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 (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.
Yamada
T
T
Protein Engineering Research Institute, Osaka, Japan.
Uyeda
A
A
Takao
T
T
Shimonishi
Y
Y
Matsushima
M
M
Kikuchi
M
M
D000595:Amino Acid Sequence; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D006031:Glycosylation; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry*; D013912:Threonine / Q000378:metabolism; D015003:Yeasts / Q000235:genetics
D015815:Cell Adhesion Molecules; D009842:Oligopeptides; D013912:Threonine; C047981:arginyl-glycyl-aspartic acid; D009113:Muramidase