Synthesis of low buoyant density proteoglycans by human chondrocytes in culture

Abstract

Human chondrocyte strains were derived from explant outgrowth of nonarthritic or osteoarthritic human cartilage. Chondrocytes radiolabeled with [35SO4] or [35S)-methionine were used to measure the biosynthesis of proteoglycans recovered from the most buoyant fraction (A4) of a CsCl density gradient centrifugation performed under associative conditions. The proteoglycans isolated from the A4 fraction (ϱ < 1.47 g/ml) were hydrodynamically small and contained both large and small glycosaminoglycan chains. When assessed by SDS/PAGE using 3–16% gradient gels, two subpopulations of small proteoglycans (smPG) were identified. The larger of the two species (smPG-I) migrated slower than the 200 kDa marker protein; when reassessed on 3–5% acrylamide gels, its apparent molecular mass was larger than the 480kDa and 440 kDa α and β heavy chains of dynein. We estimated the apparent molecular size of this smPG to be approximately 520 kDa. The smaller smPG (smPG-II) had an apparent average molecular mass of 180 kDa (range 170–210 kDa) after 3–16% SDS/PAGE. Three monoclonal antibodies, 1C6, 5D4, and S103L, reactive with the hyaluronic acid binding region of the aggregating proteoglycan core protein, keratan sulfate, and a core protein domain in the chondroitin sulfate attachment region, respectively, reacted with a single protein (apparent molecular mass, 180 kDa) that was similar in size to smPG-II.