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.