In vitro reconstitution of the Clostridium botulinum type D progenitor toxin
In vitro reconstitution of the Clostridium botulinum type D progenitor toxin
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DOI：
10.1074/jbc.m106762200
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发表时间：
2002-01-25
影响因子：
4.8
通讯作者：
Ohyama, T
中科院分区：
文献类型：
--
作者：
Kouguchi, H;Watanabe, T;Ohyama, T
D 型肉毒梭菌菌株 4947 产生两种不同大小的祖毒素（M 和 L），它们是未经蛋白水解加工的完整形式。 M 毒素由神经毒素 (NT) 和无毒非血凝素 (NTNHA) 组成，而 L 毒素由 M 毒素和血凝素 (HA) 亚成分（RA-70、HA-17 和 HA-33）组成。在变性剂存在下，通过色谱法将 RA-70 亚组分和 HA-33/17 复合物与 L 毒素分离，使其接近均质。我们首次证明了通过混合纯化的 M 毒素、HA-70 和 HA-33/17 形成的 L 毒素的体外重构。重组和天然 L 毒素的特性在凝胶过滤特性、天然 PAGE 特性、血凝活性、红细胞结合活性以及对小鼠的口服毒性方面无法区分。用胰蛋白酶处理制备的含有带切口的NTNHA的M毒素不能再重构为具有HA亚成分的L毒素，而用蛋白酶处理的L毒素则不能降解为M毒素和HA亚成分。我们得出结论，M 毒素首先通过 NT 与 NTNHA 组装形成，随后通过与 HA-70 和 HA-33/17 组装转化为 L 毒素。
Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing. The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (RA-70, HA-17, and HA-33). The RA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents. We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17. The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice. M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents. We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17.