Intracellular Maturation of the Mouse Metalloprotease Disintegrin MDC15*
- From the ‡Cellular Biochemistry and Biophysics Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Abstract
Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that play a role in fertilization, myoblast fusion, neuronal development, and cleavage of the membrane-anchored cytokine tumor necrosis factor-α. Here, we report the cloning and cDNA sequencing of the mouse metalloprotease disintegrin MDC15 and an analysis of its processing in the secretory pathway. A notable difference between mMDC15 and its putative human orthologue (hMDC15, metargidin) is the presence of the peptide sequence TDDC instead of the RGDC found in the disintegrin domain of hMDC15. In a Western blot analysis the majority of mMDC15 was found to lack the pro-domain in all mouse tissues examined. Pulse-chase experiments in transiently transfected COS-7 cells suggest that mMDC15 is processed by a pro-protein convertase in a late Golgi compartment, since (i) addition of brefeldin A or monensin blocks pro-domain removal, (ii) all detectable processed mMDC15 is endoglycosidase H -resistant, and (iii) a recombinant soluble form of the trans-Golgi network pro-protein convertase furin can mimic mMDC15 processing in vitro.Cell-surface trypsinization revealed that more than half of mature mMDC15 is intracellular. Immunolocalization provided evidence for a strong perinuclear accumulation in a region resembling the trans-Golgi network and/or endosomal compartments. This study provides the first characterization of the intracellular processing of a metalloprotease disintegrin, and highlights the potential role of pro-protein convertases in removal of the inhibitory pro-domain. These results further suggest possible intracellular functions for mMDC15, such as in protein maturation, in addition to a potential role in cell-surface proteolysis or cell adhesion.
Footnotes
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↵* This work was funded in part by National Science Foundation Grant MCB-9631601 (to C. P. B.) and by 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF006196.
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↵§ Participant in the tri-institutional (Cornell/Rockefeller University/Memorial Sloan-Kettering Cancer Center) M.D./Ph.D. Training Program, New York, NY 10021.
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↵¶ Supported in part by National Institutes of Health Training Grant 5T32GM07739-17 and by the Robert Wood Johnson, Jr., Charitable Trust Endowment Fund.
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↵‖ Present address: Mojave Therapeutics Inc., 715 Old Saw Mill River Rd., Tarrytown, NY 10591.
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↵** 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.
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↵2 R. Fuller, personal communication.
- Abbreviations:
- endo H
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endoglycosidase H
- PBS
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phosphate-buffered saline
- PAGE
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polyacrylamide gel electrophoresis
- PNGase F
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peptide:N-glycosidase F
- MES
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4-morpholineethanesulfonic acid
- ConA
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concanavalin A
- GST
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glutathioneS-transferase.
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- Received March 12, 1998.
- Revision received July 22, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
