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 在多种炎性疾病的病理生理学中的募集和激活。所有 趋化因子可逆地以单体和二聚体形式存在，但关于趋化因子的作用机制知之甚少。 趋化因子单体和二聚体结合GAG的分子机制和结构基础， 这些相互作用介导体内功能。三大瓶颈阻碍了实现这一目标的努力 知识-i）由于趋化因子单体和二聚体的异质性，2）天然趋化因子的复杂多样性， 发生的GAG，和3）NMR和X射线方法的局限性。在项目III中，我们将开发方法， 克服这些瓶颈，并表征HS结合三个 嗜中性粒细胞活化趋化因子：人IL-8和NAP-2，以及小鼠KC。我们将利用这些知识， 设计GAG/趋化因子诱饵并测试它们在各种动物炎症和异种移植模型中的功效。 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 相互作用，即趋化因子在溶液中以单体和二聚体形式以及以GAG结合形式存在的能力 是耦合的，受到严格的调控，这一过程中的失调是直接导致 观察临床症状。这一假设将通过追求三个具体目标来检验，即： 表征HS与趋化因子单体和二聚体结合的分子特性; 2）确定HS与趋化因子单体和二聚体结合的分子特性。 HS结合的趋化因子单体和二聚体的溶液结构;和3）设计和测试GAG， 在小鼠炎症模型和各种炎症模型中应抑制中性粒细胞募集的趋化因子诱饵 异种移植相关试验和动物模型（项目IV）。这些目标将通过三种方法实现： 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， 二聚体。策略2-化学酶促合成尺寸确定的化学均质GAG。PL-I，谁是 该方法的专家将合成GAG，包括统一的和选择性标记的（第一个 它们的种类）^^N和^^C-GAG，它们对于溶液NMR结构研究至关重要。策略3-NMR结构 使用来自化学位移扰动，顺磁弛豫增强（PRE）， 残余偶极耦合（RDC）、N-弛豫和分子间NOE实验。新方法包括 对于RDC使用选择性的13C标记的GAG，对于PRE实验使用自旋标记的GAG。 相关性（参见说明）： 本研究的主要成果有两个方面：（1）了解了生物体的基本结构/分子结构， GAG结合趋化因子单体和二聚体的原理，以及（2）基于GAG的鉴定 用于趋化因子介导的炎性疾病的抑制剂。

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.