FUNCTIONAL PROPERTIES OF HUMAN P68 (BIG H3) PROTEIN IN CORNEA

P68/BIGH3(P68)蛋白分泌到角膜细胞外间隙。该蛋白质有两个碱性氨基酸簇，一个是Arg-Gly-Asp序列，另一个是Fasciclin 1-like重复序列。这些特征中的每一个都存在于细胞和轴突黏附和迁移的蛋白质中。P68的这些结构特征导致了p68在角膜细胞黏附和迁移中发挥重要作用的假说。蛋白质分级实验表明，p68与VI型胶原和微纤维蛋白相互作用，表明它可能形成细胞外基质(ECM)分子，这是健康、透明的角膜的基本特性。有趣的是，五个常染色体显性突变中的任何一个的患者都会发展成角膜营养不良，导致透明度降低和失明。突变导致R124C、R124H、P501T、R555Q和R555W的氨基酸发生变化。这些突变导致R124C、R124H、P501T、晶格营养不良、晶格I型、颗粒-晶格结合、晶格III型氨基酸改变。Reis-Buckler和Groenouw Type I。这些营养不良的部分特征是角膜中含有p68的沉积。这导致了第二种假设，即p68是角膜沉积形成的原因，导致部分或完全失明。目前，关于正常p68在视觉中的功能，或者关于p68促进角膜沉积形成的机制的信息是有限的。本项目的目的是检测P68的生理病理特性。第一个特定目的是测试重组p68底物，以确定它是否在伤口愈合、支持角膜细胞黏附和迁移以及轴突延伸方面发挥作用。活性将通过量化细胞黏附来记录。第二个目的是引入轴突延伸。活性将通过量化细胞黏附来记录。第二个具体目的是将点突变引入碱性残基簇和Ar-Gly-Asp位点，并删除Fasiclin 1-like重复序列。这些突变体将接受粘附性改变的测试。第三个目标是将每一个与角膜营养不良相关的突变引入p68cdna，产生五个不同的突变。这些突变体同样将在细胞黏附实验中进行测试。此外，角质形成细胞将与含有表位标记的野生型p68的质粒共转染，而含有五种突变型p68蛋白之一和另一种突变型p68的质粒将进行免疫检测和放射免疫分析。最后，将测试与角膜营养不良相关的突变p68蛋白与角膜细胞和角膜ECM分子的结合变化。这些实验的结果将提供新的信息，将导致更好地了解p68的生理病理功能和新的疾病，以帮助治疗这些角膜营养不良的患者。

The protein P68/BIGH3 (p68) is secreted to the extracellular space of corneal cells. This protein has two clusters of basic amino acids, an Arg- Gly-Asp sequence and fasciclin 1-like repeats. Each of these features are found in proteins that function in cell and axon adhesion and migration. These structural traits in p68 have led to the hypothesis that p68 plays important roles in corneal cell adhesion and migration. Protein fractionation experiments suggest p68 interacts with type VI collagen and microfibril proteins, suggesting it may organize extracellular matrix (ECM) molecules, a fundamental property of healthy, transparent cornea. Interestingly, patients with any one of five p68 autosomal dominant mutations develop corneal dystrophies resulting in reduced transparency and blindness. The mutations result in the amino acid changes R124C, R124H, P501T, R555Q and R555W. These mutations result in the amino acid changes R124C, R124H, P501T, dystrophies Lattice type I, Combined granular-lattice, Lattice type IIIA. Reis-Bucklers and Groenouw type I, respectively. These dystrophies are partly characterized by p68-containing depositions within the cornea. This has led to a second hypothesis that p68 is a causative in the formation of corneal depositions, leading to partial or complete blindness. Currently, limited, if any, information is available about the function of normal p68 in vision, or about the mechanism by which p68 contributes to the formation of corneal depositions. The objective of this project is to test for physiopathologic properties of p68. The first specific aim is to test recombinant p68 substrate to determine whether it may function in wound healing, supporting corneal cell adhesion and migration and neurite extension. Activity will be documented by quantifying cell adhesion. The second aim is to introduce neurite extension. Activity will be documented by quantifying cell adhesion. The second specific aim is to introduce point mutations into basic residue clusters and into the Ar-Gly-Asp site and to delete fasiclin 1-like repeats. These mutants will be tested for altered adhesion properties. A third aim is to introduce each of the corneal dystrophy-related mutations into p68 cDNA, generating each of the five different mutants. These mutants likewise will be tested in cell adhesion experiments. Also, keratocytes will be co-transfected with a plasmid containing epitope-tagged wild type p68 and a plasmid that contains one of the five mutant p68 proteins with a different mutant p68 will be tested for immunodetection and radioimmunoassays. Finally, corneal dystrophy- related mutant p68 proteins will be tested for altered binding with corneal cells and corneal ECM molecules. The results of these experiments should provide new information that will lead to a better understanding of the physiopathologic function of p68 and new disease to help treat patients with these corneal dystrophies.