Identification of Core α1,3-Fucosylated Glycans and Cloning of the Requisite Fucosyltransferase cDNA from Drosophila melanogaster: POTENTIAL BASIS OF THE NEURAL ANTI-HORSERADISH PEROXIDASE EPITOPE

Gustáv Fabini,Angelika Freilinger,Friedrich Altmann,Iain B.H Wilson

doi:10.1074/jbc.m100573200

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 of Drosophila melanogaster. This epitope is considered to be of carbohydrate origin, but no glycan structure from Drosophila has yet been isolated that could account for this cross-reactivity. Here we report that N-glycan core alpha1,3-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 alpha1,3- and alpha1,6-linked fucose residues on the proximal core GlcNAc. Moreover, we have isolated three cDNAs encoding alpha1,3-fucosyltransferase homologues from Drosophila. One of the cDNAs, when transformed into Pichia pastoris, was found to direct expression of core alpha1,3-fucosyltransferase activity. This recombinant enzyme preferred as substrate a biantennary core alpha1,6-fucosylated N-glycan carrying two non-reducing N-acetylglucosamine residues (GnGnF6; Km 11 microm) over the same structure lacking a core fucose residue (GnGn; Km 46 microm). The Drosophila core alpha1,3-fucosyltransferase enzyme was also shown to be able to fucosylate N-glycan structures of human transferrin in vitro, this modification correlating with the acquisition of binding to anti-horseradish peroxidase antibody.

Highlights

For many years, polyclonal antibodies raised against the plant glycoprotein horseradish peroxidase have been used to stain the neural and male reproductive tissue of Drosophila melanogaster
As compared with the amount of information known on the genetics and genome of Drosophila, relatively little is known about the structures of its glycoconjugates; there exists one report from 1991 on the N-linked oligosaccharides of larval membrane glycoproteins in which only data on oligomannosidic and core ␣1,6-fucosylated N-glycans and no data on glycan structures that could explain anti-HRP binding were presented [16]
We report the specific inhibition of anti-HRP staining of Drosophila embryonic nervous system by a neoglycoconjugate carrying core ␣1,3-fucose

Summary

EXPERIMENTAL PROCEDURES

Immunostaining of Whole-mount Drosophila Embryos—D. melanogaster embryos (Canton S) were collected and staged at 25 °C. The chemical defucosylation to yield MUX glycopeptide was estimated by sugar content analysis to have been 95% effective These neoglycoconjugates in the concentration range 0.004 – 40 ␮M in terms of GlcNAc (or 0.04 – 400 ␮g/ml) were allowed to preabsorb the rabbit anti-HRP antibodies (1 ␮g/ml) for 1 h at room temperature before applying as primary antibody in whole-mount embryo stainings. The subsequent fucosyltransferase assays were performed as follows; 2 ␮l of concentrated FucTA supernatant was incubated with 0.2 nmol of dansylated glycopeptide and 10 nmol of GDP-Fuc in a final volume of 20 ␮l. For Km determinations, dansyl glycopeptides derived from IgG (GnGn and GnGnF6) were used with 2 ␮l of concentrated FucTA supernatant (16-h incubation time). Clean-up, and analysis by MALDI-TOF MS of N-glycans were performed as described [23]

RESULTS

Percentage occurrence

TABLE II Substrate specificity of Drosophila FucTA

Relative conversion

DISCUSSION