The project involves investigation of a human enzyme responsible for desulfation of heparan sulfate and heparin. The enzyme, (N-acetyl) glucosamine 6-sulfatase (G6S), is not particularly well characterized at the molecular level, but the Gloster group has recently obtained the structure of G6S using X-ray crystallography1. Gaining further insights into G6S activity is important for industrial biotechnology and biomedical applications.
Heparin, which is obtained from pig tissue, has been used as an anti-coagulant for a century. However, a contamination scare in 2008, which caused fatalities worldwide, has amplified the need for homogenous synthetic heparins. Given the difficulties often associated with chemical synthesis of large polysaccharides, enzymatic approaches are much more tractable. Ultra low molecular weight heparins (ULMWHs) have been demonstrated to work effectively as anti-coagulants. However, ULMWHs have one drawback as they can't be neutralized by protamine sulfate, meaning there is no antidote if a patient is given an overdose of anti-coagulant. Others2 have shown that G6S desulfates ULMWHs, leading to molecules that are unable to bind to anti-thrombin III and are thus devoid of their anti-coagulation effect. Treatment with G6S could, therefore, be effective in the case of an overdose of anti-coagulant. However, given its lysosomal origin, G6S does not work effectively at physiological pH.
The aim of this project is to develop a robust assay to measure G6S activity, using novel synthetic substrates that will be synthesized in collaboration with chemists. This, in addition to the structural information already obtained, will be used as the basis for engineering G6S, using rational site directed mutagenesis, in order to obtain an enzyme that will work at higher pH. Such an engineered enzyme could then be explored used therapeutically as an antidote to anti-coagulant overdose.
The project is highly multi-disciplinary and will provide a rich training ground for a PhD student. This will include exposure to synthetic chemistry, molecular biology, protein biochemistry, enzymology, structural biology and cell biology.
2. X Zhou, L Li, RJ Linhardt, J Liu. FEBS J, 2013, 280, 2523-32.