Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates.
Core alpha1,6-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 alpha1,6-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 alpha1,6-fucosyltransferases, as well as for native Caenorhabditis and Schistosoma mansoni core alpha1,6-fucosyltransferases. On the other hand, these alpha1,6-fucosyltransferases could not act on N-glycopeptides already carrying core alpha1,3-fucose residues, whereas recombinant Drosophila and native Schistosoma core alpha1,3-fucosyltransferases were able to use core alpha1,6-fucosylated glycans as substrates. Lewis-type fucosylation was observed with native Schistosoma extracts and could take place after core alpha1,3-fucosylation, whereas prior Lewis-type fucosylation precluded the action of the Schistosoma core alpha1,3-fucosyltransferase. Overall, we conclude that the strict order of fucosylation events, previously determined for fucosyltransferases in crude extracts from insect cell lines (core alpha1,6 before core alpha1,3), also applies for recombinant Drosophila core alpha1,3- and alpha1,6-fucosyltransferases as well as for core fucosyltransferases in schistosomal egg extracts.
Paschinger
Katharina
K
Department für Chemie der Universität für Bodenkultur, Muthgasse 18, 1190 Wien, Austria.
Staudacher
Erika
E
Stemmer
Ute
U
Fabini
Gustáv
G
Wilson
Iain B H
IB
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004331:Drosophila melanogaster / Q000378:metabolism; D005643:Fucose / Q000378:metabolism*; D005647:Fucosyltransferases / Q000378:metabolism*; D007448:Invertebrates / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D012547:Schistosoma / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D005643:Fucose; D005647:Fucosyltransferases