title: Alpha2-macroglobulin is a novel substrate for ADAMTS-4 and ADAMTS-5 and represents an endogenous inhibitor of these enzymes.
abstract: Osteoarthritis is characterized by the loss of aggrecan and collagen from the cartilage extracellular matrix. The proteinases responsible for the breakdown of cartilage aggrecan include ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). Post-translational inhibition of ADAMTS-4/-5 activity may be important for maintaining normal homeostasis of aggrecan metabolism, and thus, any disruption to this inhibition could lead to accelerated aggrecan breakdown. To date TIMP-3 (tissue inhibitor of matrix metalloproteinases-3) is the only endogenous inhibitor of ADAMTS-4/-5 that has been identified. In the present studies we identify alpha(2)-macroglobulin (alpha(2)M) as an additional endogenous inhibitor of ADAMTS-4 and ADAMTS-5. alpha(2)M inhibited the activity of both ADAMTS-4 and ADAMTS-5 in a concentration-dependent manner, demonstrating 1:1 stoichiometry with second-order rate constants on the order of 10(6) and 10(5) m(-1) s(-1), respectively. Inhibition of the aggrecanases was mediated by proteolysis of the bait region within alpha(2)M, resulting in physical entrapment of these proteinases. Both ADAMTS-4 and ADAMTS-5 cleaved alpha(2)M at Met(690)/Gly(691), representing a novel proteinase cleavage site within alpha(2)M and a novel site of cleavage for ADAMTS-4 and ADAMTS-5. Finally, the use of the anti-neoepitope antibodies to detect aggrecanase-generated alpha(2)M-fragments in synovial fluid was investigated and found to be uninformative.
authors.affiliation: Pfizer Global Research and Development, Chesterfield, Missouri 63017, USA. tortorella@pfizer.com
authors.forename: Micky D
authors.initials: MD
authors.lastname: Tortorella
authors.forename: Elizabeth C
authors.initials: EC
authors.lastname: Arner
authors.forename: Robert
authors.initials: R
authors.lastname: Hills
authors.forename: Alan
authors.initials: A
authors.lastname: Easton
authors.forename: Jennifer
authors.initials: J
authors.lastname: Korte-Sarfaty
authors.forename: Kam
authors.initials: K
authors.lastname: Fok
authors.forename: Arthur J
authors.initials: AJ
authors.lastname: Wittwer
authors.forename: Rui-Qin
authors.initials: RQ
authors.lastname: Liu
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Malfait
mesh_terms: D051722:ADAM Proteins; D000071121:ADAMTS4 Protein; D000071116:ADAMTS5 Protein; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D004791:Enzyme Inhibitors / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008744:Methylamines / Q000737:chemistry; D008969:Molecular Sequence Data; D009300:Nasal Septum / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011348:Procollagen N-Endopeptidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013582:Synovial Fluid / Q000378:metabolism; D013997:Time Factors; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism / Q000502:physiology*
chemical_list: D004791:Enzyme Inhibitors; D000939:Epitopes; D008744:Methylamines; D010455:Peptides; D019717:Tissue Inhibitor of Metalloproteinase-3; D000511:alpha-Macroglobulins; D018698:Glutamic Acid; C027451:methylamine; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D008666:Metalloendopeptidases; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human