The murine goblet cell protein mCLCA3 is a zinc-dependent metalloprotease with autoproteolytic activity

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.

Fig. 1.. Inhibition of precursor cleavage in E157Q mutants. Expression of wild-type proteins and E157Q mutants of both mCLCA3 and pCLCA1 in cell lysates of transiently transfected HEK293 cells was examined by immunoblot. The precursor molecules had a size of 110 kDa while the subunits were detected at 75 kDa.

Fig. 2.. Uncleaved mCLCA3E157Q precursor passes through the Golgi and is released into the cell supernatant. Immunoblot analyses of cell lysates and supernatants of HEK293 cells transfected with wild-type mCLCA3-pcDNA3.1 or the mCLCA3E157Q mutant. Samples were treated with Endo H (H) or PNGase F (F) or remained untreated (-). The precursor molecule of 110 kDa shifted to 100 kDa in size while the amino-terminal subunit shifted from 75 to 72 kDa.

Fig. 3.. Cleavage of the mCLCA3 precursor protein is zinc-dependent. Upper panel, samples from membrane preparations of transiently mCLCA3-overexpressing HEK293 cells were incubated with either 1.0 mM magnesium (Mg²⁺), calcium (Ca²⁺), zinc (Zn²⁺) or a combination of these ions, separated by SDS-PAGE and examined by immunoblot using the anti-mCLCA3-amino-terminal antibody α-p3b2. Lower panel, cleavage of the mCLCA3 precursor was quantified by chemiluminescence using Quantity One software for digital quantification of relative band intensities. Data represent the mean values and standard deviations from three independent experiments. *= p < 0.01.

Fig. 4.. Cleavage inhibition of the mCLCA3 precursor by metalloprotease inhibitors. (A) Upper panel, immunoblot of crude membrane extracts of transiently mCLCA3-overexpressing HEK293 cells incubated in PBS supplemented with Mg²⁺, Ca²⁺ and Zn²⁺ and the indicated inhibitors. EDTA, EGTA, 1,10-phenanthroline and TPEN are chelating agents that inhibit metalloproteases, and proteoblock ™ (w/o EDTA) is a broad-spectrum inhibitor of serine, cysteine and aspartic proteases. Lower panel, cleavage of the mCLCA3 precursor was quantified by chemiluminescence using Quantity One software. (B) Upper panel, inhibition of the cleavage of the mCLCA3 precursor in crude membrane extracts by 1.0 mM marimastat, a broad-spectrum matrix metalloprotease inhibitor, was analyzed by immunoblot. Samples treated with EDTA and proteoblock™ served as controls. Lower panel, cleavage of the mCLCA3 precursor was quantified by chemiluminescence using Quantity One software. (C) Immunoblot analysis of cell lysates (L) and supernatants (S) of transiently mCLCA3-overexpressing HEK293 cells treated with TPEN or marimastat. *= p < 0.01.

Fig. 5.. mCLCA3-YFP is capable of cleaving mCLCA3E157Q via intermolecular auto-proteolysis. Immunoblot analysis of supernatants of HEK293 cells co-transfected with different combinations of plasmids encoding mutant mCLCA3 E157Q, mCLCA3-YFP, wild-type mCLCA3 or vector alone (pcDNA3.1; mock). The predicted sizes of the uncleaved precursor of the mutant mCLCA3E157Q and the cleaved carboxyterminal subunits of mCLCA3 and mCLCA3- YFP protein are depicted in the upper panel. The results are representative of three independent experiments.

Fig. 6.. mCLCA3 is cleaved between R695 and A696. (A) Section of a MALDI-TOF mass spectrum of the precursor and the amino-terminal subunit after digestion with AspN protease. The peptide with 1308.74 m/z was identified by MS/MS sequencing as amino acids 685-695 of mCLCA3. a.u. = arbitrary unit. (B) Edman-degra-dation of the immunoprecipitated mCLCA3-YFP carboxyterminal subunit identified A696 to lead the carboxy-terminal subunit.