Fundamental biochemistry of N-glycosylationand the dolichol cycle

The addition of sugars to proteins is an essential and highly regulated process in healthy humans. N-glycosylation describes the process in which sugar moieties are added to asparagine residues in a protein, and has important functions in protein stability, localisation, and function, with defects in many of the steps in N-glycosylation leading to congenital disorders of glycosylation. Sugar molecules destined to be used in N-glycosylation as well as in other cellular processes, are docked to the membrane of the endoplasmic reticulum inside human cells, on a lipid molecule called dolichol. Many of the enzymes involved in the early stages of N-glycosylation and in the synthesis and recycling of dolichol carrier remain uncharacterised in the literature, with most work focussing on yeast homologs. In many cases this is due to the difficulty of accessing either the enzyme or substrates. This project aims to address this by making substrate derivatives and using them for biochemical, biophysical and structural analysis of key enzymes. By looking at the mechanisms of each catalyst alone and also together in complexes, we will assess the dynamics between the enzymes and build a biochemical model of events in early N-glycosylation and the dolichol cycle.