Dataset Information

Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (Arapaima gigas).

ABSTRACT: 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 β 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 βTSH (ag-βTSH), 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-βTSH sequence by reverse transcriptase-polymerase chain reaction (RT-PCR) and transient expression in human embryonic kidney (HEK293F) cells. Molecular cloning of ag-βTSH 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 ΔG_bind and ΔG_PMF 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.

SUBMITTER: Freire RP

PROVIDER: S-EPMC9926385 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (<i>Arapaima gigas</i>).

Freire Renan Passos RP Hernandez-Gonzalez Jorge Enrique JE Lima Eliana Rosa ER Suzuki Miriam Fussae MF de Oliveira João Ezequiel JE Torai Lucas Simon LS Bartolini Paolo P Soares Carlos Roberto Jorge CRJ

Bioinformatics and biology insights 20230212

<i>Arapaima gigas</i>, 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 β whose main activity is related to the synthesis of thyroid hormones (THs)-T3 and T4. In this work, we present a combination of bioinfor ...[more]

PMID: 36798082

Similar Datasets

Project description:In air-breathing fish a reduction of gill surface area reduces the danger of losing oxygen taken up in the air-breathing organ (ABO) to hypoxic water, but it also reduces the surface area available for ion exchange, so that ion regulation may at least in part be transferred to other organs, like the kidney or the gut. In the air-breathing Arapaima gigas, gill lamellae regress as development proceeds, and starting as a water-breathing embryo Arapaima turns into an obligate air-breathing fish with proceeding development, suggesting that ion regulation is shifted away from the gills as the fish grows. In Arapaima the kidney projects medially into the ABO and thus, probably a unique situation among fishes, is in close contact to the gas of the ABO. We therefore hypothesized that the kidney would be predestined to adopt an increased importance for ion homeostasis, because the elevated ATP turnover connected to ion transport can easily be met by aerobic metabolism based on the excellent oxygen supply directly from the ABO. We also hypothesized that in gill tissue the reduced ion regulatory activity should result in a reduced metabolic activity. High metabolic activity and exposure to high oxygen tensions are connected to the production of reactive oxygen species (ROS), therefore the tissues exposed to these conditions should have a high ROS defense capacity. Using in vitro studies, we assessed metabolic activity and ROS production of gill, kidney and ABO tissue, and determined the activity of ROS degrading enzymes in small (~ 5g, 2-3 weeks old) and larger (~ 670 g, 3-4 months old) A. gigas. Comparing the three tissues revealed that kidney tissue oxygen uptake by far exceeded the uptake measured in gill tissue or ABO. ROS production was particularly high in gill tissue, and all three tissues had a high capacity to degrade ROS. Gill tissue was characterized by high activities of enzymes involved in the glutathione pathway to degrade ROS. By contrast, the tissues of the ABO and in particular the kidney were characterized by high catalase activities, revealing different, tissue-specific strategies in ROS defense in this species. Overall the differences in the activity of cells taken from small and larger fish were not as pronounced as expected, while at the tissue level the metabolic activity of kidney cells by far exceeded the activity of ABO and gill cells.

Dataset Information

Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (Arapaima gigas).

Publications

Molecular Cloning and AlphaFold Modeling of Thyrotropin (ag-TSH) From the Amazonian Fish Pirarucu (<i>Arapaima gigas</i>).

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets