Aggrecan (PG) was isolated from Swarm rat chondrosarcoma and the chondroitin 4-sulfate removed with chondroitinase ABC (ABC) or ACII (AC), leaving a 4-deoxy-beta-d-gluc-4-enuronosyl (DeltaGlcA) residue on the nonreducing terminus of the attached chondroitin sulfate chains. Mercuric acetate (as low as 5 mm) removed the DeltaGlcA from the PG-ABC within 10 min at 25 degrees C at pH 5.0, and the rate was pH independent between pH 3.0 and 5.0. The reaction was readily monitored by following the loss of reactivity to the monoclonal antibodies specific for 4-sulfated and nonsulfated unsaturated disaccharides in ELISA. After mercury treatment, there was a loss of carbazole-positive material and a decrease in the size of the linkage region oligosaccharides consistent with DeltaGlcA being removed. Aside from the loss of DeltaGlcA, neutral sugar composition and sialic acid content remained unchanged. After electrophoresis in a 4% polyacrylamide gel, Hg-treated PG-ABC and PG-AC migrated as single major bands, but with reduced mobilities, which is consistent with a loss of charge. There was a loss of reactivity to specific monoclonal antibodies. Treated aggrecan did not bind hyaluronic acid. This loss was not completely prevented by being present in a complex with link protein and hyaluronic acid. However, link protein could partially restore the hyaluronic acid interaction, so the effect of mercuric acetate on biological function will have to be assessed on an individual basis. Treatment with mercuric acetate is an effective, rapid, reproducible way of removing DeltaGlcA from both chondroitinase ABC and ACII-digested proteoglycan.
Aggrecans Animals