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 to the 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.