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CLCA proteins, originally termed chloride channels, calcium activated, have on the one hand been described to play a modulatory role in diseases with secretory dysfunctions, predominantly cystic fibrosis (CF), asthma or chronic obstructive pulmonary disease, and, on the other hand, in cancer. They possess a broad tissue expression pattern including mucous membranes of various organs. Members of the CLCA protein family modulate endogenous chloride conductances in cultured cells in a still elusive way. The cellular processing of CLCA proteins may indicate their role as signalling molecules because CLCA proteins are either fully secreted proteins or possess one single transmembrane domain in the carboxy-terminal subunit, while the amino-terminal subunit undergoes ectodomain shedding. A recently identified HEXXH zinc binding motif indicates that CLCA proteins might act as metalloproteases, implying a putative channel activating function.
This study addresses the question of whether CLCA proteins might indeed represent metalloproteases. As all CLCA proteins undergo post-translational cleavage, the cleavage process of murine mCLCA3, a secreted CLCA family member relevant for CF as an example for secretory diseases, was investigated with regard to an autoproteolytic activity. Furthermore, this study includes the comparison of the cleavage processes of a secreted murine CLCA family member with a murine CLCA family member of CF relevant tissues possessing a transmembrane domain. The results were supposed to either corroborate or neglect the hypotheses that CLCA proteins are metalloproteases (hypothesis I) and that the cleavage processes of secreted CLCA proteins differ from those of transmembrane CLCA proteins due to the transmembrane domain (hypothesis II). Therefore, this study will set the stage for investigating the proteolytic role of the putative CLCA proteases in the fields of secretory disorders or cancer.
The tissue expression pattern and the role of the secreted murine CLCA family member mCLCA3 in CF mouse models have been intensively studied in the past. To identify a murine CLCA family member with a transmembrane domain in CF relevant tissues, the tissue expression patterns and cellular processing of murine mCLCA5 and mCLCA6 were investigated. The mCLCA5 protein was expressed in keratinizing keratinocytes of stratified squamous epithelium of skin, cervix, stomach and other organs. Though cell culture experiments and computational analyses suggested a transmembrane domain in the carboxy-terminal subunit, this study failed to identify the carboxy-terminal subunit associated with the plasma membrane in immunohistochemical analyses. The mCLCA5 protein was rather associated with keratohyaline granules. In contrast, the mCLCA6 protein was identified at the apical plasma membrane of non-goblet cell enterocytes in both the small and large intestine but in no other organs. In addition, the presence of a transmembrane domain of mCLCA6 was corroborated via acid treatment. Thus, mCLCA6 was used as a murine transmembrane CLCA family member expressed in intestine as a CF relevant tissue and its cleavage process was compared with that of the secreted mCLCA3 expressed in intestinal goblet cells.
In metalloproteases, the HEXXH zinc-binding amino acid motif is involved in the catalytic process. Mutation E157Q of the HEXXH motif of mCLCA3 or mCLCA6 abrogated cleavage of both proteins in the endoplasmic reticulum, consistent with the previously reported data for hCLCA1. In contrast to mCLCA3E157Q whose uncleaved precursor was fully secreted similar to the wild-type protein, the precursor molecule of mCLCA6E157Q was cleaved at the plasma membrane instead of the endoplasmic reticulum. Both the cleavage of mCLCA3 in the endoplasmic reticulum and the cleavage of mCLCA6E157Q at the plasma membrane were zinc-dependent. In contrast to mCLCA3, however, which was capable of intermolecular autoproteolytic cleavage, the cleaving agent of mCLCA6E157Q at the plasma membrane remains unidentified. The cleavage may therefore be performed by a metalloprotease or represents an autoproteolytic process. Interestingly, the delayed cleavage of mCLCA6E157Q does not require membrane association via a transmembrane domain, raising the question of whether the same is true for other transmembrane CLCA or whether it is specific for mCLCA6.
The results of this study support the hypothesis that at least secreted CLCA proteins represent metalloproteases because their cleavage is zinc-dependent, abrogated after mutation of the HEXXH motif and they are capable of intermolecular proteolysis, possibly even intramolecular cleavage. The in vivo substrates of CLCA proteases of cluster 1 as well as their role in secretory disorders or cancer remain to be established. Furthermore, the mutant mCLCA6E157Q protein underwent rescue cleavage while the mutant mCLCA3E157Q protein did not. This rescue cleavage was not dependent on the transmembrane domain of the protein. Protein characteristics other than the transmembrane domain might therefore be responsible for the different cleavage processes of the mCLCA3E157Q and the mCLCA6E157Q proteins. These protein characteristics should be addressed in the future.
CLCA proteases of cluster 1 may play a role in diseases with secretory dysfunctions including CF as well as in tumor biology. Roles of other metalloproteases in CF include activation of chloride channels, hypersecretion and degradation of mucus. The exact role of CLCA proteases in CF and other diseases requires knowledge on potential substrates and the substrate specificity of CLCA proteases. Investigation of the conserved amino acids at the cleavage site of mCLCA3 could give a first hint towards the substrate specificity of CLCA proteases of cluster 1 and might provide the basis for potential therapeutic interventions in the future.