Biochemical Characterization of Human Collagenase-3 (*)
- From the (1)Strangeways Research Laboratory, Department of Cell and Molecular Biology and
- (2)Department of Cell Adhesion and Signalling, Worts' Causeway, Cambridge CB1 4RN, United Kingdom, the
- (3)Universidad de Oviedo, Departamento de Biologia Funcional, 33006 Oviedo, Spain,
- (4)Celltech Ltd., 216 Bath Rd., Slough SL1 4EN, United Kingdom
- § To whom correspondence should be addressed. Tel.: 44-1223-243231; Fax: 44-1223-411609.
Abstract
The cDNA of a novel matrix metalloproteinase, collagenase-3 (MMP-13) has been isolated from a breast tumor library (Freije,
J. M. P., Diez-Itza, I., Balbin, M., Sanchez, L. M., Blasco, R., Tolivia, J., and López-Otin, C.(1994) J. Biol. Chem. 269, 16766-16773), and a potential role in tumor progression has been proposed for this enzyme. In order to establish the
possible role of collagenase-3 in connective tissue turnover, we have expressed and purified recombinant human procollagenase-3
and characterized the enzyme biochemically. The purified procollagenase-3 was shown to be glycosylated and displayed a M
of 60,000, the N-terminal sequence being LPLPSGGD, which is consistent with the cDNA-predicted sequence. The proenzyme was
activated by p-aminophenylmercuric acetate or stromelysin, yielding an intermediate form of M
50,000, which displayed the N-terminal sequence L
EVTGK. Further processing resulted in cleavage of the Glu
-Tyr
peptide bond to the final active enzyme (M
48,000). Trypsin activation of procollagenase-3 also generated a Tyr
N terminus, but it was evident that the C-terminal domain was rapidly lost, and hence the collagenolytic activity diminished.
Analysis of the substrate specificity of collagenase-3 revealed that soluble type II collagen was preferentially hydrolyzed,
while the enzyme was 5 or 6 times less efficient at cleaving type I or III collagen. Fibrillar type I collagen was cleaved
with comparable efficiency to the fibroblast and neutrophil collagenases (MMP-1 and MMP-8), respectively. Unlike these collagenases,
gelatin and the peptide substrates Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH
and Mca-Pro-Cha-Gly-Nva-His-Ala-Dpa-NH
were efficiently hydrolyzed as well, as would be predicted from the similarities between the active site sequence of collagenase-3
(MMP-13) and the gelatinases A and B. Active collagenase-3 was inhibited in a 1:1 stoichiometric fashion by the tissue inhibitors
of metalloproteinases, TIMP-1, TIMP-2, and TIMP-3. These results suggest that in vivo collagenase-3 could play a significant role in the turnover of connective tissue matrix constituents.
Footnotes
-
↵* This work was supported in part by the Arthritis and Rheumatism Council, United Kingdom, by a Wellcome Trust Travelling Fellowship (to V. K.) and by Comision Interministerial de Ciencia y Tecnologia Spain Project SAF94-0892 (to C. L.-O.). 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
- TIMP
-
tissue inhibitor of metalloproteimase
- Mca
-
(7-methoxycoumarin-4-yl)acetyl
- Cha
-
3-cyclohexylalanyl
- Nva
-
norvalyl
- Dpa
-
N-3-(2, 4-dinitrophenyl)-L-2,3-diaminopropionyl
- Dnp
-
2,4-dinitrophenyl
- Nma
-
Nmethylanthranilyl
- APMA
-
p-aminophenylmercuric acetate
- PAGE
-
polyacrylamide gel electrophoresis
- CT1399
-
N4-hydroxy-N1-(1-(S-(morpholinosulfonylaminoethylaminoacarbonyl)-2-cyclohexylethyl)-2-(R)(4-chlorophenylpropyl)succinamide
- CT1847
-
N4-hydroxy-N1-(1-(S)methylaminocarbonyl-2-methylthiopropyl)-2-(R)-(2-methylpropyl)succinamide
- HPLC
-
high performance liquid chromatography
- NSO
-
nonsecreter zero
- TPCK
-
L-tosylamido-2-phenylethyl chloromethyl ketone.
-
↵2V. Knäuper and G. Murphy, unpublished results.
-
↵3V. Knäuper, C. Lopez-Otin, and G. Murphy, manuscript in preparation.
-
↵4J. O'Connell, personal communication.
-
- Received July 14, 1995.
- Revision received October 24, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
