Biosynthesis of helminth N-glycoproteins in insect cells

Haemonchus contortus is one of the major helminthic parasites, which infects sheep and goats resulting in economic loss of the ruminant industry worldwide. In comparison to the usage of anthelmintic reagents which can cause resistance among parasitic nematodes, vaccination represents a sustainable and effective approach. Vaccination with a mixture of native Haemonchus gut glycoproteins (namely H11 antigens) has shown effective protection in lambs. However, attempts to produce these antigens recombinantly in various expression hosts and use them as vaccines resulted in either low or no protection in animal trials. As the native H11 antigens are known to carry additional modifications with sugars on the surface of proteins (termed glycosylation), which plays an important role in the biological function of antigens, investigation on how to mimic these natural sugar modifications on the recombinant H11 became necessary and will facilitate the production of effective antigens. The sugar structures on the native H11 antigens possess a special core modified with up to three fucose residues, which are enzymatically synthesised by three fucosyltransferases. FUT-1 and FUT-8 are the fucosyltransferases which only modify the innermost GlcNAc residue with fucose to the 3- and 6-positions respectively. In this proposal, I hypothesise that a Golgi a1,6-mannosidase activity is essential for the fucosylation of the distal GlcNAc residue by a third fucosyltransferase (FUT-6), thereby enabling the ultimate formation of core tri- fucosylation. A commercial insect cell line (Hi5) is a suitable host for glycoengineering using recombinant baculovirus to introduce genes encoding Caenorhabditis elegans glyco-enzymes, which can be used to effectively remodel the sugars on the selected helminth reporter. Full length nematode glyco-enzyme encoding sequences are sufficient for Golgi targeting and realising glycoengineering in insect cells. The biological role of C. elegans glyco-enzymes will be investigated by studying the N-glycomes of relevant mutants using HPLC and mass spectrometers as major tools. Recombinant baculoviruses carrying genes encoding two C. elegans glyco-enzymes and DNA sequences encoding Haemonchus H11 (expression targets) will be prepared using molecular biology approaches to produce H11 antigens in Hi5 insect cells. In addition to the protein sequences and peptidase activities, the biochemical properties of glyco-engineered H11 antigens will be evaluated focusing on the sugar modifications and other potential protein modifications. This is probably the first attempt to express helminth antigens modified with authentic sugars in insect cells. The glyco-engineered recombinant H11 antigens are expected to better mimic the native H11 antigens; therefore they may serve as a vaccine candidate in animal trials to protect ruminants from Haemonchus infection.