O-GLYCOSYLATION OF THE THR70 RESIDUE OF CELL-ADHESIVE LYSOZYME IN YEAST

The cell-adhesive protein Cys-RGD4 has been constructed using a yeast expression system by inserting the sequence Cys-Arg-Gly-Asp-Ser-Cys (C RGDSC) between Val74 and Asn75 of human lysozyme [Yamada, T., Uyeda, A ., Kidera, A. & Kikuchi, M. (1994b) Biochemistry 33, 11678-11683]. The Cys(74a) Arg(74b), Gly(74c), Asp(74d), Ser(74e), Cys(74f)-lysozyme mu tant, purified from the yeast culture supernatant contained glycosylat ed variants, in addition to the unglycosylated form. Peptide mapping a nalyses 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 v ariants. All of these hexose residues were identified as mannose by an alysis of the oligosaccharide mixture obtained by mild alkaline treatm ent of the variants. No other glycosylation was observed, although the Cys-RGD4 molecule possesses a total of 12 threonine and serine residu es. In addition, the Thr70 residue is not glycosylated in either nativ e lysozyme or the Arg-Gly-Asp-Ser (RGDS)-inserted mutant, RGD4 [Yamada , T., Matsushima, M., Inaka, K., Ohkubo, T., Uyeda, A., Maeda, T., Tit ani, K., Sekiguchi, K. & Kikuchi, M. (1993) J. Biol. Chem. 268, 10588- 10592]. Thus, this O-glycosylation seems to be specific far both the m utant lysozyme molecule and the site of the threonine residue. Structu ral analyses of these lysozymes by X-ray crystallography suggest that the conformation of the serine-containing or threonine-containing regi on can affect the specificity of yeast O-glycosylation.