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

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  • Linda Troeberg, Denmark
  • Kazunari Fushimi, Denmark
  • Simone D Scilabra, Denmark
  • Hiroyuki Nakamura, Denmark
  • Vincent Dive, Denmark
  • Ida B Thøgersen, Denmark
  • Jan J Enghild
  • Hideaki Nagase, Denmark
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
JournalMatrix Biology
Volume28
Issue8
Pages (from-to)463-9
Number of pages6
ISSN0945-053X
DOIs
Publication statusPublished - 2009

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