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 (Pendas, A.M., Knauper, V., Puente, X. S., Llano, E. , Mattei, M. G., Apte, S., Murphy, G., and Lopez-Otin, C. (1997) J. Biol. C hem. 272, 4281-4286). A recombinant COOH-terminal deletion mutant of MMP-19 (pro Delta(260-508)MMP-19), comprising the propeptide and the catalytic do main, was expressed in Escherichia coil, refolded, and purified. Interestin gly, we found that pro Delta(260-508)MMP-19 has the tendency to autoactivat e, whereby the Lys(97)-Tyr(98) peptide bond is hydrolyzed, resulting in fre e 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 specifi city revealed that the catalytic domain of MMP-19 was able to hydrolyze the general MMP substrate Mca-Pro-Leu-Gly-Dpa-Ala-Arg-NH, and, with higher eff iciency, the stromelysin substrate Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2. Kin etic analysis of the interactions of the catalytic domain of MMP-19 with th e natural MMP inhibitors, the tissue inhibitors of metalloproteinases (TIMP s), showed strong inhibition using TIMP-2, TIMP-3, and TIMP-4, while TIMP-1 was less efficient. We also demonstrated that synthetic hydroxamic acid-ba sed compounds efficiently inhibited the enzyme. The catalytic domain of MMP -19 was able to hydrolyze the basement membrane components type TV collagen , laminin, and nidogen, as well as the large tenascin-C isoform, fibronecti n, and type I gelatin in vitro, suggesting that MMP-19 is a potent proteina se capable of hydrolyzing a broad range of extracellular matrix components. Neither the catalytic domain nor the full-length MMP-19 was able to degrad e triple-helical collagen. Finally, and in contrast to studies with other M MPs, MMP-19 catalytic domain was not able to activate any of the latent MMP s tested in vitro.