Isoform-specific O-glycosylation of osteopontin and bone sialoprotein by polypeptide N-acetylgalactosaminyltransferase-1.

Mucin-type O-glycan biosynthesis is regulated by the family of UDP-GalNAc polypeptide:N-acetylgalactosaminlytransfersases (ppGalNAcTs) that catalyzes the first step in the pathway by transferring GalNAc to Ser or Thr residues in a protein from the sugar donor UDP-GalNAc. Because not all Ser/Thr residues are glycosylated, rules must exist that signal which hydroyxamino acids acquire sugar. To date, no universal consensus signal has emerged. Therefore, strategies to deduce the subset of proteins that will be glycosylated by distinct ppGalNAcTs must be developed. Mucin-type O-glycoproteins are present abundantly in bone, where we found multiple ppGalNAcT isoforms, including ppGalNAcT-1, to be highly expressed. Thus, we compared glycoproteins expressed in wild-type and Galnt1-null mice to identify bone-associated proteins that were glycosylated in a ppGalNAcT-1-dependent manner. A reduction in the apparent molecular masses of two SIBLINGs (small integrin binding ligand N-linked glycoproteins), osteopontin (OPN) and bone sialoprotein (BSP) in the Galnt1-null mice relative to those of the wild-type was observed. Several synthetic peptides derived from OPN and BSP sequences were designed to include either known or predicted (in silico) glycosylation sites. In vitro glycosylation assays of these peptides with recombinant ppGalNAcT-1, ppGalNAcT-2, or ppGalNAcT-3 demonstrated that both SIBLINGs contained Thr/Ser residues that were preferentially glycosylated by ppGalNAcT-1. In addition, lysates prepared from wild-type, but not those from Galnt1-null derived osteoblasts, could glycosylate these peptides efficiently, suggesting that OPN and BSP contain sites that are specific for ppGalNAcT-1. Our study presents a novel and systematic approach for identification of isoform-specific substrates of the ppGalNAcT family and suggests ppGalNAcT-1 to be indispensable for O-glycosylation at specific sites of the bone glycoproteins OPN and BSP.