The Matrix Metalloproteinase 9 (MMP-9) Hemopexin Domain Is a Novel Gelatin Binding Domain and Acts as an Antagonist

• Published in: The Journal of biological chemistry 2002-12, Vol.277 (52), p.50326-50332
• Main Authors: Roeb, Elke, Schleinkofer, Karin, Kernebeck, Thomas, Pötsch, Stephan, Jansen, Bettina, Behrmann, Iris, Matern, Siegfried, Grötzinger, Joachim
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Cells, Cultured; Circular Dichroism; Cloning, Molecular; Escherichia coli; Gelatin - metabolism; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Hemopexin - chemistry; Hemopexin - isolation & purification; Kinetics; Matrix Metalloproteinase 9 - chemistry; Matrix Metalloproteinase 9 - genetics; Matrix Metalloproteinase 9 - metabolism; Matrix Metalloproteinase Inhibitors; Mice; Molecular Sequence Data; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M207446200

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathioneS-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be Kd = 2,4 × 10−8m, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.