Structural and functional analysis of heparan sulfate sulfotransferases.

摘要

Heparan sulfate (HS), a major polysaccharide component of the vascular system, is involved in regulating a number of functions of the blood vessel wall including blood coagulation, cell differentiation, and the inflammatory response. The wide range of biological functions makes HS an attractive therapeutic target. The long term goal of our research involves utilizing an enzyme-based approach to develop HS-based therapeutics for treating thrombotic diseases, cancer and excessive inflammatory responses. The biosynthesis of HS involves multiple specialized sulfotransferases such as 2-O-sulfotransferase (2OST) and 6-O-sulfotransferase (6OST), which are essential for preparing HS with activities in regulating vascular development and blood coagulation. The substrate specificity of the HS sulfotransferases controls the sulfation patterns of HS, permitting HS to exhibit a specific function, however, limited knowledge regarding the mechanism of these enzymes has hindered our ability to prepare functionally-specific HS. We aim to understand the mechanism of action of these two enzymes in hopes of developing heparin/HS with improved anticoagulant efficacy.;In this dissertation, we present successful crystallization of 2OST in complex with 3'-phosphoadenosine 5'-phosphate (PAP). The substrate recognition mechanism of 2OST was examined by way of extensive structurally guided mutational analysis. Several residues were identified, including Arg-189, Tyr-94, and His-106, that are responsible for dictating the substrate specificity of 2OST. Despite success with the crystallization of 2OST, the crystallization of 6OST has been an ongoing process. A promising expression construct for crystallization purposes has been identified for 6OST-1. Using a homology model of 6OST-3 with structurally known 3OST-3, several residues involved in PAPS and substrate binding were proposed.