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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

Both antibodies against the amino-terminus of CLCA4b detect two specific bands in CLCA4b transfected cell lysate.(A) Two antibodies against CLCA4b were generated, both against peptides corresponding to the proposed amino-terminal cleavage product of the protein. The length and mass of the theoretically truncated protein CLCA4b were calculated, and antibody p4b-N-2 was designed to also recognize a truncated CLCA4b protein in the immunoblot. SS = signal sequence; TMD = transmembrane domain; C = carboxy-terminus; N = amino-terminus. (B) Antibody p4b-N-1 recognized an approx. 140 kDa precursor protein as well as a 120 kDa amino-terminal cleavage product in the cell lysate of HEK293 cells transiently transfected with the CLCA4b ORF in pcDNA3.1 (left). No specific bands were detected when cells were transfected with the pcDNA3.1 vector alone (mock). Preabsorption of antibody p4b-N-1 using 20 μg/ml of its specific peptide resulted in complete abolishment of the specific bands (right pattern). (C) Antibody p4b-N-1 failed to detect any specific bands in cell lysates of cells transfected with other porcine CLCA family members, CLCA1, CLCA2 or CLCA4b. In cells transfected with the CLCA4b clone carrying the additional intron sequence due to the genomic deletion (CLCA4b mut), no specific protein bands were detected, arguing for a complete inhibition of protein translation in these cells. In comparison, both the precursor protein and the amino-terminal cleavage product are clearly visible when cells were transfected with the intact CLCA4b clone without a deletion (CLCA4b wt). (D) The results of the antibody p4b-N-1 were confirmed by antibody p4b-N-2 which recognized two band of the same size in cell lysates from HEK293 cells transiently transfected with the CLCA4b ORF in pcDNA3.1 (left pattern). No specific bands were detected in lysates from mock transfected cells. Preimmune serum served as negative control and failed to reveal any specific bands (right pattern). (E) No specific bands were detected by antibody p4b-N-2 when cells transfected with CLCA1, CLCA2, or CLCA4a clones were subjected to immunoblotting. Interestingly, although this antibody would be capable of detecting a possibly shortened CLCA4b protein, no specific bands were detected in cell lysates from cells transfected with the CLCA4b (mut) clone, arguing for a complete translation stop of the CLCA4b protein in case of the mutation.
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pone.0140050.g003: Both antibodies against the amino-terminus of CLCA4b detect two specific bands in CLCA4b transfected cell lysate.(A) Two antibodies against CLCA4b were generated, both against peptides corresponding to the proposed amino-terminal cleavage product of the protein. The length and mass of the theoretically truncated protein CLCA4b were calculated, and antibody p4b-N-2 was designed to also recognize a truncated CLCA4b protein in the immunoblot. SS = signal sequence; TMD = transmembrane domain; C = carboxy-terminus; N = amino-terminus. (B) Antibody p4b-N-1 recognized an approx. 140 kDa precursor protein as well as a 120 kDa amino-terminal cleavage product in the cell lysate of HEK293 cells transiently transfected with the CLCA4b ORF in pcDNA3.1 (left). No specific bands were detected when cells were transfected with the pcDNA3.1 vector alone (mock). Preabsorption of antibody p4b-N-1 using 20 μg/ml of its specific peptide resulted in complete abolishment of the specific bands (right pattern). (C) Antibody p4b-N-1 failed to detect any specific bands in cell lysates of cells transfected with other porcine CLCA family members, CLCA1, CLCA2 or CLCA4b. In cells transfected with the CLCA4b clone carrying the additional intron sequence due to the genomic deletion (CLCA4b mut), no specific protein bands were detected, arguing for a complete inhibition of protein translation in these cells. In comparison, both the precursor protein and the amino-terminal cleavage product are clearly visible when cells were transfected with the intact CLCA4b clone without a deletion (CLCA4b wt). (D) The results of the antibody p4b-N-1 were confirmed by antibody p4b-N-2 which recognized two band of the same size in cell lysates from HEK293 cells transiently transfected with the CLCA4b ORF in pcDNA3.1 (left pattern). No specific bands were detected in lysates from mock transfected cells. Preimmune serum served as negative control and failed to reveal any specific bands (right pattern). (E) No specific bands were detected by antibody p4b-N-2 when cells transfected with CLCA1, CLCA2, or CLCA4a clones were subjected to immunoblotting. Interestingly, although this antibody would be capable of detecting a possibly shortened CLCA4b protein, no specific bands were detected in cell lysates from cells transfected with the CLCA4b (mut) clone, arguing for a complete translation stop of the CLCA4b protein in case of the mutation.

Mentions: In silico analyses of the CLCA4b amino acid sequence using several prediction programs suggested a transmembrane domain between aa 882 and 908, with a carboxy-terminal intracellular domain and an amino-terminal extracellular tail (Fig 3A).


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)

Both antibodies against the amino-terminus of CLCA4b detect two specific bands in CLCA4b transfected cell lysate.(A) Two antibodies against CLCA4b were generated, both against peptides corresponding to the proposed amino-terminal cleavage product of the protein. The length and mass of the theoretically truncated protein CLCA4b were calculated, and antibody p4b-N-2 was designed to also recognize a truncated CLCA4b protein in the immunoblot. SS = signal sequence; TMD = transmembrane domain; C = carboxy-terminus; N = amino-terminus. (B) Antibody p4b-N-1 recognized an approx. 140 kDa precursor protein as well as a 120 kDa amino-terminal cleavage product in the cell lysate of HEK293 cells transiently transfected with the CLCA4b ORF in pcDNA3.1 (left). No specific bands were detected when cells were transfected with the pcDNA3.1 vector alone (mock). Preabsorption of antibody p4b-N-1 using 20 μg/ml of its specific peptide resulted in complete abolishment of the specific bands (right pattern). (C) Antibody p4b-N-1 failed to detect any specific bands in cell lysates of cells transfected with other porcine CLCA family members, CLCA1, CLCA2 or CLCA4b. In cells transfected with the CLCA4b clone carrying the additional intron sequence due to the genomic deletion (CLCA4b mut), no specific protein bands were detected, arguing for a complete inhibition of protein translation in these cells. In comparison, both the precursor protein and the amino-terminal cleavage product are clearly visible when cells were transfected with the intact CLCA4b clone without a deletion (CLCA4b wt). (D) The results of the antibody p4b-N-1 were confirmed by antibody p4b-N-2 which recognized two band of the same size in cell lysates from HEK293 cells transiently transfected with the CLCA4b ORF in pcDNA3.1 (left pattern). No specific bands were detected in lysates from mock transfected cells. Preimmune serum served as negative control and failed to reveal any specific bands (right pattern). (E) No specific bands were detected by antibody p4b-N-2 when cells transfected with CLCA1, CLCA2, or CLCA4a clones were subjected to immunoblotting. Interestingly, although this antibody would be capable of detecting a possibly shortened CLCA4b protein, no specific bands were detected in cell lysates from cells transfected with the CLCA4b (mut) clone, arguing for a complete translation stop of the CLCA4b protein in case of the mutation.
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Related In: Results  -  Collection

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

pone.0140050.g003: Both antibodies against the amino-terminus of CLCA4b detect two specific bands in CLCA4b transfected cell lysate.(A) Two antibodies against CLCA4b were generated, both against peptides corresponding to the proposed amino-terminal cleavage product of the protein. The length and mass of the theoretically truncated protein CLCA4b were calculated, and antibody p4b-N-2 was designed to also recognize a truncated CLCA4b protein in the immunoblot. SS = signal sequence; TMD = transmembrane domain; C = carboxy-terminus; N = amino-terminus. (B) Antibody p4b-N-1 recognized an approx. 140 kDa precursor protein as well as a 120 kDa amino-terminal cleavage product in the cell lysate of HEK293 cells transiently transfected with the CLCA4b ORF in pcDNA3.1 (left). No specific bands were detected when cells were transfected with the pcDNA3.1 vector alone (mock). Preabsorption of antibody p4b-N-1 using 20 μg/ml of its specific peptide resulted in complete abolishment of the specific bands (right pattern). (C) Antibody p4b-N-1 failed to detect any specific bands in cell lysates of cells transfected with other porcine CLCA family members, CLCA1, CLCA2 or CLCA4b. In cells transfected with the CLCA4b clone carrying the additional intron sequence due to the genomic deletion (CLCA4b mut), no specific protein bands were detected, arguing for a complete inhibition of protein translation in these cells. In comparison, both the precursor protein and the amino-terminal cleavage product are clearly visible when cells were transfected with the intact CLCA4b clone without a deletion (CLCA4b wt). (D) The results of the antibody p4b-N-1 were confirmed by antibody p4b-N-2 which recognized two band of the same size in cell lysates from HEK293 cells transiently transfected with the CLCA4b ORF in pcDNA3.1 (left pattern). No specific bands were detected in lysates from mock transfected cells. Preimmune serum served as negative control and failed to reveal any specific bands (right pattern). (E) No specific bands were detected by antibody p4b-N-2 when cells transfected with CLCA1, CLCA2, or CLCA4a clones were subjected to immunoblotting. Interestingly, although this antibody would be capable of detecting a possibly shortened CLCA4b protein, no specific bands were detected in cell lysates from cells transfected with the CLCA4b (mut) clone, arguing for a complete translation stop of the CLCA4b protein in case of the mutation.
Mentions: In silico analyses of the CLCA4b amino acid sequence using several prediction programs suggested a transmembrane domain between aa 882 and 908, with a carboxy-terminal intracellular domain and an amino-terminal extracellular tail (Fig 3A).

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