BASEMENT MEMBRANE PROTEOGLYCAN

基底膜蛋白多糖

• 批准号: 2734685
• 负责人: JOHN ROBERT HASSELL
• 金额: $ 16.46万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734685
• 关键词: antibody formation; basement membrane; cell adhesion; collagen; entactin; extracellular matrix; genetic manipulation; genetically modified animals; growth /development; heparan sulfate; laboratory mouse; laboratory rabbit; laminin; membrane structure; molecular cloning; mutant; protein structure function; proteoglycan; radiotracer; recombinant proteins

Basement membranes are thin sheets of extracellular matrix that separate endothelial, epithelial and muscle cells, as well as the nervous system, from adjacent cells and connective tissues. All basement membranes are composed primarily of collagen IV, laminin, entactin and perlecan, a large heparan sulfate containing proteoglycan. These components interact to provide the fundamental architectural framework of basement membranes. Perlecan is a critical participant in a number of diverse biological functions such as ionic filtration, cell-attachment and matrix assembly that are needed for the development and maintenance of tissues and organs. Perlecan's 396 kDa core protein consists of five major domains; one with a potential site for heparan sulfate attachment and the remaining four showing homology to LDL receptor, laminin, N-CAM and agrin. The long term goals of this project are to determine the specific roles of the different domains in heparan sulfate synthesis, cell adhesion, matrix interaction and embryonic development. We will do this by using the cDNA clones to perlecan to express individual domains, selected groups of domains and full length perlecan as recombinant proteins and then evaluating their activities and properties. The first aim is to identify the domain that initiates heparan sulfate synthesis and the domains that influence this activity by biosynthetically radiolabeling cells producing the recombinant proteins and measuring the amount of heparan sulfate on the protein. The second aim is to identify the domains that regulate cell adhesion by purifying the recombinant proteins, coating the proteins on dishes and measuring the number and type of cells that attach to the dish. The third aim is to identify the domains that interact with collagen IV, laminin and entactin by mixing the purified radiolabeled recombinant proteins with each of these components, capturing the complex with an antibody and measuring the amount of protein in the complex. The fourth aim is to identify new biological functions for perlecan by producing a perlecan dominant negative mutation in mice and evaluating its consequences on growth and development. The information obtained from these studies will identify the biological functions of each domain of perlecan and provide new insights into the role of perlecan in growth and development.

地下膜是细胞外基质的薄片 内皮，上皮和肌肉细胞以及神经系统， 来自相邻的细胞和结缔组织。 所有地下室膜都是 主要由胶原蛋白IV，层粘连蛋白，Entactin和perlecan组成 硫酸乙酰肝素含有蛋白聚糖。 这些组件与 提供地下室膜的基本建筑框架。 Perlecan是许多不同生物学的关键参与者 离子过滤，细胞跟踪和基质组件等功能 开发和维护组织和器官所需的。 Perlecan的396 kDa核心蛋白质由五个主要领域组成。一个 硫酸乙酰肝素附着和其余四个的潜在部位 与LDL受体，层粘连蛋白，N-CAM和Agrin显示同源。 长期 该项目的目标是确定不同的特定作用 硫酸乙酰肝素合成，细胞粘附，基质相互作用和 胚胎发展。 我们将通过使用cDNA克隆来做到这一点 佩尔坎表达单个域，选定的域和完整的组 长度perlecan作为重组蛋白，然后评估其 活动和特性。 第一个目的是确定 启动硫酸乙酰肝素的合成和影响这一点的领域 通过生物合成的放射性标记细胞的活性，产生重组 蛋白质并测量蛋白质上硫酸乙酰肝素的量。 这 第二个目的是确定通过 纯化重组蛋白，将蛋白涂在菜肴上， 测量附着在盘子上的细胞的数量和类型。 第三 目的是确定与胶原蛋白IV，层粘连蛋白和 通过将纯化的放射性标记的重组蛋白与每种混合混合 在这些成分中，用抗体捕获复合物并测量 复合物中的蛋白质量。 第四个目标是确定新的 perlecan的生物学功能通过产生perlecan显性阴性 小鼠突变，并评估其对生长和发育的后果。 从这些研究中获得的信息将确定生物学 perlecan的每个领域的功能，并为角色提供新的见解 Perlecan的增长和发展。