Molecular probes for the characterisation of lysosomal oligosaccharide transport – MOCALOST

Context. Oligosaccharide fragments derived from pathogens, and those derived from host glycoconjugates, can be immunogenic. In mammalian cells, free oligosaccharides (fOS) are produced normally during the biosynthesis of N-glycoproteins in the endoplasmic reticulum (ER), but under certain circumstances, become proinflammatory after partial demannosylation. How and where these proinflammatory fOS are demannosylated remains unclear. It is known that after transport of fOS out of the ER into the cytoplasm, cytoplasm-to-lysosome fOS transport (LOST: Lysosomal OligoSaccharide Transport) enables lysosomal mannosidases to access fOS. So, our hypothesis is that LOST is involved in processing fOS into proinflammatory mediators. This hypothesis is difficult to test because LOST is an orphan activity whose underlying protein(s)/gene(s) have not been identified.
Objectives. Identify LOST proteins using oligosaccharide-based photoactivable cross-linking probes and quantitative proteomics. After identification of encoding gene(s), LOST expression will be manipulated genetically in cells to understand its role in normal and pathological conditions. Also, we will generate novel fluorogenic probes suitable for real time assay of LOST and visualization of LOST action using fluorescence microscopy.
Preliminary data. We demonstrated that small chitooligosaccharides (COS) and sugar-containing peptidoglycan (PG) fragments of bacterial origin, which present easier targets than fOS for chemical manipulation, are LOST inhibitors and that small COS are likely LOST substrates. LOST requires oligosaccharide substrates to have an intact reducing terminus, which precludes simple chemical derivatisation. Using innovative bioengineering and chemistry the partners provided proof of principle that COS substituted at their non-reducing termini interact with LOST and can be transported into lysosomes.
Methods. Based on our preliminary results, COS- and PG fragment-based biotinylated x-link affinity probes and fluorogenic substrates for LOST will be synthetized by the chemistry teams. Using the affinity probes, the biology team will covalently x-link LOST proteins in preparations of partially purified intact lysosomes. Proteins labeled in this way will be purified using magnetic beads coated with the biotin binding-protein streptavidin. Quantitative proteomics will be used to identify the harvested proteins. LOST candidates will be assessed by examining fOS catabolism in cells in which their expression has been manipulated genetically. LOST protein(s), confirmed in this way, will be downregulated in cells that generate proinflammatory fOS to determine the role of LOST in this pathophysiological situation. The novel fluorogenic LOST substrates will be synthesised and used to develop simple real-time transport assays and visualize LOST action in semi-intact cell systems using fluorescence microscopy.
Expected results. Novel chemical tools to study lysosomal oligosaccharide transport. Identification of LOST protein(s). Fluorescence-based real-time assays and fluorescence microscopy techniques to study LOST. An understanding of the role of LOST genes in lysosomal oligosaccharide transport in normal and pathophysiological processes.
Impact and Benefits. LOST may not just be involved in fOS metabolism but may regulate other oligosaccharides such as COS and bacterial PG fragments. Our research into LOST will be of interest to glycoscientists as well as scientists in the fields of pathogen/host interactions, innate immunity, and inflammation. Finally, several rare inherited diseases are associated with altered fOS metabolism, and the identification of LOST at the molecular level will be important for clinicians and scientists involved with potential LOST-deficient patients.