Biochemical characterization of the catalytic domain of human matrix metalloproteinase 19. Evidence for a role as a potent basement membrane degrading enzyme.
We have recently cloned MMP-19, a novel matrix metalloproteinase, which, due to unique structural features, was proposed to represent the first member of a new MMP subfamily (Pendás, A. M., Knäuper, V. , Puente, X. S., Llano, E., Mattei, M. G., Apte, S., Murphy, G., and López-Otin, C. (1997) J. Biol. Chem. 272, 4281-4286). A recombinant COOH-terminal deletion mutant of MMP-19 (proDelta(260-508)MMP-19), comprising the propeptide and the catalytic domain, was expressed in Escherichia coli, refolded, and purified. Interestingly, we found that proDelta(260-508)MMP-19 has the tendency to autoactivate, whereby the Lys(97)-Tyr(98) peptide bond is hydrolyzed, resulting in free catalytic domain. Mutation of two residues (Glu(88) --> Pro and Pro(90) --> Val) within the propeptide latency motif did not prevent autoactivation but the autolysis rate was somewhat reduced. Analysis of the substrate specificity revealed that the catalytic domain of MMP-19 was able to hydrolyze the general MMP substrate Mca-Pro-Leu-Gly-Dpa-Ala-Arg-NH(2) and, with higher efficiency, the stromelysin substrate Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH(2). Kinetic analysis of the interactions of the catalytic domain of MMP-19 with the natural MMP inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), showed strong inhibition using TIMP-2, TIMP-3, and TIMP-4, while TIMP-1 was less efficient. We also demonstrated that synthetic hydroxamic acid-based compounds efficiently inhibited the enzyme. The catalytic domain of MMP-19 was able to hydrolyze the basement membrane components type IV collagen, laminin, and nidogen, as well as the large tenascin-C isoform, fibronectin, and type I gelatin in vitro, suggesting that MMP-19 is a potent proteinase capable of hydrolyzing a broad range of extracellular matrix components. Neither the catalytic domain nor the full-length MMP-19 was able to degrade triple-helical collagen. Finally, and in contrast to studies with other MMPs, MMP-19 catalytic domain was not able to activate any of the latent MMPs tested in vitro.
Stracke
J O
JO
School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom. j.stracke@uea.ac.uk
Hutton
M
M
Stewart
M
M
Pendás
A M
AM
Smith
B
B
López-Otin
C
C
Murphy
G
G
Knäuper
V
V
D000595:Amino Acid Sequence; D001485:Basement Membrane / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D053505:Matrix Metalloproteinases, Secreted; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D053505:Matrix Metalloproteinases, Secreted; D008666:Metalloendopeptidases; C108362:matrix metalloproteinase 19