Enzymatic Activity of Human Corneal GlcNAc 6-O Sulfotransferase (hCGn6ST) In Vitro and In Vivo; a Key Enzyme for Sulfation of Keratan Sulfate

Abstract

Abstract: : Purpose:CHST6, which encodes corneal GlcNAc 6-O sulfotransferase (CGn6ST), is the causative gene for a human hereditary disease, macular corneal dystrophy. To understand the involvement of the gene product for keratan sulfate biosynthesis, we analyzed the enzymatic activity of CGn6ST and other homologous sulfotransferases in vitro and in vivo. Methods:Soluble form sulfotransferase was prepared from transfected HeLa cells which produce a catalytic domain of the enzyme into culture medium. The culture medium was concentrated and used as an enzyme source. Chemically synthesized carbohydrate substrates were mixed with the enzyme and a sulfate donor, [₃₅S] phosphoadenosine phosphosulfate and the incorporation of ₃₅S to the substrates was measured by scintillation counting. Production of highly sulfated keratan sulfate in transfected HeLa cells was also analyzed using monoclonal antibody, 5D4. Results:Human CGn6ST (hCGn6ST), human intestinal GlcNAc 6-O sulfotransferase (hIGn6ST) are mouse intestinal GlcNAc 6-O sulfotransferase (mIGn6ST) were analyzed their sulfotransferase activities. When hCGn6ST is expressed with human keratan sulfate Gal-6 sulfotransferase (hKSG6ST) in HeLa cells, the cells produced highly sulfated keratan sulfate. The same result was observed on mIGn6ST but not on hIGn6ST. By in vitro sulfotransferase analysis, all three sulfotransferases were observed their enzymatic activity to short carbohydrate substrates which have GlcNAc on their non-reducing terminus. hCGn6ST and mIGn6ST also had sulfotransferase activity over long carbohydrate substrates, however, hIGn6ST had no activity to the substrates. A carbohydrate substrate that has sulfate on internal Gal was not suitable substrate for hCGn6ST enzyme. Conclusion:By in vitro and in vivo analysis, hCGn6ST and mIGn6ST, but not hIGn6ST, are indicated to be involved in the production of keratan sulfate. Substrate specificity of hCGn6ST suggested that sulfation step of GlcNAc in keratan sulfate biosynthesis is cooperated with the elongation step of the carbohydrate chain by GlcNAc- and Gal-transferases, and the sulfation of Gal residue occurs after the GlcNAc sulfation in the pathway.

Keywords: 529 proteoglycans/glycosaminoglycans • 403 extracellular matrix • 370 cornea: basic science