Billington Stephen J, Songer J Glenn, Jost B Helen
Department of Veterinary Science and Microbiology, The University of Arizona, 1117 East Lowell Street, Tucson, AZ 85721, USA1.
Microbiology (Reading). 2002 Dec;148(Pt 12):3947-3954. doi: 10.1099/00221287-148-12-3947.
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.C(492)) 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.
胆固醇依赖细胞毒素（CDCs）的特征是存在一个十一肽序列（ECTGLAWEWWR），该序列位于这些蛋白质的C末端附近且在结构域4内。化脓隐秘杆菌的CDC——化脓菌素（PLO）具有一个变体十一肽序列（EATGLAWDPWW）。在含有六聚组氨酸标签的重组PLO分子（His-PLO）的十一肽残基中构建了定点突变体。三个十一肽色氨酸残基中每个残基的突变都导致溶血活性降低，证实了这些残基在蛋白质中的重要性。删除插入PLO中的一个脯氨酸残基（P(499)），或将该残基替换为苯丙氨酸或甘氨酸，导致突变蛋白的溶血活性无法检测到或很低，表明P(499)对His-PLO的溶血活性至关重要。用一个共有十一肽替换PLO的十一肽序列，得到的His-PLO蛋白活性仅为0.1%，证实了变体PLO十一肽是该毒素完全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.C(492)）或存在于共有序列中时，会产生在还原化合物存在下被激活的His-PLO分子，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与溶血活性相似，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增强。尽管溶血活性和胆固醇结合能力降低，但所有突变蛋白仍能与红细胞膜结合，这表明PLO的其他区域可能通过除胆固醇以外的受体识别宿主细胞膜。
