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This paper describes the first report of the production and use of an artificial recombinant protein substrate to study "aggrecanase" activity. The substrate (rAgg1) is composed of the complete interglobular domain (IGD) of human aggrecan flanked by the "marker" sequences FLAGTM at the amino terminus and the human immunoglobulin G1 constant region at the carboxyl terminus. The expressed protein occurs as large multimolecular aggregates (>120 kDa) that, upon reduction, consist of a major isoform of 72 kDa (containing the IGD) and a minor 39-kDa species that through alternative splicing has had the IGD deleted. Using this recombinant substrate we developed a novel agarose cell culture system containing either rat chondrosarcoma or bovine chondrocytes that could be used in studies of the biochemical characterization of aggrecanase activities. These studies showed the following. (i) rAgg1 is a suitable substrate for aggrecanase proteolysis. (ii) Aggrecanase activity was specifically induced by exposing chondrocytes to retinoic acid. (iii) A considerable time period was required to synthesize and/or activate aggrecanase, with considerable differences in that found in rat chondrosarcoma versus bovine chondrocyte culture systems. (iv) Aggrecanase cleavage of the aggrecan IGD does not require the presence of the G1 or G2 globular domains or keratan sulfate post-translational modification in the IGD. (v) Aggrecanase is a diffusible activity that does not require association with the chondrocyte plasma membrane or immediate pericellular matrix for its action.

The release of aggrecan catabolites from cartilage is an early event in the pathogenesis of degenerative joint diseases. The enzymes involved in this process are unknown, controversial, and the subject of intense investigation. In this paper we have utilized a recombinant substrate containing the interglobular domain (IGD) of aggrecan to study specifically aggrecanase versus matrix metalloproteinase (MMP) catabolism in this domain of aggrecan. Our studies have shown that (i) there are species differences in the expression of latent versus active MMP activity on the aggrecan IGD; (ii) interleukin-1alpha exposure induces both aggrecanase and MMP activities, whereas retinoic acid induces only aggrecanase activity and inhibits the MMP activity on the aggrecan IGD; (iii) activators of latent MMP activity (p-aminophenylmercuric acetate and trypsin) significantly reduce aggrecanase activity; (iv) the time course of the appearance of aggrecanase versus the MMP catabolism of aggrecan IGD differs; (v) aggrecanase is a protease with metalloprotease characteristics; however (vi) the physiological (tissue) inhibitors of MMPs show weak inhibition (TIMP-1) or no inhibition (TIMP-2) of aggrecanase activity. Collectively, these studies show that aggrecanase and MMP catabolism of the aggrecan IGD are independent and uncoupled.