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

Abstract

The common gonadotrophic hormone alpha-subunit (GTHa) has been previously isolated by our research group from A. gigas pituitaries; in the present work the cDNA sequences encoding FSHB- and LHB-subunits have also been isolated from the same species of fish. The FSHB-subunit consists of 126 amino acids with a putative 18 amino acid signal peptide and a 108 amino acid mature peptide, while the LHB-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 FSHB and of Cypriniformes (76%) for LHB, followed by Siluriformes, 53% for FSHB and 75% for LHB. Interestingly, the identity with the corresponding human amino acid sequences was still remarkable: 45.1% for FSHB and 51.4% for LHB. 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 B-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. (AU)

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