Bone Morphogenetic Protein 1 Is an Extracellular Processing Enzyme of the Laminin 5 γ2 Chain

doi:10.1074/jbc.M002345200

Bone Morphogenetic Protein 1 Is an Extracellular Processing Enzyme of the Laminin 5 γ2 Chain*

From the ^‡MGH/Harvard Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, the ^§§Life Science Research Laboratories, Shiseido Research Center, Yokohama, 236-8643 Japan, the ^¶Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53706, the ^**Shriners Hospital for Children, Portland, Oregon 97201

Abstract

Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (α3β3γ2) within the α3 and γ2 chains (1). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first α3 chain cleavage (200-l65 kDa α3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa α3) occurs within the lIla domain, 11 residues N-terminal to the start of domain II. The γ chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the γ2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (2). Recombinant BMP-1 cleaves γ2 in vitro,both within intact laminin 5 and at the predicted site of a recombinant γ2 short arm. α3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin α3 and γ2 chains to be substrates for BMP-1 in vitro. We speculate that γ2 cleavage is required for formation of the laminin 5–6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermal-epidermal junction basement membrane in vivo.

Footnotes

↵* This work was supported by U.S. Public Health Service Grants AR 35689 and AR 38923 (to R. E. B.) and AR 43621 and GM 46846 (to D. S. G.), by a grant from FibroGen, Inc. (South San Francisco, CA), and by the Cutaneous Biology Research Center, which is partially supported by a grant from Shiseido Co. Ltd., Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Present address: Life Science Research Laboratories, Shiseido Research Center, Yokohama, 236-8643 Japan.
↵‖ Present address: Dept. of Pharmachology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854.
↵‡ Present address: Inst. of Urology and Nephrology, University College of London, WIP 7PN United Kingdom.
↵¶¶ To whom correspondence should be addressed: MGH/Harvard Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, MA 02129. Tel.: 617-726-4186; E-mail: bob.burgeson@cbrc2.mgh.harvard.edu.
Published, JBC Papers in Press, May 9, 2000, DOI 10.1074/jbc.M002345200
↵2 H. Welgus and R. E. Burgeson, unpublished results.
↵3 M.-F. Champliaud and R. E. Burgeson, unpublished data.
Abbreviations:

BMP-1

bone morphogenetic protein-1

EGF

epidermal growth factor

DMEM

Dulbecco's modified Eagle's medium

RIPA

radioimmunoprecipitation assay

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PCR

polymerase chain reaction
- Received March 20, 2000.
- Revision received April 28, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.