PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 °C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.
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May 1999
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Research Article|
April 26 1999
Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain
Joseph F. SUCIC;
Joseph F. SUCIC
1
*Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington VT 05405, U.S.A.
1To whom correspondence should be addressed. Present address: Biology Department, University of Michigan-Flint, 303 East Kearsley St., Flint, MI 48502-1950, U.S.A. (e-mail jsucic@flint.umich.edu).
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Joan M. MOEHRING;
Joan M. MOEHRING
*Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington VT 05405, U.S.A.
†Vermont Cancer Center, University of Vermont, Burlington, VT 05405, U.S.A.
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Noel M. INOCENCIO;
Noel M. INOCENCIO
2
*Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington VT 05405, U.S.A.
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Jason W. LUCHINI;
Jason W. LUCHINI
*Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington VT 05405, U.S.A.
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Thomas J. MOEHRING
Thomas J. MOEHRING
*Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington VT 05405, U.S.A.
†Vermont Cancer Center, University of Vermont, Burlington, VT 05405, U.S.A.
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Publisher: Portland Press Ltd
Received:
August 14 1998
Revision Received:
January 22 1999
Accepted:
February 12 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 339 (3): 639–647.
Article history
Received:
August 14 1998
Revision Received:
January 22 1999
Accepted:
February 12 1999
Citation
Joseph F. SUCIC, Joan M. MOEHRING, Noel M. INOCENCIO, Jason W. LUCHINI, Thomas J. MOEHRING; Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain. Biochem J 1 May 1999; 339 (3): 639–647. doi: https://doi.org/10.1042/bj3390639
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