Biosynthetic pathway of corneal keratan sulfate

描述(申请人提供)：硫酸角蛋白多糖是角膜基质的主要成分之一，约占角膜总厚度的90%，对维持角膜透明度很重要。黄斑性角膜营养不良(MCD)是一种遗传性眼病，患者的角膜基质呈斑点状混浊。由于角膜混浊致盲，患者最终需要接受角膜移植。通过对与MCD相关的糖基转移酶基因CHST6的研究，以及对该基因产物hCGn6ST的功能分析，我们得出结论：hCGn6ST参与了角膜中硫酸角蛋白碳水化合物的硫酸盐化。 根据这些发现，我们推测MCD的表型是由于角膜组织中缺乏hCGn6ST活性的角膜细胞产生的未硫化的硫酸角蛋白多糖所致。我们还假设CHST6中存在一个角膜组织特异性基因调控元件(S)，该元件上的突变导致了CHST6在角膜组织中的表达中断。为了解决这些问题，我们提出了以下具体目标； 1)建立角膜硫酸角蛋白的生物合成机制 2)鉴定CHST6中的角膜组织特异性基因调控元件 3)建立人类MCD的动物模型--基因敲除小鼠 这些结果将为我们提供有关硫酸角蛋白碳水化合物参与维持角膜透明度的信息。本项目获得的数据有望通过组织工程、药物治疗和基因治疗为MCD和其他角膜疾病的进一步临床应用奠定基础。

DESCRIPTION (provided by applicant): Keratan sulfate proteoglycan is one of the major component of the corneal stroma, which composes about 90% of the total corneal thickness, and is important to maintain corneal transparency. Macular corneal dystrophy (MCD) is a hereditary eye disease, of which the patients show spotted opacity in their corneal stroma. The patients eventually need to be subjected to keratoplasty because of blindness by cornea clouding. Based on the studies of a carbohydrate sulfotransferase gene, CHST6, which is responsible for MCD, and the functional analysis of the gene product named human corneal GlcNAc 6-O sulfotransferase (hCGn6ST), we concluded that hCGn6ST is involved in the sulfation of keratan sulfate carbohydrate in the cornea. From these findings, we hypothesize that MCD phenotype is caused by the accumulation of unsulfated keratan sulfate proteoglycan, which is produced by the corneal cells lacking hCGn6ST activity, in the corneal tissue. We also hypothesize the presence of a corneal tissue-specific gene regulatory element(s) in CHST6 and mutations on the element result in the disruption of CHST6 expression in the corneal tissue. To address these issues, we propose the following specific aims; 1) To establish biosynthetic mechanism of corneal keratan sulfate 2) To identify the corneal tissue-specific gene regulatory element in CHST6 3) To produce the gene knockout mouse that is an animal model of MCD in human These results will provide us with information upon the involvement of keratan sulfate carbohydrate in the maintenance of the corneal transparency. Data obtained from this project are expected to form the basis for further clinical applications for MCD and the other corneal diseases through tissue engineering, medication and gene therapy.