Molecular Characterization of Putative Calcitonin Gene-Related Peptide Receptors and Expression of Calcitonin Gene-Related Peptide and its Receptor in the Early Development of Flounder, Paralichthys olivaceus
Molecular Characterization of Putative Calcitonin Gene-Related Peptide Receptors and Expression of Calcitonin Gene-Related Peptide and its Receptor in the Early Development of Flounder, Paralichthys olivaceus
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发表时间：
2018
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通讯作者：
Sekiguchi Toshio;S. Tohru;Kurokawa Tadahide;Amornsakun Thumronk;H. Tran;Ngoc;K. SrivastavAjai;Suzuki Nobuo
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作者：
Sekiguchi Toshio;S. Tohru;Kurokawa Tadahide;Amornsakun Thumronk;H. Tran;Ngoc;K. SrivastavAjai;Suzuki Nobuo
降钙素基因相关肽（CGRP）是一种由37个氨基酸组成的神经肽，已在硬骨鱼和哺乳动物中发现。降钙素受体样受体（CGRP）形成CGRP受体。在硬骨鱼中，CGRP在血管舒张中起作用并且与渗透调节相关。然而，CGRP的胚胎功能仍不清楚。因此，我们研究了降钙素基因相关肽在比目鱼早期发育中的作用。我们首先从比目鱼基因组数据库中识别出两个CLR。根据分子系统发育分析，将两个CLR归入由硬骨鱼类CLR 1和2组成的类群。它们在河豚鱼CLR 1 -3和人的peptide之间表现出高度的氨基酸序列同一性。我们还从比目鱼基因组中鉴定了另一个候选基因。分子系统发育分析表明，该基因位于脊椎动物CTR形成的分支，这些CTR是同源的。该基因推导的蛋白质还具有4个结合结构域，这是硬骨鱼CTR的典型特征，与包括比目鱼类CLR在内的硬骨鱼类的结合结构域完全不同。这些数据有力地表明，比目鱼拥有两个推定的CGRP受体。最后，我们进行了比目鱼CGRP和两个CLRs在比目鱼胚胎中的表达分析。从受精后96小时开始检测到CGRP mRNA和两种CGRP mRNA的表达。这些结果表明CGRP在96 hpf具有一定的功能。在这个阶段，胚胎已经孵化，循环系统功能正常，而消化器官仍在继续发育。综上所述，这些发现表明CGRP肽可能在循环系统中发挥作用，如在成鱼中，或者它可能与消化道的发育有关。
Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that has been identified in teleosts and mammals. Calcitonin receptor-like receptor (CLR) forms the CGRP receptor. In teleost fish, CGRP plays a role in vasodilation and is associated with osmotic regulation. However, the embryonic function of CGRP remains unclear. Therefore, we have investigated CGRP function related to early development in the flounder, Paralichthys olivaceus. We first identified two CLRs from the flounder genome database. According to molecular phylogenetic analysis, two CLRs are classified in the group formed by teleost CLR1 and 2. They represented high amino acid sequence identity among mefugu CLR1-3 and human CLR. We also identified another CLR/CTR candidate from the flounder genome. Molecular phylogenetic analysis revealed that this gene was located at the clade formed by vertebrate CTRs that are CLR paralogs. The deduced protein of the gene also had four hormone-binding domains, which are the typical feature of teleost CTR, and was completely different from the CLR of teleosts, including flounder CLRs. These data strongly suggest that flounder possess two putative CGRP receptors. Finally, we performed the expression analysis of flounder CGRP and two CLRs in the flounder embryo. Both expressions of CGRP mRNA and two CLR mRNAs were detected from 96 hours post fertilization (hpf). These results demonstrate that CGRP has some functions at 96 hpf. At that stage, embryos have already hatched and the circulatory system functions, whereas digestive organs still continue to develop. Taken together, these findings suggest that the CGRP peptide may play a role in the circulatory system, as in adult fish, or it may be associated with the development of the digestive tract.