Human corneal GlcNAc 6-O-sulfotransferase and mouse intestinal GlcNAc 6-O-sulfotransferase both produce keratan sulfate
Human corneal GlcNAc 6-O-sulfotransferase and mouse intestinal GlcNAc 6-O-sulfotransferase both produce keratan sulfate
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DOI：
10.1074/jbc.m009995200
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发表时间：
2001-05-11
影响因子：
4.8
通讯作者：
Fukuda, MN
中科院分区：
文献类型：
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作者：
Akama, TO;Nakayama, J;Fukuda, MN
人角膜N-乙酰葡糖胺6-O-磺基转移酶（hCGn 6ST）已通过位置候选方法被鉴定为负责黄斑角膜营养不良（MCD）的基因。由于其与碳水化合物磺基转移酶具有高度同源性，并且在角膜和血清中缺乏硫酸化硫酸角质素的MCD患者中存在该基因的突变，因此认为hCGn 6ST蛋白是一种磺基转移酶，可催化硫酸角质素中GlcNAc的硫酸化。在这份报告中，我们通过在培养细胞中表达hCGn 6ST来分析其酶活性。从用完整形式的hCGn 6ST cDNA转染的HeLa细胞制备的裂解物或从用分泌形式的hCGn 6ST cDNA转染的细胞制备的培养基在体外显示出将硫酸根转移到合成寡糖底物的GlcNAc的C-6的活性。当hCGn 6ST与人硫酸角质素Gal-6-磺基转移酶（hKSG 6ST）一起表达时，HeLa细胞产生通过抗硫酸角质素抗体5D4检测到的高度硫酸化的碳水化合物。这些结果表明hCGn 6ST将硫酸根转移至硫酸角质素中GlcNAc的C-6。与MCD患者中发现的错义突变导致的变化相同的hCGn 6ST中的氨基酸取代废除了酶活性。此外，小鼠肠道GlcNAc 6-O-磺基转移酶具有与hCGn 6ST相同的活性。这一观察结果表明，小鼠肠道GlcNAc 6-O-磺基转移酶是hCGn 6ST的直向同源物，并作为磺基转移酶在角膜中产生硫酸角质素。
Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been identified by the positional candidate approach as the gene responsible for macular corneal dystrophy (MCD). Because of its high homology to carbohydrate sulfotransferases and the presence of mutations of this gene in MCD patients who lack sulfated keratan sulfate in the cornea and serum, hCGn6ST protein is thought to be a sulfotransferase that catalyzes sulfation of GlcNAc in keratan sulfate. In this report, we analyzed the enzymatic activity of hCGn6ST by expressing it in cultured cells. A lysate prepared from HeLa cells transfected with an intact form of hCGn6ST cDNA or culture medium from cells transfected with a secreted form of hCGn6ST cDNA showed an activity of transferring sulfate to C-6 of GlcNAc of synthetic oligosaccharide substrates in vitro. When hCGn6ST was expressed together with human keratan sulfate Gal-6-sulfotransferase (hKSG6ST), HeLa cells produced highly sulfated carbohydrate detected by an anti-keratan sulfate antibody 5D4. These results indicate that hCGn6ST transfers sulfate to C-6 of GlcNAc in keratan sulfate. Amino acid substitutions in hCGn6ST identical to changes resulting from missense mutations found in MCD patients abolished enzymatic activity. Moreover, mouse intestinal GlcNAc 6-O-sulfotransferase had the same activity as hCGn6ST. This observation suggests that mouse intestinal GlcNAc 6-O-sulfotransferase is the orthologue of hCGn6ST and functions as a sulfotransferase to produce keratan sulfate in the cornea.