title: The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3.
abstract: We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better K(i) value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3.
authors: Kennedy Institute of Rheumatology, Imperial College London, Hammersmith, London, W6 8LH, UK.
authors: Linda
authors: L
authors: Troeberg
authors: Kazunari
authors: K
authors: Fushimi
authors: Simone D
authors: SD
authors: Scilabra
authors: Hiroyuki
authors: H
authors: Nakamura
authors: Vincent
authors: V
authors: Dive
authors: Ida B
authors: IB
authors: Thøgersen
authors: Jan J
authors: JJ
authors: Enghild
authors: Hideaki
authors: H
authors: Nagase
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071121:ADAMTS4 Protein; D000071116:ADAMTS5 Protein; D055162:Biocatalysis / Q000187:drug effects; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D011348:Procollagen N-Endopeptidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection
chemical_list: D061965:Matrix Metalloproteinase Inhibitors; D011994:Recombinant Proteins; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D011348:Procollagen N-Endopeptidase; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human