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.

IL-13–induced airway mucus production is attenuated by MAPK13 inhibition

“…Moreover, subsequent findings were more consistent with the proposal that CLCA1 effects on chloride transport may be based on increasing conductance of endogenous CaCCs (40). It further appears (based on analysis of crude membrane preparations) that secreted CLCA proteins are first processed intracellularly into N-terminal and C-terminal peptide fragments (18,39,(41)(42)(43)(44). The present findings support this alternative proposal as well, in that CLCA1 is colocalized within the cell along with MUC5AC in secretory granules and is secreted to the apical cell surface in response to IL-13.…”

Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation

“…First, we have demonstrated that CLCA proteins represent a novel class of zincin metalloproteases capable of self-and crosscleavage. It should be noted that previous studies have attempted to address the issue of CLCA metalloprotease activity (28,29); however, these studies were based on experiments utilizing CLCA proteins found in crude membrane fractions. The authors claimed that purified CLCA proteins could not be produced.…”

hCLCA1 DNA Vaccine Suppresses Cell Hyperplasia and Mucin Expression of Goblet Cells in vitro

“…Secreted hCLCA1 and mCLCA3 had zinc-dependent autoproteolytic activity which might cleave other proteins [23,24]. Thus, CLCA proteins are no longer thought to function as chloride channels and might serve crucial roles in calcium signaling.…”

“…Both mCLCA3 and hCLCA1 were reported to be cleaved into two subunits (a ∼90-kDa N-terminal and ∼30-kDa C-terminal products) that are secreted soluble proteins [20,21] and Yurtsever et al [22] have demonstrated that CLCA family proteins have self-cleavage as well as cross-cleavage zinc-dependent proteolytic activity which unmasked the N-terminal fragment of CLCA to activate CLCA-mediated chloride transport. Recent studies demonstrated that the secreted hCLCA1 and mCLCA3 had zinc-dependent autoproteolytic activity that might cleave other proteins [23,24]. In the process of asthma, AECs are able to produce numerous cytokines in an autocrine or paracrine manner, which in turn regulates asthma pathology.…”