Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype

Abstract

The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

A global analysis of CNVs in swine using whole genome sequence data and association analysis with fatty acid composition and growth traits

“…The porcine CLCA4 gene has recently been shown to be duplicated into two separated genes, CLCA4a and CLCA4b [55]. …”

Interspecies diversity of chloride channel regulators, calcium-activated 3 genes

“…Of note, the CLCA4 cluster also showed a high evolutionary dynamic with several events of gene duplication in certain species. The tissue expression pattern showed similarities but also marked differences between the species [ 17 ] with duplicates occupying different cellular niches which had been shown for the porcine CLCA4a and 4b [ 18 ]. In contrast to CLCA3 , pseudogene formation seems not to be a common event in the cluster 4 as the murine CLCA4c is described to be the only putative pseudogene in the mouse database.…”

“…In contrast to CLCA3 , pseudogene formation seems not to be a common event in the cluster 4 as the murine CLCA4c is described to be the only putative pseudogene in the mouse database. Interestingly, our group had identified a naturally occurring deletion mutant of porcine CLCA4b among contemporary and ancient pig breeds in high percentages of animals [ 18 ]. This protein silencing did not result in any obvious phenotype.…”

“…For example, we have recently identified a duplication of the CLCA 4 gene that appears to be unique to the pig [ 11 ] as a model for CF research. In contrast to the human CLCA4 protein which is designated CLCA4a in the pig and expressed at the apical membrane of enterocytes at the villus tips and respiratory epithelial cells [ 17 ], the porcine-specific duplication product CLCA4b is selectively expressed in intestinal crypt epithelial cells and has thus adopted a novel cellular expression pattern unlike any other known CLCA protein in other species [ 18 ].…”

Soluble mucus component CLCA1 modulates expression of leukotactic cytokines and BPIFA1 in murine alveolar macrophages but not in bone marrow-derived macrophages

“…Interestingly, in the mouse, two gene duplication events resulted in three apparently functional CLCA3 proteins which are expressed in different cellular niches (Mundhenk et al 2018 ; Patel et al 2009 ). The Clca4 gene is duplicated in the pig and the mouse with two or three, respectively, apparently distinctly regulated proteins, expressed in different cell types and functional niches (Patel et al 2009 ; Plog et al 2015 ). Other mammals appear to possess only a single CLCA4 protein (Plog et al 2015 ).…”