The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist.
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.
Roeb
Elke
E
Medizinische Klinik III and the Institut für Biochemie der Rheinisch-Westfälischen Technischen Hochschule Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany. eroeb@ukaachen.de
Schleinkofer
Karin
K
Kernebeck
Thomas
T
Pötsch
Stephan
S
Jansen
Bettina
B
Behrmann
Iris
I
Matern
Siegfried
S
Grötzinger
Joachim
J
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
D061965:Matrix Metalloproteinase Inhibitors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005780:Gelatin; D006466:Hemopexin; D005982:Glutathione Transferase; D020780:Matrix Metalloproteinase 9