Human corneal GlcNAc 6-O-sulfotransferase and mouse intestinal GlcNAc 6-O-sulfotransferase both produce keratan sulfate

Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been i dentified by the positional candidate approach as the gene responsible for macular corneal dystrophy (MCD). Because of its high homology to carbohydra te sulfotransferases and the presence of mutations of this gene in MCD pati ents who lack sulfated keratan sulfate in the cornea and serum, hCGn6ST pro tein 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 cel ls transfected with an intact form of hCGn6ST cDNA or culture medium from c ells transfected with a secreted form of hCGn6ST cDNA showed an activity of transferring sulfate to C-6 of GlcNAc of synthetic oligosaccharide substra tes in vitro. When hCGn6ST was expressed together with human keratan sulfat e Gal-6-sulfotransferase (hKSG6ST), HeLa cells produced highly sulfated car bohydrate detected by an anti-keratan sulfate antibody 5D4. These results i ndicate that hCGn6ST transfers sulfate to C-6 of GlcNAc in keratan sulfate. Amino acid substitutions in hCGn6ST identical to changes resulting from mi ssense mutations found in MCD patients abolished enzymatic activity. Moreov er, mouse intestinal GlcNAc 6-O-sulfotransferase had the same activity as h CGn6ST. This observation suggests that mouse intestinal GlcNAc 6-O-sulfotra nsferase is the orthologue of hCGn6ST and functions as a sulfotransferase t o produce keratan sulfate in the cornea.