Glycogenomics of the social amoeba 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.

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 this project, the protein-linked carbohydrates were studied using modern techniques of analytical biochemistry and molecular biology; particularly, in order to elucidate the role of fucose, we focussed on genes related to the biosynthesis of fucose. Specifically, a mutant (modC) with a defect in fucose incorporation was analysed in order to determine the exact molecular and biochemical nature of the mutation. The relevant molecular and biological phenotypes of the mutant included glycan structures, cross-reactivity with anti-horseradish peroxidase and growth rates the fucosylation defect was indeed rescued with fucose added exogenously to the medium. Mass spectrometric methods were the key to examine glycomic differences between different Dictyostelium strains and also during the life cycle. Furthermore, the molecular defect in a slime mould model of human Congenital Defect of Glycosylation IL was pinpointed to a premature stop codon in a mannosyltransferase gene; this mutation results in a definite shift in the glycomic profile of the affected strain, which displays a reduced number of charged modifications of its N-glycans. For this purpose, refined methodologies were required in order to effectively analyse the highly unusual sulphated and methylphosphorylated N-glycans of this organism.