Heparan sulfate binds many growth factors, chemokines, morphogens, extracellular matrix proteins, enzymes and enzyme inhibitors via relatively short sulfated saccharide sequences. One relatively rare modification to the chains, the addition of sulfate to carbon-3 of glucosamine residues, is poorly understood in terms of its biosynthesis and function. A family of seven glucosaminyl 3-O-sulfotransferases (Hs3st) exists in vertebrates. Although the enzymes have been cloned and partially characterized, little is known about the context in which these modifications occur and even less is known about the endogenous ligands that bind to heparan sulfate chains that contain 3-O-sulfate groups. The central hypothesis of this proposal is that the Hs3sts act selectively on subsequences in heparan sulfate and thereby generate specific binding sites for endogenous protein ligands. This proposal focuses on (i) methods to structurally characterize 3-O-sulfated heparan sulfate, (ii) the specificity of Hs3st-1 and Hs3st-2 as model 3-O-sulfotransferases, (iii) the discovery and characterization of natural ligands that bind to Hs3st modified chains, and (iv) examination of heparan sulfate derived from mice altered in Hs3st-1 and Hs3st-2. Towards these goals, we have the following specific aims:
Aim 1 : Develop a quantitative method for determining 3-O-sulfation of disaccharide and tetrasaccharide subunits in heparan sulfate.
Aim 2 : Generate functionally active 3-O-sulfated heparan sulfate using Hs3st-1 and Hs3st-2.
Aim 3 : Capture and identify protein ligands that bind to 3-O-sulfated heparan sulfate.
Aim 4 : Examine the formation of binding sites by Hs3st-1 and Hs3st-2 in vivo. We expect that this project will significantly enhance our understanding on how this class of sulfotransferases influences biological processes and pathological states, and validate them as potential targets for pharmacological intervention.
The overarching goal of this proposal is to understand the function of 3-O-sulfation of heparan sulfate. The specific objectives include the development of analytical tools for assessing the presence of 3-O-sulfated motifs in heparan sulfate, to compare the specificity of Hs3st-1 and Hs3st-2 as candidate members of the AT-type and gD-type sulfotransferases, to generate affinity matrices using recombinant forms of these enzymes, to fractionate tissue extracts to search for endogenous ligands, and to analyze how altering the expression of Hs3st-1 and Hs3st-2 in vivo affects the formation of ligand binding sites. The work will also provide a set of tools that other investigators can exploit for studies of model organisms and human disorders in which 3-O-sulfation of heparan sulfate may play a role.
|Thacker, Bryan E; Seamen, Emylie; Lawrence, Roger et al. (2016) Expanding the 3-O-Sulfate Proteome--Enhanced Binding of Neuropilin-1 to 3-O-Sulfated Heparan Sulfate Modulates Its Activity. ACS Chem Biol 11:971-80|
|van Wijk, Xander M; Lawrence, Roger; Thijssen, Victor L et al. (2015) A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3). FASEB J 29:2993-3002|
|Wen, Jianzhong; Xiao, Junyu; Rahdar, Meghdad et al. (2014) Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis. Proc Natl Acad Sci U S A 111:15723-8|
|Lawrence, Roger; Brown, Jillian R; Lorey, Fred et al. (2014) Glycan-based biomarkers for mucopolysaccharidoses. Mol Genet Metab 111:73-83|
|Wang, Raymond Y; Aminian, Afshin; McEntee, Michael F et al. (2014) Intra-articular enzyme replacement therapy with rhIDUA is safe, well-tolerated, and reduces articular GAG storage in the canine model of mucopolysaccharidosis type I. Mol Genet Metab 112:286-93|
|Xu, Ding; Esko, Jeffrey D (2014) Demystifying heparan sulfate-protein interactions. Annu Rev Biochem 83:129-57|
|Thacker, Bryan E; Xu, Ding; Lawrence, Roger et al. (2014) Heparan sulfate 3-O-sulfation: a rare modification in search of a function. Matrix Biol 35:60-72|
|Gasimli, Leyla; Glass, Charles A; Datta, Payel et al. (2014) Bioengineering murine mastocytoma cells to produce anticoagulant heparin. Glycobiology 24:272-80|
|Lawrence, Roger; Brown, Jillian R; Lorey, Fred et al. (2013) WITHDRAWN: Glycan-based biomarkers for mucopolysaccharidoses. Dis Markers :|
|van Wijk, Xander M; Thijssen, Victor L; Lawrence, Roger et al. (2013) Interfering with UDP-GlcNAc metabolism and heparan sulfate expression using a sugar analogue reduces angiogenesis. ACS Chem Biol 8:2331-8|
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