Biosynthesis of novel methylated and fucosylated N- and O-glycans in Caenorhabditits elegans

The model organism, Caenorhabditis elegans, is being much studied at the genetic level; however, its glycosylation is poorly understood. Recent data from this and other laboratories suggest that the carbohydrates covalently-bound to nematode glycoproteins share some basic features with those found in mammals, but have some novel decorations. In particular, the presence of asparagine-linked glycan structures with up to four fucose residues and two O-methyl substitutions is highly intriguing. O-methylated residues have also been found on the nematode`s O- linked oligosaccharides. Some of these features are shared, at least in part, by parasitic nematodes and so Caenorhabditis elegans is potentially a useful model for developing novel strategies in the study of these types of oligosaccharides with potential benefits in combatting nematode parasites. In the proposed study, it is intended to complete the analyses of the glycan structures and to initiate a study of their biosynthesis. In particular, the relevant fucosyl- and methyltransferases will be assayed from nematode extracts. Further, purification of these enzymes will be performed and their peptide maps compared to the protein sequences predicted from the complete genomic sequence. The corresponding cDNAs will be cloned and expressed and the enzymatic activity of the recombinant proteins tested. Recombinant enzymes will be used in synthesis of key N- and O-linked structures and these will be conjugated to protein in order to raise antibodies against the putatively immunogenic oligosaccharides. These antibodies could then be used to examine a wide range of extracts from parasitic nematodes. In particular, we will apply our findings to Haemonchus contortus, a parasite of sheep and cattle.

For over twenty years, antibodies raised against the plant glycoprotein horseradish peroxidase have been used to recognise the neural tissue of invertebrates. In this project we have solved the problem as to how the glycoproteins of the soil nematode (and genetic model organism) Caenorhabditis elegans can be recognised by this antiserum: by using genetic, molecular biological and biochemical means, we have shown that an enzyme transferring fucose to N-glycans of the nematode generates the recognised epitope. We have also studied other aspects of the nematode`s glycosylation pathway such as addition of phosphorylcholine and a hexosaminidase involved in N-glycan biosynthesis. Since also parasitic nematodes share some features with Caenorhabditis, we believe that the use of fucose-deficient mutant nematodes will in the future shed light on the immunological responses to parasites and so perhaps help define why people with parasitic infections are not so susceptible to allergy.