Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates

Core alpha 1,6-fucosylation is a conserved feature of animal N-linked oligosaccharides being present in both invertebrates and vertebrates. To prove that the enzymatic basis for this modification is also evolutionarily conserved, cDNAs encoding the catalytic regions of the predicted Caenorhabditis elegans and Drosophila melanogaster homologs of vertebrate alpha 1,6-fucosyltransferases (E.C. 2.4.1.68) were engineered for expression in the yeast Pichia pastoris. Recombinant forms of both enzymes were found to display core fucosyltransferase activity as shown by a variety of methods. Unsubstituted nonreducing terminal GlcNAc residues appeared to be an obligatory feature of the substrate for the recombinant Caenorhabditis and Drosophila alpha 1,6-fucosyltransferases, as well as for native Caenorhabditis and Schistosoma mansoni core alpha 1,6-fucosyltransferases. On the other hand, these alpha 1,6-fucosyltransferases could not act on N-glycopeptides already carrying core alpha 1,3-fucose residues, whereas recombinant Drosophila and native Schistosoma core alpha 1,3-fucosyltransferases were able to use core alpha 1,6-fucosylated glycans as substrates. Lewis-type fucosylation was observed with native Schistosoma extracts and could take place after core alpha 1,3-fucosylation, whereas prior Lewis-type fucosylation precluded the action of the Schistosoma core alpha 1,3-fucosyltransferase. Overall, we conclude that the strict order of fucosylation events, previously determined for fucosyltransferases in crude extracts from insect cell lines (core alpha 1,6 before core alpha 1,3), also applies for recombinant Drosophila core alpha 1,3- and alpha 1,6-fucosyltransferases as well as for core fucosyltransferases in schistosomal egg extracts.