Proteolytic degradation of aggrecan is a hallmark of the pathology of arthritis, yet the identity of the enzyme(s) in cartilage responsible for this degradation is unknown. Previous studies have suggested that the matrix metalloproteinases (MMPs) may be involved but there has been no definitive evidence for their direct action in the proteolysis of aggrecan in human arthritis. We now show unequivocally that aggrecan fragments derived from the specific action of MMPs can be detected in synovial fluids from patients with both inflammatory and noninflammatory arthritis, with a neoepitope monoclonal antibody AF-28 that detects the NH2-terminal sequence F342FGVG.... The synovial fluid MMP fragments were of low buoyant density and distributed exclusively at the top of cesium chloride density gradients, suggesting that these fragments lacked chondroitin sulfate chains. AF-28 immunoblotting of synovial fluid aggrecan fragments revealed a population of small AF-28 fragments of 30-50 kD. Based on their size relative to characterized products of an MMP-8 digest (Fosang, A.J., K. Last, P. Gardiner, D.C. Jackson, and L. Brown. 1995, Biochem. J. 310:337-343), these AF-28 fragments were derived from proteinase cleavage at, or near, the ...ITEGE373 / ARGSV... aggrecanase site. Immunodetection with polyclonal anti-ITEGE antiserum revealed that these fragments lacked the ...ITEGE374 COOH terminus and were not therefore products of aggrecanase action. The same fluid samples contained a broad 68-90-kD G1 fragment that contained the COOH-terminal ...ITEGE374 neoepitope. The results suggest that in some circumstances, despite extensive proteolysis of the core protein, aggrecan molecules may be cleaved by MMPs or aggrecanase in the interglobular domain, but not both.

The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.

Pain is the predominant symptom of osteoarthritis, but the connection between joint damage and the genesis of pain is not well understood. Loss of articular cartilage is a hallmark of osteoarthritis, and it occurs through enzymatic degradation of aggrecan by cleavage mediated by a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS-4) or ADAMTS-5 in the interglobular domain (E ³⁷³ – ³⁷⁴ A). Further cleavage by MMPs (N ³⁴¹ – ³⁴² F) releases a 32-amino-acid aggrecan fragment (32-mer). We investigated the role of this 32-mer in driving joint pain. We found that the 32-mer excites dorsal root ganglion nociceptive neurons, both in culture and in intact explants. Treatment of cultured sensory neurons with the 32-mer induced expression of the proalgesic chemokine CCL2. These effects were mediated through TLR2, which we demonstrated was expressed by nociceptive neurons. In addition, intra-articular injection of the 32-mer fragment provoked knee hyperalgesia in WT but not Tlr2 -null mice. Blocking the production or action of the 32-mer in transgenic mice prevented the development of knee hyperalgesia in a murine model of osteoarthritis. These findings suggest that the aggrecan 32-mer fragment directly activates TLR2 on joint nociceptors and is an important mediator of the development of osteoarthritis-associated joint pain.