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. 2012 Mar;33(3):251-7.
doi: 10.1007/s10059-012-2217-1. Epub 2012 Feb 15.

Impaired autoproteolytic cleavage of mCLCA6, a murine integral membrane protein expressed in enterocytes, leads to cleavage at the plasma membrane instead of the endoplasmic reticulum

Melanie K Bothe  1 , Lars Mundhenk, Carol L Beck, Matthias Kaup, Achim D Gruber
Affiliations

Impaired autoproteolytic cleavage of mCLCA6, a murine integral membrane protein expressed in enterocytes, leads to cleavage at the plasma membrane instead of the endoplasmic reticulum

Melanie K Bothe et al. Mol Cells. 2012 Mar.

Abstract

CLCA proteins (calcium-activated chloride channel regulators) have been linked to diseases involving secretory disorders, including cystic fibrosis (CF) and asthma. They have been shown to modulate endogenous chloride conductance, possibly by acting as metalloproteases. Based on the differential processing of the subunits after posttranslational cleavage, two subgroups of CLCA proteins can be distinguished. In one subgroup, both subunits are secreted, in the other group, the carboxy-terminal subunit possesses a transmembrane segment, resulting in shedding of only the amino-terminal subunit. Recent data on the post-translational cleavage and proteolytic activity of CLCA are limited to secreted CLCA. In this study, we characterized the cleavage of mCLCA6, a murine CLCA possessing a transmembrane segment. As for secreted CLCA, the cleavage in the endoplasmic reticulum was not observed for a protein with the E157Q mutation in the HEXXH motif of mCLCA6, suggesting that this mutant protein and secreted CLCA family members share a similar autoproteolytic cleavage mechanism. In contrast to secreted CLCA proteins with the E157Q mutation, the uncleaved precursor of the mCLCA6E157Q mutant reached the plasma membrane, where it was cleaved and the amino-terminal subunit was shed into the supernatant. Using crude membrane fractions, we showed that cleavage of the mCLCA6E157Q protein is zinc-dependent and sensitive to metalloprotease inhibitors, suggesting secondary cleavage by a metalloprotease. Interestingly, anchorage of mCLCA6E157Q to the plasma membrane is not essential for its secondary cleavage, because the mCLCA6(Δ™)E157Q mutant still underwent cleavage. Our data suggest that the processing of CLCA proteins is more complex than previously recognized.

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Figures

Fig. 1.
Fig. 1.
Reduction but not elimination of the cleavage of the precursor of the mutant protein mCLCA6E157Q. HEK293 cells were transfected with plasmids expressing wild-type mCLCA6 or the mCLCA6E157Q mutant. Cells were lysed after 24 h and analyzed by immunoblotting with antibodies directed against the aminoterminal (αm6-N-1ap) or carboxy-terminal (αm6-C-1b) subunit of the mCLCA6 protein, respectively. Asterisk (*) = immature precursor molecule.
Fig. 2.
Fig. 2.
The uncleaved mCLCA6E157Q precursor passes through the Golgi and undergoes cleavage in a post-Golgi-compartment, and the amino-terminal subunit is shed into the supernatant. (A) The lysates of HEK293 cells transfected with an expression vector encoding mCLCA6 wild-type or the mCLCA6E157Q mutant were treated with Endo H (H) or PNGase F (F) or were left untreated (−). (B) HEK293 cells transiently transfected with an expression vector encoding wild-type mCLCA6 or mCLCA6E157Q or with the pcDNA3.1 vector alone (mock) were subjected to surface biotinylation, lysed and precipitated using high-capacity streptavidin beads. Precipitates were analyzed by immunoblotting using the anti-amino-terminal mCLCA6 antibody. (C) Cells were incubated at pH 2.5 to release non-covalently associated proteins from the plasma membrane into the supernatant. Incubation with PBS served as a negative control.
Fig. 3.
Fig. 3.
Cleavage of the mature mCLCA6E157Q precursor is zinc dependent. (A) The cleavage of the mCLCA6E157Q precursor in membrane preparations upon incubation with 1.0 mM magnesium (Mg2+), calcium (Ca2+), zinc (Zn2+) or a combination of these ions was analyzed by immunoblotting. (B) Immunoblot analysis of the cleavage of the mCLCA6E157Q precursor in membrane preparations was inhibited by various protease inhibitors in the presence of Mg2+, Ca2+ and Zn2+. Incubation in the presence of Mg2+, Ca2+ and Zn2+ without inhibitor served as a negative control.
Fig. 4.
Fig. 4.
Anchorage of mCLCA6E157Q with a transmembrane segment to the plasma membrane is not essential for the cleavage of mCLCA6E157Q. (A) The cleavage of the mCLCA6E157Q precursor in crude membrane extracts upon incubation with 1.0 mM magnesium (Mg2+), calcium (Ca2+) and zinc (Zn2+) with or without the addition of Triton X-100 (Triton) to solubilize membranes was analyzed by immunoblotting. (B) A truncated mCLCA6 protein lacking the transmembrane domain (mCLCA6Δ™) was generated and expressed in HEK293 cells. Samples were analyzed by immunoblotting using the anti-mCLCA6-carboxy-terminal antibody αm6-C-1b. Asterisk (*) = truncated precursor of mCLCA6Δ™ (approximately 120 kDa). (C) Deglycosylation of mCLCA6Δ™ and mCLCA6Δ™ E157Q expressed by HEK293 cells in the cell lysates and in the supernatant. Samples were analyzed by immunoblotting using the anti-mCLCA6-amino-terminal antibody αm6-N-1ap.
Fig. 5.
Fig. 5.
Schematic comparison of the cleavage processes of the wild-type and E157Q mutant forms of mCLCA3 and mCLCA6.
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