Attachment of phosphorylcholine to a nematode glycoprotein

Phosphorylcholine (PC) is an immunodominant epitope present on many proteins secreted (ES) by filarial nematodes and is thought to be involved in parasite survival by modulating the host immune response. Based on the postulate that nematodes producing ES lacking PC would have diminished survival time, studies were undertaken to investigate the mechanism of PC addition to ES. ES-62 is the major PC-containing ES product of Acanthocheilonema viteae (a rodent model of filariasis) and by investigating this molecule we have shown PC to be attached to the protein backbone via an asparagine-linked glycan. This type of association between PC and carbohydrate is absent from humans and thus the PC-glycans of ES may pose a novel target for chemotherapy. Pulse-chase experiments have shown that PC addition to ES-62 is a fairly early event during intracellular trafficking occurring within 40-60 min of protein synthesis. Investigation employing inhibitors of intracellular trafficking and also oligosaccharide processing, has revealed three reagents capable of preventing attachment of PC to ES-62: Brefeldin A, which blocks transport from the ER to the golgi; 1-deoxynojirimycin (dNM), which inhibits alpha-glucosidase I in the ER; and 1-deoxymannojirimycin, which inhibits alpha-mannosidase I in the cis Golgi. Swainsonine, an inhibitor of alpha-mannosidase II, does not interfere with PC transfer. Taken together these observations suggest that PC attachment is a post-ER event probably occurring in the medial Golgi and that the substrate for transfer is Man(5)GlcNAc(3) or Man(3)GlcNAc(3). Data from mass spectrometry are consistent with this interpretation and reveal that PC is attached to glycans containing a trimannosyl core, with or without core fucosylation and carrying between one and four additional GlcNAc residues.