Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides
Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides
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DOI：
10.1111/j.1348-0421.1997.tb01888.x
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发表时间：
1997-01-01
影响因子：
2.6
通讯作者：
Okabe, A
中科院分区：
文献类型：
--
作者：
Minami, J;Katayama, S;Okabe, A
检查了产气荚膜梭菌的嗜热菌蛋白酶样金属蛋白酶 lambda-毒素对同一生物体产生的无活性ε-原毒素的影响。当纯化的ε-原毒素与纯化的lambda-毒素在37°C下温育2小时时，32.5-kDa的ε-原毒素被加工成30.5-kDa多肽，如通过SDS-聚丙烯酰胺凝胶电泳。小鼠致死试验表明，该处理激活了原毒素：经过和未经 lambda 毒素处理的原毒素的 50% 致死剂量 (LD50) 分别为 110 ng/kg 体重和 70,000 ng/kg 体重。 λ-毒素激活的原毒素的致死活性与胰蛋白酶加胰凝乳蛋白酶相当，并高于单独的胰蛋白酶：胰蛋白酶和胰蛋白酶加胰凝乳蛋白酶处理的原毒素的LD50分别为320和65ng/kg体重。 ε-毒素基因被克隆并测序。测定每种活化的ε-原毒素的N-末端氨基酸序列表明，λ-毒素在原毒素N-末端的第10和第11个氨基酸残基之间裂解，而胰蛋白酶和胰蛋白酶加胰凝乳蛋白酶在第13和第14个氨基酸残基之间裂解。还通过基质辅助激光测定了每种活化的ε-原毒素的分子量解吸/电离飞行时间质谱，从分子量推算的C端位于原毒素C端第23或30个氨基酸残基，表明不仅N端肽被去除，C端肽也被去除是原毒素激活的原因。
The effect of lambda-toxin, a thermolysin-like metalloprotease of Clostridium perfringens, on the inactive epsilon-prototoxin produced by the same organism was examined, When the purified epsilon-prototoxin was incubated with the purified lambda-toxin at 37 C for 2 hr, the 32.5-kDa epsilon-prototoxin was processed into a 30.5-kDa polypeptide, as determined by SDS-polyacrylamide gel electrophoresis. A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD50) of the prototoxin with and without lambda-toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. The lethal activity of the prototoxin activated by lambda-toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD50 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. The epsilon-toxin gene was cloned and sequenced. Determination of the N-terminal amino acid sequence of each activated epsilon-prototoxin revealed that lambda-toxin cleaved between the 10th and 11th amino acid residues from the N-terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues, The molecular weight of each activated epsilon-prototoxin was also determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, The C-terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C-terminus of the prototoxin, suggesting that removal of not only N-terminal but also C-terminal peptide is responsible for activation of the prototoxin.