Gene inactivation confirms the identity of enzymes involved in nematode phosphorylcholine-N-glycan synthesis.
An unusual feature of nematodes is the covalent attachment of immunomodulatory phosphorylcholine (PC) moieties to N-type glycans. Our previous work on the filarial nematode glycoprotein ES-62 has enabled us to predict the identity of enzymes necessary for PC-N-glycan biosynthesis. Here, we addressed these predictions using gene knockout technology applied to C. elegans and present two pieces of confirmatory data. Employing a triple null mutant worm lacking all three genes that encode active UDP-N-acetyl-D-glucosamine: alpha-3-D-mannoside beta1, 2-N-acetylglucosaminyltransferase I (GnT I) we have confirmed our earlier prediction that a crucial step in the generation of the substrate for PC transfer is addition of terminal GlcNAc to the alpha1-3-linked mannose residue of the glycan by GnT I. Second, by silencing genes responsible for expressing enzymes of the Kennedy pathway of phosphatidylcholine biosynthesis by RNA interference (RNAi), we have confirmed our belief for a role for diacylglycerol: choline phosphotransferase (CPT) in PC-N-glycan biosynthesis.
Houston
Katrina M
KM
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, Strathclyde, United Kingdom.
Sutharsan
Ratneswary
R
Steiger
Christina N
CN
Schachter
Harry
H
Harnett
William
W
D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D002798:Diacylglycerol Cholinephosphotransferase / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D010767:Phosphorylcholine / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D011134:Polysaccharides; D010767:Phosphorylcholine; D017351:N-Acetylglucosaminyltransferases; C446926:UDP-GlcNAc alpha-D-mannoside beta-1,2-N-acetylglucosaminyltransferase; D002798:Diacylglycerol Cholinephosphotransferase