Neuron-specific fucosylation in Drosophila

The use of antibodies raised against a plant protein (horseradish peroxidase) has become a well-established method for specifically labelling neurons in Drosophila melanogaster and other insects. Recently we determined that this labelling is due to the presence of a specific carbohydrate (core alpha1,3-linked fucose) previously found to occur on some bee venom proteins and on some plant allergens. The specific occurrence of this structure on insect neurons creates a unique chance to study a cell-type specific form of glycosylation in a model organism.We have previously also found that a specific enzyme (a fucosyltransferase named FucTA) from Drosophila can create the core alpha1,3-fucosylated structure in vitro. In the proposed work we will use a number of methods to establish the role of this enzyme in vivo. The availability of a neuronal cell line, BG2-c6, which expresses the anti-horseradish peroxidase epitope and has been shown in preliminary work to express both the specific fucosyltransferase FucTA and core alpha1,3-fucose containing asparagine-linked (N-linked) oligosaccharides, gives us access to an easily- manipulable system to investigate the mechanisms resulting in expression of this structure. Specifically we will perform RNA interference in BG2-c6 cells to verify the role of FucTA and other proteins of fucose metabolism in the expression of the specific glycan structure, examine the transcription of FucTA and determine its intracellular localisation. Ectopic expression of FucTA in Schneider cells, which do not express the structure, will also be performed to examine whether this enzyme is a pre-requisite for expression of the epitope. In collaboration, we will also study mutant Drosophila embryos which do not express the epitope.Since a carbohydrate epitope can only have a biological function when it is recognised by another molecule, the interaction networks involving core alpha1,3-fucose containing glycans will also be a topic of the study. In particular we will examine whether a candidate lectin receptor is indeed specific for this structure and determine whether this lectin is expressed in the right place and at the right time in order to act in vivo as a receptor for core alpha1,3-fucose.Overall, this study will bring us, beyond the standard identication of glycosyltransferases and glycan structures, onto a higher plane of complexity - i.e., the actual in vivo or nature-identical interactions of glycans and their partners within the context of a cell-specific type of glycosylation in a model organism.

For over two decades, antibodies raised against a plant protein were used to specifically stain nerve cells in fruit flies. In this project, the molecular basis for the recognition of nerve structures by this antibody was determined. The relevant enyzme was identified and, through a number of different and complementary experiments (including work with cell lines), shown to be indispensable for synthesis of the structure recognized by the antibody. The structure recognized by the antibody is first of that kind to be related to nerve-specific environment in invertebrates. Interestingly, this structure is actually a single sugar unit bound in a specific way to a sugar chain (termed "N-glycan"), which is, in turn, attached to a protein. Although roughly 20 proteins are known to carry that structure, the interactions of this structure with other molecules in nerve function and development are yet to be discovered, and are one the aims of our future projects. The results of this project opened an undisclosed avenue of research in insects neurological function and development, with currently unknown impact on counterparts that might play a role in normal and/or pathological neurological states in humans.