Cloning and Characterization of ADAMTS11 , an Aggrecanase from the ADAMTS Family

Abstract

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...

Inhibition of furin‐like enzymes blocks interleukin‐1α/oncostatin M–stimulated cartilage degradation

“…The aggrecanases ADAM-TS4 and ADAM-TS5 are considered to be the principal proteinases responsible for cartilage proteoglycan degradation, and these enzymes contain the typical furin recognition motif (9,10). This study shows that the addition of a furin inhibitor to resorbing cartilage can partially block the breakdown of the proteoglycan matrix.…”

“…Aggrecan fragments cleaved at this bond have been identified in cultures undergoing cartilage matrix degradation (5,6) and in arthritic synovial fluids (7,8). Two cartilage-derived aggrecanases have so far been identified that belong to a disintegrin and metalloproteinase with thrombospondin motifs (ADAM-TS) subfamily of proteinases; these are ADAM-TS4 (aggrecanase 1) (9) and ADAM-TS5 (aggrecanase 2) (10).…”

“…MT1-MMP, MT3-MMP, and MMP-11 can all be activated by furin (31)(32)(33). In addition, the aggrecanases ADAM-TS4 and ADAM-TS5 contain this furin cleavage site (9,10), and the pro-domain of ADAM-TS4 can be removed by furin (34). Furin has been detected in normal cartilage, and elevated levels are found in osteoarthritic cartilage (35).…”

Fibroblast growth factor 2 is an intrinsic chondroprotective agent that suppresses ADAMTS‐5 and delays cartilage degradation in murine osteoarthritis

“…It was initially described as a constitutively expressed aggrecanase in bovine (16,17) and human (18) chondrocytes, although we and others have demonstrated regulation by inflammatory cytokines in murine (4), bovine (19), and human (9) tissue. The putative promoter region of the gene has predicted binding sites for a number of transcription factors, including NF-B, which are likely to be involved in gene expression following activation of inflammatory signaling (20).…”

Sodium and T1ρ MRI for molecular and diagnostic imaging of articular cartilage

“…The same study also showed upregulation of the aggrecanases ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2) by IL-1. These proteins belong to the disintegrin and metalloprotease with thrombospondin motifs family (61), and are the only enzymes that have been shown to cleave aggrecan at a site that generates the Glu373-Ala374 fragments (62,63) seen in interleukin-1 stimulated bovine explant cultures (64) and human synovial fluid of OA patients (65).…”