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Activation of Epithelial Sodium Channels by Mouse Channel Activating Proteases (mCAP) Expressed in Xenopus Oocytes Requires Catalytic Activity of mCAP3 and mCAP2 but not mCAP1
- Source :
- Journal of the American Society of Nephrology. 17:968-976
- Publication Year :
- 2006
- Publisher :
- Ovid Technologies (Wolters Kluwer Health), 2006.
-
Abstract
- Mouse channel activating proteases 1, 2, and 3 (mCAP1, mCAP2, and mCAP3) were described recently as regulators of the epithelial sodium channel (ENaC). The mCAP are membrane-bound serine proteases that are synthesized as inactive proenzymes. To mature into active proteases, they undergo intramolecular cleavage by auto- and/or heterocatalytic processing. Specific antibodies against each mCAP were developed to distinguish between proenzyme and active protease by Western blot analysis. Various point mutations were introduced in the catalytic or protein–protein interacting domains of mCAP and wild-type and mutant enzymes were expressed in the Xenopus oocyte expression system to test for ability to activate ENaC. In mCAP3, an intact catalytic triad was necessary for activation of ENaC but not for intramolecular cleavage of the protease. This suggests a heterocatalytic mechanism. Mutating the catalytic triad of mCAP2 not only abolished ENaC activation completely but also impeded cleavage of the protease. Processing of mCAP2 therefore seems to be autocatalytic. Furthermore, mutations in conserved residues of mCAP2 located in two protein–protein interacting domains significantly modulated ENaC activation. Surprisingly, mCAP1 catalytically inactive mutants were still able to fully activate ENaC, and no evidence of mCAP1 intramolecular cleavage was seen. The presence of an intact glycosylphosphatidylinositol anchor, however, was required. It is concluded that auto- and heterocatalytic requirements are specific for each CAP and that endogenous partners are a necessity for activation of ENaC by mCAP in the Xenopus oocyte expression system.
- Subjects :
- Epithelial sodium channel
Proteases
Recombinant Fusion Proteins
medicine.medical_treatment
Xenopus
In Vitro Techniques
Biology
Cleavage (embryo)
Antibodies
Sodium Channels
Serine
Mice
Xenopus laevis
Catalytic Domain
Catalytic triad
medicine
Animals
Epithelial Sodium Channels
Serpins
chemistry.chemical_classification
Protease
urogenital system
Serine Endopeptidases
Membrane Proteins
General Medicine
biology.organism_classification
Enzyme
Biochemistry
chemistry
Nephrology
Mutagenesis, Site-Directed
Oocytes
Female
Subjects
Details
- ISSN :
- 10466673
- Volume :
- 17
- Database :
- OpenAIRE
- Journal :
- Journal of the American Society of Nephrology
- Accession number :
- edsair.doi.dedup.....78e76977ce2bbec257aff2a5e2a7d57c