CHARGE-DISTRIBUTION OF FLANKING AMINE 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 accepters, it appears that the presence of charged amino acids near serine/threonine residues reduces the lik elihood of O-glycosylation by UDP-GalNAc polypeptide:N-acetylgalactosa minyl-transferases (ppGaNTases). Previously, we demonstrated that the in vivo O-glycosylation of a sequence derived from a known glycosylati on site of human von Willebrand factor (PHMAQVTVGPGL) was markedly red uced when charged residues were substituted at position -1 and +3 rela tive to the single threonine, In contrast, acidic residues at position s -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 ppG aNTase, we have analyzed the in vivo O-glycosylation of six secreted r ecombinant 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 contai ns a single site of O-glycosylation, was O-glycosylated in a cell-back ground-specific manner; digestion with O-glycanase and alpha-N-acetylg alactosaminidase following mild acid hydrolysis suggested that simple type II core structures were acquired, However, in COS7 cells, one rep orter 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 markedl y diminished the level of mucin-type O-glycosylation, Charge distribut ion would appear, therefore, to play a more general role in determinin g the extent to which solitary O-glycosylation sites are modified.