Biochemical characterization of the catalytic domain of human matrix metalloproteinase 19. Evidence for a role as a potent basement membrane degrading enzyme.
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Stracke JO, Hutton M, Stewart M, Pendas AM, Smith B, Lopez-Otin C, Murphy G, Knauper V
Biochemical characterization of the catalytic domain of human matrix metalloproteinase 19. Evidence for a role as a potent basement membrane degrading enzyme.
J Biol Chem. 2000 May 19;275(20):14809-16.
- PubMed ID
- 10809722 [ View in PubMed]
- Abstract
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 (Pendas, A. M., Knauper, V. , Puente, X. S., Llano, E., Mattei, M. G., Apte, S., Murphy, G., and Lopez-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.