Metalloproteinase Activity Secreted by Fibrogenic Cells in the Processing of Prolysyl Oxidase
POTENTIAL ROLE OF PROCOLLAGEN C-PROTEINASE (*)
- Maria V. Panchenko(1),
- William G. Stetler-Stevenson(2),
- Olga V. Trubetskoy(3),
- Stephen N. Gacheru(1) and
- Herbert M. Kagan(1)(§)
- From the (1)Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, the
- (2)Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892, and
- (3)OsteoArthritis Sciences, Inc., Cambridge, Massachusetts 02139
- § To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118. Tel.: 617-638-4064; Fax: 617-638-4459.
Abstract
Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme
in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase
was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase
activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor
of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested
for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned
fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH
-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The
C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence
(Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in
the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify
inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.
Footnotes
-
↵* This work was supported by National Institutes of Health Grants HL 13262 and HL 46902. 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:
- DMEM
-
Dulbecco's modified Eagle's medium
- RFL6
-
rat fetal lung fibroblasts
- SMC
-
neonatal rat smooth muscle cells
- S4 cells
-
RS485 cells stably transfected with the lysyl oxidase expression vector
- MMP
-
matrix metalloproteinase
- MMP-1
-
interstitial collagenase
- MMP-3
-
stromelysin
- MMP-9
-
92-kDa gelatinase
- MMP-2
-
72-kDa gelatinase
- C-proteinase
-
carboxyl-terminal procollagen proteinase
- PMSF
-
phenylmethylsulfonyl fluoride
- FP
-
fusion protein
- TIMP-2
-
tissue inhibitor of metalloproteinase-2
- PAGE
-
polyacrylamyde gel electrophoresis
- HS peptide
-
HS-CH
-CH(CH
-CH (CH
))
-CO-Phe-Ala-HH
.
-
- Received August 17, 1995.
- Revision received January 9, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
