Altered Matrix-Cells and Intermolecular Interactions

描述(申请人提供)：这项建议将利用一个受控良好的实验系统，涉及重组II型胶原和软骨细胞，以研究纤维胶原蛋白的突变如何影响细胞的行为，改变细胞的空间排列，以及影响受影响组织中细胞外基质的组装。突变对突变胶原蛋白与其他大分子相互作用的影响也将被分析。目前的应用基于以下观察：(I)软骨细胞的附着和运动取决于它们与II型胶原特定结构域的相互作用，(Ii)表达突变的人COL2A1基因的小鼠的II型胶原纤维密度、肥大软骨细胞的组织和软骨细胞分泌囊泡的形态因Arg-α-519替换的半胱氨酸而改变，以及(Iii)II型胶原中的Arg-αL-519的半胱氨酸改变了其与正常的第IX型胶原的相互作用。本提议的总体目标是检验这样的假设，即纤维胶原的突变不仅影响细胞外，还影响突变的蛋白质与其他大分子的相互作用。改变细胞外基质的结构，改变细胞的空间排列。为了验证这些假设，我们将使用一个利用软骨细胞和重组II型胶原蛋白的细胞-基质实验系统。为了消除正常的内源性II型胶原对细胞行为的影响，将利用现有的COL2al基因失活的转基因小鼠来获得不产生II型前胶原但表达其他软骨大分子的软骨细胞。这些软骨细胞将被种植在包覆有突变的重组胶原II的工程化三维纳米纤维支架上。在另一系列实验中，不表达Col2al但表达正常人类C0L2A1的小鼠软骨细胞将被导入突变的DNA构建体，以表达除正常链之外还包含突变链的II型前胶原。表达突变蛋白的细胞将在长期悬浮培养中分析它们组装软骨细胞外基质的能力。此外，还将研究类似于细胞内、未折叠的胶原蛋白的单个突变胶原蛋白a链与其他大分子的相互作用。其具体目的是：(1)确定细胞外信号如何影响细胞的行为和空间组织。(2)分析细胞分泌和加工的突变型前胶原组装成的胶原纤维的密度和分布。(3)研究突变型纤维蛋白与其他分子的细胞内外相互作用。

DESCRIPTION (provided by applicant): This proposal will exploit a well-controlled experimental system that involves recombinant Collagen II and chondrocytes to study how mutations in fibrillar collagens influence the behavior of cells, change their spatial arrangement, and influence the assembly of the extracellular matrix in affected tissues. The effect of mutations on the interaction of mutant collagens with other macromolecules will also be analyzed. The present application is based on the following observations: (i) attachment and motility of chondrocytes depends on their interaction with specific domains of Collagen II, (ii) density of Collagen II fibrils, organization of hypertrophic chondrocytes, and morphology of chondrocytes secretory cysternae in mice expressing the mutated human COL2A1 gene are altered because of the Cys for Arg-alpha -519 substitution, and (iii) the Cys for Arg-alpha l -519 substitution in Collagen II alters its interaction with normal Collagen IX The overall goal of this proposal is to test hypotheses that mutations in fibrillar collagens affect not only extracellular but also affect the intracellular interaction of mutant proteins with other macromolecules, alter the structure of the extracellular matrix, and change the spatial arrangement of cells. To test these hypotheses, a cell-matrix experimental system, which exploits chondrocytes and recombinant Collagen II will be employed. To eliminate the influence of normal endogenous Collagen II on the behavior of cells, the existing transgenic mice with an inactivated Col2al gene will be exploited to obtain chondrocytes that do not produce procollagen II but express other cartilaginous macromolecules. These chondrocytes will be seeded onto engineered three-dimensional nanofibrillar scaffolds coated with mutated recombinant Collagen II. In another series of experiments, the mouse chondrocytes that do not express Col2al but express the normal human C0L2A1 will be transfected with mutant DNA constructs to express procollagen II that contains mutant chains in addition to normal ones. The cells that express the mutant protein will be analyzed for their ability to assemble a cartilaginous extracellular matrix in a long-term suspension culture. Moreover, the interaction of single mutant Collagen a-chains that resemble intracellular, non-folded Collagen, with other macromolecules will be studied. The Specific Aims are: (1) To determine how extracellular signals from mutant fibrillar collagens deposited in extracellular matrix influence the behavior and spatial organization of cells. (2) To analyze the density and distribution of Collagen fibrils assembled from mutant procollagen secreted and processed by cells. (3) To study intra- and extracellular interactions of mutant fibrillar collaqen with other molecules.