The structural, biosynthetic and genetic basis of anti-horseradish peroxidase carbohydrate epitopes in Drosophila melanogaster and Caenorhabditis elegans.

For many years, there have been reports that antibodies raised against plant glycoproteins either IgG or IgE cross-react with some insect glycoproteins. In particular, the anti-horseradish peroxidase has become an accepted means to allow the staining of neurons in Drosophila and grasshopper. More recently, it has become apparent that a proportion of neurons in Caenorhabditis elegans can also be stained by the same method. However, in neither case has the basis of this cross-reaction been explained, even though a very high proportion of anti-horseradish peroxidase antibodies are anti-carbohydrate. To determine the basis of this specific neuronal staining, we propose to study the structure and biosynthesis of N-linked oligosaccharides in Drosophila and Caenorhabditis, with a special emphasis on seeking the glycans, and the relevant enzymes, that could account for this cross-reaction, the primary candidate being core a1,3-linked fucose, a modification reported in plants, insects and helminths. To this end, using immunoaffinity chromatography, we will select for the glycoproteins expressing the anti-horseradish peroxidase epitope and analyse their N-linked oligosaccharides and peptide maps by HPLC and MALDI mass spectrometry. In addition, we will seek Drosophila DNAs encoding fucosyltransferases and clone, sequence and express them, as well as assay extracts of Drosophila and Caenorhabditis for fucosyltransferase activities. We will also examine the cross-reaction between anti-horseradish peroxidase and Drosophila and Caenorhabditis proteins by inhibition with specifically-synthesised a1,3-fucosylated neoglycoconjugates.