Phosphatidylcholine requirement for the N-glycosylation of synthetic peptides by detergent-solubilized oligosaccharyltransferase.
The ability of dolichyl-P-P-oligosaccharide:peptide oligosaccharyltransferase to use exogenous substrates (a previously labeled oligosaccharide lipid and an Asn-X-Thr containing heptapeptide) is shown to require phospholipid. The enzyme was extracted from porcine thyroid rough microsomes using NaCl-Nonidet P-40. When measured at low concentration, in a neutral detergent-containing medium, it undergoes a rapid loss of activity, which renders impossible quantitative estimates in the range of 0-50 micrograms microsomal protein/50 microliters assay. We observed that inactivation could be prevented by supplementing the assay with a previously heat-treated suspension of microsomes in neutral detergent, or with the corresponding extract. Further investigation revealed that phospholipids are responsible for this enzyme stabilization, since phospholipase A2 and phospholipase C treatments were both able to abolish this effect. When individual phospholipids were compared for their protective efficiency, egg yolk phosphatidylcholine was found to be by far the most efficient. Phosphatidylglycerol, phosphatidylinositol and phosphatidylserine were only slightly effective, while phosphatidylethanolamine and lysophosphatidylcholine had no effect at all. Of those tested, partly unsaturated phosphatidylcholines with 16-18 carbon atom acyl chains were the most active, at an optimal concentration of 1-2 mM. Under these conditions a Km of 15 microM was measured for the acceptor, a synthetic ribonuclease heptapeptide, and a Km of 0.55 microM for the donor, dolichyl-P-P-GlcNAc2-Man9-Glc2-3. These findings were confirmed by subjecting a sodium deoxycholate extract to depletion of endogenous lipids by gel filtration. Enzyme activity was totally abolished and then restored (up to now only partially) by addition of phosphatidylcholine.
D000818:Animals; D003902:Detergents; D005227:Fatty Acids / Q000032:analysis; D006602:Hexosyltransferases; D007700:Kinetics; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology; D017830:Octoxynol; D010455:Peptides / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D011092:Polyethylene Glycols; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism; D012995:Solubility; D013552:Swine; D013961:Thyroid Gland / Q000201:enzymology; D014166:Transferases / Q000378:metabolism*
D003902:Detergents; D005227:Fatty Acids; D008565:Membrane Proteins; D010455:Peptides; D010713:Phosphatidylcholines; D011104:Polyisoprenyl Phosphate Oligosaccharides; C024962:dolichyl diphosphate oligosaccharides; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase