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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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ADAM Proteins / antagonists & inhibitors
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ADAM Proteins / genetics
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ADAM Proteins / metabolism*
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ADAMTS4 Protein
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ADAMTS5 Protein
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Biocatalysis / drug effects
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Cell Line
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Glycosylation
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Humans
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Kinetics
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Matrix Metalloproteinase 1 / genetics
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Matrix Metalloproteinase 1 / metabolism
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Matrix Metalloproteinase 2 / genetics
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Matrix Metalloproteinase 2 / metabolism
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Matrix Metalloproteinase 3 / genetics
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Matrix Metalloproteinase 3 / metabolism
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Matrix Metalloproteinase Inhibitors
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Procollagen N-Endopeptidase / antagonists & inhibitors
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Procollagen N-Endopeptidase / genetics
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Procollagen N-Endopeptidase / metabolism*
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Protein Binding / physiology
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Protein Interaction Domains and Motifs / physiology*
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Recombinant Proteins / pharmacology
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Tissue Inhibitor of Metalloproteinase-3 / biosynthesis
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Tissue Inhibitor of Metalloproteinase-3 / genetics
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Tissue Inhibitor of Metalloproteinase-3 / metabolism*
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Tissue Inhibitor of Metalloproteinase-3 / pharmacology
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Transfection
Substances
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Matrix Metalloproteinase Inhibitors
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Recombinant Proteins
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TIMP3 protein, human
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Tissue Inhibitor of Metalloproteinase-3
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ADAM Proteins
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ADAMTS5 Protein
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ADAMTS5 protein, human
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Procollagen N-Endopeptidase
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Matrix Metalloproteinase 3
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Matrix Metalloproteinase 2
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Matrix Metalloproteinase 1
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ADAMTS4 Protein
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ADAMTS4 protein, human