Top

Research Article

Split Viewer

Mol. Cells 2011; 32(6): 535-541

Published online December 31, 2011

https://doi.org/10.1007/s10059-011-0158-8

© The Korean Society for Molecular and Cellular Biology

The Murine Goblet Cell Protein mCLCA3 Is a Zinc-Dependent Metalloprotease with Autoproteolytic Activity

Melanie K. Bothe, Lars Mundhenk, Matthias Kaup1, Christoph Weise2, and Achim D. Gruber*

Department of Veterinary Pathology, Freie Universitaet Berlin, Germany, 1Department of Laboratory Medicine and Pathobiochemistry, Charit? Berlin, Germany, 2Institute of Chemistry and Biochemistry, Freie Universitaet Berlin, Germany

Correspondence to : *Correspondence: gruber.achim@vetmed.fu-berlin.de

Received: July 29, 2011; Revised: September 13, 2011; Accepted: September 15, 2011

Abstract

Several members of the CLCA family of proteins, originally named chloride channels, calcium-activated, have been shown to modulate chloride conductance in various cell types via an unknown mechanism. Moreover, the human (h) hCLCA1 is thought to modulate the severity of disease in asthma and cystic fibrosis (CF) patients. All CLCA proteins are post-translationally cleaved into two subunits, and recently, a conserved HEXXH zinc-binding amino acid motif has been identified, suggesting a role for CLCA proteins as metalloproteases. Here, we have characterized the cleavage and autoproteolytic activity of the murine model protein mCLCA3, which represents the murine orthologue of human hCLCA1. Using crude membrane fractions from transfected HEK293 cells, we demonstrate that mCLCA3 cleavage is zinc-dependent and exclusively inhibited by cation-chelating metalloprotease inhibitors. Cellular transport and secretion were not affected in response to a cleavage defect that was introduced by the insertion of an E157Q mutation within the HEXXH motif of mCLCA3. Interspecies conservation of these key results was further confirmed with the porcine (p) orthologue of hCLCA1 and mCLCA3, pCLCA1. Importantly, the mCLCA3E157Q mutant was cleaved after co-transfection with the wild-type mCLCA3 in HEK293 cells, suggesting that an intermolecular autoproteolytic event takes place. Edman degradation and MALDI-TOF-MS of the protein fragments identified a single cleavage site in mCLCA3 between amino acids 695 and 696. The data strongly suggest that secreted CLCA proteins have zinc-dependent autoproteolytic activity and that they may cleave additional proteins.

Keywords CLCA, cystic fibrosis, HEXXH motif, intermolecular auto-proteolysis, metalloprotease

Article

Research Article

Mol. Cells 2011; 32(6): 535-541

Published online December 31, 2011 https://doi.org/10.1007/s10059-011-0158-8

Copyright © The Korean Society for Molecular and Cellular Biology.

The Murine Goblet Cell Protein mCLCA3 Is a Zinc-Dependent Metalloprotease with Autoproteolytic Activity

Melanie K. Bothe, Lars Mundhenk, Matthias Kaup1, Christoph Weise2, and Achim D. Gruber*

Department of Veterinary Pathology, Freie Universitaet Berlin, Germany, 1Department of Laboratory Medicine and Pathobiochemistry, Charit? Berlin, Germany, 2Institute of Chemistry and Biochemistry, Freie Universitaet Berlin, Germany

Correspondence to:*Correspondence: gruber.achim@vetmed.fu-berlin.de

Received: July 29, 2011; Revised: September 13, 2011; Accepted: September 15, 2011

Abstract

Several members of the CLCA family of proteins, originally named chloride channels, calcium-activated, have been shown to modulate chloride conductance in various cell types via an unknown mechanism. Moreover, the human (h) hCLCA1 is thought to modulate the severity of disease in asthma and cystic fibrosis (CF) patients. All CLCA proteins are post-translationally cleaved into two subunits, and recently, a conserved HEXXH zinc-binding amino acid motif has been identified, suggesting a role for CLCA proteins as metalloproteases. Here, we have characterized the cleavage and autoproteolytic activity of the murine model protein mCLCA3, which represents the murine orthologue of human hCLCA1. Using crude membrane fractions from transfected HEK293 cells, we demonstrate that mCLCA3 cleavage is zinc-dependent and exclusively inhibited by cation-chelating metalloprotease inhibitors. Cellular transport and secretion were not affected in response to a cleavage defect that was introduced by the insertion of an E157Q mutation within the HEXXH motif of mCLCA3. Interspecies conservation of these key results was further confirmed with the porcine (p) orthologue of hCLCA1 and mCLCA3, pCLCA1. Importantly, the mCLCA3E157Q mutant was cleaved after co-transfection with the wild-type mCLCA3 in HEK293 cells, suggesting that an intermolecular autoproteolytic event takes place. Edman degradation and MALDI-TOF-MS of the protein fragments identified a single cleavage site in mCLCA3 between amino acids 695 and 696. The data strongly suggest that secreted CLCA proteins have zinc-dependent autoproteolytic activity and that they may cleave additional proteins.

Keywords: CLCA, cystic fibrosis, HEXXH motif, intermolecular auto-proteolysis, metalloprotease

Mol. Cells
Sep 30, 2022 Vol.45 No.9, pp. 603~672
COVER PICTURE
The Target of Rapamycin Complex (TORC) is a central regulatory hub in eukaryotes, which is well conserved in diverse plant species, including tomato (Solanum lycopersicum). Inhibition of TORC genes (SlTOR, SlLST8, and SlRAPTOR) by VIGS (virus-induced gene silencing) results in early fruit ripening in tomato. The red/ orange tomatoes are early-ripened TORC-silenced fruits, while the green tomato is a control fruit. Top, left, control fruit (TRV2-myc); top, right, TRV2-SlLST8; bottom, left, TRV2-SlTOR; bottom, right, TRV2-SlRAPTOR(Choi et al., pp. 660-672).

Share this article on

  • line
  • mail

Molecules and Cells

eISSN 0219-1032
qr-code Download