Glycogenomics of Dictyostelium discoideum

Dictyostelium discoideum (slime mould) is a valuable non-pathogenic model for the study of cell-cell interactions since it has a novel life-cycle in which single cell amoeba form, upon starvation, multicellular fruiting bodies. Their protein-linked carbohydrate chains contain the deoxyhexose sugar fucose, which in general is implicated in cell-cell interactions in many organisms, including man. In the proposed project, the protein-linked carbohydrates will be studied using modern techniques of analytical biochemistry and molecular biology; particularly, in order to elucidate the role of fucose, we will focus on genes related to the biosynthesis of fucose and its transfer to oligosaccharides. Specifically, mutants with defects in fucose incorporation will be analysed in order to determine the exact molecular and biochemical nature of the mutation. Based on hypotheses and analyses, the defective genes will be complemented to verify if the molecular and biological phenotypes of the mutants can be rescued. Markers will include glycan structures, cross-reactivity with anti-horseradish peroxidase and growth rates. Furthermore, other mutants will be constructed by targetted knock-out of putative fucosyltransferase genes in order to investigate other aspects of fucosylation in this organism. Mass spectrometric methods will be used to examine changes in the N-glycans. The proposed experiments will contribute towards a better understanding of the glycosylation of this organism, thereby opening new avenues in the optimisation of its potential use as a model for parasitic organisms or for biotechnological production of recombinant proteins.