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T. O. Akama, K. Kitayama, Y. Hayashida, K. Nishida; Responsible Enzymes for Elongation of Corneal Keratan Sulfate Carbohydrate Backbone. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4295.
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Keratan sulfate (KS) 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. KS carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine (GlcNAc) and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. To establish corneal KS biosynthesis, we analyzed enzymatic activity of two glycosyltransferases, β1,3-N-acetylglucosaminyltansferase-7 (β3GnT7) and β1,4-galactosyltransferase-4 (β4GalT4), in production of KS carbohydrate in vitro.
We prepared soluble form recombinant enzymes of β3GnT7, β4GalT4, corneal GlcNAc 6-O sulfotransferase (CGn6ST) and KS galactose 6-O sulfotransferase (KSG6ST) and analyzed sulfated KS production in vitro. We also investigated suppression effect of the glycosyltransferase mRNAs over sulfated KS production in human corneal cells by introducing specific siRNAs into the cultured cells.
Mixture of two glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than 4 repeats of GlcNAc-sulfated N-acetyllactosamine unit in presence of CGn6ST. Moreover, we detected production of highly sulfated KS by a two-step reaction in vitro with a mixture of β3GnT7/β4GalT4/CGn6ST followed by KSG6ST treatment. We also observed that production of highly sulfated KS in cultured human corneal cells was dramatically reduced when expression of β3GnT7 or β4GalT4 was suppressed by siRNAs.
β3GnT7 and β4GalT4 are indicating that these glycosyltransferases are responsible for elongation of the KS carbohydrate backbone and may have critical role on maintenance of corneal extracellular matrix structure.
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