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

Abstract

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,

Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS‐4 (aggrecanase 1) or ADAMTS‐5 (aggrecanase 2)

“…In this context, whereas aggrecanase activity is responsible for generating most of the "free" G1 (i.e., G1-NITEGE 373 ) that is detectable in human synovial fluid, MMP-generated G1 (i.e., G1-VDIPEN 341 ) can be detected in synovial fluid from older individuals and from patients with end-stage OA (13), although this likely represents secondary "trimming" of preexisting G1-NITEGE 373 by MMPs (37). It has also been demonstrated that relatively high concentrations of ADAMTS-4 can cleave aggrecan secondarily, at the Asn 341 -Phe 342 site (21). However, given the correlation between the release of sGAG and G1-NITEGE 373 in response to ADAMTS-4 or ADAMTS-5 treatment observed in the current study, it is unlikely that G1-VDIPES 341 fragments were generated under the conditions tested.…”

Low molecular weight isoforms of the aggrecanases are responsible for the cytokine‐induced proteolysis of aggrecan in a porcine chondrocyte culture system

“…The zymogen form of the protein was predicted to have a molecular weight of 90.2 kd, and the furin-cleaved isoform was predicted to have a molecular weight of 67.9 kd (9). Furin-cleaved recombinant human ADAMTS-4 (rHuADAMTS-4) has been reported as having a molecular weight of 68 kd (10) and a molecular weight of 70 kd (11). The thrombospondin motif of ADAMTS-4 is required for aggrecan binding and cleavage (mediated via the GAG chains of aggrecan) (12).…”

Proprotein Convertase Furin Interacts with and Cleaves Pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi Network

“…Compared with the constitutively expressed ADAMTS5, ADAMTS4 is induced dramatically by IL-1, tumor necrosis factor ␣, and type ␤ transforming growth factor in rheumatoid arthritis (RA) and osteoarthritis, suggesting that ADAMTS4 plays a more important role in cartilage breakdown (6,7). In addition to arthritic joints, ADAMTS4 is also expressed in ovary, spinal cord, uterus, brain, and heart and can mediate the degradation of other extracellular matrices such as versican and brevican (4,8). Although the biochemical properties of ADAMTS4 are well characterized, the cellular mechanisms by which ADAMTS4 is activated remain largely unknown.…”