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

Plog S, Klymiuk N, Binder S, Van Hook MJ, Thoreson WB, Gruber AD, Mundhenk L - PLoS ONE (2015)

Bottom Line: 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.Moreover, the naturally occurring variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathology, Faculty of Veterinary Medicine, Freie Universit├Ąt Berlin, Berlin, Germany.

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 variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

No MeSH data available.


Related in: MedlinePlus

The porcine CLCA proteins CLCA1, CLCA4a and CLCA4b occupy different cellular niches in the intestinal tract.The CLCA4a protein (blue) had previously been detected exclusively in small intestinal villus enterocytes. In contrast, its paralog CLCA4b (orange) is expressed only in small and large intestinal crypt epithelial cells where the porcine CFTR protein (green) is also expressed. Moreover, a third CLCA protein, the fully secreted CLCA1 (brown) is expressed by goblet cells. Thus, each porcine CLCA protein appears to occupy a distinct functional niche in small and large intestine. Interestingly, the combination of CLCA4a (blue) and CLCA4b (orange) represent the expression pattern of their single murine ortholog CLCA4a (compare Bothe et al. 2008). G = goblet cell, NG = non-goblet enterocyte.
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pone.0140050.g006: The porcine CLCA proteins CLCA1, CLCA4a and CLCA4b occupy different cellular niches in the intestinal tract.The CLCA4a protein (blue) had previously been detected exclusively in small intestinal villus enterocytes. In contrast, its paralog CLCA4b (orange) is expressed only in small and large intestinal crypt epithelial cells where the porcine CFTR protein (green) is also expressed. Moreover, a third CLCA protein, the fully secreted CLCA1 (brown) is expressed by goblet cells. Thus, each porcine CLCA protein appears to occupy a distinct functional niche in small and large intestine. Interestingly, the combination of CLCA4a (blue) and CLCA4b (orange) represent the expression pattern of their single murine ortholog CLCA4a (compare Bothe et al. 2008). G = goblet cell, NG = non-goblet enterocyte.

Mentions: Interestingly, the murine Clca4a protein, formerly termed mClca6, is expressed in enterocytes both at the villous tips and deep crypts [17], arguing that both microenvironments are served by the same Clca protein in mice. The duplication of the two porcine CLCA4 genes and the cellular expression of their products in the same tissue environment (Fig 6) thus support the notion of species-specific divergences of CLCA genes which has previously been observed in other species and for other members of the CLCA family of proteins [36]. Whether the change of expressing cell type goes along with a similar or changed protein function or regulatory pathways will have to be established in the future. A second obvious difference between the pig CLCA4a and CLCA4b genes and the mouse Clca4 ortholog clearly lies in their different expressions in the respiratory tract: while the porcine CLCA4a was convincingly detected at the apical membranes of tracheal and bronchial epithelial cells [28], neither the duplicated CLCA4b protein nor the mouse CLCA4 orthologous proteins [17] were found to be expressed in the trachea or lungs. Details on the cellular expression of the human CLCA4 protein have not yet been reported and await further studies. The porcine CLCA1 is another CLCA protein expressed in the intestine which, however, has been found in mucus-producing goblet cells only but not in non-goblet cell enterocytes [27]. The close proximity of the two CLCA4 variants in pigs to this third CLCA protein further supports the notion of different CLCA members covering different cellular and probably functional niches in the intestine [17] (Fig 6).


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

Plog S, Klymiuk N, Binder S, Van Hook MJ, Thoreson WB, Gruber AD, Mundhenk L - PLoS ONE (2015)

The porcine CLCA proteins CLCA1, CLCA4a and CLCA4b occupy different cellular niches in the intestinal tract.The CLCA4a protein (blue) had previously been detected exclusively in small intestinal villus enterocytes. In contrast, its paralog CLCA4b (orange) is expressed only in small and large intestinal crypt epithelial cells where the porcine CFTR protein (green) is also expressed. Moreover, a third CLCA protein, the fully secreted CLCA1 (brown) is expressed by goblet cells. Thus, each porcine CLCA protein appears to occupy a distinct functional niche in small and large intestine. Interestingly, the combination of CLCA4a (blue) and CLCA4b (orange) represent the expression pattern of their single murine ortholog CLCA4a (compare Bothe et al. 2008). G = goblet cell, NG = non-goblet enterocyte.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608703&req=5

pone.0140050.g006: The porcine CLCA proteins CLCA1, CLCA4a and CLCA4b occupy different cellular niches in the intestinal tract.The CLCA4a protein (blue) had previously been detected exclusively in small intestinal villus enterocytes. In contrast, its paralog CLCA4b (orange) is expressed only in small and large intestinal crypt epithelial cells where the porcine CFTR protein (green) is also expressed. Moreover, a third CLCA protein, the fully secreted CLCA1 (brown) is expressed by goblet cells. Thus, each porcine CLCA protein appears to occupy a distinct functional niche in small and large intestine. Interestingly, the combination of CLCA4a (blue) and CLCA4b (orange) represent the expression pattern of their single murine ortholog CLCA4a (compare Bothe et al. 2008). G = goblet cell, NG = non-goblet enterocyte.
Mentions: Interestingly, the murine Clca4a protein, formerly termed mClca6, is expressed in enterocytes both at the villous tips and deep crypts [17], arguing that both microenvironments are served by the same Clca protein in mice. The duplication of the two porcine CLCA4 genes and the cellular expression of their products in the same tissue environment (Fig 6) thus support the notion of species-specific divergences of CLCA genes which has previously been observed in other species and for other members of the CLCA family of proteins [36]. Whether the change of expressing cell type goes along with a similar or changed protein function or regulatory pathways will have to be established in the future. A second obvious difference between the pig CLCA4a and CLCA4b genes and the mouse Clca4 ortholog clearly lies in their different expressions in the respiratory tract: while the porcine CLCA4a was convincingly detected at the apical membranes of tracheal and bronchial epithelial cells [28], neither the duplicated CLCA4b protein nor the mouse CLCA4 orthologous proteins [17] were found to be expressed in the trachea or lungs. Details on the cellular expression of the human CLCA4 protein have not yet been reported and await further studies. The porcine CLCA1 is another CLCA protein expressed in the intestine which, however, has been found in mucus-producing goblet cells only but not in non-goblet cell enterocytes [27]. The close proximity of the two CLCA4 variants in pigs to this third CLCA protein further supports the notion of different CLCA members covering different cellular and probably functional niches in the intestine [17] (Fig 6).

Bottom Line: 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.Moreover, the naturally occurring variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathology, Faculty of Veterinary Medicine, Freie Universit├Ąt Berlin, Berlin, Germany.

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 variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

No MeSH data available.


Related in: MedlinePlus