2001 Volume 129 Issue 1 Pages 107-118
The glycosaminoglycan (GAG)-protein linkage regions of various proteoglycans share the common tetrasaccharide GlcA-Gal-Gal-Xyl-attached to Ser residues in the core proteins. In previous analysis we demonstrated unique modifications by epimerization, sulfation and phosphorylation of the component sugars. Here we developed a sensitive analytical method for the linkage region oligosaccharides to detect or monitor structural variations and changes. This will be useful for investigation of their biological roles, which are largely unknown, but they have been implicated in biosynthesis. A variety of linkage region-derived hexasaccharides was first prepared as reducing sugar chains from peptide chondroitin/dermatan sulfate of whale cartilage, shark cartilage, and bovine aorta by means of chondroitinase digestion in conjunction with β-elimination in the absence of reducing reagents, but involving a mild alkali, 0.5M LiOH, at 4°C to prevent peeling reactions. The structures of these oligosaccharides were determined by the combination of HPLC, enzymatic digestion, matrix-assisted laser desorption ion-ization time-of-flight (MALDI-TOF) mass spectrometry, and 1H NMR spectroscopy, which revealed eleven different hexasaccharides including a novel structure, ΔHexAα1-3GalNAcβ1-4IdoAα1-3Gal (4-O-sulfate) β1-3Galβ1-4Xyl (ΔHexA and IdoA represent unsaturated hexuronic acid and L-iduronic acid, respectively). These oligosaccharides were labeled with a fluorophore, 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 separated by HPLC and successfully analyzed by MALDI-TOF mass spectrometry. The principle of the method should be applicable to the analysis of the linkage region oligosaccharides derived from heparin and heparan sulfate as well.
