*EASTBIO* Understanding protein C-mannosylation in Drosophila

BBSRC Theme: World-Class Underpinning Bioscience

Carbohydrates are ubiquitous throughout nature and perform a number of important functions in our cells. A single or several sugar moieties can be appended to other biomolecules such as proteins and lipids and are important in cell processes such as signalling and defence against pathogens. The structure and sequence of carbohydrates is complex and highly variable, but unlike DNA there is no genetic code that can be read to determine how it should exist. Instead, carbohydrate structure and sequence is defined only by the enzymes that synthesize and degrade the carbohydrate molecules.

We are interested in an unusual eukaryotic post-translational modification called C-mannosylation1. C-mannosylation involves the addition of a mannose residue to a tryptophan in proteins, with the formation of a covalent carbon-carbon bond. There is, however, very little known about the function of this modification in vivo or its biological significance. The glycosyltransferase (C-mannosyltransferase) responsible for the modification in Caenorhabditis elegans was identified recently2, which has opened up the field for research. Sequence alignments predict two splice variants of a C-mannosyltransferase in Drosophila melanogaster, which have not been studied to date.

We aim to understand these enzymes from a molecular, cellular and organismal viewpoint, which we anticipate will aid our understanding of the human homologues and their potential role in disease. Molecular biology and protein biochemistry will be used to clone and over-express the enzymes recombinantly in insect cells and their activity will be tested in vitro using an established assay. Structural biology (primarily X-ray crystallography) will be used to understand the enzymes at the molecular level and how the enzymes interact with their substrates. Complementing these approaches, Drosophila will be used to study the role of C-mannosyltransferases and their putative substrates in the living organism. Protein localisation and gene function will be examined using various genetic techniques. Mutant phenotypes will be analysed by immunohistochemistry as well as by in vivo microscopy. This will be a highly multi-disciplinary project, which will include learning aspects of cell biology, biochemistry, molecular biology, structural biology and developmental biology.