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化糖序列结合许多生长因子、趋化因子、形态发生素、细胞外基质蛋白、酶和酶抑制剂。一种相对罕见的链修饰，即在葡糖胺残基的碳-3上添加硫酸盐，在其生物合成和功能方面知之甚少。在脊椎动物中存在一个由7个葡糖胺基3-O-磺基转移酶（Hs 3st）组成的家族。虽然酶已被克隆和部分表征，很少有人知道的情况下，这些修改发生，甚至更少的是已知的内源性配体结合到硫酸乙酰肝素链，含有3-O-硫酸基团。该提议的中心假设是，Hs 3sts选择性地作用于硫酸乙酰肝素中的酶活性，从而产生内源性蛋白配体的特异性结合位点。该提案的重点是（i）方法，以结构表征3-O-硫酸乙酰肝素硫酸酯，（ii）的特异性Hs 3st-1和Hs 3st-2作为模型3-O-磺基转移酶，（iii）的发现和表征的天然配体结合Hs 3st修饰链，和（iv）检查硫酸乙酰肝素从小鼠Hs 3st-1和Hs 3st-2改变。为了实现这些目标，我们有以下具体目标：目标1：建立一种定量测定硫酸乙酰肝素中二糖和四糖亚基3-O-硫酸化的方法。目的2：使用Hs 3st-1和Hs 3st-2产生功能活性的3-O-硫酸化硫酸乙酰肝素。目的3：捕获并鉴定与3-O-硫酸化硫酸乙酰肝素结合的蛋白质配体。目的4：检测Hs 3st-1和Hs 3st-2在体内结合位点的形成。我们期望，这个项目将显着提高我们的理解，这类磺基转移酶如何影响生物过程和病理状态，并验证它们作为潜在的药物干预的目标。 公共卫生相关性：本提案的总体目标是了解硫酸乙酰肝素3-O-硫酸化的功能。具体目标包括开发用于评估硫酸乙酰肝素中3-O-硫酸基序的存在的分析工具，以比较Hs 3st-1和Hs 3st-2作为AT型和GD型磺基转移酶的候选成员的特异性，以使用这些酶的重组形式产生亲和基质，以破碎组织提取物以寻找内源性配体，并分析在体内改变Hs 3st-1和Hs 3st-2的表达如何影响配体结合位点的形成。这项工作还将提供一套工具，其他研究人员可以利用这些工具来研究模型生物和人类疾病，其中硫酸乙酰肝素的3-O-硫酸化可能发挥作用。

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. PUBLIC HEALTH RELEVANCE: 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.