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.

Abstract

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.