Site-specific O-glycosylation of cell adhesive lysozyme in yeast.
The cell adhesive protein RGD8 has been constructed using a yeast expression system by inserting eight amino acid residues (TGRGDSPA) between Val74 and Asn75 of human lysozyme [Yamada et al. (1993) J. Biol. Chem. 268, 10588-10592]. Purified RGD8 from yeast culture supernatant was found to contain glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the inserted Thr residue in the RGD8 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of four or five hexose residues in the glycosylated variants. Only mannose was detected in the sugar analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants, and the structures of these carbohydrate chains were identified as Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha and Man alpha 1-3Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha by 1H-NMR spectroscopy. No other glycosylation was found, although the RGD8 molecule possesses a total of 13 Thr and Ser residues. In addition, no O-glycosylation was observed when the RGD8 protein was expressed in mouse L-cells. Thus, this O-glycosylation looks specific for yeast and the site of the Thr residue. The O-glycosylated variants of RGD8 exhibited a high level of adhesion activity to baby hamster kidney cells, which was almost comparable to that of the unglycosylated form.
Yamada
T
T
Protein Engineering Research Institute, Osaka, Japan.
Uyeda
A
A
Otsu
M
M
Matsushima
M
M
Sekiguchi
K
K
Kikuchi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006031:Glycosylation; D006801:Humans; D007739:L Cells; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid; D009113:Muramidase