Variation in the nature of attachment of phosphorylcholine to excretory-secretory products of adult Brugia pahangi.

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.

The use of inhibitors of N-linked glycosylation and oligosaccharide processing to produce monoclonal antibodies against non-phosphorylcholine epitopes of Brugia pahangi excretory-secretory products.

Adult Brugia pahangi were cultured with [3H]-choline in both the absence and the presence of either tunicamycin or 1-deoxymannojirimycin (dMM), inhibitors of N-linked glycosylation and N-linked oligosaccharide processing, respectively. Excretory-secretory products (ES) were recovered from the spent medium and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography. Both inhibitors were found to prevent radiolabeling of ES, a result consistent with blockage of the addition of the highly immunodominant phosphorylcholine (PC) group. Tunicamycin/dMM-treated ES were subsequently employed to immunise a mouse in an attempt to produce monoclonal antibodies (mAbs) against non-PC epitopes of ES. Three mAbs were isolated, each of which reacted with ES but not with PC.

Some preliminary data on the nature/structure of the PC-glycan of the major excretory-secretory product of Acanthocheilonema viteae (ES-62).

The structure of the PC-glycan of the major excretory-secretory product (ES-62) of Acanthocheilonema viteae has been investigated using endoglycosidases and lectins. Results obtained raise the possibility that it may be of the high mannose type. This, and the insensitivity of the PC-glycan to treatments which remove PC or choline from bacterial PC-glycans, suggests that it may be more analogous to fungal, than to bacterial PC-containing glycans.