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The CLCA (chloride channel regulators, calcium-activated) family contains a group of heterologous proteins which induce a Ca2+-dependent chloride current at epithelial inter-faces. The CLCA do not represent real chlorid channels but, according to new insights, they have an indirect effect on so far unknown proteins.
The human CLCA4 which is pre-dominantly located in the intestinal tract and in the respiratory tract seems to play an important role in cystic fibrosis (CF). Possible functional mechanisms of hCLCA4 have not been investigated to date. Its murine ortholog mCLCA6 is colocalized with the murine cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of colonic crypt cells, which may argue for a potential role as modulator of the ―alternative chloride current― in CF. However, considering the expression patterns of these two closely related CLCA members, species-specific differences are obvious because mCLCA6 is, unlike hCLCA4, not expressed in CF-relevant respiratory epithelial cells. Thus, functional differences of these two CLCA orthologs have to be assumed at the moment. The limited transferability of data from one species to the other restricts the use of animal models for translational research in human diseases such as CF. The CF mouse model which has been used as an animal model for the investigation of CF so far shows considerable variations compared to the human CF phenotype. The predominant CF lung phenotype of human patients is nearly totally missing in the murine CF model. The usefulness of the CF mouse model for the investigation of this severe disease is thus limited. Recently, CF pig models have been successfully generated which seem to reflect the human CF phenotype more closely.
The intention of this work was to characterize biochemical properties of the porcine ortholog to hCLCA4 and mCLCA6, pCLCA4a, and to determine its expression patterns. With this knowledge, a comparison between the species should be possible, and species-specific differences of CLCA orthologs will become obvious. RT-PCR analyses revealed strong pCLCA4a mRNA expression in the upper respiratory tract, in the intestinal tract and considerably lower in the eye and in the uterus. Using new generated specific anti-pCLCA4a antibodies, the pCLCA4a protein was immunohisto-chemically detected in the bronchial ciliated epithelium of the upper respiratory tract and in the brush border of enterocytes in the apical third of small intestine villi. Computer-aided 100 predictions of the protein structure as well as biochemical investigations by immunoblotting analyses suggest a way of intracellular movement for pCLCA4a that is similar to the secretory pathway of mCLCA6. According to this, pCLCA4a is posttranscriptionally regulated similar to other CLCA proteins, being cleaved into a 110 kDa-amino-terminal and two 52 kDa- and 42 kDa-carboxy-terminal products and glycosylated in the endoplasmic reticulum and Golgi apparatus. pCLCA4a is obviously transported to the apical cell membrane where it is found anchored by a carboxy-terminal transmembrane domain. Only the amino-terminal cleavage product of the pCLCA4a protein was detected in the extracellular medium. Despite some species-specific differences between the pCLCA4a expression pattern and that of its human and murine orthologs, the expression patterns of hCLCA4 and pCLCA4a in CF relevant tissues have more in common than hCLCA4 and mCLCA6 have.
This supports the idea that porcine CF models are more suitable to study the role of hCLCA4 in CF than currently available murine models are. In this respect, the results of this work could help in investigating the species-specific differences of the CF phenotype. Future studies could use these results as a basis to investigate the functional mechanisms of these proteins in the transepithelial anion conductance and to discover their potential modulatory functions in diseases such as cystic fibrosis.