Alpha2-macroglobulin is a novel substrate for ADAMTS-4 and ADAMTS-5 and represents an endogenous inhibitor of these enzymes.
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.
Tortorella
Micky D
MD
Pfizer Global Research and Development, Chesterfield, Missouri 63017, USA. tortorella@pfizer.com
Arner
Elizabeth C
EC
Hills
Robert
R
Easton
Alan
A
Korte-Sarfaty
Jennifer
J
Fok
Kam
K
Wittwer
Arthur J
AJ
Liu
Rui-Qin
RQ
Malfait
Anne-Marie
AM
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*
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