Regulation of Mammalian Protein O-Mannosylation PREFERENTIAL AMINO ACID SEQUENCE FOR O-MANNOSE MODIFICATION

Abstract O-Mannosyl glycans are important in muscle and brain development. Protein O-mannosyltransferase (POMT) catalyzes the initial step of O-mannosyl glycan biosynthesis. To understand which serine (Ser) and threonine (Thr) residues POMT recognizes for mannosylation, we prepared a series of synthetic peptides based on a mucin-like domain in α-dystroglycan (α-DG), one of the best known O-mannosylated proteins in mammals. In α-DG, the mucin-like domain spans amino acid residues 316 to 489. Two similar peptide sequences, corresponding to residues 401–420 and 336–355, respectively, were strongly mannosylated by POMT, whereas other peptides from α-DG and peptides of various mucin tandem repeat regions were poorly mannosylated. Peptides 401–420 and 336–355 contained four and six Ser and Thr residues, respectively. Substitution of Ala residues for the Ser or Thr residues showed that Thr-414 of peptide 401–420 and Thr-351 of peptide 336–355 were prominently modified by O-mannosylation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Edman degradation analysis of the mannosylated peptide 401–420 indicated that Thr-414 was the Thr residue that was most prominently modified by O-mannosylation and that O-mannosylation occurred sequentially rather than at random. Based on these results, we propose a preferred amino acid sequence for mammalian O-mannose modification.