Sjöbring U, Björck L, Kastern W
Department of Medical Microbiology, University of Lund, Sweden.
J Biol Chem. 1991 Jan 5;266(1):399-405.
Protein G was solubilized from 31 human group C and G streptococcal strains with the muralytic enzyme mutanolysin. As judged by the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the binding patterns of the solubilized protein G molecules in Western blot experiments, the strains could be divided into three groups, represented by the group G streptococcal strains G148 and G43 and the group C streptococcal strain C40. The 65-kDa G148 protein G and the 58-kDa C40 protein G showed affinity for both immunoglobulin G (IgG) and human serum albumin (HSA), whereas the 40-kDa G43 protein G bound only IgG. Despite the different molecular patterns, the three protein G species had identical NH2-terminal amino acid sequences. Apart from the 65-kDa peptide, digestion of G148 streptococci with mutanolysin also produced a 52-kDa IgG- and HSA-binding peptide and a 14-kDa HSA-binding peptide. It was demonstrated that these peptides resulted from cleavage of 65-kDa protein G by proteolytic components in the mutanolysin preparation. The protein G genes of the C40 and G43 strains were cloned and sequenced, and their structure was compared to the previously published sequence of the G148 protein G gene. As compared to G148, both the C40 and G43 genes lacked a 210-base pair fragment in the IgG-binding region, accounting for the 10-fold lower affinity of these proteins for IgG. The G43 gene also lacked a 450-base pair fragment in the 5'-end of the gene, explaining why the G43 protein G did not bind HSA. The differences in protein G structure did not correlate with the clinical origin of the strains used in this study. The IgG-binding region of protein G was further mapped. Thus, a peptide corresponding to a single IgG-binding unit was obtained by the cloning and expression of a 303-base pair polymerase chain reaction-generated DNA fragment. The affinity of this 11.5-kDa peptide for human IgG was 8.0 x 10(7) M-1, as determined by Scatchard plots. Finally, a 55-amino acid-long synthetic peptide, corresponding to one of the three repeated domains in the COOH-terminal half of strain G148 protein G, effectively blocked binding of protein G to IgG.
用溶菌酶从31株人C组和G组链球菌中溶解出蛋白G。根据十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳中的迁移率以及蛋白质印迹实验中溶解的蛋白G分子的结合模式判断，这些菌株可分为三组，以G组链球菌菌株G148和G43以及C组链球菌菌株C40为代表。65 kDa的G148蛋白G和58 kDa的C40蛋白G对免疫球蛋白G（IgG）和人血清白蛋白（HSA）均有亲和力，而40 kDa的G43蛋白G仅结合IgG。尽管分子模式不同，但这三种蛋白G具有相同的NH2末端氨基酸序列。除了65 kDa的肽段外，用溶菌酶消化G148链球菌还产生了一个52 kDa的与IgG和HSA结合的肽段以及一个14 kDa的与HSA结合的肽段。已证明这些肽段是由溶菌酶制剂中的蛋白水解成分切割65 kDa蛋白G产生的。克隆并测序了C40和G43菌株的蛋白G基因，并将它们的结构与先前发表的G148蛋白G基因序列进行了比较。与G148相比，C40和G43基因在IgG结合区域均缺少一个210碱基对的片段，这解释了这些蛋白对IgG的亲和力低10倍的原因。G43基因在基因的5'端还缺少一个450碱基对的片段，这解释了为什么G43蛋白G不结合HSA。蛋白G结构的差异与本研究中使用的菌株的临床来源无关。进一步绘制了蛋白G的IgG结合区域。因此，通过克隆和表达303碱基对聚合酶链反应产生的DNA片段获得了一个对应于单个IgG结合单位的肽段。通过Scatchard图测定，该11.5 kDa肽段对人IgG的亲和力为8.0×10(7) M-1。最后，一个55个氨基酸长的合成肽，对应于G148菌株蛋白G羧基末端一半的三个重复结构域之一，有效地阻断了蛋白G与IgG的结合。
