Invertebrate models of Golgi trafficking disorders

Glycans cover the surfaces of all cells and are biosynthesised by specific biochemical pathways in intracellular compartments known as the endoplasmic reticulum and Golgi apparatus prior to transport to the cell surface. Genetic defects in the trafficking between and within these compartments result in altered cell surface glycosylation resulting often in neurological and other phenotypes in human patients. In this project we will investigate trafficking in a model nematode (Caenorhabditis elegans) and in insect cell lines. Mutant strains will be generated and examined in terms of altered glycosylation and altered localisation of key biosynthetic enzymes using modern mass spectrometric, array-based and microscopic methods; in the case of mutant nematode lines, also their behaviour will be analysed using a high-throughput video system. Particularly the data from C. elegans will yield a basic understanding of trafficking in a model organism with relevance to human disease. Wider research context / theoretical framework: Glycans cover the surfaces of all cells and the Golgi apparatus is key to their biosynthesis in eukaryotes. The intracellular trafficking of Golgi resident enzymes ensures that they act on glycan substrates in the correct order. Alterations to trafficking caused by mutations in the Conserved Oligomeric Golgi (COG) and the Trafficking Protein Particle (TRAPP) complexes thereby result in shifted glycomes in human, which can result in biological phenotypes as observed in some trafficking-dependent congenital disorders of glycosylation. Hypotheses/research questions /objectives: Based on previous work by us and others, we hypothesise that knocking-out genes encoding components of the COG and TRAPP complexes has variable impacts on N- and O-glycosylation in Caenorhabditis elegans and in insect cell lines due to alterations in the localisation of key glycan-modifying enzymes. This affects also their interactions with lectins and the behaviour of the mutants. Approach/methods The proposed methods fall into foue parts: (i) identification or design of COG and TRAPP mutants using C. elegans or Sf9 cells together with glycomic analyses; (ii) glycan array experiments focussed on addressing lectin specificity towards wild-type and mutant N-glycans; (iii) examination of altered Golgi localisation of key enzymes by microscopic and proteomic techniques; (iv) behavioural analyses of C. elegans COG and TRAPP mutants. Level of originality / innovation To date, there have been few studies on the impact of mutations affecting COG and TRAPP complexes in either nematodes or insects. The glycomic studies will be combined uniquely with behavioural studies on mutant C. elegans as well as determining the impact on recombinant protein glycosylation in Sf9 cells. Particularly the data from C. elegans will yield a basic understanding of trafficking in a model organism with relevance to human disease. Primary researchers involved The project will be led by Dr. Iain Wilson with Prof. Dominique Glauser as international partner.