FUNCTIONAL IMPORTANCE OF DOMAIN OF TYPE IV COLLAGEN

IV 型胶原蛋白结构域的功能重要性

基本信息

批准号：
3463111
负责人：
PHOTINI C TSILIBARY
金额：
$ 9.82万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-04-01 至 1993-03-31
项目状态：
已结题

项目摘要

Basement membranes (BM) are specialized extracellular matrices which are composed of distinctive macromolecules, such as laminin, type IV (t.IV) collagen, proteopheparen sulfate and nidogen. In the microvasulature, endothelial cells and their BM form the wall across which nutrients and proteins exchange. In the kidney, the glomerular basement membrane (GBM) form the only barrier to the extravasation of plasma proteins and serves as a selective filter. This function of perselectively becomes impaired in nephrotic syndromes and excessive protein leakage occurs. The molecular mechanisms which lead to such defects of the GBM are not understood. In diabetes, t.IV collagen is chemically modified by non-enzymatic binding of glucose to the epsilon-amino groups of lysine residues. In Goodpasture nephritis, an auto-antibody forms to the main non-collagenous, NC1 domain of t.IV collagen. In Alport's nephritis there is a lack of recognition of the Goodpasture (NC1) antigen. However, it is not known whether structural or other changes of t.IV collagen play a causal role in the development of the above mentioned nephrotic syndromes. It is the purpose of this study to examine the structural and functional changes of t.IV collagen and the NCI domain in one of these pathological conditions. T.IV collagen has the ability to self-assemble by end-to-end and lateral interactions to a complex network. The NC1 domain is required for network formation because a) it binds to an adjacent NC1 and forms dimers b) it binds to the collagenous part of adjacent t.IV collagen molecules and initiates complex formation. In the proposed study the binding of NC1 to t.IV collagen and the formation of networks will be assessed in 1) control, 2) diabetic conditions. The effect of principally in vitro and to a lesser extent in vivo non-enzymatic glucosylation of t.IV collagen will be explore. Network formation by control and glucosylated t.IV collagen will be examined by rotary shadowing. The binding of control and glucosylated NC1 domain to diabetically modified t.IV collagen and to control or glucosylated triple-helical domain of type IV collagen will be evaluated, by rotary shadowing, zonal rate, velocity sedimentation, solid phase binding assays and turbidometry. Furthermore, chemically or proteolytically derived fragments from the NC1 and triple-helical domains as well as synthetic peptides will be used to identify active binding sites which mediate assembly of t.IV collagen. These studies will examine the possibility that supromolecular assembly of t.IV collagen in altered following non-enzymatic glucosylation.

基底膜 (BM) 是特殊的细胞外基质它们由独特的大分子组成，例如层粘连蛋白、IV 型 (t.IV) 胶原蛋白、硫酸 proteopheparen 和尼多根。在微血管系统中，内皮细胞及其 BM 形成营养物质和蛋白质交换的墙壁。在肾脏、肾小球基底膜 (GBM) 形成血浆蛋白外渗的唯一屏障，并可作为选择性过滤器。这个函数选择性地变成肾病综合征和蛋白质过多渗漏受损发生。导致此类缺陷的分子机制 GBM 不被理解。在糖尿病中，t.IV 胶原蛋白是通过葡萄糖非酶促结合进行化学修饰赖氨酸残基的ε-氨基。在古德帕斯特肾炎中，针对主要非胶原蛋白 NC1 结构域形成自身抗体 t.IV 胶原蛋白。奥尔波特肾炎缺乏 Goodpasture (NC1) 抗原的识别。然而，它并不是已知 t.IV 胶原蛋白的结构或其他变化是否发挥作用与上述肾病发生的因果关系综合症。本研究的目的是检验 t.IV 胶原蛋白和 NCI 的结构和功能变化这些病理状况之一的域。 T.IV 胶原蛋白具有通过端到端和横向交互进行自组装的能力到一个复杂的网络。网络需要 NC1 域形成，因为 a) 它与相邻的 NC1 结合并形成二聚体 b) 它与相邻 t.IV 胶原蛋白的胶原部分结合分子并引发复合物形成。在拟议的研究中 NC1 与 t.IV 胶原蛋白的结合以及网络的形成将评估 1) 对照，2) 糖尿病状况。效果主要是体外的，其次是体内非酶促的将探索 t.IV 胶原蛋白的糖基化。网络形成通过对照和糖基化 t.IV 胶原蛋白将通过旋转阴影。对照和糖基化 NC1 的结合域糖尿病修饰的 t.IV 胶原蛋白并控制或 IV型胶原的糖基化三螺旋结构域将是通过旋转阴影、区域速率、速度进行评估沉降、固相结合测定和浊度测定。此外，化学或蛋白水解衍生的片段来自 NC1 和三螺旋结构域以及合成肽将用于识别活性结合位点介导 t.IV 胶原蛋白的组装。这些研究将检验 t.IV 胶原蛋白超分子组装的可能性非酶促糖基化后发生改变。