Sevilhano Thais, Carvalho Roberto Feitosa de, Oliveira Nélio Alessandro de Jesus, Oliveira João Ezequiel, Maltarollo Vinicius Gonçalves, Trossini Gustavo, Garcez Riviane, Bartolini Paolo
Biotechnology Department, IPEN-CNEN/SP, Cidade Universitária, São Paulo, SP, Brazil.
Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
PLoS One. 2017 Aug 28;12(8):e0183545. doi: 10.1371/journal.pone.0183545. eCollection 2017.
The common gonadotrophic hormone α-subunit (GTHα) has been previously isolated by our research group from A. gigas pituitaries; in the present work the cDNA sequences encoding FSHβ and LHβ subunits have also been isolated from the same species of fish. The FSH β-subunit consists of 126 amino acids with a putative 18 amino acid signal peptide and a 108 amino acid mature peptide, while the LH β-subunit consists of 141 amino acids with a putative 24 amino acid amino acid signal peptide and a 117 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the order of Anguilliformes (61%) for FSHβ and of Cypriniformes (76%) for LHβ, followed by Siluriformes, 53% for FSHβ and 75% for LHβ. Interestingly, the identity with the corresponding human amino acid sequences was still remarkable: 45.1% for FSHβ and 51.4% for LHβ. Three dimensional models of ag-FSH and ag-LH, generated by using the crystal structures of h-FSH and h-LH as the respective templates and carried out via comparative modeling and molecular dynamics simulations, suggested the presence of the so-called "seat-belt", favored by a disulfide bond formed between the 3rd and 12th cysteine in both β-subunits. The sequences found will be used for the biotechnological synthesis of A. gigas gonadotrophic hormones (ag-FSH and ag-LH). In a first approach, to ascertain that the cloned transcripts allow the expression of the heterodimeric hormones, ag-FSH has been synthesized in human embryonic kidney 293 (HEK293) cells, preliminarily purified and characterized.
常见的促性腺激素α亚基（GTHα）此前已由我们的研究小组从巨蛎垂体中分离出来；在本研究中，编码促卵泡激素β亚基（FSHβ）和促黄体生成素β亚基（LHβ）的cDNA序列也从同一鱼类中分离得到。FSHβ亚基由126个氨基酸组成，带有一个推测的18个氨基酸的信号肽和一个108个氨基酸的成熟肽，而LHβ亚基由141个氨基酸组成，带有一个推测的24个氨基酸的信号肽和一个117个氨基酸的成熟肽。基于氨基酸序列，发现与鳗鲡目FSHβ的同源性最高为61%，与鲤形目LHβ的同源性最高为76%，其次是鲶形目，FSHβ为53%，LHβ为75%。有趣的是，与相应人类氨基酸序列的同源性仍然很高：FSHβ为45.1%，LHβ为51.4%。通过使用人促卵泡激素（h-FSH）和人促黄体生成素（h-LH）的晶体结构作为各自模板，并通过比较建模和分子动力学模拟生成的巨蛎促卵泡激素（ag-FSH）和巨蛎促黄体生成素（ag-LH）的三维模型表明，在两个β亚基的第3个和第12个半胱氨酸之间形成的二硫键有利于所谓“安全带”的存在。所发现的序列将用于巨蛎促性腺激素（ag-FSH和ag-LH）的生物技术合成。在第一步中，为了确定克隆的转录本是否允许异二聚体激素的表达，已在人胚肾293（HEK293）细胞中合成了ag-FSH，进行了初步纯化和表征。
