RESUMO
Arapaima gigas, known as Pirarucu in Brazil, is one of the largest freshwater fish in the world. Some individuals could reach 3 m in length and weight up to 200 kg. Due to extinction risks and its economic value, the species has been a focus for preservation and reproduction studies. Thyrotropin (TSH) is a glycoprotein hormone formed by 2 subunits α and ß whose main activity is related to the synthesis of thyroid hormones (THs)-T3 and T4. In this work, we present a combination of bioinformatics tools to identify Arapaima gigas ßTSH (ag-ßTSH), modeling its molecular structure and express the recombinant heterodimer form in mammalian cells. Using the combination of computational biology, based on genome-related information, in silico molecular cloning and modeling led to confirm results of the ag-ßTSH sequence by reverse transcriptase-polymerase chain reaction (RT-PCR) and transient expression in human embryonic kidney (HEK293F) cells. Molecular cloning of ag-ßTSH retrieved 146 amino acids with a signal peptide of 21 amino acid residues and 6 disulfide bonds. The sequence has a similarity to 39 fish species, ranging between 43.1% and 81.6%, whose domains are extremely conserved, such as cystine knot motif and N-glycosylation site. The Arapaima gigas thyrotropin (ag-TSH) model, solved by AlphaFold, was used in molecular dynamics simulations with Scleropages formosus receptor, providing similar values of free energy ΔGbind and ΔGPMF in comparison with Homo sapiens model. The recombinant expression in HEK293F cells reached a yield of 25 mg/L, characterized via chromatographic and physical-chemical techniques. This work shows that other Arapaima gigas proteins could be studied in a similar way, using the combination of these techniques, recovering more information from its genome and improving the reproduction and preservation of this prehistoric fish.
RESUMO
In a previous work, the common gonadotrophic hormone α-subunit (ag-GTHα), the ag-FSH ß- and ag-LH ß-subunit cDNAs, were isolated and characterized by our research group from A. gigas pituitaries, while a preliminary synthesis of ag-FSH was also carried out in human embryonic kidney 293 (HEK293) cells. In the present work, the cDNA sequence encoding the ag-growth hormone (ag-GH) has also been isolated from the same giant Arapaimidae Amazonian fish. The ag-GH consists of 208 amino acids with a putative 23 amino acid signal peptide and a 185 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the Elopiformes (82.0%), followed by Anguilliformes (79.7%) and Acipenseriformes (74.5%). The identity with the corresponding human GH (hGH) amino acid sequence is remarkable (44.8%), and the two disulfide bonds present in both sequences were perfectly conserved. Three-dimensional (3D) models of ag-GH, in comparison with hGH, were generated using the threading modeling method followed by molecular dynamics. Our simulations suggest that the two proteins have similar structural properties without major conformational changes under the simulated conditions, even though they are separated from each other by a >100 Myr evolutionary period (1 Myr = 1 million years). The sequence found will be used for the biotechnological synthesis of ag-GH while the ag-GH cDNA obtained will be utilized for preliminary Gene Therapy studies.