Characterization of the 46-kDa Intermediates of Matrix Metalloproteinase 3 (Stromelysin 1) Obtained by Site-directed Mutation of Phenylalanine 83 (*)
- From the (1)Departments of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160-7421 and the
- (2)Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48202
- **To whom correspondence should be addressed: Dept. of Biochemistry & Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160-7421. Tel.: 913-588-7079; Fax: 913-588-7440.
Abstract
The precursor of matrix metalloproteinase 3 (MMP-3/stromelysin 1) is activated in vitro by proteinases or mercurial compounds by stepwise processes which include the initial formation of short-lived intermediates
and the subsequent intermolecular cleavage of the His
-Phe
bond to generate the fully activated mature MMP-3 (Nagase, H., Enghild, J. J., Suzuki, K., and Salvesen, G.(1990) Biochemistry 29, 5783-5789). To study the enzymatic properties of the intermediates we have mutated either His
or Phe
to Arg to obtain a stable MMP-3 intermediate. The mutant proteins were expressed in Chinese hamster ovary K-1 cells using
a mammalian expression system. The proMMP-3(H82R) mutant was activated by chymotrypsin, elastase, and 4-aminophenylmercuric
acetate to the 45-kDa MMP-3 with similar mechanism and kinetics as the wild-type. In contrast, the activation of the proMMP-3(F83R)
mutant by proteinases or 4-aminophenylmercuric acetate resulted in 46-kDa forms, which retained 13, 14, or 15 amino acids
of the pro-domain depending on the activators. The proteinase-activated MMP-3(F83R) intermediates exhibited little enzymatic
activity, but they were partially active after treatment with SH-reacting reagents. These molecules could bind to the tissue
inhibitor of metalloproteinases-1 and α
-macroglobulin. However, the SH group of Cys
of the intermediates was not modified by SH-reagents, indicating that the enzymatic activity generated by SH-reagents resulted
from molecular perturbation of the enzyme rather than their interaction with Cys
. When gelatin and transferrin were digested with the 46-kDa intermediates the products were different from those generated
by the wild-type MMP-3, suggesting an alteration in substrate specificity. The treatment of proMMP-3 with trypsin resulted
in the formation of a 45-kDa MMP-3 with an NH
-terminal Thr
, whose activity and substrate specificity were similar to those of the 46-kDa MMP-3(F83R) obtained from the proMMP-3(F83R)
mutant. These observations indicate that the correct processing at the His
-Phe
bond is critical for expression of the full activity and the specificity of MMP-3.
Footnotes
-
↵* This work was supported by National Institute of Health Grants AR39189 and AR40994 (to H. N.) and HD24442 (to M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- MMP
-
matrix metalloproteinase
- APMA
-
4-aminophenylmercuric acetate
- α
M -
α
-macroglobulin
- Cm-Tf
-
reduced, carboxymethylated transferrin
- DFP
-
diisopropyl fluorophosphate
- HNE
-
human neutrophil elastase
- DTNB
-
5,5′-dithio-bis(2-nitrobenzoic acid)
- NFF-3
-
(7-methoxycoumarin-4-yl)acetyl-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys-(2,4-dinitrophenyl)-NH
- TIMP
-
tissue inhibitor of metalloproteinase
- CHO
-
Chinese hamster ovary
- PAGE
-
polyacrylamide gel electrophoresis.
-
- Received October 24, 1995.
- Revision received February 12, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
