title: The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist.
abstract: 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 glutathione S-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 K(d) = 2,4 x 10(-8) m, 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.
authors.affiliation: Medizinische Klinik III and the Institut für Biochemie der Rheinisch-Westfälischen Technischen Hochschule Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany. eroeb@ukaachen.de
authors.forename: Elke
authors.initials: E
authors.lastname: Roeb
authors.forename: Karin
authors.initials: K
authors.lastname: Schleinkofer
authors.forename: Thomas
authors.initials: T
authors.lastname: Kernebeck
authors.forename: Stephan
authors.initials: S
authors.lastname: Pötsch
authors.forename: Bettina
authors.initials: B
authors.lastname: Jansen
authors.forename: Iris
authors.initials: I
authors.lastname: Behrmann
authors.forename: Siegfried
authors.initials: S
authors.lastname: Matern
authors.forename: Joachim
authors.initials: J
authors.lastname: Grötzinger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D002942:Circular Dichroism; D003001:Cloning, Molecular; D005780:Gelatin / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006466:Hemopexin / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D061965:Matrix Metalloproteinase Inhibitors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005780:Gelatin; D006466:Hemopexin; D005982:Glutathione Transferase; D020780:Matrix Metalloproteinase 9