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Sequence analysis and identification of new isoform of EP4 receptors in different atlantic salmon tissues (Salmo salar L.) and its role in PGE2 induced immunomodulation in vitro.

Guo TC, Gamil AA, Koenig M, Evensen Ø - PLoS ONE (2015)

Bottom Line: In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5' untranslated region was accompanied by silent mutation at nt 668.While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product.We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation.

View Article: PubMed Central - PubMed

Affiliation: Norwegian University of Life Sciences, Faculty of Veterinary Medicine and Biosciences, Sea Lice Research Centre, P.O. Box 8146 Dep., 0033 Oslo, Norway.

ABSTRACT
PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR) called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4) are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5' untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation.

No MeSH data available.


Related in: MedlinePlus

Nucleotide sequences of two EP4 subtypes.Comparison of nucleotide sequence of two subtypes of EP4 gene identified in Atlantic salmon.
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pone.0120483.g001: Nucleotide sequences of two EP4 subtypes.Comparison of nucleotide sequence of two subtypes of EP4 gene identified in Atlantic salmon.

Mentions: While the full length EP4 sequence was easily obtained from several tissues, it could not be amplified from TO cells. We obtained the first half of EP4 when reverse primers were designed in the middle of the published EP4 sequence (data not shown). For the second part of the sequence, we designed several primers using published Atlantic salmon EP4 sequence and based on the fragmented sequences obtained, three PCR fragments constituting most of the sequence of EP4 gene in TO cells were amplified (excluding the 5’ and 3’ end sequences). The sequence for the 5’ and 3’ ends were then attained by 5’ and 3’ RACE as described in materials and methods. The result of full sequencing of EP4 gene in TO cells revealed a different isoform of EP4 (Fig. 1). We therefore designated the first published variant as As-EP4a while the second identified sequence in TO cells was named as As-EP4b. The length of the As-EP4b was found to be 2346 nucleotides compared to 2423 nucleotides for As-EP4a. While the first half of As-EP4b sequence was found to be identical to As-EP4a, many single nucleotide mutations and segmental deletions were found from nt 1027, which is located almost half through the EP4 gene, and onwards (Fig. 1). The coding region was located from nt 267 to nt 1694 and, although the length of nucleotide for both isoforms were different at C-terminal tail, the encoded length of amino acids were found to be identical, 473 amino acid for both isoforms (Fig. 2). Three amino acids were deleted at positions 389–401 in As-EP4b, whereas another three amino acid deletion was found at position 429–431 As-EP4a. Amino acid sequences for both isoforms were also aligned to EP4 amino acid sequences from several species (Fig. 3). Putative transmembrane domains (TM) 1–7 were noted based on a previous study [10]. Alignment result showed that EP4 genes from Atlantic salmon are in general conserved compared to other species at their seven transmembrane domains while differences in N- and C-terminals were remarkable (Fig. 3). High homology was found between intracellular domain 1 and 2 while in intracellular 3, a deletion of 20–30 amino acids was found only in non-mammalian species (fish and chicken).


Sequence analysis and identification of new isoform of EP4 receptors in different atlantic salmon tissues (Salmo salar L.) and its role in PGE2 induced immunomodulation in vitro.

Guo TC, Gamil AA, Koenig M, Evensen Ø - PLoS ONE (2015)

Nucleotide sequences of two EP4 subtypes.Comparison of nucleotide sequence of two subtypes of EP4 gene identified in Atlantic salmon.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383505&req=5

pone.0120483.g001: Nucleotide sequences of two EP4 subtypes.Comparison of nucleotide sequence of two subtypes of EP4 gene identified in Atlantic salmon.
Mentions: While the full length EP4 sequence was easily obtained from several tissues, it could not be amplified from TO cells. We obtained the first half of EP4 when reverse primers were designed in the middle of the published EP4 sequence (data not shown). For the second part of the sequence, we designed several primers using published Atlantic salmon EP4 sequence and based on the fragmented sequences obtained, three PCR fragments constituting most of the sequence of EP4 gene in TO cells were amplified (excluding the 5’ and 3’ end sequences). The sequence for the 5’ and 3’ ends were then attained by 5’ and 3’ RACE as described in materials and methods. The result of full sequencing of EP4 gene in TO cells revealed a different isoform of EP4 (Fig. 1). We therefore designated the first published variant as As-EP4a while the second identified sequence in TO cells was named as As-EP4b. The length of the As-EP4b was found to be 2346 nucleotides compared to 2423 nucleotides for As-EP4a. While the first half of As-EP4b sequence was found to be identical to As-EP4a, many single nucleotide mutations and segmental deletions were found from nt 1027, which is located almost half through the EP4 gene, and onwards (Fig. 1). The coding region was located from nt 267 to nt 1694 and, although the length of nucleotide for both isoforms were different at C-terminal tail, the encoded length of amino acids were found to be identical, 473 amino acid for both isoforms (Fig. 2). Three amino acids were deleted at positions 389–401 in As-EP4b, whereas another three amino acid deletion was found at position 429–431 As-EP4a. Amino acid sequences for both isoforms were also aligned to EP4 amino acid sequences from several species (Fig. 3). Putative transmembrane domains (TM) 1–7 were noted based on a previous study [10]. Alignment result showed that EP4 genes from Atlantic salmon are in general conserved compared to other species at their seven transmembrane domains while differences in N- and C-terminals were remarkable (Fig. 3). High homology was found between intracellular domain 1 and 2 while in intracellular 3, a deletion of 20–30 amino acids was found only in non-mammalian species (fish and chicken).

Bottom Line: In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5' untranslated region was accompanied by silent mutation at nt 668.While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product.We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation.

View Article: PubMed Central - PubMed

Affiliation: Norwegian University of Life Sciences, Faculty of Veterinary Medicine and Biosciences, Sea Lice Research Centre, P.O. Box 8146 Dep., 0033 Oslo, Norway.

ABSTRACT
PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR) called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4) are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5' untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation.

No MeSH data available.


Related in: MedlinePlus