The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity
The variant undecapeptide sequence of the Arcanobacterium pyogenes haemolysin, pyolysin, is required for full cytolytic activity
复制标题
DOI：
10.1099/00221287-148-12-3947
复制
发表时间：
2002-12-01
期刊：
影响因子：
2.8
通讯作者：
Jost, BH
中科院分区：
文献类型：
--
作者：
Billington, SJ;Songer, JG;Jost, BH
胆固醇依赖性溶细胞素 (CDC) 的特征在于位于这些蛋白质的 C 末端附近和结构域 4 内的十一肽序列 (ECTGLAWEWWR)。化脓菌溶血素 (PLO) 是化脓秘杆菌的 CDC，具有变异的十一肽序列 (EATGLAWDPWW)。在含有六组氨酸标签 (His-PLO) 的重组 PLO 分子的十一肽残基中构建定点突变体。三个十一肽色氨酸残基中的每一个的突变都会导致低溶血活性，证实了这些残基在蛋白质中的重要性。删除插入 PLO 中的脯氨酸残基 (P-499)，或用苯丙氨酸或甘氨酸取代该残基，会产生溶血活性不可检测或较低的突变蛋白，表明 P-499 对于 His-PLO 溶血活性至关重要。用共有十一肽替换 PLO 十一肽序列导致 His-PLO 蛋白活性仅为 0.1%，证实变体 PLO 十一肽是该毒素的完整细胞溶解活性所必需的。保守的十一肽半胱氨酸残基单独存在 (His-PLO,CM) 或在共有序列中导致 His-PLO 分子在还原化合物存在下被激活，证实了该残基在许多 CDC 毒素的硫醇激活性质中的重要性。 His-PLO突变蛋白结合胆固醇的能力模仿了溶血活性，但His-PLO.C-492除外，尽管其溶血活性降低，但与His-PLO相比，其结合胆固醇的能力有所增强。尽管溶血活性和胆固醇结合减少，但所有突变蛋白仍然能够与红细胞膜结合，这表明 PLO 的其他区域可能通过胆固醇以外的受体识别宿主细胞膜。
The cholesterol-dependent cytolysins (CDCs) are characterized by an undecapeptide sequence (ECTGLAWEWWR) that is located near the C terminus and within domain 4 of these proteins. Pyolysin (PLO), the CDC of Arcanobacterium pyogenes, has a variant undecapeptide sequence (EATGLAWDPWW). Site-directed mutants were constructed in undecapeptide residues in a recombinant PLO molecule containing a hexahistidine tag (His-PLO). Mutations in each of the three undecapeptide tryptophan residues resulted in low haemolytic activity, confirming the importance of these residues in the protein. Deletion of a proline residue (P-499), inserted in PLO, or substitution of this residue with either phenylalanine or glycine resulted in mutant proteins with undetectable or low haemolytic activities, indicating that P-499 is essential for His-PLO haemolytic activity. Substitution of the PLO undecapeptide sequence with a consensus undecapeptide resulted in a His-PLO protein with only 0.1 % activity, confirming that the variant PLO undecapeptide is required for the full cytolytic activity of this toxin. The presence of the conserved undecapeptide cysteine residue either alone (His-PLO,CM) or in a consensus sequence resulted in His-PLO molecules which were activated in the presence of reducing compounds, confirming the importance of this residue in the thiol-activated nature of many CDC toxins. The ability of His-PLO mutant proteins to bind cholesterol mimicked haemolytic activity, with the exception of His-PLO.C-492, which, despite having reduced haemolytic activity, showed an increased ability to bind cholesterol compared to His-PLO. Despite reductions in haemolytic activity and cholesterol-binding, all mutant proteins were still able to bind to erythrocyte membranes, suggesting that other regions of PLO may recognize host-cell membranes, through receptors other than cholesterol.