Membrane‐associated hyaluronate‐binding activity of chondrosarcoma chondrocytes

Abstract

The association of hyaluronate with the surface of chondrocytes was examined by several approaches using primary cultures of chondrocytes derived from the Swarm rat chondrosarcoma. In culture, chondrosarcoma chondrocytes produced large pericellular coats, which can be visualized by particle exclusion, and which can be removed by Streptomyceshyaluronidase. Exposure of chondrocytes, which had been metabolically labelled with ³H-acetate, to exogenous hyaluro-nate or to Streptomyceshyaluronidase resulted in the release of 36–38% of the endogenous, labelled chondroitin sulfate from the cell layer into the incubation solution. These results imply that at least 37% of the cell layer chondroitin sulfate proteoglycan is retained there by an interaction with hyaluronate. Thus membranes were prepared from cultured chondrocytes and examined for sites which bind³H-hyaluronate. Binding was observed and found to be saturable, specific for hyaluronate, of high affinity (K_d =∼10⁻¹⁰M),and destroyed by treating the membranes with trypsin. The ³ H-hyaluronate-binding activity was inhibited competitively by hyaluronate decasaccharides but not by hexasaccharides or octasaccharides, indicating that the binding sites recognize a sequence of hyalu-ronate composed of five disaccharide repeats. The binding activity was partially purified from a detergent extract of chondrocyte membranes by ion exchange chromatography on DEAE-cellulose, followed by affinity chromatography on wheat germ agglutinin-agarose. Analysis of the partially purified binding activity by SDS-PAGE revealed five protein bands of 48,000–66,000 daltons in silver-stained gels. SDS-PAGE followed by Western blotting and exposure to mono-clonal antibodies which recognize epitopes present in link protein and in the hyaluronate-binding region of cartilage proteoglycan revealed no immunoreac-tive protein bands in the partially purified material. We conclude that one mechanism by which hyaluronate associates with the chondrocyte surface may be via interaction with a membrane-bound hyaluronate-binding protein which is distinct from link protein and proteoglycan.