The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large M r chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ionexchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following β-elimination of the protein core, the liberated low M r oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz 1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc → Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: ▪ The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAcα(2 → 3)Galβ(1 → 3)GalNAcol, [NeuAcα(2 → 3)Galβ(1 → 3)[NeuAcα(2 → 6)]GalNAc-ol, and NeuAcα-(2 → 3) Galβ(1 → 3)[NeuAcα(2 → 3)Galβ(1 → 4)GlcNAcβ(1 → 6)]GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): Δ 4,5GlcUAβ(1 → 3)GalNAcβ(1 → 4)GlcUAβ(1 → 3)Galβ(1 → 3)Galβ(1 → 4)Xyl-ol.