Keratan sulfate disaccharide composition determined by FACE analysis of keratanase II and endo-β-galactosidase digestion products

Abstract

Many tissues contain glycoproteins and proteoglycans, which are substituted with N-or O-linked keratan sulfate, a glycosaminoglycan in which the lactosamine (-galβ1,4glcNAc-) disaccharide backbone is variably modified by sulfation, fucosylation, and sialylation. We report here a rapid, sensitive, and quantitative procedure for obtaining a complete disaccharide compositional analyses for keratan sulfates after FACE separation of products generated by hydrolysis of the glycosaminoglycans with B. fragillis keratanase II and E. freundii endo-β-galactosidase. Seven digestion end products are separable in a single electrophoretic step using Monosaccharide™ composition gels. These are: the unsulfated disaccharide, glcNAcβ1,3gal, the fucosylated trisaccharide, galβ1,2[fucα1,3]glcNAc6S, the mono- and disulfated disaccharides, galβ1, 4glcNAc6S or gal6Sβ1, 4glcNAc6S from the chain interior, and the sialylated mono- and disulfated trisaccharides neuAα2,3galβ1, 4glcNAc6S or neuAα2,3gal6Sβ1,4glcNAc6S from the nonreducing terminus. FACE analyses also revealed the presence of a contaminant β-galactosidase activity in keratanase II enzyme preparations which cleaves the disaccharide, galβ1,4glcNAc6S to its constituent monosaccharides, gal and glcNAc6S. It was particularly prominent at enzyme concentrations > 2 mU per nmole substrate glcNH2 or after prolonged digestion times (> 12 h), and was not inhibitable by thiogalactosides or N-acetyl-lactosamine. As these monosaccharide products would not be detectable using the commonly described analytical methods for KS hydrolase products, such as 1H-NMR and HPLC analyses, our data illustrate that the FACE procedure represents an improved approach for accurate compositional microanalyses of corneal and skeletal keratan sulfates, especially applicable to experimentation involving small amounts (1-2 μg) of this glycosaminoglycan.