We have studied aggrecan catabolism mediated by matrix metalloproteinases (MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN 341 Aggrecan is the major proteoglycan present in articular cartilage, and it is the molecule that endows cartilage with its intrinsic capacity to bear load and resist compression. This weight-bearing capacity is dependent on the structure and organization of collagen and aggrecan within the extracellular matrix. Normal turnover of aggrecan is a conservative process in which the rate of breakdown and release of fragments from the tissue does not exceed the rate at which it is replaced by newly synthesized molecules. In pathology, an increased rate of degradation results in a net loss of aggrecan, and the tissue becomes thin and mechanically weak.Aggrecan is lost from cartilage following proteolysis of the core protein. Aggrecanases are thought to be the enzymes primarily responsible for this proteolysis. Two ADAM 1 proteins with thrombospondin motifs, ADAMTS-4 (aggrecanase-1) (1) and ADAMTS-5 (aggrecanase-2, also known as ADAMTS-11) (2, 3) have been purified from bovine cartilage, and the corresponding human enzymes have been cloned and shown to exhibit the same specificity for aggrecan substrates as the purified bovine enzymes. The most widely documented activity of aggrecanase is hydrolysis of the Glu 373 2Ala 374 bond (numbering based on the human sequence) (4) in the interglobular domain (IGD) (Fig. 1c); however aggrecanases also cleave within highly conserved regions of the chondroitin sulfate attachment region (5, 6). Fragments resulting from aggrecanase action and containing the 374 ARGSV N terminus (Fig. 1c) have been detected in conditioned culture medium by sequence analysis of isolated fragments or by detection with specific neoepitope antibodies. Aggrecanase-derived 374 ARGSV fragments are the major aggrecan products found in synovial fluids from arthritis patients (7,8). Furthermore, the aggrecanase-derived ITEGE 373 neoepitope at the C terminus of the G1 domain has been detected in human (9, 10), bovine (11, 12), pig (12), and rat (11) cartilage and in mice with experimental arthritis (13,14).In addition to cleavage by aggrecanase, there is convincing evidence for the direct involvement of the matrix metalloproteinase (MMP) family of enzymes in aggrecanolysis, albeit at much lower levels. The specific products of MMP activity have been detected in vivo (10, 15, 16) and in vitro (17-19) by sequencing and more recently by cleavage site-specific neo-