ADAM‐8 isolated from human osteoarthritic chondrocytes cleaves fibronectin at Ala 271

• Published in: Arthritis and rheumatism Vol. 60; no. 9; pp. 2704 - 2713
• Main Authors: Zack, Marc D., Malfait, Anne‐Marie, Skepner, Adam P., Yates, Matthew P., Griggs, David W., Hall, Troii, Hills, Robert L., Alston, James T., Nemirovskiy, Olga V., Radabaugh, Melissa R., Leone, Joseph W., Arner, Elizabeth C., Tortorella, Micky D.
• Format: Journal Article
• Language: English
• Published: 01.09.2009
• ISSN: 0004-3591; 1529-0131
• DOI: 10.1002/art.24753

Abstract Objective Fibronectin fragments are thought to play a critical role in the initiation and progression of cartilage degradation in arthritis. In a recent study, fibronectin neoepitopes resulting from cleavage of intact fibronectin at the Ala 271 /Val 272 scissile bond, generating an ∼30‐kd fragment with the new C‐terminus VRAA 271 and an ∼50–85‐kd fragment with the new N‐terminus 272 VYQP, were identified in osteoarthritis (OA) cartilage. The present study was undertaken to isolate the enzymes responsible for this cleavage from human OA chondrocytes. Methods Fibronectin‐degrading activity in human OA chondrocyte–conditioned medium (OACCM) was purified using conventional chromatography. A fluorescent peptide was developed based on the fibronectin scissile bond 269 RAA↓Val 272 , and this peptide was used to track fibronectinase activity during purification. Western blotting with antibodies that detect the fibronectin neoepitopes VRAA 271 and 272 VYQP was used to confirm cleavage of intact fibronectin by the enzymatically active fractions. Mass spectrometry was used to identify the proteins found in the fibronectinase‐enriched fractions, with further confirmation by Western blotting. In addition, a recombinant enzyme identified by mass spectrometry was tested by Western blotting and dimethylmethylene blue assay for its ability to produce fibronectin neoepitopes in OA cartilage. Results Purification of OACCM by chromatography resulted in isolation of a fibronectin‐degrading enzyme, and mass spectrometry identified ADAM‐8 as the fibronectinase present in these preparations. Furthermore, treatment of OA cartilage with recombinant human ADAM‐8 promoted cartilage catabolism. Conclusion The results of this study identify ADAM‐8 as a fibronectinase in human OA chondrocytes. Because ADAM‐8 is capable of producing the fibronectin neoepitopes VRAA 271 and 272 VYQP in human OA cartilage, this enzyme may be an important mediator of cartilage catabolism.