title: TIMP-3 inhibition of ADAMTS-4 (Aggrecanase-1) is modulated by interactions between aggrecan and the C-terminal domain of ADAMTS-4.
abstract: ADAMTS-4 (aggrecanase-1) is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In this study, we developed a sensitive fluorescence resonance energy transfer peptide assay with a K(m) in the 10 microm range and utilized this assay to demonstrate that inhibition of full-length ADAMTS-4 by full-length TIMP-3 (a physiological inhibitor of metalloproteinases) is enhanced in the presence of aggrecan. Our data indicate that this interaction is mediated largely through the binding of glycosaminoglycans (specifically chondroitin 6-sulfate) of aggrecan to binding sites in the thrombospondin type 1 motif and spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. The results of this study therefore indicate that the cartilage environment can modulate the function of enzyme-inhibitor systems and could have relevance for therapeutic approaches to aggrecanase modulation.
authors.affiliation: GlaxoSmithKline, New Frontiers Research Park, Harlow, Essex CM19 5AW, United Kingdom.
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Wayne
authors.forename: Su-Jun
authors.initials: SJ
authors.lastname: Deng
authors.forename: Augustin
authors.initials: A
authors.lastname: Amour
authors.forename: Satty
authors.initials: S
authors.lastname: Borman
authors.forename: Rosalie
authors.initials: R
authors.lastname: Matico
authors.forename: H Luke
authors.initials: HL
authors.lastname: Carter
authors.forename: Gillian
authors.initials: G
authors.lastname: Murphy
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors*; D000071121:ADAMTS4 Protein; D053674:Aggrecans / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002809:Chondroitin Sulfates / Q000737:chemistry; D010450:Endopeptidases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011348:Procollagen N-Endopeptidase / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000737:chemistry / Q000502:physiology*
chemical_list: D053674:Aggrecans; D004791:Enzyme Inhibitors; D019717:Tissue Inhibitor of Metalloproteinase-3; D002809:Chondroitin Sulfates; D010450:Endopeptidases; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human; C083641:aggrecanase