Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans

• Published in: The Journal of biological chemistry 2007-10, Vol.282 (41), p.30085-30096
• Main Authors: Kitayama, Kazuko, Hayashida, Yasutaka, Nishida, Kohji, Akama, Tomoya O.
• Format: Article
• Language: English
• Subjects: Amino Sugars - chemistry; Carbohydrate Sulfotransferases; Chromatography, Gel; Chromatography, High Pressure Liquid - methods; Cornea - metabolism; Disaccharides - chemistry; Epithelial Cells - metabolism; Genetic Vectors; Glycosaminoglycans - chemical synthesis; Glycosaminoglycans - chemistry; Hexosaminidase A - chemistry; Humans; Keratan Sulfate - chemistry; N-Acetylglucosaminyltransferases - metabolism; N-Acetyllactosamine Synthase - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering - metabolism; Sulfotransferases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M703695200

Keratan sulfate 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. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, β1,3-N-acetylglucosaminyltansferase-7 (β3GnT7) and β1,4-galactosyltransferase-4 (β4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of β3GnT7/β4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of β3GnT7 or β4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.