A new method for sequence analysis of glycosaminoglycans from heavily substituted proteoglycans reveals non-random positioning of 4- and 6-O-sulphated N-acetylgalactosamine in aggrecan-derived chondroitin sulphate.
We have developed a new procedure for the sequence analysis of glycosaminoglycans, which is particularly suitable for the analysis of chains from heavily substituted proteoglycans. The procedure has been applied to various aggrecan-derived chondroitin sulphates. The glycans are released from the core protein by alkaline scission of the xylose-serine bond, subjected to reductive amination using p-aminobenzoic acid and finally radioiodinated at an acidic pH. Sequence analysis is performed by using various enzymic degradations, partial or complete, followed by high-resolution polyacrylamide gel electrophoresis, blotting and autoradiography to identify segments extending from the labelled reducing end to the point of cleavage. By using chondroitin C lyase to identify the location of 6-O-sulphated hexosamines, we find that chondroitin sulphate from tracheal cartilage has its 6-O-sulphated repeats concentrated to the extreme non-reducing terminal portion of the chain. In chondroitin sulphates derived from intervertebral discs (nucleus pulposus), the 6-O-sulphated repeats have a biphasic distribution; they occur mostly near the linkage region (i.e. the reducing end), but also in the non-reducing portion of the chain. Chondroitin sulphate from nasal cartilage, which is mostly 4-O-sulphated, displays considerable heterogeneity in the linkage region. Three or possibly more charge variants are observed.
Cheng
F
F
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
Yoshida
K
K
Heinegård
D
D
Fransson
L A
LA
D010129:4-Aminobenzoic Acid; D000116:Acetylgalactosamine / Q000737:chemistry*; D053674:Aggrecans; D001345:Autoradiography; D002240:Carbohydrate Sequence; D002808:Chondroitin Lyases; D002809:Chondroitin Sulfates / Q000737:chemistry*; D004187:Disaccharides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans / Q000737:chemistry*; D007457:Iodine Radioisotopes; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000737:chemistry*; D012091:Repetitive Sequences, Nucleic Acid; D017421:Sequence Analysis / Q000379:methods*; D013431:Sulfates / Q000737:chemistry
D053674:Aggrecans; D004187:Disaccharides; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D007457:Iodine Radioisotopes; D037181:Lectins, C-Type; D011509:Proteoglycans; D013431:Sulfates; D002809:Chondroitin Sulfates; D002808:Chondroitin Lyases; D000116:Acetylgalactosamine; D010129:4-Aminobenzoic Acid