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 severaldifferent sugars including N-acetylglucosamine, galactose, and mannose. Inthis report we have reexamined the glycosylation of T antigen and foundthat while we could detect modification with N-acetylglucosamine, we couldnot detect any other sugars on the protein. Surprisingly, even though[3H]galactose could be metabolically incorporated into the protein,analysis showed that all of the radioactivity in T antigen had beenconverted to other species. The N-acetylglucosamine was demonstrated to belinked to the protein in the form of O-linked N- acetylglucosamine, thebest characterized form of nuclear and cytoplasmic glycosylation inmammalian systems. We have localized the major site of glycosylation to theamino terminal portion of the molecule. Analysis of mutated T antigen whereserines 111/112 were substituted with alanine suggest that these residuesconstitute a glycosylation site on the protein. These two serines fallwithin a typical O-linked N-acetylglucosamine glycosylation site (PSS) andare also known to be phosphorylated. Thus, it is likely that competitionbetween phosphorylation and glycosylation occurs at this site.