Cleavage of native cartilage aggrecan by neutrophil collagenase (MMP-8) is distinct from endogenous cleavage by aggrecanase.
Cleavage of aggrecan core protein at the Glu373-Ala374 site by the unidentified enzyme, "aggrecanase," is thought to play an important role in cartilage degradation. To examine aggrecan cleavage by MMP-8 at this aggrecanase site, we evaluated the release of fragments with the N terminus ARGSVIL from freeze-thawed bovine nasal cartilage using the monoclonal antibody BC-3. Recombinant human MMP-8 catalytic domain cleaved native aggrecan in a concentration-related manner between 0.2 and 2 microg/ml, with complete release of glycosaminoglycan at 2 microg/ml or greater. Cleavage at the aggrecanase site was observed only at MMP-8 concentrations resulting in complete release of glycosaminoglycan from the cartilage, suggesting that preferential cleavage occurs at a different site. Time course studies indicated that only following depletion of substrate containing the preferred clip site did MMP-8 rapidly cleave at the aggrecanase site. Finally, MMP-8 resulted in a different pattern of BC-3-reactive fragments from that produced by endogenous aggrecanase in live cartilage, and SA751(N-(1(R)-carboxyethyl) -alpha-(S)-(4-phenyl-3-butynyl)glycyl-L-O-methyltyrosine, N-methylamide), a potent inhibitor of MMP-8 (Ki = 2 nM) which was effective in blocking cleavage by MMP-8 at the aggrecanase site with an IC50 in the nanomolar range, did not prevent aggrecan degradation or specific cleavage at this site by endogenously generated aggrecanase at concentrations up to 100 microM. Taken together these data suggest that MMP-8 does not represent cartilage aggrecanase.
Arner
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EC
Inflammatory Diseases Research, The DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880, USA.
Decicco
C P
CP
Cherney
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R
Tortorella
M D
MD
D053674:Aggrecans; D000409:Alanine; D000818:Animals; D001665:Binding Sites; D002356:Cartilage / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D005973:Glutamine; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D037181:Lectins, C-Type; D020784:Matrix Metalloproteinase 8; D011092:Polyethylene Glycols / Q000494:pharmacology; D011509:Proteoglycans / Q000378:metabolism*
D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D007375:Interleukin-1; D037181:Lectins, C-Type; D011509:Proteoglycans; D005973:Glutamine; D011092:Polyethylene Glycols; D010450:Endopeptidases; D017364:Collagenases; D020784:Matrix Metalloproteinase 8; C083641:aggrecanase; D000409:Alanine