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M.M. Mann, M.L. Funderburgh, J.L. Funderburgh; Identifying the Enzymes and Genes Involved in Keratan Sulfate Biosynthesis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1590.
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© ARVO (1962-2015); The Authors (2016-present)
Keratan sulfate (KS) is a unique glycosaminoglycan of the corneal stroma important for corneal transparency. Biosynthesis of corneal KS involves multiple enzymes. During polymerization, galactose and N–acetylglucosamine are sequentially added by glycosyltransferases. Both sugars are subsequently sulfated. Defects in a gene for glucosamine–6–sulfotransferase (Gn6ST) cause human macular corneal dystrophy due to under–sulfation of corneal KS, but genes for other enzymes required for KS biosynthesis are still unknown. The purpose of this study was to identify these enzymes by reducing expression of genes for candidate enzymes using siRNA.
Primary bovine keratocytes secrete high levels of KS in substratum–free culture. These cells were transfected using Lipofectamine 2000 with synthetic siRNA for beta–1,3–N–acetylglucosaminyltransferase (iGnT), beta–1,4–galactosyltransferase–1 (B4GalT1), KS galactose 6–O–sulfotransferase (KSGal6ST), and N–acetylglucosamine 6–O–sulfotransferase–6 (Gn6ST). KS synthesis was determined 4–5 days later by western blotting, fluorophore–assisted carbohydrate electrophoresis (FACE), and by labeling with radioactive sulfate.
Western blotting with J19, a monoclonal antibody against sulfated KS, showed that siRNA against each of the four target genes markedly reduced KS biosynthesis in bovine keratocytes. FACE analysis confirmed reduction in KS–derived disaccharides in response to siRNA against sulfotransferases. Analysis of KS labeled with radioactive sulfate showed only moderate reduction in the size and amount of KS in the presence of the siRNAs.
Western blotting studies, the most sensitive assay for KS, implicated all four targeted enzymes to be involved in corneal KS biosynthesis. These data represent the first link of specific genes to KS biosynthesis other than Gn6ST. FACE and radioactive studies supported involvement of the two targeted sulfotransferases, but failed to show significant alteration in KS amount or size by targeting elongation genes. Studies are underway to determine if siRNA achieves full knockdown of these glycosyltransferases or if other elongation enzymes may also be involved.
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