Structural Studies of N-Glycans of Filarial Parasites

Abstract

N-Type glycans containing phosphorylcholine (PCglycans), unusual structures found in the important human pathogens filarial nematodes, represent a novel target for chemotherapy. Previous work in our laboratories produced compositional information on the PCglycan of ES-62, a secreted protein of the rodent parasite Acanthocheilonema viteae. In particular, we established using fast atom bombardment mass spectrometry (MS) analysis that PC was attached to a glycan with a trimannosyl core, with and without core fucosylation, carrying between one and four additional N-acetylglucosamine residues. In the present study, we demonstrate that this structure is conserved among filarial nematodes, including the parasite of humans, Onchocerca volvulus, for which new drugs are most urgently sought. Furthermore, by employing a variety of procedures, including collision-activated dissociation MS-MS analysis and matrix-assisted laser desorption MS analysis, we reveal that surprisingly, filarial nematodes also contain N-linked glycans, the antennae of which are composed of chito-oligomers. To our knowledge, this is the first report of such structures in a eukaryotic glycoprotein.

N-Glycans of Caenorhabditis elegans Are Specific to Developmental Stages

“…This result is consistent with our recent observation that the phosphatidylcholine:oligosaccharide phosphorylcholine transferase of C. elegans preferentially uses as its in vitro substrate oligosaccharides containing Man disubstituted with GlcNAc (7). Rare Pc oligosaccharides with up to five phosphorylcholine substitutions have been reported in some nematodes (31). We did not observe any of these structures in the present study.…”

Carbohydrates and glycosylation

“…Phosphorylcholine (PCho)-substituted N-glycans have been identified in a high mannose structure carrying three PCho groups (Cipollo et al, 2002) and on complex type glycans where PCho is attached at GlcNAc (Haslam and Dell, 2003). This last structure has been previously found in several parasitic nematodes (Haslam et al, 1999;Haslam et al, 1997;Morelle et al, 2000).…”

The Fine Structure of Caenorhabditis elegans N-Glycans

“…These N-glycans have been reported in Acanthocheilonema viteae, Onchocerca volvulus, and Trichinella spiralis (17,18,47,48). The Pc-containing oligosaccharides are able to modulate B-lymphocyte activation through activation of protein tyrosine kinase and mitogen-activating protein kinase signal transduction pathways resulting in desensitization to downstream activation (49).…”

“…These oligosaccharides suggest that GNT II and IV type activities are present. Also, LacdiNAc-(43, 44) and chito-oligomer branches (17) have been reported in nematodes glycans. Therefore, it is possible that C. elegans produces these oligosaccharides.…”

“…Permethylation tends to impart similar chemical properties over a wide range of oligosaccharides, which translates into similar detector response with added sensitivity in MALDI-TOF MS analysis. HF treatment has been used in combination with permethylation to aid in characterizing oligosaccharides in complex mixtures, where some components contain Pc (17,18). As C. elegans protein extracts bind TEPC-15, we anticipated that some glycoconjugates would contain Pc and therefore used oligosaccharide permethylation, with and without prior hydrolysis with HF, to aid glycan detection.…”