Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

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 publication

DOI

10.1016/j.matbio.2009.07.005

Type

Journal article

Journal

Matrix Biol

Publication Date

10/2009

Volume

28

Pages

463 - 469

Keywords

ADAM Proteins, ADAMTS4 Protein, ADAMTS5 Protein, Biocatalysis, Cell Line, Glycosylation, Humans, Kinetics, Matrix Metalloproteinase 1, Matrix Metalloproteinase 2, Matrix Metalloproteinase 3, Matrix Metalloproteinase Inhibitors, Procollagen N-Endopeptidase, Protein Binding, Protein Interaction Domains and Motifs, Recombinant Proteins, Tissue Inhibitor of Metalloproteinase-3, Transfection