Saunders F K, Mitchell T J, Walker J A, Andrew P W, Boulnois G J
Department of Microbiology, University of Leicester, United Kingdom.
Infect Immun. 1989 Aug;57(8):2547-52. doi: 10.1128/iai.57.8.2547-2552.1989.
The role of the single cysteine residue in the activity of the thiol-activated toxin pneumolysin was investigated using oligonucleotide-mediated, site-directed mutagenesis. Three modified toxins in which the cysteine residue was changed to an alanine, a serine, or a glycine residue were purified to homogeneity and examined for activity. The Cys-428----Ala modified toxin was indistinguishable from the wild-type recombinant toxin in terms of hemolytic activity and lytic and inhibitory effects on human polymorphonuclear leukocytes (PMN), indicating that the cysteine residue is not essential for toxin activity. The Cys-428----Ser and Cys-429----Gly modified toxins had reduced activity on erythrocytes and polymorphonuclear leukocytes, being 6 and 20 times less active than the wild type, respectively. However, all the modified toxins formed oligomers in erythrocyte membranes to the same extent as the wild-type recombinant toxin. This suggests that the cysteine residue at position 428 is involved in neither the binding of toxin to membranes nor its insertion into the membrane, and also that the formation of oligomers is not by itself sufficient for toxin activity.
利用寡核苷酸介导的定点诱变技术，研究了单个半胱氨酸残基在硫醇激活毒素肺炎球菌溶血素活性中的作用。将半胱氨酸残基分别突变为丙氨酸、丝氨酸或甘氨酸残基的三种修饰毒素纯化至同质，并检测其活性。Cys-428→Ala修饰毒素在溶血活性以及对人多形核白细胞（PMN）的裂解和抑制作用方面与野生型重组毒素无差异，表明半胱氨酸残基对毒素活性并非必不可少。Cys-428→Ser和Cys-429→Gly修饰毒素对红细胞和多形核白细胞的活性降低，分别比野生型低6倍和20倍。然而，所有修饰毒素在红细胞膜中形成寡聚体的程度与野生型重组毒素相同。这表明428位的半胱氨酸残基既不参与毒素与膜的结合，也不参与其插入膜中，而且寡聚体的形成本身不足以产生毒素活性。
