Heparan Sulfate - Chemokine Interactions and Inflammation

糖胺聚糖(GAG)-硫酸乙酰肝素(HS)在趋化因子介导的中性粒细胞中起关键作用 在多种炎症性疾病的病理生理学中的招募和激活。全 趋化因子以单体和二聚体的形式可逆地存在，但值得注意的是，人们对趋化因子 趋化因子单体和二聚体结合GAG的分子机制和结构基础 这些相互作用调节着体内的功能。三个主要瓶颈阻碍了实现这一目标的努力 知识-i)趋化因子单体和二聚体引起的异质性，2)天然的复杂多样性 3)核磁共振和X射线方法的局限性。在项目III中，我们将开发方法来 克服这些瓶颈，并表征三种HS结合的结构/分子基础 中性粒细胞激活趋化因子：人IL-8和NAP-2，以及小鼠KC。我们将利用这一知识来 设计Gag/趋化因子诱饵，并在各种动物炎症和异种移植模型中测试它们的有效性。 我们的中心假设是，中性粒细胞募集的差异一定是由于不同的GAG所致 相互作用，趋化因子以单体和二聚体形式在溶液中和以Gag结合形式存在的能力 是相互关联的，并且受到严格的监管，这一过程中的失调直接导致了 观察临床症状。这一假设将通过追求三个具体目标来检验：1) 表征HS与趋化因子单体和二聚体结合的分子性质；2)测定 HS结合的趋化因子单体和二聚体的溶液结构；3)设计和测试Gag和 趋化因子诱饵应抑制中性粒细胞在小鼠炎症模型和各种 与异种移植相关的分析和动物模型(项目四)。这些目标将通过3个途径实现： 策略1-使用蛋白质工程方法，设计和合成捕获的趋化因子单体和 二聚体。策略2-化学酶法合成大小确定、化学均一的GAG。PL-I，谁是 这一方法的专家将合成GAG，包括统一的和选择性地标记的(第一个 它们的种类)^N和^C-GAG对于溶液核磁共振结构研究是至关重要的。策略3-核磁共振结构 使用来自化学位移微扰、顺磁弛豫增强(PRE)、 剩余偶极耦合(RDC)、^N驰豫和分子间NOE实验。新方法包括 使用选择性^C标记的GAG用于RDC和自旋标记的GAG用于预实验。 相关性(请参阅说明)： 这项工作的主要成果将是两个方面-(1)对基本结构/分子的理解 GAG与趋化因子单体和二聚体结合的原理以及(2)基于GAG的鉴定 趋化因子介导的炎症性疾病的抑制剂。

The glycosaminoglycan (GAG) heparan sulfate (HS) plays a critical role in chemokine-mediated neutrophil recruitment and activation in the pathophysiology of a wide variety of inflammatory diseases. All chemokines exist reversibly as monomers and dimers, but remarkably very little is known regarding the molecular mechanisms and structural basis by which chemokine monomers and dimers bind GAGs, and how these interactions mediate in vivo function. Three major bottlenecks have stymied efforts to obtain this knowledge - i) heterogeneity due to chemokine monomers and dimers, 2) the complex diversity of naturally occurring GAGs, and 3) limitations to NMR and X-ray methods. In Project III, we vsdll develop methods to overcome these bottlenecks, and characterize the structural/molecular basis of HS binding for three neutrophil-activating chemokines: human IL-8 and NAP-2, and mouse KC. We will use this knowledge to design GAG/chemokine decoys and test their efficacy in various animal inflammation and xenograft models. Our Central Hypothesis is that differences in neutrophil recruitment must be due to differential GAG interactions, that chemokines' ability to exist as monomers and dimers in solution and in GAG-bound forms are coupled and tightly regulated, and that dysregulation in this process is directly responsible for the observed clinical symptoms. This hjrpothesis v^ll be tested by pursuing three Specific Aims, to: 1) characterize the molecular properties of HS binding to chemokine monomers and dimers; 2) determine the solution structures of HS-bound chemokine monomers and dimers; and 3) design and test GAG and chemokine decoys that should inhibit neutrophil recruitment in mouse inflammation models and in various xenograft-related assays and animal models (Project IV). These Aims will be accomplished via 3 approaches: Strategy 1 - Using protein engineering methods, design and synthesize trapped chemokine monomers and dimers. Strategy 2 - Chemoenzymatic synthesis of size-defined, chemically homogeneous GAG. PL-I, who is an expert in this methodology, will synthesize the GAGs, including uniform and selectively labeled (first of their kind) ^^N and ^^C-GAGs that are critical for solution NMR structural studies. Strategy 3 -NMR structure determination using data from chemical shift perturbation, paramagnetic relaxation enhancement (PRE), residual dipolar coupling (RDC), ^^N-relaxation, and intermolecular NOE experiments. Novel methods include using selective ^^C-labeled GAG for RDC and spin-labeled GAG for PRE experiments. RELEVANCE (See instructions): Major achievements from this work will be two fold - (1) an understanding of the basic structural/molecular principles by which GAGs bind chemokine monomers and dimers, and (2) identification of GAG-based inhibitors for chemokine-mediated inflammatory diseases.