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 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.
Highlights
O-Mannosyl glycans are important in muscle and brain development
We have found that protein O-mannosyltransferase 1 (POMT1) and its homolog POMT2 are responsible for the catalysis of the first step in O-mannosyl glycan synthesis [6]
Mutations in POMT1 and POMT2 genes are considered to be the cause of Walker-Warburg syndrome (WWS: OMIM 236670), an autosomal recessive developmental disorder associated with congenital muscular dystrophy, neuronal migration defects, and ocular abnormalities [7, 8]
Summary
Chemicals—Synthetic peptides were purchased by madeto-order system on the web site of Sigma-Aldrich (www. genosys.jp, Tokyo, Japan), and the quality of the synthesis was ascertained by HPLC analysis and mass spectrometry. The reaction mixture containing 20 mM Tris-HCl, pH 8.0, 100 nM mannosylphosphoryldolichol (125,000 dpm/pmol), 2 mM dithiothreitol, 10 mM EDTA, 0.5% n-octyl--D-thioglucoside, 0.25– 4 mM synthetic peptide; 40 g of microsomal membrane fraction in 20 l of total volume was incubated for 60 min at 25 °C. The reaction mixture containing 20 mM Tris-HCl, pH 8.0, 300 M unlabeled mannosylphosphoryldolichol, 2 mM dithiothreitol, 10 mM EDTA, 0.5% n-octyl--D-thioglucoside, 1 mM peptide 401– 420; 120 g of microsomal membrane fraction in 30 l of total volume was incubated for 15–120 min at 25 °C. The bound fraction was obtained by eluting with 250 l of 200 mM ␣-methylmannoside in 20 mM Tris-HCL, pH 7.4, 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, and 1 mM MgCl2 and separated by reversed-phase HPLC as described above. For MS/MS spectra, parent ion was selected Ϯ 15 Da from the observed MHϩ value using time gate and re-accelerated (LIFT mode)
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