| Abstract: | The chondroitin sulfate proteoglycan of rat brain was digested with Pronase, and after removal of glycosaminoglycans, the resulting glycopeptides were treated with alkaline borohydride to release O-glycosidically linked oligosaccharides. These were fractionated by ion exchange chromatography, gel filtration, and preparative thin layer chromatography, and their structural properties were studied by specific enzymatic degradations, methylation analysis, and gas-liquid chromatography-mass spectrometry of disaccharides as their trimethylsilylated and permethylated derivatives. In addition to the previously characterized N-acetyl-galactosamine-linked oligosaccharides and neutral mannitol-containing oligosaccharides [GlcNAc(beta 1-3) Manol and Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Manol] (where Fuc is fucose), we have now identified the sialylated tetrasaccharide NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc (beta 1-3)Manol, which accounts for approximately 20% of the mannitol-containing oligosaccharides. The proteoglycan also contains mannose-linked keratan sulfate chains (with a molecular size of 3,000 to 10,000 Da) composed of disaccharide repeating units consisting of Gal(beta 1-4)GlcNAc-6-O-SO4(beta 1-3), with a small proportion of branch points at C-6 of galactose residues. There is approximately one keratan sulfate chain per four chondroitin sulfate chains of 18,000-19,000 Da. After alkaline borohydride treatment of the neutral and monosialyl glycopeptide fractions, the combined decrease in mannose and N-acetylgalactosamine was very close to the observed destruction of serine + threonine and was accompanied by an equimolar increase in alanine and alpha-aminobutyric acid. One half of the mannose was destroyed by alkaline borohydride treatment of the glycopeptides and stoichiometrically converted to mannitol, while there were only small changes in the relative amounts of the other sugars and amino acids. The data demonstrate that over half of the carbohydrate-peptide linkages in the proteoglycan are of the mannosyl-O-serine/threonine type. |
