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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 biology : journal of the International Society for Matrix Biology

Publication Date

10/2009

Volume

28

Pages

463 - 469

Addresses

Kennedy Institute of Rheumatology, Imperial College London, Hammersmith, London, W6 8LH, UK.

Keywords

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