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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

Comparison of the genomic structures of CLCA4a and CLCA4b, their promoter regions and the encoded proteins sequences.(A) Albeit having longer intronic sequences than CLCA4a (top panel), the genomic structure of CLCA4b (bottom panel) was highly similar to its paralog with the same number of exons (boxes) and has an open reading frame according to its ortholog. Due to a deletion of one amino acid in exon 2 and a gap of 5 amino acids in exon 14 the translated CLCA4b protein is slightly shorter than the CLCA4a protein with 921 vs. 927 amino acids (aa). (B) The intergenic region between CLCA1 and CLCA4a showed high homology to the orthologous bovine and human regions (top panel). In the intergenic region between CLCA4a and 4b, only the sequence downstream to CLCA4a showed homology to the corresponding bovine and human regions (I) followed by a sequence with no similarity to any sequence of mammalian CLCA loci investigated (II). In contrast, the region proximal to CLCA4b had homology to the intergenic region upstream from the human and bovine CLCA4 gene (III) (bold style: regions with similarity to both species, faint style: regions without similarity to any of the species, boxes: repetitive elements). In regions I and III, parts are conserved among the examined mammalian species and contain numerous putative transcription factor binding sites (TFB). Sequences not fully conserved at TFB sites in distinct species are shaded; sequences not matching the prerequisites of the binding sites in distinct species are densely shaded. C) Alignment of 11 protein sequences clustering to the CLCA4 family revealed three conserved domains, the CLCA_N domain, the van Willebrand-factor A domain and a domain of unknown function (DUF). Sites of positive selection were defined by the codonML software, using the model algorithms 3 (discrete, naïve empirical bayes) and 8 (beta & omega > 1, naïve empirical bayes). Sites that are proposed to be positively selected are marked with a rhombus, sites that are proposed as significantly (p > 95%) or highly significantly (p > 99%) positively selected in both algorithms have two or three rhombi, respectively. In comparison, positions that are unique for CLCA4a or CLCA4b are labeled with triangulars or circles, respectively. A conserved region covering parts of the vWFA domain as well as the downstream sequence is shown in detail. Of the 38 unique sites in pCLCA4b in this region, 15 are located at sites under putative positive selection.
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pone.0140050.g001: Comparison of the genomic structures of CLCA4a and CLCA4b, their promoter regions and the encoded proteins sequences.(A) Albeit having longer intronic sequences than CLCA4a (top panel), the genomic structure of CLCA4b (bottom panel) was highly similar to its paralog with the same number of exons (boxes) and has an open reading frame according to its ortholog. Due to a deletion of one amino acid in exon 2 and a gap of 5 amino acids in exon 14 the translated CLCA4b protein is slightly shorter than the CLCA4a protein with 921 vs. 927 amino acids (aa). (B) The intergenic region between CLCA1 and CLCA4a showed high homology to the orthologous bovine and human regions (top panel). In the intergenic region between CLCA4a and 4b, only the sequence downstream to CLCA4a showed homology to the corresponding bovine and human regions (I) followed by a sequence with no similarity to any sequence of mammalian CLCA loci investigated (II). In contrast, the region proximal to CLCA4b had homology to the intergenic region upstream from the human and bovine CLCA4 gene (III) (bold style: regions with similarity to both species, faint style: regions without similarity to any of the species, boxes: repetitive elements). In regions I and III, parts are conserved among the examined mammalian species and contain numerous putative transcription factor binding sites (TFB). Sequences not fully conserved at TFB sites in distinct species are shaded; sequences not matching the prerequisites of the binding sites in distinct species are densely shaded. C) Alignment of 11 protein sequences clustering to the CLCA4 family revealed three conserved domains, the CLCA_N domain, the van Willebrand-factor A domain and a domain of unknown function (DUF). Sites of positive selection were defined by the codonML software, using the model algorithms 3 (discrete, naïve empirical bayes) and 8 (beta & omega > 1, naïve empirical bayes). Sites that are proposed to be positively selected are marked with a rhombus, sites that are proposed as significantly (p > 95%) or highly significantly (p > 99%) positively selected in both algorithms have two or three rhombi, respectively. In comparison, positions that are unique for CLCA4a or CLCA4b are labeled with triangulars or circles, respectively. A conserved region covering parts of the vWFA domain as well as the downstream sequence is shown in detail. Of the 38 unique sites in pCLCA4b in this region, 15 are located at sites under putative positive selection.

Mentions: The duplication of the CLCA4 gene into CLCA4a and CLCA4b appears to be a unique event in the pig and has not been observed in any other species analyzed so far [28]. A systematic analysis revealed an overall highly similar genetic structure to CLCA4a, albeit with longer intron sequences (Fig 1A). Like CLCA4a and other mammalian CLCA4 genes [29], CLCA4b is also composed of 14 exons and the protein is only six amino acids shorter than pCLCA4a, due to a single amino acid deletion in exon 2 and a second deletion of 5 amino acids in exon 14. To determine the evolutionary history of the porcine CLCA4 duplication, the genomic region between CLCA1 and CLCA3 genes which contain CLCA4a and CLCA4b in the pig and CLCA4 in all other examined mammalian species were compared (Fig 1B). Throughout the entire length of 29 kb, the region between CLCA1 and CLCA4a showed high homology with the corresponding regions in humans and cattle. The downstream region of the CLCA4a gene showed similarity to the corresponding regions between mammalian CLCA4 and CLCA3 genes for 11.6 kb, but then in the pig a region follows that lack any similarity to other mammalian CLCA loci and rather comprises repetitive elements. Further downstream of porcine CLCA4a, only a short region of 0.5 kb, quite upstream of the CLCA4b transcription start site, showed similarity to the region upstream of the bovine CLCA4, suggesting that CLCA4b resulted from a duplication of an initial porcine CLCA4 ancestor gene. Thus, while CLCA4a comprises the original gene with the entire regulatory elements of the ancestor, the regulatory properties of CLCA4b might have been altered as a result of the duplication. Although we have found numerous potential binding sites for transcription factors (TFB) in the upstream region of pCLCA4b, the length of this potential proximal promoter is much shorter than the conserved putative promoters of other mammalian CLCA4, including porcine CLCA4a. On the other hand, putative unique enhancer elements of porcine CLCA4b were identified as conserved and potential regulatory elements that are otherwise located downstream of CLCA4 in other mammalian species are positioned upstream of the porcine CLCA4b as a result of the gene duplication.


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)

Comparison of the genomic structures of CLCA4a and CLCA4b, their promoter regions and the encoded proteins sequences.(A) Albeit having longer intronic sequences than CLCA4a (top panel), the genomic structure of CLCA4b (bottom panel) was highly similar to its paralog with the same number of exons (boxes) and has an open reading frame according to its ortholog. Due to a deletion of one amino acid in exon 2 and a gap of 5 amino acids in exon 14 the translated CLCA4b protein is slightly shorter than the CLCA4a protein with 921 vs. 927 amino acids (aa). (B) The intergenic region between CLCA1 and CLCA4a showed high homology to the orthologous bovine and human regions (top panel). In the intergenic region between CLCA4a and 4b, only the sequence downstream to CLCA4a showed homology to the corresponding bovine and human regions (I) followed by a sequence with no similarity to any sequence of mammalian CLCA loci investigated (II). In contrast, the region proximal to CLCA4b had homology to the intergenic region upstream from the human and bovine CLCA4 gene (III) (bold style: regions with similarity to both species, faint style: regions without similarity to any of the species, boxes: repetitive elements). In regions I and III, parts are conserved among the examined mammalian species and contain numerous putative transcription factor binding sites (TFB). Sequences not fully conserved at TFB sites in distinct species are shaded; sequences not matching the prerequisites of the binding sites in distinct species are densely shaded. C) Alignment of 11 protein sequences clustering to the CLCA4 family revealed three conserved domains, the CLCA_N domain, the van Willebrand-factor A domain and a domain of unknown function (DUF). Sites of positive selection were defined by the codonML software, using the model algorithms 3 (discrete, naïve empirical bayes) and 8 (beta & omega > 1, naïve empirical bayes). Sites that are proposed to be positively selected are marked with a rhombus, sites that are proposed as significantly (p > 95%) or highly significantly (p > 99%) positively selected in both algorithms have two or three rhombi, respectively. In comparison, positions that are unique for CLCA4a or CLCA4b are labeled with triangulars or circles, respectively. A conserved region covering parts of the vWFA domain as well as the downstream sequence is shown in detail. Of the 38 unique sites in pCLCA4b in this region, 15 are located at sites under putative positive selection.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140050.g001: Comparison of the genomic structures of CLCA4a and CLCA4b, their promoter regions and the encoded proteins sequences.(A) Albeit having longer intronic sequences than CLCA4a (top panel), the genomic structure of CLCA4b (bottom panel) was highly similar to its paralog with the same number of exons (boxes) and has an open reading frame according to its ortholog. Due to a deletion of one amino acid in exon 2 and a gap of 5 amino acids in exon 14 the translated CLCA4b protein is slightly shorter than the CLCA4a protein with 921 vs. 927 amino acids (aa). (B) The intergenic region between CLCA1 and CLCA4a showed high homology to the orthologous bovine and human regions (top panel). In the intergenic region between CLCA4a and 4b, only the sequence downstream to CLCA4a showed homology to the corresponding bovine and human regions (I) followed by a sequence with no similarity to any sequence of mammalian CLCA loci investigated (II). In contrast, the region proximal to CLCA4b had homology to the intergenic region upstream from the human and bovine CLCA4 gene (III) (bold style: regions with similarity to both species, faint style: regions without similarity to any of the species, boxes: repetitive elements). In regions I and III, parts are conserved among the examined mammalian species and contain numerous putative transcription factor binding sites (TFB). Sequences not fully conserved at TFB sites in distinct species are shaded; sequences not matching the prerequisites of the binding sites in distinct species are densely shaded. C) Alignment of 11 protein sequences clustering to the CLCA4 family revealed three conserved domains, the CLCA_N domain, the van Willebrand-factor A domain and a domain of unknown function (DUF). Sites of positive selection were defined by the codonML software, using the model algorithms 3 (discrete, naïve empirical bayes) and 8 (beta & omega > 1, naïve empirical bayes). Sites that are proposed to be positively selected are marked with a rhombus, sites that are proposed as significantly (p > 95%) or highly significantly (p > 99%) positively selected in both algorithms have two or three rhombi, respectively. In comparison, positions that are unique for CLCA4a or CLCA4b are labeled with triangulars or circles, respectively. A conserved region covering parts of the vWFA domain as well as the downstream sequence is shown in detail. Of the 38 unique sites in pCLCA4b in this region, 15 are located at sites under putative positive selection.
Mentions: The duplication of the CLCA4 gene into CLCA4a and CLCA4b appears to be a unique event in the pig and has not been observed in any other species analyzed so far [28]. A systematic analysis revealed an overall highly similar genetic structure to CLCA4a, albeit with longer intron sequences (Fig 1A). Like CLCA4a and other mammalian CLCA4 genes [29], CLCA4b is also composed of 14 exons and the protein is only six amino acids shorter than pCLCA4a, due to a single amino acid deletion in exon 2 and a second deletion of 5 amino acids in exon 14. To determine the evolutionary history of the porcine CLCA4 duplication, the genomic region between CLCA1 and CLCA3 genes which contain CLCA4a and CLCA4b in the pig and CLCA4 in all other examined mammalian species were compared (Fig 1B). Throughout the entire length of 29 kb, the region between CLCA1 and CLCA4a showed high homology with the corresponding regions in humans and cattle. The downstream region of the CLCA4a gene showed similarity to the corresponding regions between mammalian CLCA4 and CLCA3 genes for 11.6 kb, but then in the pig a region follows that lack any similarity to other mammalian CLCA loci and rather comprises repetitive elements. Further downstream of porcine CLCA4a, only a short region of 0.5 kb, quite upstream of the CLCA4b transcription start site, showed similarity to the region upstream of the bovine CLCA4, suggesting that CLCA4b resulted from a duplication of an initial porcine CLCA4 ancestor gene. Thus, while CLCA4a comprises the original gene with the entire regulatory elements of the ancestor, the regulatory properties of CLCA4b might have been altered as a result of the duplication. Although we have found numerous potential binding sites for transcription factors (TFB) in the upstream region of pCLCA4b, the length of this potential proximal promoter is much shorter than the conserved putative promoters of other mammalian CLCA4, including porcine CLCA4a. On the other hand, putative unique enhancer elements of porcine CLCA4b were identified as conserved and potential regulatory elements that are otherwise located downstream of CLCA4 in other mammalian species are positioned upstream of the porcine CLCA4b as a result of the gene duplication.

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