Proteomics Of The Erythrocyte Ghost Proteins And Low-Temperature/Anoxic Tolerance Of Red-eared Turtle(Trachemys Scripta)

Turtles in the animal kingdom belong to the chordates, vertebrate subphylum, reptilia, turtle subclass, chelonia. Turtles are living fossil creatures during the evolutionary history. Turtles are famous as longevity and its super-tolerance to low-temperature and hypoxia are impressing. Scientists have fascinated at the mechanism of low-temperature and hypoxia tolerance for many years. The research on low-temperature/hypoxia tolerance mechanism has very important significance. Such as prevent the cells be damaged by ice crystal during freezing, In vitro, organ transplants fields, preserve isolated organ long-term without failure, which caused by ischemia/hypoxia, avoid the free-radical damage during the process of re-oxygenation or ischemia and reperfusion, improve the survival rate of injured personnel who have to be transported over a long distance to medical care, etc. And even some scientists hope to prolong lifespan similar to low-temperature/hypoxia cellaring.The turtles have some very specific body composition, such as their erythrocytes. Turtle erythrocyte has the same oval-shaped, huge, and nuclear as the other red blood cell (RBC) of low vertebrates, however, mature trutle RBCs does not contain mitochondria, which is a common existence in most of the low vertebrate RBCs. Some researchers consider that the turtle erythrocyte is closest with mammals RBC during evolutionary history. Erythrocyte of turtle between the RBCs of low vertebrates and higher vertebrates. However, the researchers still know very little about the components of turtle erythrocyte. Especially about cell membrane which plays an important role in cell protection, signal transduction, material transport, etc. in cell. The special structure of turtle erythrocyte may have direct correlation with the super endurance. Red-eared turtles were used to analyze the erythrocyte ghost proteomics in this study. We hope to partially explain the mechanism of low-temperature and hypoxia tolerance of turtle.First, we focused on how to obtain high-purity red-eared turtle erythrocyte ghost (TEG). As nucleated cells, turtle erythrocytes have a complexity of somatic cell; on the other hand, as RBCs, its plasma membrane has the fragility as mature human RBCs. Purification method of somatic cell and mature mammalian erythrocyte ghosts is unsuitable for the TEG. Therefore, how to purify TEG is a huge challenge. Before this study, we did not find any detailed information on how to separate and purify TEG. Based on existing knowledge and combined with our repeated experiments, eventually we summarized an effective separation method for the preparation of TEG. Namely, after hypotonic lysis with specific buffer, forcing through a syringe with specially shaped needle, low speed homogenizes and differential centrifugation, highly purified TEG fractions were separated effectively, which is a key to successful TEG proteomics. The TEG proteins were digested and dissociated in4different methods combined with different mass spectrum analysis; a total of169TEG proteins was identified. After further bioinformatic analyzing of these identified proteins, tubulins, heat shock proteins were identified in the TEG, which provided important insights into the low-temperature and hypoxia tolerance of Trachemys scripta. We also identified membrane located F-type ATP synthase subunits, which closely related with the production of ATP. The proteomics analysis of TEG opened a new direction of super-tolerance research of red-eared turtle.Secondly, we studied the important biochemical indicators energy metabolism of red-eared turtle by short-term low-temperature and diving exposure. We found anaerobic glycolysis and gluconeogenesis simultaneously during this short term low-temperature/hypoxic exposure. This finding gave a good explanation for why liver glycogen and muscle glycogen reserved but blood glucose elevated during a shot-term low-temperature and diving exposure.Finally, we combined the characteristics of the energy metabolism of short-term low temperature and diving exposure, blood ATP deprivation experiment in vitro and previous studies; we found that red-eared turtle red blood cells can generate ATP to the extracellular. The possible mechanism was high concentration of H+promoting the RBC plasma membrane located ATP synthase generated ATP. Meanwhile, we propose a model of RBCs energy supply for short-term low-temperature/hypoxia exposure.Therefore, in this study, an effective isolation method to prepare TEG was developed in combination with predissociation, enzymolysis and CapLC-MS/MS identification. A data set of TEG proteins were provided, which allows for more comprehensive characterization of TEG. Furthermore, we studied the energy metabolism mechanism of low-temperature/hypoxia red-eared turtle. We opened a new research field for understanding the super-tolerance of turtles when they face severe environment.