Generation and Characterization of Aggrecanase

Aggrecan is the major cartilage hyalectan (1), which, together with the collagen network, provides this tissue with its unique mechanical properties of compressibility and stiffness (2-4). Extraction of aggrecan in its native form (5) and subsequent structural analysis (6) have revealed that the molecular organization of aggrecan is perfectly suited to its central functional role in articular cartilage. The N-terminal region of aggrecan is composed of two globular domains (G1 1 and G2) separated by the interglobular domain (IGD). G1 interacts with hyaluronan and link protein, thereby keeping the aggrecan molecule anchored within the cartilage tissue. Further interactions with other matrix components such as tenascin-R and fibulin-1 and fibulin-2 (7, 8) may occur through a third globular domain (G3) at the extreme C terminus of the core protein. The extended core protein between G2 and G3 is composed of a short keratan sulfate-rich region followed by a longer chondroitin sulfate-substituted domain. The charge repulsion and hydration of the long negatively charged glycosaminoglycan (GAG) chains are thought to maintain the C-terminal portions of aggrecan in an extended conformation (9). The swelling pressure of the aggrecan-link protein complex with hyaluronan is restrained by the tension in the collagen network; and together, these components form a fiber-reinforced concentrated gel within the cartilage, which transmits forces across the articular joint.In diseases characterized by cartilage degradation such as rheumatoid arthritis and osteoarthritis, increased aggrecan release from the cartilage occurs early (10, 11) and before the bulk of the collagen network is degraded (12). Proteolytic cleavage of aggrecan within the IGD separates the GAG-rich region from the hyaluronan-anchored G1 domain, resulting in GAG release from the cartilage matrix to the synovial fluid. Biomechanical tests on cartilage discs have shown that proteolysis within the IGD of aggrecan, and not cleavages near the C terminus, is primarily responsible for the loss of compressive resistance that accompanies interleukin-1-mediated degradation of the tissue (13). Identification of the proteinases responsible for this "destructive" cleavage of aggrecan has therefore been a major focus of experimentation in arthritis-related research.In this regard, two major cleavage sites that occur in vivo have been identified in the IGD of human aggrecan. One is a matrix metalloproteinase (MMP)-sensitive site at VDIPEN 341 2F 342 FGVGG, which can be cleaved at neutral pH by any one of a range of MMPs, including MMP-1-3, -7-9, -13,

Section: Discussionmentioning

confidence: 68%

ADAMTS4 Cleaves at the Aggrecanase Site (Glu373-Ala374) and Secondarily at the Matrix Metalloproteinase Site (Asn341-Phe342) in the Aggrecan Interglobular Domain

Westling

Fosang

Thompson

et al. 2002

“…TIMPs have also recently been shown to inhibit metalloproteinases other than MMPs. An IC 50 value of 110 nM (0.11 M) has been reported for TIMP-3 inhibition of TACE (32), and an IC 50 value of 210 nM has been estimated for TIMP-1 inhibition of aggrecanase (13). Both TACE and aggrecanase are recently identified ADAM family members.…”

Section: Medium Conditioned By Human Brain Tumor Cells Contains a Metmentioning

confidence: 99%

Post-translational Proteolytic Processing of Procollagen C-terminal Proteinase Enhancer Releases a Metalloproteinase Inhibitor

Mott¹,

Thomas²,

Rosenbach³

et al. 2000

119

Activity of matrix metalloproteinases (MMP) is regulated by a family of proteins called tissue inhibitors of metalloproteinases (TIMP). Four TIMPs have been cloned, and their molecular weights range from 29,000 to 20,000. By reverse zymography, we have observed a metalloproteinase inhibitor with an apparent molecular weight of 16,500 from medium conditioned by human brain tumor cells. Antibodies directed against TIMPs failed to react with the 16,500 molecular weight inhibitor, indicating that it was not a truncated form of a known TIMP. The inhibitor was isolated from conditioned medium using affinity and ion exchange chromatography. N-terminal sequences of the inhibitor matched amino acid sequences within the C-terminal domain of a protein known as procollagen C-terminal proteinase enhancer (PCPE). Thus, the inhibitor was named CT-PCPE. Comparison of the N-terminal domain of TIMP with CT-PCPE revealed that both contained six cysteine residues. As in the case of TIMP, reduction and alkylation abolished the inhibitory activity of CT-PCPE. Purified CT-PCPE inhibited MMP-2 with an IC 50 value much greater than that of TIMP-2. This implies that MMPs may not be the physiologic targets for CT-PCPE inhibition. However, these results suggest that CT-PCPE may constitute a new class of metalloproteinase inhibitor.

“…Determination of Aggrecan Cleaving Activity-Aggrecanase activity was measured by incubating bovine aggrecanase or recombinant human ADAMTS11 with purified bovine aggrecan monomers as described (27). Reactions were terminated with 20 mM EDTA and Western blotted.…”

Section: Methodsmentioning

confidence: 99%

“…Reactions were terminated with 20 mM EDTA and Western blotted. Glu 373 -Ala 374 cleavage products were detected using the BC33monoclonal antibody (28) IC 50 values were determined for the inhibitors using conditioned media from stimulated bovine nasal cartilage explant cultures and recombinant human ADAMTS11 as described previously (27).…”

Section: Methodsmentioning

confidence: 99%

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

Abbaszade¹,

Liu²,

Yang³

et al. 1999

Self Cite

470

325

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...