BIGH3 WILD TYPE AND MUTANT PROTEINS

一些角膜营养不良[颗粒性角膜营养不良(GCD)、晶格型角膜营养不良(LCD)I型和IIIA型、混合型UCD-LCD I型、Thiel-Behnke营养不良和Reis-Buckler营养不良]都是由BIGH3基因突变引起的。这些情况的特点是角膜内沉积了与β-ig-h3抗体(BIGH3基因的蛋白产物)反应的蛋白质。对于每一种营养不良，积聚都有其特有的超微结构。本研究的目的是从不同BIGH3角膜营养不良患者的血浆以及COS-7、CHO-K1和杆状病毒感染的昆虫细胞的细胞培养中纯化和鉴定BIGH3角膜营养不良的β-ig-h3。我们将对野生型和突变型β-ig-h3蛋白进行比较，以检验这样一种假设，即BIGH3角膜营养不良患者中积累在角膜内的蛋白质的结构与突变蛋白有关，不同表型与BIGH3基因类型以及表达的突变蛋白数量之间存在关系。在某些BIGH3角膜营养不良中积聚的淀粉样蛋白的性质和产生的原因将被调查。我们将确定BIGH3相关的淀粉样蛋白是否是突变的β-ig-h3的特定片段。由于β-ig-h3与细胞外基质成分相互作用，我们将尝试确定可能导致BIGH3角膜营养不良的积聚的蛋白质-蛋白质相互作用。将在兔角膜上皮细胞中表征β-ig-h3的生物合成。

Several corneal dystrophies [granular corneal dystrophy (GCD), lattice corneal dystrophy (LCD) types I and IIIA, combined UCD-LCD type I, Thiel-Behnke dystrophy and Reis-Bucklers dystrophy] have been found to result from mutations in BIGH3. These conditions are characterized by the deposition within the cornea of proteins that react with antibodies to beta-ig-h3 (the protein product of the BIGH3 gene). For each dystrophy the accumulations have a characteristic ultrastructure. This proposal is to purify and characterize beta-ig-h3 from plasma of persons with and without different BIGH3 corneal dystrophies and from cell cultures of COS- 7, CHO-K1 and baculovirus infected insect cells transfected with vectors containing the mutations that produce BIGH3 corneal dystrophies. Wild-type and mutant beta-ig-h3 proteins will be compared to test the hypothesis that the structure of the proteins that accumulate within the cornea in the BIGH3 corneal dystrophies are related to the mutated protein and that a relationship exists between different phenotypes and the BIGH3 genotypes and between the amount of the mutant protein that is expressed. The nature of the amyloid that accumulates in certain BIGH3 corneal dystrophies and the reason for its production will be investigated. We will determine whether BIGH3 related amyloid is a specific fragment of mutant beta-ig-h3. Because beta-ig-h3 interacts with components of the extracellular matrix we will attempt to identify protein- protein interactions that may account for the accumulations that characterize the BIGH3 corneal dystrophies. The biosynthesis of beta-ig-h3 will be characterized in rabbit corneal epithelial cells.