Structure and Function of 3-O-sulfation in Heparan Sulfate

描述（由申请人提供）：硫酸乙酰肝素通过相对较短的硫酸糖苷序列结合多种生长因子、趋化因子、形态因子、细胞外基质蛋白、酶和酶抑制剂。在葡萄糖胺残基的碳-3上添加硫酸盐是一种相对罕见的链修饰，但就其生物合成和功能而言，人们对其了解甚少。在脊椎动物中存在一个由7个氨基葡萄糖3- o -硫基转移酶（Hs3st）组成的家族。尽管这些酶已被克隆并部分表征，但对这些修饰发生的背景知之甚少，甚至对与含有3- o -硫酸盐基团的硫酸肝素链结合的内源性配体知之甚少。该建议的中心假设是Hs3sts选择性地作用于硫酸肝素的子序列，从而产生内源性蛋白质配体的特异性结合位点。本提案的重点是(i)结构表征3- o -硫酸化硫酸肝素的方法，（ii） Hs3st-1和Hs3st-2作为模型3- o -硫转移酶的特异性，（iii）结合Hs3st修饰链的天然配体的发现和表征，以及（iv）检测Hs3st-1和Hs3st-2改变小鼠的硫酸肝素。为了实现这些目标，我们有以下具体目标：目标1：建立一种定量方法来测定硫酸肝素中双糖和四糖亚基的3- o -硫酸化。目的2：利用Hs3st-1和Hs3st-2生成具有功能活性的3- o -硫酸乙酰肝素。目的3：捕获和鉴定与3- o -硫酸乙酰肝素结合的蛋白质配体。目的4：观察Hs3st-1和Hs3st-2在体内结合位点的形成。我们期望这个项目将大大提高我们对这类硫转移酶如何影响生物过程和病理状态的理解，并验证它们作为药物干预的潜在靶点。

DESCRIPTION (provided by applicant): 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.