Effect of phospholipids on thyroid oligosaccharyltransferase activity and orientation. Evaluation of structural determinants for stimulation of N-glycosylation.

Oligosaccharyltransferase solubilized by Nonidet P-40 was found to have a highly specific lipid requirement which is consistent with the lability of the enzyme when removed from its membrane association. Enzyme activity as measured by the N-glycosylation of a hexapeptide acceptor was greatly stimulated and stabilized by phosphatidylcholine (PC) while other naturally occurring phosphoglycerides had minimal effect. The quaternary ammonium group of PC was observed to be involved in the interaction with the enzyme as modification of the choline moiety by removal of methyl groups resulted in a progressive loss of the stimulatory effect (choline greater than N,N-dimethylethanolamine greater than N-monomethylethanolamine greater than ethanolamine) which was reflected primarily in the Vmax rather than the Km values. Evaluation of a number of PC and choline derivatives indicated that the nonpolar domain of the lipid also played an important specifying role. Two hydrophobic chains attached to the phosphoglycerol backbone were found to be essential, and furthermore the length and degree of unsaturation of the fatty acid substituents as well as their position of attachment on the glycerol moiety greatly affected the extent of activation. Since the L-isomer of PC brought about a 3-fold greater stimulation than the D-isomer the interaction of the enzyme with the phospholipid appears to be stereoselective. Upon chromatography of the PC-stabilized enzyme on concanavalin A-agarose almost complete retention occurred at 0.4% Nonidet P-40, while no binding took place at a detergent concentration of 0.075%; this suggested that upon dilution in the presence of PC, the oligosaccharyltransferase was reconstituted into vesicles in an asymmetric fashion with its N-linked carbohydrate located internally. Enzymatic assay of these vesicles demonstrated that the active site of the enzyme was also oriented toward the interior. These studies indicate that the activity as well as the membrane insertion of the oligosaccharyltransferase are to a large measure influenced by its interaction with PC.