title: Analysis of the erythropoietin of a Tibetan Plateau schizothoracine fish (Gymnocypris dobula) reveals enhanced cytoprotection function in hypoxic environments.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  The 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 d N/d S 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Key Laboratory of Sustainable Exploitation of Oceanic Fisheries Resources, Ministry of Education, College of Marine Sciences, Shanghai Ocean University, Shanghai, China. qhxu@shou.edu.cn.|Collaborative Innovation Center for Distant-water Fisheries, Shanghai, China. qhxu@shou.edu.cn.
authors.forename: Qianghua
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Key Laboratory of Sustainable Exploitation of Oceanic Fisheries Resources, Ministry of Education, College of Marine Sciences, Shanghai Ocean University, Shanghai, China. wkzhangchi@yeah.net.
authors.forename: Chi
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Aquaculture Resources and Utilization, Ministry of Education, College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai, China. dszhang@shou.edu.cn.
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Sustainable Exploitation of Oceanic Fisheries Resources, Ministry of Education, College of Marine Sciences, Shanghai Ocean University, Shanghai, China. 865260594@qq.com.
authors.forename: Huapeng
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Key Laboratory of Aquaculture Resources and Utilization, Ministry of Education, College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai, China. shpeng@shou.edu.cn.
authors.forename: Sihua
authors.initials: S
authors.lastname: Peng
authors.affiliation: Key Laboratory of Sustainable Exploitation of Oceanic Fisheries Resources, Ministry of Education, College of Marine Sciences, Shanghai Ocean University, Shanghai, China. y_liu@shou.edu.cn.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China. zhaokai@nwipb.cas.cn.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Sustainable Exploitation of Oceanic Fisheries Resources, Ministry of Education, College of Marine Sciences, Shanghai Ocean University, Shanghai, China. ccwang@shou.edu.cn.
authors.forename: Congcong
authors.initials: C
authors.lastname: Wang
authors.affiliation: Key Laboratory of Aquaculture Resources and Utilization, Ministry of Education, College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai, China. lbchen@shou.edu.cn.
authors.forename: Liangbiao
authors.initials: L
authors.lastname: Chen
mesh_terms: D000064:Acclimatization; D000222:Adaptation, Physiological; D000818:Animals; D005075:Biological Evolution; D003530:Cyprinidae / Q000235:genetics / Q000502:physiology*; D019610:Cytoprotection; D017753:Ecosystem; D004921:Erythropoietin / Q000235:genetics / Q000502:physiology*; D010100:Oxygen; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000502:physiology; D018609:Tibet
chemical_list: D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D010100:Oxygen