Enzymes responsible for synthesis of corneal keratan sulfate glycosaminoglycans.
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, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), 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 beta3GnT7/beta4GalT4/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 beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.
Kitayama
Kazuko
K
Glycobiology Program, Burnham Institute for Medical Research, La Jolla, California 92037, USA.
Hayashida
Yasutaka
Y
Nishida
Kohji
K
Akama
Tomoya O
TO
D000606:Amino Sugars / Q000737:chemistry*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003315:Cornea / Q000378:metabolism; D004187:Disaccharides / Q000737:chemistry; D004847:Epithelial Cells / Q000378:metabolism; D005822:Genetic Vectors; D006025:Glycosaminoglycans / Q000138:chemical synthesis* / Q000737:chemistry*; D054818:Hexosaminidase A / Q000737:chemistry; D006801:Humans; D007632:Keratan Sulfate / Q000737:chemistry*; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D009237:N-Acetyllactosamine Synthase / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015238:Sulfotransferases / Q000378:metabolism; D000097811:Carbohydrate Sulfotransferases
D000606:Amino Sugars; D004187:Disaccharides; D006025:Glycosaminoglycans; D054818:Hexosaminidase A; D007632:Keratan Sulfate; D017351:N-Acetylglucosaminyltransferases; D009237:N-Acetyllactosamine Synthase; D034741:RNA, Small Interfering; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases; C000458:N-acetyllactosamine; C499227:B3GNT7 protein, human