Biological function of keratan sulfate glycosaminoglycan for corneal extracellula

硫酸角质素蛋白聚糖（Keratan sulfate，KS）是角膜的主要成分之一，对角膜的发育和维持组织的透明性具有重要作用。KS-PG由两种不同的组分组成，即核心蛋白和称为糖胺聚糖（GAG）的附着碳水化合物。据推测，KS-PG核心蛋白与胶原原纤维结合并调节原纤维的直径，而KS-GAG调节原纤维间的间距。通过使用包括基因敲除小鼠在内的几种方法，已经很好地研究了KS-PG核心蛋白（如Lumican和keratocan）在角膜发育中的重要性，然而，由于尚未建立碳水化合物的生物合成途径，因此尚未广泛研究KS-GAG链的生物学功能。 KS-GAG是由3Gal-1-4GlcNAc-1-（Gal，半乳糖; GlcNAc，N乙酰葡糖胺）的重复二糖单元组成的线性延长的糖链，在Gal和GlcNAc的6-0位上具有硫酸酯。我们研究了角膜KS碳水化合物的生物合成，并鉴定了4种高尔基体化酶，~1，3-N-乙酰葡糖胺基转移酶7，~1，4-半乳糖基转移酶4，KS半乳糖6-0磺基转移酶和角膜N-乙酰葡糖胺6-0磺基转移酶，在体外和体内都是KS-GAG产生所必需的。我们还发现KS碳水化合物硫酸化的缺乏改变了小鼠角膜中的角膜细胞外基质（ECM）结构，并证明了KS-GAG链在角膜中的功能重要性。 基于这些发现，我们推测KS碳水化合物的分子结构，如长度和硫酸化程度，影响角膜ECM结构的形成。换句话说，我们可以通过操纵KS碳水化合物结构来重塑角膜ECM结构，这可以通过抑制和/或诱导角膜细胞中KS合成所需的酶活性来控制。为了验证这个假设，我们将致力于两个目标。

Keratan sulfate (KS) proteoglycan (PG) is one of the major components of the cornea, and suggested to have important roles for corneal development and maintenance of transparency of the tissue. KS-PG is consists of two different components, core proteins and attached carbohydrates called glycosaminoglycans (GAGs). It has been postulated that KS-PG core proteins bind to collagen fibrils and regulate diameter of the fibrils, and KS-GAGs regulate interfibrillar spacing. By using several approaches including gene-knockout mice, importance of KS-PG core proteins, such as lumican and keratocan, over corneal development has been well investigated, however, biological function of KS-GAG chains has not been extensively studied, since biosynthetic pathway of the carbohydrate has not been established. KS-GAG is a linearly elongated carbohydrate chain that consists of repeating disaccharide units of 3Gal~1-4GlcNAc~1- (Gal, galactose; GlcNAc, Nacetylglucosamine), with sulfate on 6-0 position of Gal and GlcNAc. We have studied biosynthesis of corneal KS carbohydrate, and identified that 4 Golgi-Ioca/ized enzymes, ~1 ,3-N-acetylglucosaminyltransferase 7, ~1 ,4-galactosyltransferase 4, KS galactose 6-0 sulfotransferase and corneal N-acetylglucosamine 6-0 sulfotransferase, are required for KS-GAG production in vitro as well as in vivo. We also found that lack of sulfation of KS carbohydrate alters corneal extracellular matrix (ECM) structure in mouse cornea, and demonstrated functional importance of KS-GAG chain in the cornea. Based on these findings, we hypothesized that molecular structure, such as length and sulfation degree, of KS carbohydrate affects to the formation of organized corneal ECM structure. In other words, we may be able to remodel the corneal ECM structure by manipulating KS carbohydrate structure, which can be controlled by suppression and/or induction of enzymatic activity required for KS synthesis in corneal cells. To test this hypothesis, we will work on the two aims.