STRUCTURE/FUNCTION OF HYALURANAN BINDING PROTEINS/RECEPT

透明质酸结合蛋白/受体的结构/功能

• 批准号: 2904408
• 负责人: PAUL H WEIGEL
• 金额: $ 42.48万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2904408
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; Streptococcus; affinity labeling; carbohydrate biosynthesis; carbohydrate receptor; confocal scanning microscopy; electron spin resonance spectroscopy; endocytosis; fluorescence microscopy; glucuronosyltransferase; hyaluronate; isozymes; laboratory mouse; laboratory rat; lectin; liver cells; molecular cloning; monoclonal antibody; mutant; protein purification; protein structure function; virulence

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.

描述（改编自申请人的摘要）：透明质酸（HA）是一种 脊椎动物中普遍存在的 ECM 成分。 HA是一种强大的细胞调节剂 行为并在细胞迁移、发育、癌症、伤口中发挥关键作用 愈合和血管生成。指导该项目的长期假设是 必须严格控制 HA 的合成和降解以实现正常的体内平衡 和健康。出生缺陷、溃疡性伤口、关节炎和癌症可能是 通过HA合成或降解的改变而促进。该项目包含 两个子项目正在研究 HA 合成和 HA 周转。第一个 子项目正在研究链球菌酶 HA 合酶 (HAS)，我们 没有纯化。这种HAS使A组和C组细胞的HA胶囊， 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 的工作原理以及 HA 合成是如何调节的。第二个 子项目正在研究哺乳动物 HA 周转的最后阶段，即 肝内皮细胞 (LEC) 对 HA 的内吞作用。单克隆抗体 (MoAb) 与大鼠 LEC HA 受体 (HAR) 的结合使我们能够通过以下方式阻断 HA 的摄取： LEC 并亲和纯化两个 175kD 和 ~300kD 的 HAR。我们隔离了一个 17kD HAR 的 3.9kb 部分 cDNA，编码由 我们的一些 MoAb。我们在这个子项目中的假设是 LEC 包含 两个高度相似的 HA 同工受体，每个 HAR 可能专门用于 与较小或较大的 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) 表征链球菌 HAS 机制和 使用生物物理技术的结构-功能关系。 2）创建 具有特定功能缺陷的链球菌 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 175kD HAR。 4) 克隆并表达亚基的cDNA LEC 的 300kD HAR 复合体。 5）阐明结构与功能的关系 17kD 和 300kD HAR。这个项目的结果将使我们能够 首次研究 LEC HAR 在各种病理中的作用 和疾病，并可能产生治疗链球菌疾病的新药。