Metalloprotease-Disintegrin MDC9: Intracellular Maturation and Catalytic Activity

doi:10.1074/jbc.274.6.3531

Metalloprotease-Disintegrin MDC9: Intracellular Maturation and Catalytic Activity*

From the ^‡Cellular Biochemistry and Biophysics Program, ^**Molecular Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, ^¶Cell Biology and Molecular Biology Program, Graduate School of the Cornell University Medical College, New York, New York 10021, the ^§Department of Molecular Biochemistry and the ^§§Department of Analytical Chemistry, Glaxo Wellcome Research and Development Inc., Research Triangle Park, North Carolina 27709, and the ^‡Laboratori de Recerca Oncologica, Hospital General, Psg. Vall d’Hebron, 08035 Barcelona, Spain

Abstract

Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that are known to function in fertilization, myoblast fusion, neurogenesis, and ectodomain shedding of tumor necrosis factor (TNF)-α. Here we report the analysis of the intracellular maturation and catalytic activity of the widely expressed metalloprotease disintegrin MDC9. Our results suggest that the pro-domain of MDC9 is removed by a furin-type pro-protein convertase in the secretory pathway before the protein emerges on the cell surface. The soluble metalloprotease domain of MDC9 cleaves the insulin B-chain, a generic protease substrate, providing the first evidence that MDC9 is catalytically active. Soluble MDC9 appears to have distinct specificities for cleaving candidate substrate peptides compared with the TNF-α convertase (TACE/ADAM17). The catalytic activity of MDC9 can be inhibited by hydroxamic acid-type metalloprotease inhibitors in the low nanomolar range, in one case with up to 50-fold selectivity for MDC9 versus TACE. Peptides mimicking the predicted cysteine-switch region of MDC9 or TACE inhibit both enzymes in the low micromolar range, providing experimental evidence for regulation of metalloprotease disintegrins via a cysteine-switch mechanism. Finally, MDC9 is shown to become phosphorylated when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate, a known inducer of protein ectodomain shedding. This work implies that removal of the inhibitory pro-domain of MDC9 by a furin-type pro-protein convertase in the secretory pathway is a prerequisite for protease activity. After pro-domain removal, additional steps, such as protein kinase C-dependent phosphorylation, may be involved in regulating the catalytic activity of MDC9, which is likely to target different substrates than the related TNF-α-convertase.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant R55GM51988, by a grant from Glaxo Wellcome (to C. P. B.), by National Science Foundation Grant DBI-9420123 (to P. T.), and by the Memorial Sloan-Kettering Cancer Center Support Grant NCI-P30-CA-08748.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.
↵‖ Supported in part by the Ford Foundation.
↵¶¶ To whom correspondence should be addressed: Cellular Biochemistry and Biophysics Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, Box 368, 1275 York Ave., New York, NY 10021. Tel.: 212-639-2915; Fax: 212-717-3047; E-mail:c-blobel{at}ski.mskcc.org.
↵2 M. Moss, submitted for publication.
↵3 R. Black, personal communication.
↵4 M. Moss, unpublished observation.
Abbreviations:

MDC

metalloprotease/disintegrin/cysteine-rich proteins

ADAMs

a disintegrinand metalloprotease

TNF

tumor necrosis factor

TNFR

TNF receptor

PMA

phorbol 12-myristate 13-acetate

TACE

TNF-α convertase

β-APP

β-amyloid precursor protein

PAGE

polyacrylamide gel electrophoresis

PNGase F

peptideN-glcosidase

PBS

phosphate-buffered saline

TGF-α

transforming growth factor α

MMP

matrix metalloproteinase

MP

metalloprotease

endo H

endoglycosidase H

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

SPA

scintillation proximity assay

Dnp

dinitrophenyl

MALDI-TOF

matrix-assisted laser-desorption/ionization time-of-flight

CHO

Chinese hamster ovary.
- Received October 6, 1998.
- Revision received November 18, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.