Complete subunit structure of the Clostridium botulinum type D toxin complex via intermediate assembly with nontoxic components
Complete subunit structure of the Clostridium botulinum type D toxin complex via intermediate assembly with nontoxic components
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DOI：
10.1021/bi034996c
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发表时间：
2003-09-23
期刊：
影响因子：
2.9
通讯作者：
Ohyama, T
中科院分区：
文献类型：
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作者：
Mutoh, S;Kouguchi, H;Ohyama, T
肉毒梭菌D型菌株通常产生两种类型的稳定毒素复合体，即300 kDa M(M-TC)和660 kDa L(L-TC)毒素复合体。我们以前为这两个TC提出了组装路径[Kouguchi，H.，et al.(2002)J.Biol.化学。：M-TC是由神经毒素(NT)和无毒非血凝素(NTNHA)缔合而成；M-TC与三种辅助类型的血凝素亚组分(HA-33、HA-17和HA-70)结合形成L-TC。在本研究中，我们在D型4947菌株的培养上清液中发现了三种TC种，即410、540和610 kDa TC种。540kDa和610kDa TC在SDSPAGE上显示出与L TC相似的条带，但HA-33和HA-17的染色强度低于L TC，表明它们是L-TC组装途径中的中间物种。相比之下，410 kDa的TC物种由M-TC和两个HA-70分子组成。除L-TC外，其余菌株均无血凝活性。当中间TC物种与分离的HA-33/17复合体混合时，每个TC物种都转化为具有完全血凝活性的650 kDa L-TC，并且具有与L-TC相同的分子组成。根据HA-33/17络合物的滴定分析，估算了L-TC、610 kDa和540 kDa TC物种中HA-33/17络合物分子的化学计量比分别为4、3和2。推测成熟的L-TC的完整亚基组成是由一个NT、一个NTNHA、两个HA-70、四个HA-33和四个HA-17分子组装而成的十二聚体。
Clostridium botulinum serotype D strains usually produce two types of stable toxin complex (TC), namely, the 300 kDa M (M-TC) and the 660 kDa L (L-TC) toxin complexes. We previously proposed assembly pathways for both TCs [Kouguchi, H., et al. (2002) J. Biol. Chem. 277, 2650-2656]: M-TC is composed by association of neurotoxin (NT) and nontoxic nonhemagglutinin (NTNHA); conjugation of M-TC with three auxiliary types of hemagglutinin subcomponents (HA-33, HA-17, and HA-70) leads to the formation of L-TC. In this study, we found three TC species, 410, 540, and 610 kDa TC species, in the culture supernatant of type D strain 4947. The 540 and 610 kDa TC species displayed banding patterns on SDS-PAGE similar to that of L-TC but with less staining intensity of the HA-33 and HA-17 bands than those of L-TC, indicating that these are intermediate species in the pathway to L-TC assembly. In contrast, the 410 kDa TC species consisted of M-TC and two molecules of HA-70. All of the TC species, except L-TC, demonstrated no hemagglutination activity. When the intermediate TC species were mixed with an isolated HA-33/17 complex, every TC species converted to 650 kDa L-TC with full hemagglutination activity and had the same molecular composition of L-TC. On the basis of titration analysis with the HA-33/17 complex, the stoichiometry of the HA-33/17 complex molecules in the L-TC, 610 kDa, and 540 kDa TC species was estimated as 4, 3, and 2, respectively. In conclusion, the complete subunit composition of mature L-TC is deduced to be a dodecamer assembled by a single NT, a single NTNHA, two HA-70, four HA-33, and four HA-17 molecules.