Phosphorylated N-glycan epitopes in simple organisms

The processing and sorting of lysosomal hydrolases occur in eukaryotic organisms in the Golgi apparatus prior to trafficking to their destination compartments. In mammals, the lysosomal proteins have a common glyco-epitope (mannose-6-phosphate), which is recognized by Golgi receptors that direct the enzymes to the lysosomes. This sorting mechanism is conserved in vertebrates, whereas in invertebrates or simple eukaryotes this pathway is not well understood. Recently, detailed analysis of the N-glycomes of simple amoebozoa, such as the non-pathogenic slime mould Dictyostelium and the facultative parasite Acanthamoeba, confirmed the presence of phosphorylated N-glycan epitopes. The biosynthesis and function of these epitopes in both organisms remain unclear. The proposed work aims to address evolutionary aspects of the biosynthesis and the recognition system of these phosphorylated epitopes. First, the amoebal key enzyme for the biosynthesis of the phosphorylated N-glycans, the N-acetylglucosamine-1- phosphotransferase, will be cloned and recombinantly expressed. The activity will be compared to the mammalian homologue in vitro using a wide range of natural substrates such as N-glycans or lysosomal proteins from amoebae. Furthermore, the phosphorylation of the N-glycome will be analysed after developmental changes of the cellular life-form induced by stress responses such as scarce food or harsh environmental conditions. Changes in the expression of the phosphorylated epitope will be determined by analysis of the acidic N-glycans using HPLC and MALDI TOF MS/MS of various life-cycle stages and compared tothe levels of N-acetylglucosamine-1- phosphotransferase transcripts quantified using RT PCR and cross-reactivity to an anti- mannose-6-phosphate antibody. Finally, enzymes modified with mannose-6-phosphate residues from Acanthamoeba and Dictyostelium will be enriched and identified using mass spectrometric approaches. The results will indicate whether phosphorylation of N- glycans is exclusively a modification of lysosomal enzymes. Initial tests for their affinity to amoebal receptors will be performed. This work will deepen our understanding regarding biosynthesis and evolution of phosphorylated N-glycan epitopes in eukaryotes.

Dictyostelium discoideum (slime mould) is a valuable non-pathogenic model for the study of cell-cell interactions since it has the ability to form upon starvation multicellular fruiting bodies from unicellular aggregating cell mounds. Their protein-linked carbohydrate chains contain sugars residues (e.g., fucose), which in general can be implicated in cell-cell interactions in many organisms, including man. In this project, the protein-linked carbohydrates were studied in detail, with an emphasis on the phosphorylated epitopes. Mass spectrometric analytical methods were the key to examine glycomic differences between Dictyostelium strains and species, but also during the life cycle and selected developmental stages such as unicellular form or multicellular fruiting body. Furthermore, the molecular defect in a slime-mould mutant strain affecting a glycoenzyme at the early stage of the N-glycan biosynthesis was pinpointed to a premature stop codon in a glucosidase gene. This mutation results in a definite shift in the glycomic profile of the affected strain, which displays an increased number of neural modifications of its N-glycans, but is not affecting the degree of the phosphorylated building blocks. Refined methodologies were required in order to effectively analyse the highly unusual sulphated and methylphosphorylated N-glycans of the wild type and mutant Dictyostelium, but also of the species D. purpureum and D. giganteum.