title: A microplate assay specific for the enzyme aggrecanase.
abstract: We have identified a 41-residue peptide, bracketing the aggrecanase cleavage site of aggrecan, that serves as a specific substrate for this enzyme family. Biotinylation of the peptide allowed its immobilization onto streptavidin-coated plates. Aggrecanase-mediated hydrolysis resulted in an immobilized product that reveals an N-terminal neoepitope, recognized by the specific antibody BC-3. This assay is highly specific for aggrecanases; MMPs were inactive in this assay. Reduction of the peptide size below 30 amino acids resulted in a significant diminution of activity. Using the immobilized 41-residue peptide as a substrate, we have developed a 96-well microplate-based assay that can be conveniently used for high-throughput screening of samples for aggrecanase activity and for discovery of inhibitors of aggrecanase activity.
authors.affiliation: The Bristol-Myers Squibb Company Pharmaceutical Research Institute, Experimental Station, Route 141 and Henry Clay Road, Wilmington, DE 19880, USA.
authors.forename: Jeffery A
authors.initials: JA
authors.lastname: Miller
authors.forename: Rui-Qin
authors.initials: RQ
authors.lastname: Liu
authors.forename: Gary L
authors.initials: GL
authors.lastname: Davis
authors.forename: Michael A
authors.initials: MA
authors.lastname: Pratta
authors.forename: James M
authors.initials: JM
authors.lastname: Trzaskos
authors.forename: Robert A
authors.initials: RA
authors.lastname: Copeland
mesh_terms: D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019921:Biotinylation; D002356:Cartilage / Q000201:enzymology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016326:Extracellular Matrix Proteins; D006868:Hydrolysis; D037181:Lectins, C-Type; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011509:Proteoglycans / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D053674:Aggrecans; D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D010618:Phenanthrolines; D011480:Protease Inhibitors; D011509:Proteoglycans; D004492:Edetic Acid; D010450:Endopeptidases; D020782:Matrix Metalloproteinases; C083641:aggrecanase; C025205:1,10-phenanthroline