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.

Keywords: AlphaFold; Arapaima gigas; in silico prediction; thyrotropin.

Figure 1.

ag-βTSH coding sequence (1–402 nt) and 3’ untranslated region (402–677 nt). The first line corresponds to the nucleotides, and the second one corresponds to the translated amino acids. * indicates stop codon.

Figure 2.

ClustalΩ alignment of 40 species with corresponding coding sequence for βTSH. Amino acids with 100% identity are highlighted. Number 1 indicates Arapaima gigas.

Figure 3.

Phylogenetic tree for coding sequence of βTSH from 37 fish species. The numbers represents the node scores.

Figure 4.

AlphaFold model for ag-TSH, with local Distance Difference Test (IDDT) score per residue and alignment error. Right upper-side: two representations for chain (α: cyan, β: green) and predicted Local Distance Difference Test (pLDDT) colorful representation (high confident values = dark blue, less confident: red). Predicted alignment error and IDDT values per residue also showed. On the left side, best AlphaFold ag-TSH model with disulfide bridges sticks representation (yellow on β chain and red in α chain). The 3-dimensional structure was visualized on Maestro 2022.2. ag-TSH indicates Arapaima gigas thyrotropin.

Figure 5.

Surface and cartoon representation of ag-TSH and 3 human glycoproteic hormones (RCSB PDB ID: 1FL7 for hFSH, RCSB PDB ID: 1HRP for hCG, and RCSB PDB ID:7T9I for hTSH). Aligned structures were performed in PyMOL 2.0. Protein surface representation had transparency set at 0.6. ag-TSH indicates Arapaima gigas thyrotropin; hFSH, human follicle-stimulating hormone; hTSH, human thyroid-stimulating hormone.

Figure 6.

Silver-stained SDS-PAGE from transient expression of ag-TSH in HEK293F cells. The samples are described as follow: (I) BlueEye PreStained Protein Ladder (Sigma-Aldrich, St. Louis, MO, USA); (II) Thyrogen^® (hTSH) 600 ng; (III) conditioned medium of non-DNA transfected HEK293F cells from fifth day—negative control; (IV) conditioned medium of transfected HEK293F cells from third day; (V) conditioned medium of transfected HEK293F cells from fifth day; (VI) conditioned medium of transfected HEK293F cells from seventh day. Except the ladder, all samples were precipitated with TCA and heated for 3 min at 85°C. ag-TSH indicates Arapaima gigas thyrotropin; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TCA, trichloroacetic acid.

Figure 7.

Super compare the concentrated conditioned medium from the fifth day after transfection, applied in RP-HPLC. Negative control from the fifth day and pituitary human LH (p-hLH) hormone were used for better identification. Retention time of p-hLH was around 42.597 min. RP-HPLC indicates reverse-phase high-performance liquid chromatography; LH, luteinizing hormone.

Figure 8.

HPSEC and SDS-PAGE chromatograms of pools #P1 and #P2 collected from the first RP-HPLC characterization step. HPSEC indicates high-performance size exclusion chromatography; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; RP-HPLC, reverse-phase high-performance liquid chromatography.