Infection with filarial nematodes produces a chronic, long-lasting illness with adult worms able to survive within human hosts for up to 15 years. A contributor to the longevity of these parasites is the presence of phosphorylcholine (PC) on components of the worms' molecular secretions (ES). PC on ES modulates host immune responses towards an anti-inflammatory phenotype thereby generating an environment favourable for parasite survival. PC is attached to nematode ES via a covalent association with carbohydrate, which, although well-documented in bacteria and fungi, is absent from humans, making it an ideal target for the development of novel drugs. In order to produce such drugs it is first necessary to understand the structure and synthesis of nematode PC-glycans. ES-62 is the major PC-ES-product of Acanthocheilonema viteae and is a homologue of PC-ES found in human filarial nematodes. We have studied the structure and biosynthesis of PC-glycans of ES-62 by a combination of pulse-chase experiments, experiments involving the use of inhibitors of each of intracellular trafficking, oligosaccharide processing and phospholipid biosynthesis and various forms of mass spectrometry. Our indications indicate that PC is transferred in the lumen of the medial Golgi to an N-type glycan consisting of a trimannosyl core with or without core fucosylation bearing between 1 and 4 N-acetyl glucosamine residues. The structure of the PC-N-glycans found in ES-62 appears to be conserved amongst filarial nematodes in that it has additionally been identified in Onchocerca volvulus and O. gibsoni. Also, similar structures have been found in non-filarial parasitic nematodes and in the free-living nematode Caenorhabditis elegans. Finally, PC has also been recently found attached to the carbohydrate moieties of nematode glycosphingolipids and the structure of these will also be considered.

Phosphorylcholine (PC) is a common constituent of proteins secreted by filarial nematodes. As this substance has been shown to interfere with immune responses, we are interested in designing strategies for blocking its attachment. Towards this end, we are investigating the mechanism of incorporation of PC into filarial molecules and in the present manuscript we describe experiments relating to elucidating the source of PC for attachment. Synthesis of phosphatidylcholine in eukaryotic organisms can occur by a mechanism involving the transfer of PC from CDP-choline to diacylglycerol (the Kennedy pathway). By (i) measuring transfer of radio-isotope labelled PC from CDP-choline to parasite molecules and (ii) employing inhibitors of CDP-choline synthesis, we have investigated whether CDP-choline can act as a source of PC for transfer to ES–62, a major secreted glycoprotein of the rodent filarial nematode Acanthocheilonema viteae. Although we can find no evidence of this, we show that attachment of PC is blocked by hemicholinium-3, an inhibitor of choline kinase, the first enzyme in the Kennedy pathway. Thus, at least the first step in this pathway – phosphorylation of choline, would appear to be necessary for attachment of PC to ES-62.

The mechanism of linkage of phosphorylcholine (PC) to excretory–secretory products (ES) of adult Brugia pahangi has been investigated. Biosynthetic radio-isotope labelling of ES with [3H]choline followed by SDS–PAGE/fluorography revealed a smear of molecular weight approximately 40–100 kDa which loses its radiolabel following exposure to N-glycosidase F, but not mild alkali. PC is thus attached to this smear of molecules via N-type glycans, a mechanism of linkage previously observed with respect to PC–ES of Acanthocheilonema viteae. Western blotting analysis of non-radiolabelled ES demonstrated the existence of additional PC–ES which were insensitive to N-glycosidase F, but not to alkali. This second group of molecules is therefore likely to contain PC linked to O-glycans. Filarial nematodes may thus utilize 2 classes of glycan for attachment of PC. Examination of B. pahangi and A. viteae whole worm extracts by Western blotting indicated that their PC content could not be cleaved by N-glycosidase F and hence the use of N-type glycans may be restricted to a subset of ES products. The implications of these findings with respect to developing inhibitors of PC attachment for use as anti-filarial drugs are discussed.