Identification of Core α1,3-Fucosylated Glycans and Cloning of the Requisite Fucosyltransferase cDNA from Drosophila melanogaster

Abstract

For many years, polyclonal antibodies raised against the plant glycoprotein horseradish peroxidase have been used to specifically stain the neural and male reproductive tissue ofDrosophila melanogaster. This epitope is considered to be of carbohydrate origin, but no glycan structure fromDrosophila has yet been isolated that could account for this cross-reactivity. Here we report that N-glycan core α1,3-linked fucose is, as judged by preabsorption experiments, indispensable for recognition of Drosophila embryonic nervous system by anti-horseradish peroxidase antibody. Further, we describe the identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and high performance liquid chromatography of two Drosophila N-glycans that, as already detected in other insects, carry both α1,3- and α1,6-linked fucose residues on the proximal core GlcNAc. Moreover, we have isolated three cDNAs encoding α1,3-fucosyltransferase homologues from Drosophila. One of the cDNAs, when transformed into Pichia pastoris, was found to direct expression of core α1,3-fucosyltransferase activity. This recombinant enzyme preferred as substrate a biantennary core α1,6-fucosylated N-glycan carrying two non-reducingN-acetylglucosamine residues (GnGnF6;Km 11 μm) over the same structure lacking a core fucose residue (GnGn; Km 46 μm). The Drosophila core α1,3-fucosyltransferase enzyme was also shown to be able to fucosylate N-glycan structures of human transferrinin vitro, this modification correlating with the acquisition of binding to anti-horseradish peroxidase antibody.