Generation and characterization of aggrecanase - A soluble, cartilage-derived aggrecan-degrading activity

A method was developed for generating soluble, active "aggreeanase" in cond itioned media from interleukin-1-stimulated bovine nasal cartilage cultures , Using bovine nasal cartilage conditioned media as a source of the aggreca nase enzyme, an enzymatic assay was established employing purified aggrecan monomers as a substrate and monitoring specific aggrecanase-mediated cleav age products by Western analysis using the monoclonal antibody, BC-3 (which recognizes the new N terminus, ARGS, on fragments produced by cleavage bet ween amino acid residues Glu(373 an)d Ala(374)). Using this assay we have c haracterized cartilage aggrecanase with respect to assay kinetics, pH; and salt optima, heat sensitivity, and stability upon storage. Aggrecanase acti vity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect, su ggesting that aggrecanase is a metalloproteinase, Sensitivity to known matr ix metalloproteinase inhibitors as well as to the endogenous tissue inhibit or of metalloproteinases, TIMP-1, further support the notion that aggrecana se is a metalloproteinase potentially related to the ADAM family or MMP fam ily of proteases previously implicated in the catabolism of the extracellul ar matrix.