Isoform-specific O-glycosylation by murine UDP-GalNAc:polypeptide N- acetylgalactosaminyltransferase-T3, in vivo

Multiple isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase (ppGaNTase) have been cloned and expressed from a variety oforganisms. In general, these isoforms display different patterns oftissue-specific expression, but exhibit overlapping substratespecificities, in vitro . A peptide substrate, derived from the sequence ofthe V3 loop of the HIV gp120 protein (HIV peptide), has previously beenshown to be glycosylated in vitro exclusively by the ppGaNTase-T3 (Bennettet al. , 1996). To determine if this isoform- specificity is maintained invivo , we have examined the glycosylation of this substrate when it isexpressed as a reporter peptide (rHIV) in a cell background (COS7 cells)which lacks detectable levels of the ppGaNTase-T3. Glycosylation of rHIVwas greatly increased by coexpression of a recombinant ppGaNTase-T3.Overexpression of ppGaNTase- T1 yielded only partial glycosylation of thereporter. We have also determined that the introduction of a prolineresidue at the +3 position flanking the potential glycosylation siteeliminated ppGaNTase- T3 selectivity toward rHIV observed both in vivo andin vitro .