SV40 LARGE T-ANTIGEN IS MODIFIED WITH O-LINKED N-ACETYLGLUCOSAMINE BUT NOT WITH OTHER FORMS OF GLYCOSYLATION

SV40 large T antigen has been reported to be modified with several dif ferent sugars including N-acetylglucosamine, galactose, and mannose, I n this report we have reexamined the glycosylation of T antigen and fo und that while we could detect modification with N-acetylglucosamine, we could not detect any other sugars on the protein, Surprisingly, eve n though [H-3]galactose could be metabolically incorporated into the p rotein, analysis showed that all of the radioactivity in T antigen had been converted to other species, The N-acetylglucosamine was demonstr ated to be linked to the protein in the form of O-linked N-acetylgluco samine, the best characterized form of nuclear and cytoplasmic glycosy lation in mammalian systems, We have localized the major site of glyco sylation to the amino terminal portion of the molecule, Analysis of mu tated T antigen where serines 111/112 were substituted with alanine su ggest that these residues constitute a glycosylation site on the prote in. These two serines fall within a typical O-linked N-acetylglucosami ne glycosylation site (PSS) and are also known to be phosphorylated, T hus, it is likely that competition between phosphorylation and glycosy lation occurs at this site.