin complex(TC) with subunit structure that non-covalently associated with five different protein components, 150 kDa neurotoxin(NT), nontoxic non-hemagglutinin(NTNHA) and three hemagglutinin subcomponents, in culture supernatant. In this study, we have clarified molecular ration of subunit components in the TC. Type D strain 4947(D-4947) produces a large amount of TC species (650 kDa L-TC,610 kDa and 540 kDa TC). They revealed same banding patterns on SDS-PAGE, but less number of HA-33/17 complexes in the TC. Based on titration experiments with HA-33/17 complex, we have concluded that complete subunit composition of the TC is a dodecamer composed of a single NT, a single NTNHA, two HA-70,four HA-33 and four HA-17 molecules. Accordingly, we have preliminary information on the analysis of the three-dimensional structure of botulinum TC by X-ray crystallography. Of the TC species, recombination of the components was found in 610 and 540 kDa TCs depending upon storage condition such as temperature, and was thus depressed by the addition of glycerol leading supply of stable TC for the experiments. Additionally, we have established new method to separate considerable amount of the NT from the D-4947 TC, which has been technically difficult to separate. Although we have attempted to crystallize TCs (M and L-TC) that were examined using dynamic light scattering from serotype C and D strains by 130 combinations of experimental condition, it has not been yet obtain large crystals for X-ray analysis.

肉毒毒素是一种650 kDa的毒素复合物（TC），其亚基结构与5种不同的蛋白质组分（150 kDa神经毒素（NT）、无毒非血凝素（NTNHA）和3种血凝素亚组分）在培养上清液中非共价结合。在本研究中，我们阐明了TC中亚基组分的分子比。D型菌株4947（D-4947）产生大量TC物质（650 kDa L-TC、610 kDa和540 kDa TC）。在SDS-PAGE上，它们显示相同的带型，但TC中HA-33/17复合物的数量较少。基于HA-33/17复合物的滴定实验，我们得出结论，TC的完整亚基组成是由单个NT、单个NTNHA、两个HA-70、四个HA-33和四个HA-17分子组成的十二聚体。因此，我们有初步的信息分析的三维结构的肉毒杆菌TC的X-射线晶体学。在TC种类中，在610和540 kDa TC中发现了组分的重组，这取决于储存条件（如温度），因此通过添加甘油而抑制，从而为实验提供稳定的TC。此外，我们还建立了新的方法，从D-4947 TC中分离出大量的NT，这在技术上是很难分离的。虽然我们已经尝试通过130种实验条件的组合来结晶使用动态光散射从血清型C和D菌株检测的TC（M和L-TC），但是尚未获得用于X射线分析的大晶体。

项目成果

期刊论文数量（37）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Complete subunit structure of the Clostridium botulinum type D toxin complex via intermediate assembly with nontoxic components

DOI：
10.1021/bi034996c
发表时间：
2003-09-23
期刊：
BIOCHEMISTRY
影响因子：
2.9
作者：
Mutoh, S;Kouguchi, H;Ohyama, T
通讯作者：
Ohyama, T

Complete Subunit Structure of Clostridium botulinum Type D Toxin Complex via Intermediate Assembly with Nontoxic Components

通过无毒成分中间组装得到肉毒梭菌 D 型毒素复合物的完整亚基结构

DOI：
发表时间：
2003
期刊：
Biochemistry Vol.37
影响因子：
0
作者：
Mutoh;S.;Kouguchi;H.;Sagane;Y.;Suzuki;T.;Hasegawa;K.;Watanabe;T.;Ohyama;T.
通讯作者：
T.

Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes

DOI：
10.1099/mic.0.26805-0
发表时间：
2004-05-01
期刊：
MICROBIOLOGY-SGM
影响因子：
2.8
作者：
Fujinaga, Y;Inoue, K;Oguma, K
通讯作者：
Oguma, K

Characterization of Toxin Complex Produced by a Unique Strain of Clostridium botulinum type D 4947.

D 4947 型肉毒梭菌独特菌株产生的毒素复合物的表征。

DOI：
发表时间：
2004
期刊：
Protein J. 23
影响因子：
0
作者：
Hasegawa K;Watanabe T;Sato H;Sagane Y;Mutoh S;Suzuki T;Yamano Y;Kouguchi H;Takeshi K;Kamaguchi A;Fujinaga Y;Oguma K;Ohyama T.
通讯作者：
Ohyama T.

Molecular characterization of GroES and GroEL homologues from Clostridium botulinum