Expression of Specialized Collagens in the Cornea

描述（由申请人提供）：在角膜中，细胞外基质（ECM） 分子提供了结构支持和将信号从 环境到细胞表面。角膜使用ECM分子，特别是 胶原蛋白，以实现和保持透明度。我们的总体目标是 了解角膜如何调节合成，组装和分化 胶原蛋白在发育过程中的表达。调控涉及转录 特定的胶原蛋白链。几种胶原蛋白正在合成 同时，选择正确的阿尔法链组装成 必须调节三聚体分子。然后胶原蛋白被分泌和组装 在细胞外环境中，并且必须定位在它们的附近。 互动伙伴我们将研究角膜如何调节一些 这些事件。在胚胎发育的第5天， 具有中断的三螺旋的胶原蛋白（FACIT），XII型和XIV型，都是 由角膜上皮合成。尽管它们相似，但它们 只组装成同源三聚体，而从不组装成异源三聚体。我们的首要目标是 鉴定非胶原羧基末端（NC1结构域）内的序列 调节三聚体α链选择的每种FACIT胶原蛋白。我们将 胶原蛋白XIV NC1结构域的相应区域取代 胶原蛋白XII。然后我们将确定角膜上皮细胞 将胶原蛋白XIV（含有小的胶原蛋白XII区域）组装成 具有正常XII型α链的异源三聚体。第二个目标将调查 XIV型胶原蛋白，它的羧基末端位于纤维上，是否可能 通过角质素将其氨基末端与相邻原纤维间接结合 硫酸化蛋白聚糖。我们将胶原蛋白XIV的氨基末端结构域， 柱，然后将硫酸角膜角质素蛋白聚糖（KSPG）的混合物应用于 允许绑定。然后将KSPG洗脱并用特异性抗体进行鉴定。 免疫印迹法检测抗体。 角膜的结构必须绝对精确才能透明。我们 假设合成和放置三个新发现的 胶原蛋白对于角膜的发育和功能是必需的，因此， 我们的第三个目的是检测它们在角膜移植中的表达模式， 发展我们克隆的一种是XX型胶原蛋白，它是胶原蛋白的一个新成员。 FACIT。另一种是XXI型胶原蛋白，一种新的纤维状胶原蛋白， 当时人们认为所有的纤维状胶原蛋白都是已知的。的 第三种是XXIII型胶原，一种假定的跨膜分子。因为 鸡角膜是发育研究的理想模型， 与人类角膜的关系，我们将尝试将特定的发育事件 这些分子的可能功能，基于表达模式。这 将通过使用特异性抗体的免疫荧光和原位 用cDNA探针杂交。我们还将进行细胞分离， 证明胶原蛋白XXIII是一种跨膜分子。

DESCRIPTION (provided by applicant): In the cornea, extracellular matrix (ECM) molecules provide structural support and the ability to transfer signal from the environment to cell surfaces. The cornea uses ECM molecules, in particular collagens, to achieve and maintain transparency. Our overall objective is to understand how the cornea regulates the synthesis, assembly, and differential expression of collagens during development. Regulation involves transcription of specific collagen type chains. Several collagens are being synthesized simultaneously, so the selection of correct alpha chains to assemble into trimeric molecules must be regulated. Collagens are then secreted and assembled within the extracellular environment and must be positioned near their interacting partners. We will investigate how the cornea regulates some of these events. At 5 days of embryonic development, the fibril associated collagens with interrupted triple helices (FACITs), types XII and XIV, are both synthesized by the corneal epithelium. Despite their similarity, they are assembled only as homotrimers, and never as heterotrimers. Our first aim is to identify the sequence within the non-collagenous carboxyl termini (NC1 domains) of each FACIT collagen that regulates trimer alpha chain selection. We will substitute regions of the collagen XIV NC1 domain with the corresponding region of collagen XII. We will then determine whether corneal epithelial cells assemble collagen XIV (containing a small collagen XII region) into heterotrimers with normal type XII alpha chains. A second aim will investigate whether type XIV collagen, which sits on fibrils with its carboxyl end, might indirectly bind its amino terminal end to an adjacent fibril via a keratan sulfate proteoglycan. We will immobilize collagen XIV amino terminal domains on columns, then apply a mix of corneal keratan sulfate proteoglycans (KSPGs) to allow binding. The KSPGs will then be eluted and identified with specific antibodies by Western blots. The architecture of the cornea must be absolutely precise to be transparent. We hypothesize that the synthesis and placement of three newly discovered collagens are essential for development and function of the cornea, therefore our third aim is to examine their expression pattern during corneal development. One which we have cloned is type XX collagen, a new member of the FACITs. Another is type XXI collagen, a new fibrillar collagen, isolated at a time when it was believed that all the fibrillar collagens were known. The third is type XXIII collagen, a putative transmembrane molecule. Because the chick cornea is an ideal model for developmental studies that are not possible with the human cornea, we will try to correlate specific developmental events with possible functions of these molecules, based on expression pattern. This will be accomplished by immunofluorescence with specific antibodies and in situ hybridization with cDNA probes. We will also perform cell fractionations to prove collagen XXIII is a transmembrane molecule.