Regulation of mammalian protein O-mannosylation: preferential amino acid sequence for O-mannose modification.
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 alpha-dystroglycan (alpha-DG), one of the best known O-mannosylated proteins in mammals. In alpha-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 alpha-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.
Manya
Hiroshi
H
Glycobiology Research Group, Tokyo Metropolitan Institute of Gerontology, Foundation for Research on Aging and Promotion of Human Welfare, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan.
Suzuki
Takehiro
T
Akasaka-Manya
Keiko
K
Ishida
Hide-Ki
HK
Mizuno
Mamoru
M
Suzuki
Yasushi
Y
Inazu
Toshiyuki
T
Dohmae
Naoshi
N
Endo
Tamao
T
D000595:Amino Acid Sequence; D001921:Brain / Q000196:embryology / Q000201:enzymology; D002460:Cell Line; D049030:Dystroglycans / Q000378:metabolism*; D006801:Humans; D008364:Mannosyltransferases / Q000378:metabolism*; D013058:Mass Spectrometry; D009077:Mucins / Q000378:metabolism; D024510:Muscle Development / Q000502:physiology; D010455:Peptides / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein
D009077:Mucins; D010455:Peptides; D011134:Polysaccharides; D049030:Dystroglycans; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase