Abstract
BackgroundErythropoietin (EPO) is a glycoprotein hormone that plays a principal regulatory role in erythropoiesis and initiates cell homeostatic responses to environmental challenges. The Qinghai-Tibet Plateau is a natural laboratory for hypoxia adaptation. Gymnocypris dobula is a highly specialized plateau schizothoracine fish that is restricted to > 4500 m high-altitude freshwater rivers and ponds in the Qinghai-Tibet Plateau. The role of EPO in the adaptation of schizothoracine fish to hypoxia is unknown.ResultsThe EPO and EPO receptor genes from G. dobula and four other schizothoracine fish from various altitudinal habitats were characterized. Schizothoracine EPOs are predicted to possess 2–3 N-glycosylation (NGS) sites, 4–5 casein kinase II phosphorylation (CK2) sites, 1–2 protein kinase C (PKC) phosphorylation sites, and four conserved cysteine residues within four helical domains, with variations in the numbers of NGS and CK2 sites in G. dobula. PAML analysis indicated a dN/dS value (ω) = 1.112 in the G. dobula lineage, and a few amino acids potentially under lineage-specific positive selection were detected within the G. dobula EPO. Similarly, EPO receptors of the two high-altitude schizothoracines (G. dobula and Ptychobarbus kaznakovi), were found to be statistically on the border of positive selection using the branch-site model (P-value = 0.096), and some amino acids located in the ligand-binding domain and the fibronectin type III domain were identified as potentially positive selection sites. Tissue EPO expression profiling based on transcriptome sequencing of three schizothoracines (G. dobula, Schizothorax nukiangensis Tsao, and Schizothorax prenanti) showed significant upregulation of EPO expression in the brain and less significantly in the gill of G. dobula. The elevated expression together with the rapid evolution of the EPO gene in G. dobula suggested a possible role for EPO in adaptation to hypoxia. To test this hypothesis, Gd-EPO and Sp-EPO were cloned into an expression vector and transfected into the cultured cell line 293 T. Significantly higher cell viability was observed in cells transfected with Gd-EPO than cells harboring Sp-EPO when challenged by hypoxia.ConclusionThe deduced EPO proteins of the schizothoracine fish contain characteristic structures and important domains similar to EPOs from other taxa. The presence of potentially positive selection sites in both EPO and EPOR in G. dobula suggest possible adaptive evolution in the ligand-receptor binding activity of the EPO signaling cascade in G. dobula. Functional study indicated that the EPO from high-altitude schizothoracine species demonstrated features of hypoxic adaptation by reducing toxic effects or improving cell survival when expressed in cultured cells, providing evidence of molecular adaptation to hypoxic conditions in the Qinghai-Tibet Plateau.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0581-0) contains supplementary material, which is available to authorized users.
Highlights
Erythropoietin (EPO) is a glycoprotein hormone that plays a principal regulatory role in erythropoiesis and initiates cell homeostatic responses to environmental challenges
Sequence analysis indicated that the deduced amino acid sequence of schizothoracine EPO included a signal peptide predicted to be cleaved between amino acids (AAs) 23 and 24 (Fig. 1)
An additional NGS (149–152) and the loss of a CK2 site were uniquely found in the highaltitude schizothoracine G. dobula and P. kaznakovi EPO structures, which differed from lower-altitude EPOs (Fig. 1b)
Summary
Erythropoietin (EPO) is a glycoprotein hormone that plays a principal regulatory role in erythropoiesis and initiates cell homeostatic responses to environmental challenges. The Qinghai-Tibet Plateau is a natural laboratory for hypoxia adaptation. Gymnocypris dobula is a highly specialized plateau schizothoracine fish that is restricted to > 4500 m high-altitude freshwater rivers and ponds in the Qinghai-Tibet Plateau. The role of EPO in the adaptation of schizothoracine fish to hypoxia is unknown. The QinghaiTibet Plateau possesses an extremely harsh environment characterized by severe hypoxia, severe coldness and strong ultraviolet radiation that has profound effects on animal survival [2]. This unique plateau climate has made the Qinghai-Tibet Plateau a global biodiversity hotspot and a natural laboratory for long-term hypoxia and cold adaptation studies [3,4,5]. Few molecular evolutionary studies have focused on plateau fishes
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