STRUCTURE/FUNCTION OF HYALURANAN BINDING PROTEINS/RECEPT

描述（改编自申请人的摘要）：Hybryonan（HA）是一种 普遍存在于脊椎动物中的ECM成分。HA是一种强有力的细胞调节剂， 行为，并在细胞迁移、发育、癌症、创伤中发挥关键作用 愈合和血管生成。指导这个项目的长期假设是， HA的合成和降解必须严格控制正常的稳态 与健康出生缺陷，溃疡伤口，关节炎和癌症可能是 通过改变HA合成或降解促进。该项目包含 两个子项目正在研究HA合成和HA周转。第一 子项目是研究链球菌酶HA合成酶（HAS），我们 没有净化。这种HAS使A组和C组细胞的HA被膜， 这是一个重要的毒力因子。我们在这个子项目中的假设是 链球菌HAS的结构-功能分析将使我们能够 了解HAS如何工作以及HA合成如何调节。第二 一个子项目正在研究哺乳动物HA周转的最后阶段， 肝内皮细胞（LEC）对HA的内吞作用。单克隆抗体 （MoAb）对大鼠LEC HA受体（HAR）的作用使我们能够通过以下途径阻断HA摄取： 经亲和层析纯化175 kD和~ 300 kD的两种HAR。我们已经分离出了一 17 kD HAR的3.9kb部分cDNA，编码一种独特的蛋白， 我们的几个MoAb我们在这个子项目中的假设是，LEC包含 两种高度相似的HA同种受体，每种HAR可能专门用于 与更小或更大的HA交互。我们将采用技术， 生物化学分子生物学和细胞生物学来解决这些假设， 以下目标。1)为了表征链球菌HAS机制， 使用生物物理技术的结构-功能关系。2)创建 具有特异性功能缺陷的链球菌HAS突变体， 结构-功能研究3)克隆并表达小鼠和人的cDNA 对于LEC的175 kD HAR。4)克隆并表达重组质粒的亚基cDNA， LEC的300 kD HAR复合物。5)阐明结构-功能关系 17 kD和300 kD的HAR。该项目的成果将使我们能够 首次研究LEC HAR在各种病理学中的作用 并可能导致治疗链球菌疾病的新药。

DESCRIPTION (Adapted from applicant's abstract): Hyaluronan (HA) is a ubiquitous ECM component in vertebrates. HA is a powerful modulator of cell behavior and plays key roles in cell migration, development, cancer, wound healing and angiogenesis. The long-term hypothesis guiding this project is that HA synthesis and degradation must be tightly controlled for normal homeostasis and health. Birth defects, ulcerating wounds, arthritis, and cancer may be promoted by alterations in HA synthesis or degradation. The project contains two subprojects that are investigating HA synthesis and HA turnover. The first subproject is studying the streptococcal enzyme HA synthase (HAS), which we have no purified. This HAS makes the HA capsule of Group A and Group C cells, which is a significant virulence factor. Our hypothesis in this subproject is that structure-function analyses of the streptococcal HASs will allow us to understand how HAS works and how HA synthesis is regulated. The second subproject is investigating the final stage in mammalian HA turnover, which is the endocytosis of HA by liver endothelial cells (LECs). Monoclonal antibodies (MoAbs) to the rat LEC HA receptor (HAR) have enable us to block HA uptake by LECs and to affinity purify two HARs of 175kD and ~300kD. We have isolated a 3.9kb partial cDNA for the 17kD HAR, encoding a unique protein recognized by several of our MoAbs. Our hypothesis in this subproject is that LECs contain two highly similar isoreceptors for HA and that each HAR may be specialized to interact with either smaller or larger HA. We will employ techniques in biochemistry molecular biology and cell biology to address these hypotheses in the following aims. 1) To characterize streptococcal HAS mechanisms and structure-function relationships using biophysical techniques. 2) To create streptococcal HAS mutants with specific functional defects for structure-function studies. 3) To clone and express the mouse and human cDNAs for the 175kD HAR of LECs. 4) To clone and express the cDNAs for subunits of the 300kD HAR complex of LECs. 5) To elucidate structure-function relationships of the 17kD and 300kD HARs. The results from this project will allow us to investigate, for the first time, the role of the LEC HAR in various pathologies and diseases and could lead to new drugs for streptococcal diseases.