Yamada T, Uyeda A, Takao T, Shimonishi Y, Matsushima M, Kikuchi M
Protein Engineering Research Institute, Osaka, Japan.
Eur J Biochem. 1995 Jun 15;230(3):965-70. doi: 10.1111/j.1432-1033.1995.tb20643.x.
The cell-adhesive protein Cys-RGD4 has been constructed using a yeast expression system by inserting the sequence Cys-Arg-Gly-Asp-Ser-Cys (CRGDSC) between Val74 and Asn75 of human lysozyme [Yamada, T., Uyeda, A., Kidera, A. & Kikuchi, M. (1994b) Biochemistry 33, 11678-11683]. The Cys74a, Arg74b, Gly74c, Asp74d, Ser74e, Cys74f-lysozyme mutant, purified from the yeast culture supernatant contained glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the Thr70 residue in the Cys-RGD4 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of two hexose residues in the major variant, and one, three, four, or five hexose residues in the minor variants. All of these hexose residues were identified as mannose by analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants. No other glycosylation was observed, although the Cys-RGD4 molecule possesses a total of 12 threonine and serine residues. In addition, the Thr70 residue is not glycosylated in either native lysozyme or the Arg-Gly-Asp-Ser (RGDS)-inserted mutant, RGD4 [Yamada, T., Matsushima, M., Inaka, K., Ohkubo, T., Uyeda, A., Maeda, T., Titani, K., Sekiguchi, K. & Kikuchi, M. (1993) J. Biol. Chem. 268, 10588-10592]. Thus, this O-glycosylation seems to be specific for both the mutant lysozyme molecule and the site of the threonine residue. Structural analyses of these lysozymes by X-ray crystallography suggest that the conformation of the serine-containing or threonine-containing region can affect the specificity of yeast O-glycosylation.
通过将半胱氨酸-精氨酸-甘氨酸-天冬氨酸-丝氨酸-半胱氨酸（CRGDSC）序列插入人溶菌酶的Val74和Asn75之间，利用酵母表达系统构建了细胞粘附蛋白Cys-RGD4[山田，T.，上田，A.，木寺，A.和菊池，M.（1994b）生物化学33，11678 - 11683]。从酵母培养上清液中纯化得到的Cys74a、Arg74b、Gly74c、Asp74d、Ser74e、Cys74f-溶菌酶突变体，除了未糖基化形式外，还含有糖基化变体。肽图谱分析表明，糖基化发生在Cys-RGD4分子中的Thr70残基上。电喷雾电离质谱分析表明，主要变体中存在两个己糖残基，次要变体中存在一个、三个、四个或五个己糖残基。通过对经温和碱处理变体得到的寡糖混合物进行分析，所有这些己糖残基均被鉴定为甘露糖。尽管Cys-RGD4分子总共含有12个苏氨酸和丝氨酸残基，但未观察到其他糖基化现象。此外，Thr70残基在天然溶菌酶或插入精氨酸-甘氨酸-天冬氨酸-丝氨酸（RGDS）的突变体RGD4中均未糖基化[山田，T.，松岛，M.，稻中，K.，大久保，T.，上田，A.，前田，T.，谷仁，K.，关口，K.和菊池，M.（1993）生物化学杂志268，10588 - 10592]。因此，这种O-糖基化似乎对突变体溶菌酶分子和苏氨酸残基位点都具有特异性。通过X射线晶体学对这些溶菌酶进行结构分析表明，含丝氨酸或含苏氨酸区域的构象可以影响酵母O-糖基化的特异性。
