Molecular cloning and characterization of pirarucu (Arapaima gigas) follicle-stimulating hormone and luteinizing hormone β-subunit cDNAs

Abstract

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.

Genetic diversity and structure in Arapaima gigas populations from Amazon and Araguaia-Tocantins river basins

“…While occasionally successful, breeding of A. gigas in captivity is not a routine practice due to complex reproductive traits and dysfunctions in the species (i.e. failure at the final oocyte maturation and ovulation stage, lack of male-female synchronization at spawning), which require further research especially for gender identification and control of spawning [33, 34]. Therefore, fingerlings are valuable in the aquaculture market, and their illegal capture from the wild is a challenge for conservation.…”

De novo transcriptome based on next-generation sequencing reveals candidate genes with sex-specific expression in Arapaima gigas (Schinz, 1822), an ancient Amazonian freshwater fish

“…While the arapaima is an ancient species of considerable economic importance in the Amazon region, scientific studies of the species have focused primarily on its ecology [ 5 , 24 , 25 ], population genetics [ 26 – 29 ], or focused on specific genes [ 30 , 31 ]. Few data are available on the functional genomics of the species.…”

Expressão e caracterização do hormônio folículo estimulante (FSH) de pirarucu (Arapaima gigas)

“…O cDNA de ag-GTHα apresentou um comprimento total de 767 pb com uma região codificante (ORF) de 348 pb que codifica um peptídeo de 115 aminoácidos. A proteína apresenta, portanto, um suposto peptídeo sinal de 24 aminoácidos e um peptídeo maduro de 91 aminácidos que, quando alinhados com outras espécies de peixes, mostram a conservação de 10 resíduos de cisteínas, 3 prolinas e 2 potenciais sítios de glicosilação conservados (Faria et al, 2013;Sevilhano et al, 2017).…”

“…Já a subunidade β de FSH apresentou um comprimento total de 913 pb com uma ORF de pb, codificando uma proteína de 126 aminoácidos com um peptídeo sinal de aminoácidos e um peptídeo maduro de 108 aminoácidos. O ag-FSHβ mostrou conter 12 resíduos de cisteínas responsáveis pela formação de 6 pontes dissulfeto, 2 prolinas e 1 sítio de glicosilação perfeitamente conservados quando comparado com outras espécies (Faria et al, 2013;Sevilhano et al, 2017).…”

“…Esses dados específicos da espécie estudada possibilitaram estudos no campo filogenético utilizando simultaneamente as sequências de GTHα, FSHβ e LHβ de diversos peixes que posicionaram o A. gigas como grupo irmão dos Clupeocephala e os Elopomorpha como grupo mais basal entre os teleósteos (Faria et al, 2013;Sevilhano et al, 2017).…”