Role of N-glycans in maintaining the activity of protein O-mannosyltransferases POMT1 and POMT2.
The complex of protein O-mannosyltransferase 1 (POMT1) and POMT2 catalyzes the initial step of O-mannosyl glycan biosynthesis. The mutations in either POMT1 or POMT2 can lead to Walker-Warburg syndrome, a congenital muscular dystrophy with abnormal neuronal migration. Here, we used three algorithms for predicting transmembrane helices to construct the secondary structural models of human POMT1 and POMT2. In these models, POMT1 and POMT2 have seven- and nine-transmembrane helices and contain four and five potential N-glycosylation sites, respectively. To determine whether these sites are actually glycosylated, we prepared mutant proteins that were defective in each site by site-directed mutagenesis. Three of the POMT1 sites and all of the POMT2 sites were found to be N-glycosylated, suggesting that these sites face the luminal side of the endoplasmic reticulum. Mutation of any single site did not significantly affect POMT activity, but mutations of all N-glycosylation sites of either POMT1 or POMT2 caused a loss of POMT activity. The loss of activity appeared to be due to the decreased hydrophilicity. These results suggest that the N-glycosylation of POMT1 and POMT2 is required for maintaining the conformation as well as the activity of the POMT1-POMT2 complex.
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, Japan. manya@tmig.or.jp
Akasaka-Manya
Keiko
K
Nakajima
Ai
A
Kawakita
Masao
M
Endo
Tamao
T
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008364:Mannosyltransferases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009136:Muscular Dystrophies / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012995:Solubility / Q000187:drug effects; D014415:Tunicamycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D011134:Polysaccharides; D014415:Tunicamycin; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase