Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue.
Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].
Dickenson
J M
JM
Division of Biological Sciences, University of Lancaster, Bailrigg, U.K.
Huckerby
T N
TN
Nieduszynski
I A
IA
D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000032:analysis; D000117:Acetylglucosamine / Q000032:analysis*; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002356:Cartilage / Q000032:analysis*; D002417:Cattle; D002845:Chromatography; D005944:Glucosamine / Q000031:analogs & derivatives*; D006025:Glycosaminoglycans / Q000032:analysis*; D006026:Glycoside Hydrolases; D007403:Intervertebral Disc; D007632:Keratan Sulfate / Q000032:analysis* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D012794:Sialic Acids / Q000032:analysis; D013431:Sulfates / Q000032:analysis*; D001616:beta-Galactosidase / Q000378:metabolism
D006025:Glycosaminoglycans; D012794:Sialic Acids; D013431:Sulfates; C011631:N-acetylgalactosaminitol; D007632:Keratan Sulfate; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D019158:N-Acetylneuraminic Acid; D000116:Acetylgalactosamine; D005944:Glucosamine; D000117:Acetylglucosamine