Human Corneal GlcNAc 6-O-Sulfotransferase and Mouse Intestinal GlcNAc 6-O-Sulfotransferase Both Produce Keratan Sulfate

Abstract

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.

Golgi Localization of Carbohydrate Sulfotransferases Is a Determinant of L-selectin Ligand Biosynthesis

“…However, this enzyme can generate 6-sulfo-sLe x oligosaccharides in cultured cells (20). The other GlcNAc6STs are also expressed in tissues lacking sulfoadhesin, such as the intestine (GlcNAc6ST-3) and cornea (GlcNAc6ST-5) (21). Consistent with a role for GlcNAc6ST-2 in sulfoadhesin biosynthesis, mice deficient in this enzyme lack MECA-79 reactivity on the luminal aspect of peripheral lymph node HEV and display significantly reduced lymphocyte homing (22).…”

Matrix morphogenesis in cornea is mediated by the modification of keratan sulfate by GlcNAc 6- O -sulfotransferase

“…Because of their high homology, CHST5 and CHST6 seem to be created by gene duplication during evolution (10). The coding sequences of the three human and mouse genes are highly homologous, and all of the gene products have sulfotransferase activity over nonreducing terminal GlcNAc (4,40). Nevertheless, only the enzymes encoded on CHST6 and Chst5 have similar substrate specificity and the ability to produce sulfated KS Fig.…”

“…Chst5 is a murine ortholog of CHST6, the carbohydrate sulfotransferase gene that in humans is causative for macular corneal dystrophy (10). Interestingly, the human genome has an additional sulfotransferase gene, CHST5, as an ortholog for Chst5 in mouse (4,10,32). Because of their high homology, CHST5 and CHST6 seem to be created by gene duplication during evolution (10).…”

“…Chst5 encodes intestinal GlcNAc 6-O-sulfotransferase, a carbohydrate sulfotransferase that is expressed in the intestine and cornea in mouse (4,32), and which acts on transferring sulfate onto the 6-O position of the nonreducing terminal GlcNAc on KS (4). This sulfation step is coupled with the elongation of the KS backbone (33,34).…”

“…Keratan sulfate (KS) is an important constituent of several collagen-rich tissues and is the major GAG in cornea where it is N-linked to asparagine residues in one of three PG core proteins: lumican (1), keratocan (2), and mimecan͞osteoglycin (3). Human corneal GlcNAc 6-O-sulfotransferase (also known as human GlcNAc6ST-5 and GST4␤) is the responsible enzyme for the synthesis of high-sulfated KS via the transfer of sulfate onto the GlcNAc 6-O position of the KS backbone (4).…”