Enzymes responsible for synthesis of corneal keratan sulfate glycosaminoglycans
Enzymes responsible for synthesis of corneal keratan sulfate glycosaminoglycans
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DOI：
10.1074/jbc.m703695200
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发表时间：
2007-10-12
影响因子：
4.8
通讯作者：
Akama, Tomoya O.
中科院分区：
文献类型：
--
作者：
Kitayama, Kazuko;Hayashida, Yasutaka;Akama, Tomoya O.
硫酸角蛋白糖胺聚糖是角膜中最丰富的碳水化合物成分之一，在维持角膜细胞外基质结构方面发挥着重要作用。角蛋白硫酸盐碳水化合物链由重复的N-乙酰乳糖胺二糖组成，在N-乙酰氨基葡萄糖和半乳糖的6-O位上进行硫酸盐化。尽管它对角膜功能很重要，但人们对碳水化合物链的生物合成途径，特别是延长步骤知之甚少。本文分析了两种糖基转移酶--β-1，3-N-乙酰氨基葡萄糖基转移酶-7(β3GnT7)和β-1，4-半乳糖基转移酶-4(β4GalT4)在体外生产硫酸角蛋白碳水化合物中的酶活性。当这些糖基转移酶在没有碳水化合物磺基转移酶的情况下与底物反应时，它们只产生短而细长的碳水化合物；然而，在角膜N-乙酰氨基葡萄糖6-O磺基转移酶(CGn6ST)的存在下，它们产生扩展的GlcNAc-硫酸多-N-乙酰乳糖胺结构，具有超过四个重复的GlcNAc-硫酸N-乙酰乳糖胺单位。此外，我们在体外用β3GnT7/β4GalT4/CGn6ST的混合物和硫酸角蛋白半乳糖6-O磺基转移酶处理的两步反应检测到了高硫酸盐化角蛋白硫酸盐的产生。我们还观察到，当β3GnT7或β4GalT4的表达被小干扰RNA抑制时，培养的人角膜上皮细胞中高硫酸盐化角蛋白硫酸盐的产生显著减少，这表明这些糖基转移酶是导致角蛋白硫酸酯碳水化合物骨架延长的原因。
Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, beta 1,3-N-acetylglucosaminyltansferase-7 (beta 3GnT7) and beta 1,4-galactosyltransferase-4 (beta 4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of beta 3GnT7/beta 4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of beta 3GnT7 or beta 4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.