STUDIES ON THE SITE AND MECHANISM OF ATTACHMENT OF PHOSPHORYLCHOLINE TO A FILARIAL NEMATODE SECRETED GLYCOPROTEIN

We have recently shown that the immunomodulatory substance phosphorylc holine (PC) is covalently attached to ES-62, a major secreted protein of the filarial nematode parasite Acanthocheilonema viteae, via an N-l inked glycan. Linkage of PC to N-glycans is previously unreported, and hence we have investigated the biochemical events underlying it. PC a ddition was found by pulse-chase experiments to be a fairly early even t during intracellular transport, occurring within 40-60 min of protei n synthesis. Biosynthetic labeling/immunoprecipitation experiments rev ealed that addition of PC to ES-62 was blocked by (i) brefeldin A, an inhibitor of trafficking of newly synthesized proteins from the endopl asmic reticulum (ER) to the Golgi, (ii) 1-deoxynorijirimycin, an inhib itor of glucosidase activity in the ER, and (iii) 1-deoxymannojirimyci n, an inhibitor of mannosidase I in the cis Golgi. Swainsonine, an inh ibitor of mannosidase II in the medial Golgi, did not affect PC additi on. Taken together these data indicate that PC attachment is a post-ER event which is dependent on generation of an appropriate substrate du ring oligosaccharide processing. Furthermore, they strongly suggest th at PC addition takes place in the medial Golgi and that the substrate for addition is the 3-linked branch of Man(5)GlcNAc(3) or Man(3)GLcNAc (3).