The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3

Linda Troeberg, Kazunari Fushimi, Simone D Scilabra, Hiroyuki Nakamura, Vincent Dive, Ida B Thøgersen, Jan J Enghild, Hideaki Nagase

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50 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)463-469
Number of pages7
JournalMatrix Biology
Issue number8
Early online date28 Jul 2009
Publication statusPublished - Oct 2009


  • ADAM Proteins/antagonists & inhibitors
  • ADAMTS4 Protein
  • ADAMTS5 Protein
  • Biocatalysis/drug effects
  • Cell Line
  • Glycosylation
  • Humans
  • Kinetics
  • Matrix Metalloproteinase 1/genetics
  • Matrix Metalloproteinase 2/genetics
  • Matrix Metalloproteinase 3/genetics
  • Matrix Metalloproteinase Inhibitors
  • Procollagen N-Endopeptidase/antagonists & inhibitors
  • Protein Binding/physiology
  • Protein Interaction Domains and Motifs/physiology
  • Recombinant Proteins/biosynthesis
  • Tissue Inhibitor of Metalloproteinase-3/biosynthesis
  • Transfection

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