Sagane Y, Watanabe T, Kouguchi H, Sunagawa H, Inoue K, Fujinaga Y, Oguma K, Ohyama T
Department of Food Science, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Japan.
J Protein Chem. 1999 Nov;18(8):885-92. doi: 10.1023/a:1020687430927.
Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.
肉毒杆菌神经毒素（NT）由肉毒梭菌合成，最初是一种约150 kDa的单链多肽。经细菌或外源性蛋白酶进行翻译后修饰，形成双链结构，该结构通过一个二硫键环连接一条50 kDa的轻链（Lc）和一条100 kDa的重链（Hc）。我们通过分析Lc的C端序列和Hc的N端序列，确定了由C型斯德哥尔摩菌株、D型CB16菌株和F型奥斯陆菌株产生的肉毒杆菌NTs环区域切割位点周围的氨基酸序列。发现C型在Arg444/Ser445和Lys449/Thr450的一个或两个位点发生切割，D型则分别在Lys442/Asn443和Arg445/Asp446位点发生切割。因此，在C型和D型轻度蛋白水解菌株的培养液中，NT以至少三种带切口的双链分子形式的混合物存在。F型蛋白水解菌株奥斯陆的NT显示Arg435为Lc的C端残基，Ala440为Hc的N端残基，这表明细菌蛋白酶切割两次（Arg435/Lys436和Lys439/Ala440），切除四个氨基酸残基。环区域切割位点的位置和切除的氨基酸残基数量可以通过分子表面氨基酸残基的暴露程度来解释，该暴露程度是根据氨基酸序列预测的表面概率。此外，观察到的这种相关性也可能适用于其他肉毒杆菌毒素类型（A、B、E和G）的切割位点。
