Biosynthetic pathway of corneal keratan sulfate

性状（申请人提供）：硫酸角质素蛋白聚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。黄斑角膜营养不良（MCD）是一种遗传性眼病，患者角膜基质呈斑点状混浊。由于角膜混浊导致失明，患者最终需要接受角膜移植术。通过对MCD的糖磺基转移酶基因CHST 6的研究，以及对人角膜GlcNAc 6-O磺基转移酶（human corneal GlcNAc 6-O sulfotransferase，hCGn 6ST）基因产物的功能分析，我们认为hCGn 6ST参与了角膜硫酸角质素糖的硫酸化。 根据这些发现，我们假设MCD表型是由角膜组织中缺乏hCGn 6ST活性的角膜细胞产生的未硫酸化硫酸角质素蛋白聚糖积累引起的。我们还假设CHST 6中存在角膜组织特异性基因调控元件，该元件上的突变导致角膜组织中CHST 6表达的破坏。为解决这些问题，我们提出以下具体目标： 1)建立硫酸角膜角质素的生物合成机制 2)鉴定CHST 6中角膜组织特异性基因调控元件 3)建立人MCD的基因敲除小鼠模型 这些结果将为我们提供硫酸角质素碳水化合物参与维持角膜透明度的信息。从该项目中获得的数据有望通过组织工程、药物和基因治疗为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.