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 of organisms. In general, these isoforms display different patterns of tissue-specific expression, but exhibit overlapping substrate specificities, in vitro . A peptide substrate, derived from the sequence of the V3 loop of the HIV gp120 protein (HIV peptide), has previously been shown to be glycosylated in vitro exclusively by the ppGaNTase-T3 (Bennett et al. , 1996). To determine if this isoform-specificity is maintained in vivo , we have examined the glycosylation of this substrate when it is expressed as a reporter peptide (rHIV) in a cell background (COS7 cells) which lacks detectable levels of the ppGaNTase-T3. Glycosylation of rHIV was greatly increased by coexpression of a recombinant ppGaNTase-T3. Overexpression of ppGaNTase-T1 yielded only partial glycosylation of the reporter. We have also determined that the introduction of a proline residue at the +3 position flanking the potential glycosylation site eliminated ppGaNTase-T3 selectivity toward rHIV observed both in vivo and in vitro .