Characterization of metalloprotease cleavage products of human articular cartilage

• Published in: Arthritis and rheumatism Vol. 58; no. 8; pp. 2420 - 2431
• Main Authors: Zhen, Eugene Y., Brittain, Isabelle J., Laska, Dennis A., Mitchell, Peter G., Sumer, Eren U., Karsdal, Morten A., Duffin, Kevin L.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.08.2008; Wiley
• Subjects: ADAM Proteins - physiology; ADAMTS4 Protein; ADAMTS5 Protein; Adult; Amino Acid Sequence; Arthritis - metabolism; Biglycan; Biological and medical sciences; Biomarkers - metabolism; Cartilage, Articular - enzymology; Collagen Type II - metabolism; Diseases of the osteoarticular system; Extracellular Matrix Proteins - metabolism; Female; Humans; Medical sciences; Metalloproteases - metabolism; Middle Aged; Molecular Sequence Data; Peptides - metabolism; Procollagen N-Endopeptidase - physiology; Proteoglycans - metabolism; Human; Articular cartilage; Rheumatology
• ISSN: 0004-3591; 1529-0131
• DOI: 10.1002/art.23654

Objective To identify, characterize, and compare proteolysis peptide products generated by metalloprotease digests of human articular cartilage. Methods Human articular cartilage was digested by the addition of exogenous metalloproteases, including matrix metalloproteinases 2, 3, 8, 9, 12, and 13 and aggrecanases ADAMTS‐4 and ADAMTS‐5. Proteolyzed peptide products were identified by proteomics methods using mass spectrometry. Results Complete sequences of the peptides proteolyzed from human articular cartilage, including N‐ and C‐termini and hydroxylated posttranslational modifications, were determined. A wide variety of peptides, originating from types I, II, and III collagen, biglycan, prolargin, fibromodulin, fibronectin, decorin, cartilage oligomeric matrix protein, cartilage intermediate‐layer protein, megakaryocyte‐stimulating factor, mimecan, aggrecan, and lumican, was analyzed following metalloprotease digestion. Release of peptides varied as a function of time, enzyme specificity, and abundance. Specific type II collagen peptide biomarkers, including those containing the three‐quarter–length fragment cleavage site and those containing the domains for helical peptide of type II collagen and C‐telopeptide of type II collagen, were observed after release by selected proteases. Conclusion The use of intact cartilage instead of purified protein substrates in the assay allowed for the identification of novel potential substrates and cleavage sites for individual enzymes under more physiologically relevant conditions. Characterization of these cartilage matrix peptides may help in the development of pharmacodynamic biomarkers of cartilage degradation, and also may contribute to an understanding of the bioactive peptides important in chondrocyte signaling.