Characterization of EPO H131S as a key mutation site in the hypoxia-adaptive evolution of Gymnocypris dobula

Erythropoietin (EPO) is a glycoprotein hormone involved in proerythropoiesis, antioxidation, and antiapoptosis. It also contributes to cellular immune function in high-altitude species, such as the schizothoracine fish Gymnocypris dobula. Six mutation sites previously identified in EPO from G. dobula (GD-EPO) were injected into zebrafish embryos, and their effects were compared with EPO from the low-altitude schizothoracine Schizothorax prenanti. The key mutation site in GD-EPO was identified as H131S. Under hypoxic conditions, the levels of superoxide dismutase and malondialdehyde were decreased, whereas that of nitric oxide was increased in zebrafish injected with GD-EPO compared with those injected with S. prenanti-EPO (SP-EPO). The results suggest that EPO in high-altitude schizothoracine species is both antioxidative and antiapoptotic, driven by the H131S mutation site. Thus, this enhanced the ability of this species to adapt to the high-altitude hypoxic environment. These results provide a basis for investigating further the hypoxia adaptation mechanisms of teleosts.


Introduction
As the highest plateau in the world, the average altitude of the Tibet Plateau is about 4300 m with a lower oxygen partial pressure (40%) compared with that at sea level (Beall 2007;Frisancho 2013). Local species exhibit physiological and morphological characteristics that have evolved to cope with this relatively extreme hypoxic environment. Gymnocypris 1 3 Vol:. (1234567890) dobula is a highly specialized schizothoracine fish that mainly inhabits shallow lakes and tributaries at an altitude > 4500 m (Wang et al. 2020). A recent study reported numerous physiological adaptations in G. dobula compared with another schizothoracine, Schizothorax prenanti, a relatively primitive fish distributed at a lower altitude in the upper reaches of the Yangtze River (Luo et al. 2016). These adaptations include relatively higher numbers of red blood cells and a stronger hypoxic respiratory response (Beall et al. 2012). However, the detailed regulatory mechanism of these changes was unclear.
Oxidative stress is caused by a variety of environmental factors, including ultraviolet stress, pathogen invasion, and hypoxia (Blokhina et al. 2003). To resist external changes, the organism has developed antioxidant and apoptosis systems (Radi et al. 2014;He et al. 2017). The antioxidant system is mainly composed of small molecular antioxidants and antioxidant enzymes. The main antioxidant enzymes are superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and so on. (Yang et al. 1999;He et al. 2017). SOD is usually more sensitive than other antioxidant enzymes (Víctor et al. 2009). Oxygen free radicals produced during oxidative stress enable unsaturated fatty acids to form lipid peroxides (MDA). The MDA level does not only reflect the rate and intensity of lipid peroxidation; it also indirectly reflects the degree of tissue peroxidation injury (Tsikas 2017). At physiological levels, nitric oxide (NO) can reduce ROS-induced damage (Wink et al. 2001). As a reactive radical, it is cytotoxic at high concentrations; in mammalian cellular immunity, damage to mitochondria exacerbates cellular lipid peroxidation damage, but at low concentrations, NO can positively and negatively regulate innate and acquire immune responses (Choudhari et al. 2013).
There are two main pathways of apoptosis, namely the mitochondrial pathway (endogenous activation pathway) and the death receptor pathway (exogenous activation pathway) (Morrill and He 2017). These pathways share a set of enzymes termed cysteineaspartic proteases (Caspases), which degrade cellular proteins. Initiator caspases include caspase-8 and caspase-9 (Riedl and Salvesen 2007;Timmer and Salvesen 2007;Obeng 2021). Following an initial apoptotic signal, these enzymes target scaffold proteins. The main executioner caspases are caspase-3, caspase-6, and caspase-7 (Morrill and He 2017;Obeng 2021). Upon cleavage of executioner caspases, a proteolytic cascade begins that will lead to the lysis of nearly all parts of the cell. Caspase-3 is one of the most important caspases (Morrill and He 2017). Moreover, the Bcl-2 family of proteins strongly regulates the intrinsic pathway (Antonsson et al. 1997). Bcl-2 is an integral membrane protein (Tsujimoto et al. 1987;Chen-Levy et al. 1989), but it can also serve as an inhibitor of cytochrome c (Cytc) release from the mitochondria as it binds to Bax and inhibits its oligomerization (Yang et al. 1997).
EPO is an acidic glycoprotein hormone of 166 amino acids and induced by hypoxia-inducible transcription factor (HIF) through the oxygen-sensing pathway (Cai et al. 1992;Tomc and Debeljak 2021). It stimulates the hematopoietic function of bone marrow, and increases red blood cell numbers to improve the oxygen-carrying capacity of blood (Browne et al. 1986;Davis et al. 1987). Meanwhile, Blixt et al. demonstrated that apoptosis, oxidative stress injury, and inflammation in neurocytes were also repressed by EPO, improving the ability to repair hypoxia-induced brain injuries (Katakura et al. 2013). Previous research confirmed the antiapoptotic and antioxidative effects of EPO on tissues or organs in mammals in response to hypoxia and ischemia (Tramontano et al. 2003;Wang et al. 2010;Chau et al. 2011;Parvin et al. 2014;Blixt et al. 2018;Dey et al. 2020). Moreover, most mammals exhibit increased levels of plasma EPO that promote erythropoiesis, thus improving the physiological response to hypoxia (Souvenir et al. 2011).
The study of hypoxic adaptation in high-altitude fish is limited. G. dobula, one of the most important fish in Tibet, is a good biomaterial to study the hypoxic adaption mechanism of high-altitude fish. We previously reported (Xu et al. 2016) six mutation sites (L117I, H131S, T133P, S138P, S139T, and L153I) in GD-EPO compared with SP-EPO. We also demonstrated that GD-EPO is involved in improving cell viability in this species. However, it was unclear which mutation site was most important in the adaptation of G. dobula to high-altitude hypoxic conditions.
In the current study, we determined the effects of the six mutation sites on zebrafish embryos. Our results provide additional insight into the regulatory mechanisms involved in the adaptation of G. dobula to their hypoxic environment.

Animal sources
Specimens of G. dobula were collected from Yadong County, Tibet (46°03.371′ N, 89°17.831′ E, altitude 4506 m; dissolved oxygen 1.9 ± 0.3 mg/L; 11 °C), and S. prenanti were collected from Ya'an, Sichuan (altitude 950 m; dissolved oxygen 9.0 ± 0.5 mg/L; 18 °C). Wild-type (WT) zebrafish (AB type), procured from Haisheng Biology, Ltd (Shanghai, China), were used as the model organism and were fed freely at 28 °C. The animal experiments were approved by the Ethics Committee of Shanghai Ocean University, and were performed in accordance with the institutional guide for the care and use of laboratory animals.

Construction of recombinant plasmids
The cDNAs of GD-EPO and SP-EPO were synthesized with mRNAs extracted from the pronephros of G. dobula (GenBank accession no. KT188754.1) and S. prenanti (GenBank accession no. KT188758.1) using PrimeScript RT reagent kit with gDNA Eraser (Takara, Dalian, China), according to the manufacturer's instructions. The PCR reactions were carried out (denaturation at 95 °C for 5 min, followed by 30 cycles of 30 s at 95 °C, 45 s at 55 °C, 90 s at 72 °C, followed by a final extension of 10 min at 72 °C) with Taq DNA polymerase (Sangon, Shanghai, China).

Enzyme activity detection
The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and nitric oxide (NO) were determined in tissue homogenate of WT zebrafish embryos and zebrafish. Zebrafish tissue was homogenized in physiological saline at a mass: volume ratio of 1:9 ratio to obtain the 10% homogenate. SOD, MDA, and NO levels were determined, respectively, using the specific kits (Jiancheng Science & Technology, Ltd., Nanjing, China), according to the manufacturer's protocol.

Western blotting
The embryos underwent hypoxia treatment for 6.5 h at 48 h post-birth. After washing the embryos with phosphate buffer saline (PBS) three times, the protein productions were extracted using RIPA buffer containing protease inhibitors Proteins were then separated by 12% SDS-PAGE gel and transferred onto a PVDF membrane. Blocking was performed using 5% nonfat dried milk. After blocking, membranes were incubated with primary antibodies against Bcl-2 (DIA. An, China), Bax (DIA. An, China), caspase-3 (DIA. An, China), 6 × His (HUABIO, China), and GAPDH (DIA. An, China) for 2.5 h at room temperature. Then, 1 × PBST was used to wash the membranes three times. Subsequently, at room temperature, a secondary antibody (DIA. An, China) was added and incubated for 1 h. The membranes were then washed three times with 1 × PBST. The membranes were developed by chemiluminescence reagents (Thermo Fisher Scientific, USA) under a Gel-Pro analyzer (Version 4.0, USA). Finally, the western blotting images were analyzed using the ImageJ 1.46 software (National institutes of Health, USA).

Statistical analysis
All results were obtained from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 8.0.2. Data were expressed as the mean ± standard deviation. One-way ANOVA was used to evaluate the differences among multiple groups. The survival rate of zebrafish was obtained by percent survival analysis. Differences were significant at P < 0.05 or P < 0.01.

GD-EPO increases the expression level of hemoglobin under hypoxia
EPO is a glycoprotein hormone with a principal regulatory role in erythropoiesis (Spivak 1989;Fried 2009). Various studies on red blood cells and hemoglobin in species from the Tibet Plateau and plain areas showed that the oxygen transport and utilization abilities of these species are stronger than those living at lower altitude areas, manifesting as large lung volume, high myoglobin concentration, and strong oxygen uptake ability (Yangzong et al. 2013;Li et al. 2018). In the current study, injection of nine overexpressed plasmids in zebrafish embryos increased the hemoglobin level in the acute hypoxic group compared with the normoxia group, suggesting that hypoxia increases the numbers of red blood cells, leading to increased levels of hemoglobin ( Fig. 1). However, the nine overexpressed plasmids in zebrafish embryos of the normoxia group had no obvious effect on hemoglobin level (Fig. 1b). By contrast, in the hypoxia groups, the expression level of hemoglobin in zebrafish embryos injected with GD-EPO was significantly elevated compared with those injected with SP-EPO (Fig. 1a). This suggests that, under hypoxia stress, GD-EPO may increase the level of hemoglobin to carry more oxygen and overcome the effects of hypoxia. In addition, the hemoglobin level was upregulated by overexpression of L117I, T133P, S138P, S139T, and L153I compared with control group and overexpression of SP-EPO, whereas there were no obvious changes in the overexpressed H131S group (Fig. 1a). Thus, the mutant site H131S might have a crucial role in enabling G. dobula to adapt to a high-altitude hypoxia environment.
H131S is a key mutation site in EPO in the hypoxia-adaptive evolution of G. dobula Six amino acid mutation sites (L117I, H131S, T133P, S138P, S139T, H131S, and L153I) were previously identified in GD-EPO compared with SP-EPO using a human EPO model (Fig. 2a). In the current study, H131S was determined to have a key role in the protective mechanism of GD-EPO against a hypoxic environment. To further validate this hypothesis, the percentage survival of zebrafish injected with GD-EPO, SP-EPO, L117I, H131S, T133P, S138P, S139T, H131S, and L153I was assessed. The 4-h percentage survival of zebrafish in the GD-EPO, L117I, T133P, S138P, S139T, and L153I groups was 100%, 100%, 80%, 60%, 70%, and 30%, respectively (Fig. 2c). By contrast, numerous zebrafish in the H131S group had died 2 h after hypoxia stimulation, with 0% survival at 4 h. This suggests that H131S overexpression in zebrafish renders them more sensitive to oxygen concentration. Previously, we reported that the EPO of G. dobula and Ptychobarbus kaznakovi (P. kaznakovi, which is also a highly specialized schizothoracines) was highly homologous. The H131S of EPO lacked a casein kinase II (CK2) phosphorylation site that was uniquely found in the high-altitude schizothoracine G. dobula and P. kaznakovi, which was different from the lower altitude Cyprinidae fish (Xu et al. 2016). CK2 is a serine/threonine protein kinase, which regulates the activity or stability of the substrate via its phosphorylation. CK2 also participates in the development and function of neurons, and in synaptic information transmission, which controls the development and longevity of synaptic connections. Therefore, the CK2 phosphorylation site might be missing at the 131 amino acid site of G. dobula, rendering it insensitive to hypoxia. Thus, mutated H131S might have enabled G. dobula to adapt to its high-altitude hypoxic environment.

GD-EPO functions as an antioxidant
The Tibet Plateau is characterized by low temperatures, hypoxia, and strong radiation (Beall 2007). Dramatic hypoxia has significant negative effects on organisms, including metabolic disorders, increasing concentrations of oxygen-derived free radicals, damage to cell membranes and nucleic acid structures, or even death (Fuhrmann and Brune 2017;Gonzalez et al. 2019). To adapt to hypoxic environments, organisms have evolved endogenous antioxidant enzyme systems and apoptosis, comprising small-molecule antioxidants and antioxidant enzymes. SOD is a cytoplasmic and mitochondrial-based antioxidant that can convert O 2 − into H 2 O 2 and O 2 (Du et al. 2017). Oxygen-derived free radicals produced during oxidative stress can convert unsaturated fatty acids to MDA (Tsikas 2017). Generally, the MDA level indirectly reflects the degree of tissue peroxidation injury (Yang et al. 1999). In addition, NO as a reactive free radical functions in nerve transmission, the immune response, and apoptosis (Ghasemi 2019). Humans living on the Tibetan Plateau have a relatively high concentration of NO, which suggest that NO has an important role in this hypoxic environment (Beall 2007). In this study, the levels of SOD, NO, and MDA in injected zebrafish embryos under hypoxia conditions were determined. Levels of SOD and MDA in zebrafish embryos injected with GD-EPO were significantly decreased compared with those of WT zebrafish embryos (P < 0.05) or those injected with SP-EPO (P < 0.05) (Fig. 3b, d). Similarly, Omrani et al. reported that the levels of SOD and MDA in rat tissues were increased significantly after hypoxia stimulation (Omrani et al. 2017). Manor et al. also confirmed that the levels of ROS and lipid oxidation in mice injected with EPO (5000 U/kg) were decreased (Manor et al. 1986). These results suggest that GD-EPO not only alleviated the oxidative damage caused by hypoxia, thus reducing the production of free radicals, but also attenuated the degree of lipid oxidation. In addition, a high level of NO occurred in zebrafish embryos injected with GD-EPO compared with WT zebrafish embryos or those injected with SP-EPO (Fig. 3c). In Tibetans, the utilization of NO is relatively high compared with plain inhabitants. Thus, the antioxidant effect of EPO might be closely associated with the hypoxic environment of the Tibet Plateau.

GD-EPO functions as a role of antiapoptosis
Under conditions of ischemia and hypoxia, the mitochondrial membrane potential is decreased and mitochondrial permeability transition pores (mPTP) open, inducing the expression of apoptosis-related genes and proteins (Chen et al. 2018). Cytc is a water-soluble protein located between the inner and outer mitochondrial membranes (Wan et al. 2019). Mitochondria release Cytc into the cytoplasmic matrix to initiate apoptosis once cells are stimulated by apoptosis signals (Wan et al. 2019). Members of the Bcl-2 protein family can interact to induce or prevent apoptosis. Among them, Bcl-2 inhibits the opening of mPTPs, thereby repressing apoptosis. By contrast, Bax promotes the opening of mPTP to accelerate apoptosis (Zhang et al. 2015). In this study, to investigate the role of GD-EPO in apoptosis, the expression levels of Cytc, caspase-3, caspase-8, caspase-9, and the Bcl-2/Bax ratio were determined. Expression levels of Cytc, caspase-3, caspase-8, and caspase-9 in the GD-EPO groups were reduced compared with those in the SP-EPO group (P < 0.01) (Fig. 4a, b), whereas the Bcl-2/ Bax ratio was higher in the GD-EPO group, as also shown by western blotting (Fig. 4c). EPO interacts with the p53 signaling pathway (Pham et al. 2019) in leukemia cells or the Akt signaling pathway (Tramontano et al. 2003) in cardiac myocytes to suppress apoptosis. Antiapoptotic treatments, such as EPO, are also suggested to improve outcomes in hypoxic brain injury (Jung et al. 2021). EPO and vitamin D3 (VD3) can be used to prevent or treat renal ischemia-reperfusion (I/R) injury, and these beneficial effects are closely related to antiinflammatory and antiapoptosis pathways (Qin et al. 2021). Thus, we hypothesize that EPO is also involved in the regulation of apoptosis in G. dobula through these pathways. However, the specific mechanisms involved remain to be fully understood.

Conclusions
In summary, the H131S mutation was identified as a key mutation affecting the cytoprotective function of G. dobula EPO during the process of adaptation to long-term hypoxia. Our results showed that EPO from high-altitude fish could increase the hemoglobin level. Furthermore, the antioxidative and antiapoptotic functions of G. dobula EPO were enhanced during evolution. Although the detailed regulatory mechanism remains unclear, these results provide a research direction to elucidate the mechanisms utilized by schizothoracine fish under long-term hypoxia conditions. The mRNA level of caspase-3, caspase-8, caspase-9, Cytc, Bax, and Bcl-2. (c) Expression level of Bcl-2, Bax, and caspase-3 by Western blotting. The results were expressed as the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01