Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates
2005
Paschinger, Katharina | Staudacher, Erika | Stemmer, Ute | Fabini, Gustáv | Wilson, Iain B. H.
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.
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