Isolation of reducing oligosaccharide chains from the chondroitin/dermatansulfate-protein linkage region and preparation of analytical probes by fluorescent labeling with 2-aminobenzamide

The glycosaminoglycan (GAG)-protein linkage regions of various proteoglycan s share the common tetrasaccharide GlcA-Gal-Gal-Xyl-attached to Ser residue s in the core proteins. In previous analysis we demonstrated unique modific ations by epimerization, sulfation and phosphorylation of the component sug ars. Here we developed a sensitive analytical method for the linkage region oligosaccharides to detect or monitor structural variations and changes. T his will be useful for investigation of their biological roles, which are l argely unknown, but they have been implicated in biosynthesis, A variety of linkage region-derived hexasaccharides was first prepared as reducing suga r chains from peptide chondroitin/dermatan sulfate of whale cartilage, shar k cartilage, and bovine aorta by means of chondroitinase digestion in conju nction with beta -elimination in the absence of reducing reagents, but invo lving a mild alkali, 0.5 M LiOH, at 4 degreesC to prevent peeling reactions . The structures of these oligosaccharides were determined by the combinati on of HPLC, enzymatic digestion, matrix-assisted laser desorption ionizatio n time-of-flight (MALDI-TOF) mass spectrometry, and H-1 NMR spectroscopy, w hich revealed eleven different hexasaccharides including a novel structure, Delta HexA alpha1-3GalNAc beta1-4IdoA alpha1-3Gal(4-O-sulfate)beta1-3Gal b eta1-4Xyl (Delta HexA and IdoA represent unsaturated hexuronic acid and L-i duronic acid, respectively). These oligosaccharides were labeled with a flu orophore, 2-aminobenzamide, to prepare analytical probes using the recently developed procedure [Kinoshita and Sugahara (1999) Anal, Biochem, 269, 367 -378], The fluorophore-tagged hexasacharides of low picomoles were well sep arated by HPLC and successfully analyzed by MALDI-TOF mass spectrometry, Th e principle of the method should be applicable to the analysis of the linka ge region oligosaccharides derived from heparin and heparan sulfate as well .