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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 Universität 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 CLCA4b protein is exclusively located in apical membranes of intestinal crypt epithelial cells and shows high variations in response to the genomic deletion of CLCA4b.(A) In wild type pigs without the genomic deletion of CLCA4b, the protein is invariably located at apical membranes of non-goblet and goblet cells in the crypts of the small (upper left) and large (lower left) intestines. In heterozygous pigs, the protein is similarly distributed in the small intestine (upper central) whereas it is completely absent from the large intestine (lower central). In pigs with the genomic deletion of CLCA4b, no CLCA4b protein was detected, neither in the small (upper right) nor in the large intestine (lower right). Immunohistochemistry using antibody CLCA4b-N-1 diluted 1:8,000. Bars: 80 μm. (B) Quantitative RT-PCR expression analysis from jejunum (black) and colon tissues (white) revealed CLCA4b expression twice as high in the jejunum than in the colon of CLCA4b wild type pigs (left). When compared to wild type pigs, CLCA4b mutant pigs (right pattern) had a markedly decreased amount of CLCA4b mRNA in both the jejunum and colon. While CLCA4b mRNA copy numbers were also reduced in the colon of heterozygous compared to wild type pigs, copy numbers of CLCA4b in the jejunum were markedly increased (central pattern). Results are mean +/- SEM.
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pone.0140050.g004: The CLCA4b protein is exclusively located in apical membranes of intestinal crypt epithelial cells and shows high variations in response to the genomic deletion of CLCA4b.(A) In wild type pigs without the genomic deletion of CLCA4b, the protein is invariably located at apical membranes of non-goblet and goblet cells in the crypts of the small (upper left) and large (lower left) intestines. In heterozygous pigs, the protein is similarly distributed in the small intestine (upper central) whereas it is completely absent from the large intestine (lower central). In pigs with the genomic deletion of CLCA4b, no CLCA4b protein was detected, neither in the small (upper right) nor in the large intestine (lower right). Immunohistochemistry using antibody CLCA4b-N-1 diluted 1:8,000. Bars: 80 μm. (B) Quantitative RT-PCR expression analysis from jejunum (black) and colon tissues (white) revealed CLCA4b expression twice as high in the jejunum than in the colon of CLCA4b wild type pigs (left). When compared to wild type pigs, CLCA4b mutant pigs (right pattern) had a markedly decreased amount of CLCA4b mRNA in both the jejunum and colon. While CLCA4b mRNA copy numbers were also reduced in the colon of heterozygous compared to wild type pigs, copy numbers of CLCA4b in the jejunum were markedly increased (central pattern). Results are mean +/- SEM.

Mentions: To elucidate the tissue and cellular expression pattern of CLCA4b, antibody p4b-N-1 was used for immunohistochemical protein localization in various formalin-fixed, paraffin-embedded tissues. In pigs carrying the wild-type form of the gene, the CLCA4b protein was exclusively detected in apical membranes of small and large intestinal crypt epithelial cells (Fig 4A) but not in any other tissue. The specificity of this signal was confirmed by pre-incubation of the antibody with its specific peptide and the use of the preimmune serum which both resulted in complete loss or strong reduction of the signal.


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 CLCA4b protein is exclusively located in apical membranes of intestinal crypt epithelial cells and shows high variations in response to the genomic deletion of CLCA4b.(A) In wild type pigs without the genomic deletion of CLCA4b, the protein is invariably located at apical membranes of non-goblet and goblet cells in the crypts of the small (upper left) and large (lower left) intestines. In heterozygous pigs, the protein is similarly distributed in the small intestine (upper central) whereas it is completely absent from the large intestine (lower central). In pigs with the genomic deletion of CLCA4b, no CLCA4b protein was detected, neither in the small (upper right) nor in the large intestine (lower right). Immunohistochemistry using antibody CLCA4b-N-1 diluted 1:8,000. Bars: 80 μm. (B) Quantitative RT-PCR expression analysis from jejunum (black) and colon tissues (white) revealed CLCA4b expression twice as high in the jejunum than in the colon of CLCA4b wild type pigs (left). When compared to wild type pigs, CLCA4b mutant pigs (right pattern) had a markedly decreased amount of CLCA4b mRNA in both the jejunum and colon. While CLCA4b mRNA copy numbers were also reduced in the colon of heterozygous compared to wild type pigs, copy numbers of CLCA4b in the jejunum were markedly increased (central pattern). Results are mean +/- SEM.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140050.g004: The CLCA4b protein is exclusively located in apical membranes of intestinal crypt epithelial cells and shows high variations in response to the genomic deletion of CLCA4b.(A) In wild type pigs without the genomic deletion of CLCA4b, the protein is invariably located at apical membranes of non-goblet and goblet cells in the crypts of the small (upper left) and large (lower left) intestines. In heterozygous pigs, the protein is similarly distributed in the small intestine (upper central) whereas it is completely absent from the large intestine (lower central). In pigs with the genomic deletion of CLCA4b, no CLCA4b protein was detected, neither in the small (upper right) nor in the large intestine (lower right). Immunohistochemistry using antibody CLCA4b-N-1 diluted 1:8,000. Bars: 80 μm. (B) Quantitative RT-PCR expression analysis from jejunum (black) and colon tissues (white) revealed CLCA4b expression twice as high in the jejunum than in the colon of CLCA4b wild type pigs (left). When compared to wild type pigs, CLCA4b mutant pigs (right pattern) had a markedly decreased amount of CLCA4b mRNA in both the jejunum and colon. While CLCA4b mRNA copy numbers were also reduced in the colon of heterozygous compared to wild type pigs, copy numbers of CLCA4b in the jejunum were markedly increased (central pattern). Results are mean +/- SEM.
Mentions: To elucidate the tissue and cellular expression pattern of CLCA4b, antibody p4b-N-1 was used for immunohistochemical protein localization in various formalin-fixed, paraffin-embedded tissues. In pigs carrying the wild-type form of the gene, the CLCA4b protein was exclusively detected in apical membranes of small and large intestinal crypt epithelial cells (Fig 4A) but not in any other tissue. The specificity of this signal was confirmed by pre-incubation of the antibody with its specific peptide and the use of the preimmune serum which both resulted in complete loss or strong reduction of the signal.

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 Universität 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