Taniyama Y, Seko C, Kikuchi M
Protein Engineering Research Institute, Osaka, Japan.
J Biol Chem. 1990 Oct 5;265(28):16767-71.
A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.
一种突变型人溶菌酶C77A（其中半胱氨酸-77被丙氨酸取代）由酿酒酵母分泌为两种具有不同比活性的蛋白质（C77A-a和C77A-b）。通过胃蛋白酶消化从C77A-a获得了一个从Val93到Ala108的肽片段，并通过快原子轰击质谱法和氨基酸分析进行了检测。结果表明，谷胱甘肽通过二硫键连接到该片段半胱氨酸95的巯基上。通过过甲酸处理C77A-a定量形成游离谷胱甘肽磺酸，证实了这一观察结果。相比之下，C77A-b的情况没有修饰。这些结果表明，C77A-a含有一种混合二硫键，谷胱甘肽连接到半胱氨酸残基95上。在C77A-b中，半胱氨酸95的游离巯基似乎被许多侧链包围，在天然条件下未被碘乙酸修饰，这表明谷胱甘肽的连接发生在折叠过程中。这些发现进一步表明，在酵母分泌的人溶菌酶二硫键形成的氧化步骤中，会与谷胱甘肽形成混合二硫键，并且即使在酵母分泌的蛋白质中也会发生谷胱甘肽的翻译后修饰。
