Identification of core alpha 1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster - Potential basis of the neural anti-horseradish peroxidase epitope

For many years, polyclonal antibodies raised against the plant glycoprotein horseradish peroxidase have been used to specifically stain the neural and male reproductive tissue of Drosophila melanogaster. This epitope is consi dered to be of carbohydrate origin, but no glycan structure from Drosophila has yet been isolated that could account for this cross-reactivity. Here w e report that N-glycan core alpha1,3-linked fucose is, as judged by pre-abs orption experiments, indispensable for recognition of Drosophila embryonic nervous system by anti-horseradish peroxidase antibody. Further, we describ e 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 bot h alpha1,3- and alpha1,6-linked fucose residues on the proximal core GlcNAc . Moreover, we have isolated three cDNAs encoding alpha1,3-fucosyltransfera se homologues from Drosophila. One of the cDNAs, when transformed into Pich ia pastoris, was found to direct expression of core alpha1,3-fucosyltransfe rase activity. This recombinant enzyme preferred as substrate a biantennary core alpha1,6-fucosylated N-glycan carrying two non-reducing N-acetylgluco samine residues (GnGnF(6), K-m 11 muM) over the same structure lacking a co re fucose residue (GnGn; K-m 46 muM). The Drosophila core alpha1,3-fucosylt ransferase enzyme was also shown to be able to fucosylate N-glycan structur es of human transferrin in. vitro, this modification correlating with the a cquisition of binding to anti-horseradish peroxidase antibody.