Mutoh Shingo, Kouguchi Hirokazu, Sagane Yoshimasa, Suzuki Tomonori, Hasegawa Kimiko, Watanabe Toshihiro, Ohyama Tohru
Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
Biochemistry. 2003 Sep 23;42(37):10991-7. doi: 10.1021/bi034996c.
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.
肉毒杆菌D型菌株通常产生两种类型的稳定毒素复合物（TC），即300 kDa的M型（M-TC）和660 kDa的L型（L-TC）毒素复合物。我们之前提出了两种毒素复合物的组装途径[Kouguchi, H., 等人（2002年）《生物化学杂志》277, 2650 - 2656]：M-TC由神经毒素（NT）和无毒非血凝素（NTNHA）结合而成；M-TC与三种辅助类型的血凝素亚组分（HA-33、HA-17和HA-70）结合导致L-TC的形成。在本研究中，我们在D型菌株4947的培养上清液中发现了三种毒素复合物，分别为410、540和610 kDa的毒素复合物。540和610 kDa的毒素复合物在SDS-PAGE上显示出与L-TC相似的条带模式，但HA-33和HA-17条带的染色强度低于L-TC，表明这些是L-TC组装途径中的中间物种。相比之下，410 kDa的毒素复合物由M-TC和两个HA-70分子组成。除L-TC外，所有毒素复合物均无血凝活性。当将中间毒素复合物与分离的HA-33/17复合物混合时，每种毒素复合物都转化为具有完全血凝活性的650 kDa L-TC，且具有与L-TC相同的分子组成。基于用HA-33/17复合物进行的滴定分析，估计L-TC、610 kDa和540 kDa毒素复合物中HA-33/17复合物分子的化学计量比分别为4、3和2。总之，成熟L-TC的完整亚基组成推断为由一个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。
