Assembly of newly synthesized proteoglycan and link protein into aggregates in cultures of chondrosarcoma chondrocytes.

The synthesis and subsequent assembly of proteoglycan monomer and link protein into aggregates were examined using cultured chondrocytes from the Swarm rat chondrosarcoma. Proteoglycans purified directly from culture medium after a 6-h label with [14C]serine contained up to 80% of the radioactivity in aggregates, 8% of which was in link protein. The addition of hyaluronic acid oligosaccharides (averaging 50 monosaccharides) to the culture medium during labeling inhibited aggregate formation by 94%. More than 90% of the labeled link molecules (about 7% of the total radioactivity in the proteoglycan fraction) was associated in ternary complexes with monomeric proteoglycan and HA approximately 50. The stoichiometry of labeled link protein to labeled proteoglycan core protein aggregated with either macromolecular hyaluronic acid or hyaluronic acid oligosaccharides was calculated to be 1:1. The effect of exogenous proteoglycan on aggregate formation was examined by incubating cultures for 4 h with up to 5 mg of exogenous monomer. Over this time, about 85 micrograms of newly synthesized proteoglycan accumulated in the culture medium. With increasing amounts of exogenous monomer, up to 80 micrograms of 35S-labeled exogenous monomer was incorporated into link-stabilized aggregate, while the aggregation of endogenous proteoglycan decreased from 73 to 44%. The total endogenous plus exogenous monomer in link-stabilized aggregate increased to about 130 micrograms, approximately 1 1/2 times the total amount of endogenous monomer in the culture medium, indicating that at least 1 1/2 times more link molecules than endogenous proteoglycan molecules were present. Further, the molar ratio of labeled link to labeled monomer molecules associated with the nonaggregated proteoglycan fraction remained constant at approximately 1:1, indicating that the exogenous proteoglycans were ineffective in displacing link molecules from endogenous monomer.link complexes. The data suggest that the formation of proteoglycan aggregates may be a sequential process in which a link.monomer complex forms and this complex subsequently binds to hyaluronic acid.