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

说明(申请人提供)：硫酸乙酰肝素通过相对较短的硫化糖序列与许多生长因子、趋化因子、形态因子、细胞外基质蛋白、酶和酶抑制剂结合。链的一种相对罕见的修饰是将硫酸盐添加到氨基葡萄糖残基的碳-3上，就其生物合成和功能而言，人们对其知之甚少。脊椎动物中存在一个由7个氨基葡萄糖3-O-磺基转移酶(Hs3st)组成的家族。虽然这些酶已经被克隆和部分鉴定，但人们对这些修饰发生的背景知之甚少，更不知道与含有3-O-硫酸酯基团的乙酰肝素链结合的内源配体。这一建议的中心假设是，Hs3st选择性地作用于硫酸乙酰肝素的亚序列，从而产生内源性蛋白配体的特定结合位点。这项建议侧重于(I)表征3-O-硫酸乙酰肝素的结构特征的方法，(Ii)Hs3st-1和Hs3st-2作为模式3-O-磺基转移酶的特异性，(Iii)发现和鉴定与Hs3st修饰链结合的天然配体，以及(Iv)检测Hs3st-1和Hs3st-2突变小鼠的硫酸乙酰肝素。针对这些目标，我们有以下具体目标：目标1：建立硫酸乙酰肝素中二糖和四糖亚基3-O-磺化的定量测定方法。目的2：利用Hs3st-1和Hs3st-2合成具有功能活性的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：检测Hs3st-1和Hs3st-2在体内形成结合位点的情况。我们期望这一项目将显著提高我们对这类磺基转移酶如何影响生物过程和病理状态的理解，并验证它们作为潜在的药物干预靶点的有效性。 与公共卫生相关：本提案的首要目标是了解硫酸乙酰肝素的3-O-硫酸盐的功能。具体目标包括开发分析工具以评估硫酸乙酰肝素中3-O-硫酸基序的存在，比较Hs3st-1和Hs3st-2作为AT型和GD型磺基转移酶候选成员的特异性，使用这些酶的重组形式产生亲和力矩阵，分离组织提取物以寻找内源性配体，以及分析Hs3st-1和Hs3st-2在体内的表达变化如何影响配体结合位点的形成。这项工作还将提供一套其他研究人员可以利用的工具，用于模式生物和人类疾病的研究，其中硫酸乙酰肝素的3-O-硫化可能起到作用。

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.