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

Many tissues contain glycoproteins and proteoglycans, which are substituted with N-or O-linked keratan sulfate, a glycosaminoglycan in which the lacto samine (-gal beta1,4glcNAc-) disaccharide backbone is variably modified by sulfation, fucosylation, and sialylation. We report here a rapid, sensitive , and quantitative procedure for obtaining a complete disaccharide composit ional analyses for keratan sulfates after FACE separation of products gener ated by hydrolysis of the glycosaminoglycans with B. fragillis keratanase I I and E. freundii endo-p-galactosidase. Seven digestion end products are se parable in a single electrophoretic step using Monosaccharide(TM) compositi on gels. These are: the unsulfated disaccharide, glcNAc beta1,3gal, the fuc osylated trisaccharide, gal beta1,2[fuc alpha1,3]glcNAc6S, the mono- and di sulfated disaccharides, gal beta1,4glcNAc6S or gal6S beta1,4glcNAc6S from t he chain interior, and the sialylated mono- and disulfated trisaccharides n euA alpha2,3gal beta1, 4glcNAc6S or neuA alpha2,3gal-6S beta1,4glcNAc6S fro m the nonreducing terminus. FACE analyses also revealed the presence of a c ontaminant P-galactosidase activity in keratanase II enzyme preparations wh ich cleaves the disaccharide, gal beta1,4glcNAc6S to its constituent monosa ccharides, gal and glcNAc6S. It was particularly prominent at enzyme concen trations > 2 mU per nmole substrate glcNH(2) or after prolonged digestion t imes (>12 h), and was not inhibitable by thiogalactosides or N-acetyl-lacto samine. As these monosaccharide products would not be detectable using the commonly described analytical methods for KS hydrolase products, such as H- 1-NMR and RPLC analyses, our data illustrate that the FACE procedure repres ents an improved approach for accurate compositional microanalyses of corne al and skeletal keratan sulfates, especially applicable to experimentation involving small amounts (1-2 mug) of this glycosaminoglyean.