Charge distribution of flanking amino acids inhibits O-glycosylation of several single-site acceptors in vivo.

From surveys of known O-glycosylation sites and in vitro glycosylation assays with synthetic peptide acceptors, it appears that the presence of charged amino acids near serine/threonine residues reduces the likelihood of O-glycosylation by UDP-GalNAc polypeptide:N-acetylgalactosaminyltransferases (ppGaNTases). Previously, we demonstrated that the in vivo O-glycosylation of a sequence derived from a known glycosylation site of human von Willebrand factor (PHMAQVTVGPGL) was markedly reduced when charged residues were substituted at position -1 and +3 relative to the single threonine. In contrast, acidic residues at positions -2, +1, and +2 had no effect (Nehrke et al., 1996), suggesting that charge distribution but not charge density was important. To determine whether the charge distribution effect on O-glycosylation is limited to a specific sequence context or restricted to unique isoforms of ppGaNTase, we have analyzed the in vivo O-glycosylation of six secreted recombinant reporter proteins in three different cell backgrounds. The differential presence of known ppGaNTase transcripts was determined in each cell type by Northern blot analysis. Each reporter, which contains a single site of O-glycosylation, was O-glycosylated in a cell-background-specific manner; digestion with O-glycanase and alpha-N-acetylgalactosaminidase following mild acid hydrolysis suggested that simple type II core structures were acquired. However, in COS7 cells, one reporter peptide acquired glycosaminoglycans in preference to mucin-type O-glycans. Regardless of cell background or the reporter examined, the substitution of glutamic acid residues at positions -1 and +3 markedly diminished the level of mucin-type O-glycosylation. Charge distribution would appear, therefore, to play a more general role in determining the extent to which solitary O-glycosylation sites are modified.