Structure and function of heparan sulfate degrading sulfatases

Abstract

Heparan sulfate (HS) is a linear glycosaminoglycan (GAG) synthesised in the Golgi of all mammalian cells and is composed of alternating glucosamine (GlcN) and iduronic acid/glucuronic acid (IdoA/GlcA) residues. Due to the number of processes HS mediates, turnover of HS is a tightly regulated and necessary procedure. The degradation of HS begins at the cell surface and finishes in the lysosome, after which components can be recycled. Deficiencies or mutations in the lysosomal enzymes that process GAGs result in Mucopolysaccharidoses disorders (MPSs), where accumulation of incomplete degradation products in the lysosome is observed. There are five known sulfatases which degrade HS in the lysosome, three of which have been studied here. The aim of this project was to further understand the structure, mechanism and function of human N-acetylglucosamine-6-sulfatase (GN6S), iduronate-2-sulfatase (IDS) and N-sulfoglucosamine-3-sulfatase (GN3S), and their associated diseases. Methods to recombinantly express and purify these three sulfatases have been established in this study, including the co-expression with the formylglycine generating enzyme (FGE). This was necessary to improve the activity of recombinant sulfatase, with the most improvement observed for IDS. The development of a coupled assay within this study has allowed the investigation of the kinetic parameters of GN6S using the substrate, 4-methylumbelliferyl N-acetylglucosamine-6-sulfate (4-MU-GlcNAc6S). The structure of GN6S has been determined here, allowing the identification of residues likely to be involved in substrate binding and the reasoning behind the pathology of three known MPS IIID-causing missense mutations. The knowledge of the human GN6S structure will allow the rational design of molecular chaperones which function to stabilise misfolded GN6S in MPS IIID patients.

Date of Award	21 Jun 2017
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Tracey Gloster (Supervisor)