The chondroitin sulphate epitope 846 of aggrecan: Its relationship to aggrecan synthesis and its partial characterization

The chondroitin sulphate epitope 846 of aggrecan is abundant in foetal cartilage, barely detectable in normal adult cartilage, but reappears in the cartilage and body fluids of arthritic patients. This epitope has been proposed to be a marker of aggrecan synthesis during new cartilage formation and repair. The purpose of the present studies was to investigate, in vitro, whether the epitope was truly reflective of aggrecan synthesis, and to understand its role in the cartilage repair process. Foetal bovine chondrocyte cultures were established to study aggrecan synthesis in order to investigate whether the epitope 846 was present on these molecules. These studies showed that the epitope was indeed present on the newly synthesised aggrecan molecules and that these were preferentially retained within the extracellular matrix. The larger size and higher epitope density of the matrix molecules, compared to those molecules which were released into culture medium, suggested a role for the 846-epitope bearing molecules in the formation of new cartilage and during repair. Normal adult articular cartilage cultures were established to investigate whether the epitope could be synthesised by this tissue under conditions where the tissue had been stimulated to repair a damaged matrix, by trypsin-treatment of the cartilage. In these studies, an increase in abundance of the epitope on newly synthesised proteoglycans was observed, further indicating a role for the epitope-bearing molecules in cartilage repair. Explant cultures of osteoarthritic cartilage demonstrated that the release of the 846-bearing molecules from the cartilage was accompanied by the release of some of the newly synthesised 35S-sulphate labelled proteoglycans from the cartilage, and that these molecules contained, in part at least, the epitope. In addition, the release of 846-bearing molecules from the cartilage into culture medium or synovial fluid was reflective of the epitope content of the tissue. The structural characterization of the 846 epitope was performed using treatment of foetal bovine aggrecan with various enzymes. The data showed that the epitope was located on the non-reducing terminal ends of chondroitin sulphate chains and that its structure involved a terminal GaINAc4S residue. Furthermore, the data demonstrated the requirement for a high epitope density for the recognition of this epitope by the monoclonal 846 IgM antibody.