Investigation of the nature of potential phosphorylcholine donors for filarial nematode glycoconjugates

Abstract

Filarial nematodes produce proteins containing phosphorylcholine (PC) covalently attached to N-linked glycans. Our previous work has suggested that transfer of PC might be dependent on a metabolite of the Kennedy pathway of phospholipid biosynthesis. In this study we have investigated whether the end product of this pathway, phosphatidylcholine, and in addition, sphingomyelin, could act as PC donors. Pulse-chase experiments employing [3H]choline as radiolabel, ruled out sphingomyelin, as the Acanthocheilonema viteae PC-containing protein, ES-62, was radiolabelled 20-30 min prior to the lipid. Phosphatidylcholine however was labelled immediately before ES-62 increasing the possibility that it could act as donor. This was further investigated by radiolabelling phosphatidylcholine synthesised via an alternative pathway such that other metabolites in the Kennedy pathway were not labelled. Specifically, we labelled the choline component of phosphatidylcholine using both [3H]serine and [3H]S-adenosyl methionine (SAM). Incubation of worms with [3H]serine failed to result in labelling of the PC component of ES-62 whereas the presence of [3H]SAM in the medium led to labelling of ES-62 but only 24 h after labelling of phosphatidylcholine. As ES-62 is labelled within minutes of phosphatidylcholine when employing [3H]choline as radiolabel, this suggests that labelling of ES-62 when using SAM is not due to direct transfer of PC from phosphatidylcholine. It is therefore concluded that neither sphingomyelin nor phosphatidylcholine act as PC donors for filarial nematode glycoproteins. The analysis of PC-containing metabolites and products from A. viteae additionally revealed the presence of PC-substituted glycolipids that were also radiolabelled by the use of [14C]choline. The kinetics of radiolabelling however differed from that observed in the case of ES-62, sphingomyelin and phosphatidylcholine in so far as labelled glycolipids were first detectable hours rather than minutes after addition of [14C]choline.