title: 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.
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.
authors.affiliation: Department of Veterinary Pathology, Freie Universitaet Berlin, 14163 Berlin, Germany.
authors.forename: Melanie K
authors.initials: MK
authors.lastname: Bothe
authors.forename: Lars
authors.initials: L
authors.lastname: Mundhenk
authors.forename: Carol L
authors.initials: CL
authors.lastname: Beck
authors.forename: Matthias
authors.initials: M
authors.lastname: Kaup
authors.forename: Achim D
authors.initials: AD
authors.lastname: Gruber
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D020895:Enterocytes / Q000201:enzymology / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D015032:Zinc / Q000494:pharmacology
chemical_list: D002614:Chelating Agents; D018118:Chloride Channels; C492979:Clca4a protein, mouse; D011498:Protein Precursors; D045726:Metalloproteases; D015032:Zinc