We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn 341 -Phe 342 and Glu 373 -Ala 374 . While several matrix metalloproteinases have been shown to cleave at Asn 341 -Phe 342 , an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu 373 -Ala 374 and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammationassociated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu 373 -Ala 374 site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.Aggrecan is the major proteoglycan of cartilage and is responsible for its compressibility and stiffness. Aggrecan contains two N-terminal globular domains, G 1 and G 2 , separated by a proteolyticaly sensitive interglobular domain, followed by a glycosaminoglycan attachment region and a C-terminal globular domain (G 3 ). The G 1 domain of aggrecan interacts with hyaluronic acid and link protein to form large aggregates containing multiple aggrecan monomers that are trapped within the cartilage matrix. Cleavage of aggrecan has been shown to occur at Asn 341 -Phe 342 and Glu 373 -Ala 374 within the interglobular domain, with the cleaved aggrecan being free to exit the matrix since it lacks the G 1 domain, which is responsible for formation of the high molecular weight complexes. Results from several studies suggest that cleavage at the Glu 373 -Ala 374 site is responsible for the increased aggrecan degradation observed in inflammatory joint disease. Products resulting from cleavage at the Glu 373 -Ala 374 site have been shown to accumulate in cartilage explants and chondrocyte cultures treated with interleukin-1 and retinoic acid (1-5) and in the synovial fluid of patients with osteoarthritis and inflammatory joint disease (6, 7). While several characterized matrix metalloproteases 1 have been shown to cleave at the Asn 341 -Phe 342 site (8 -14), they are not responsible for the observed cleavage at Glu 373 -Ala 374 . A novel proteolytic activity, termed "aggrecanase," has been hypothesized to be respo...

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity, is one of the first matrix components to undergo measurable loss in arthritic diseases. bond, this protease cleaves at four sites within the chondroitin-sulfate rich region of the aggrecan core protein, between G2 and G3 globular domains. Importantly, we show that this cleavage occurs more efficiently than cleavage within the IGD at the Glu 373 -Ala 374 bond. Cleavage occurred preferentially at the KEEE 1667-1668 GLGS bond to produce both a 140-kDa COOH-terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavage also occurred at the GELE 1480 -1481 GRGT bond to produce a 55-kDa COOH-terminal fragment and a G1-containing fragment of 320 kDa. Cleavage of this 320-kDa fragment within the IGD at the Glu 373 -Ala 374 bond then occurred to release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kDa GLGS-reactive fragment resulting from the preferential cleavage was further processed at two additional cleavage sites, at TAQE 1771 -1772 AGEG and at VSQE [1871][1872] LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of ϳ20 kDa. These data elucidate the sites and efficiency of cleavage during aggrecan degradation by aggrecanase and suggest potential tools for monitoring aggrecan cleavage in arthritis.Aggrecan is the major proteoglycan of cartilage and provides this tissue with its mechanical properties of compressibility and elasticity. Aggrecan monomers interact with hyaluronan and are usually found as part of a large aggregate containing 10 -100 monomers per hyaluronan molecule. The primary role of aggrecan is to swell and hydrate the framework of cartilage collagen fibrils thus providing cartilage with its properties of compressibility and elasticity. The NH 2 terminus of the aggrecan monomer core protein is comprised of two globular domains called G1 and G2 that are separated by an interglobular domain (IGD) 1 that spans about 150 residues in length. The G2 region is followed by a long central glycosaminoglycan (GAG) attachment region and by a COOH-terminal globular domain, G3 (2, 3).In cartilage degradation associated with diseases such as osteoarthritis and rheumatoid arthritis, aggrecan is one of the first matrix components to undergo measurable loss that ultimately leads to a loss of cartilage function. Proteolytic cleavage within the IGD is believed to be responsible for the loss of aggrecan from cartilage. Two major sites of proteolytic cleavage have been identified within the IGD, one between amino acids Asn 341 and Phe 342 and the other between Glu 373 and Ala 374 . Matrix metalloproteinases, including MMP

Aggrecanase-1 (ADAMTS-4) is a member of the a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) protein family that was recently identi-Aggrecanase-1 is one of two novel cartilage-degrading metalloproteases purified from bovine nasal cartilage cultures stimulated with interleukin-1 (1, 2). The enzyme shares 40 -50% sequence homology with aggrecanase-2 (ADAMTS-11/5), ADAMTS-1 (METH-1), and ADAMTS-8 (METH-8) as well as lower degrees of homology with other members of the a disintegrin and metalloprotease with thrombospondin motif (AD-AMTS) 1 family (3-7). All members of the ADAMTS family consist of an N-terminal propeptide domain, a metalloproteinase domain, and a disintegrin-like domain, resembling the structural elements of the reprolysin family of metalloproteases that includes the a disintegrin and metalloprotease (ADAM) and snake venom metalloproteases (8, 9). Unlike typical ADAM proteins that are membrane-anchored and have a transmembrane domain and cytoplasmic domain in the C-terminal region, the C terminus of ADAMTS proteins contains a varying number of thrombospondin type-1 (TSP-1) motifs, the sequence of which is the conserved motif in thrombospondin 1 and 2 (10).Aggrecan is a large chondroitin sulfate proteoglycan that accounts for about 10% of the dry weight of cartilage (11,12). It consists of three globular domains: G1, through which it interacts with hyaluronan (HA); G2; and G3 at the C terminus of the molecule. The core protein between G2 and G3 is highly substituted with the glycosaminoglycans (GAG) keratin sulfate and chondroitin sulfate chains. Aggrecan is usually found as part of a large aggregate with HA containing approximately 100 proteoglycan molecules/HA molecule. The molecule carries a large number of fixed negatively charged groups on the GAGs that results in high osmotic pressure in the tissue, thus allowing aggrecan to swell and hydrate the framework of collagen fibrils in cartilage, providing the tissue its properties of compressibility and resilience.The interglobular domain of aggrecan between the G1 and G2 domain has been shown to be susceptible to proteolytic cleavage. In fact, aggrecanase activity was originally defined by cleavage at the Glu 373 /Ala 374 bond within this region of the aggrecan core protein (13). We have recently demonstrated that recombinant aggrecanase-1 degrades cartilage aggrecan very efficiently and cleaves sequentially at five sites within the molecule (14).2 Several cleavages occur first in the chondroitinsulfate-rich region of the molecule followed by cleavage within the interglobular domain between residues Glu 373 and Ala 374. Fragments formed by aggrecanase-mediated cleavage are found in cartilage cultures stimulated with cytokines or retinoic acid (13,(15)(16)(17)(18)(19)(20)(21)(22)(23), 3 as well as in synovial fluid from patients

The BAR adaptor proteins encoded by the RVS167 and RVS161 genes from Saccharomyces cerevisiae form a complex that regulates actin, endocytosis, and viability following starvation or osmotic stress. In this study, we identified a human homolog of RVS161, termed BIN3 (bridging integrator-3), and a Schizosaccharomyces pombe homolog of RVS161, termed hob3؉ (homolog of Bin3). In human tissues, the BIN3 gene was expressed ubiquitously except for brain. S. pombe cells lacking Hob3p were often multinucleate and characterized by increased amounts of calcofluor-stained material and mislocalized F-actin. For example, while wild-type cells localized F-actin to cell ends during interphase, hob3⌬ mutants had F-actin patches distributed randomly around the cell. In addition, medial F-actin rings were rarely found in hob3⌬ mutants. Notably, in contrast to S. cerevisiae rvs161⌬ mutants, hob3⌬ mutants showed no measurable defects in endocytosis or response to osmotic stress, yet hob3؉ complemented the osmosensitivity of a rvs161⌬ mutant. BIN3 failed to rescue the osmosensitivity of rvs161⌬, but the actin localization defects of hob3⌬ mutants were completely rescued by BIN3 and partially rescued by RVS161. These findings suggest that hob3؉ and BIN3 regulate F-actin localization, like RVS161, but that other roles for this gene have diverged somewhat during evolution.