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

蛋白质P68/BIGH 3（p68）分泌到角膜细胞的细胞外空间。该蛋白具有两个碱性氨基酸簇，Arg-Gly-Asp序列和成束蛋白1样重复序列。这些特征中的每一个都存在于在细胞和轴突粘附和迁移中发挥作用的蛋白质中。p68的这些结构特征导致了p68在角膜细胞粘附和迁移中起重要作用的假设。蛋白质分级实验表明p68与VI型胶原蛋白和微纤维蛋白相互作用，表明它可以组织细胞外基质（ECM）分子，这是健康透明角膜的基本特性。有趣的是，具有五种p68常染色体显性突变中的任何一种的患者发展角膜营养不良，导致透明度降低和失明。突变导致氨基酸变化R124 C、R124 H、P501 T、R555 Q和R555 W。这些突变导致氨基酸变化R124 C、R124 H、P501 T、营养不良晶格I型、组合颗粒晶格、晶格IIIA型。Reis-Bucklers和Groenouw I型。这些营养不良的部分特征在于角膜内含有p68的沉积物。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在视觉中的功能或关于p68促进角膜沉积形成的机制的信息有限（如果有的话）。该项目的目的是测试p68的病理生理学特性。第一个具体目标是测试重组p68底物，以确定它是否可以在伤口愈合，支持角膜细胞粘附和迁移以及神经突延伸中发挥作用。将通过定量细胞粘附记录活性。第二个目标是引入神经突延伸。将通过定量细胞粘附记录活性。第二个具体目标是将点突变引入碱性残基簇和Ar-Gly-Asp位点，并删除fasiclin 1样重复序列。将测试这些突变体的粘附特性改变。第三个目标是将每种角膜营养不良相关突变引入p68 cDNA，产生五种不同突变体中的每一种。这些突变体同样将在细胞粘附实验中进行测试。此外，将角膜细胞与含有表位标记的野生型p68的质粒共转染，并将检测含有5种突变型p68蛋白之一和不同突变型p68的质粒，以进行免疫检测和放射免疫测定。最后，将测试角膜营养不良相关突变p68蛋白与角膜细胞和角膜ECM分子的改变的结合。这些实验的结果应该提供新的信息，这将导致更好地了解p68和新疾病的病理生理功能，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.