The Adaptive Mechanism Of Globin Family To High Altitude Hypoxia In Phrynocephalus Lizards

Hb and Mb are the bestinvestigated proteins in globin family and perform important functions in oxygen transport and storage.Over the past decade,the sequencing and analysis of vertebrate genomes have led to the discovery of many new members of the globin gene superfamily including neuroglobin(Ngb),cytoglobin(Cygb),androglobin(Adgb),globin E(GbE),globin X(GbX)and globin Y(GbY).Studies have shown that these new globins usually have Hb or Mb-like physiological functions,such as oxygen transport,ROS defense,and NO metabolism.Thus they may play a crucial role in high altitude hypoxia adaptation in animals.The aim of present study was to explore the potential adaptation mechanism of globin gene family members in the high altitude Phrynocephalus lizards.We integrated the analysis of genome,transcriptome,physiology and biochemistry,molecular evolution and protein structure to unravel the full globin repertoire of squamates,gene expression profile of lizards,the tissue-specific expression pattern and elevation-dependent expression differences of Phrynocephalus lizards living in different altitude and their adaptive evolutionary pattern in this lizard genus.The results are presented as follows:Our genomic analysis suggested that squamates retained most of globin members excepting the GbE globin gene including two copies of ? gene(?A and?D),two copies of ? gene(?1 and?2),one copy of Mb,Ngb,Cygb,GbY,GbE,GbX and Adgb.The ?A,?D,?1,?2,Mb,Ngb and Cygb were expressed in all lizard species examined,however,the GbY,GbX and Adgb showed lineage-specific expression pattern in these species.Phylogenetic relationship inference based on tetrapodian ? sequeces suggested that ?E,?D and ?A gene originated from two rounds of gene tandem replication in the stem of terapods.The ?E and ?A genes originated via tandem duplication of an ancestral proto ? gene,and the ?D gene originated subsequently via tandem duplication of the ?A gene.Gene conversion between ?D and ?E endowed the higher oxygen affinity of ?D-type Hb than ?A-type Hb in reptiles and birds.P.erythrurus living on Qinghai-Tibet plateau is the highest distributed lizard in the world.Comparaed with P.przewalskii living at lower altitude,the mechanisms of Hb adaptation to high altitude in P.erythrurus involved several aspects of changes.Firstly,the higher expression of Hb genes(?A,?D and ?1)will reslut in a increased total [Hb] which may increase the the arterial oxygen concentration and hence increasing in the blood oxygen transport efficiency.Secondly,similar to birds and turtles,Phrynocephalus lizards exhibited Hb polymorphism that contained four types of Hb isoforms with the following rank-order of oxygen affinities(low to high): HbAA1,HbAA2,HbAD1 and HbAD2.The rate of ?D/?A in P.erythrurus was significantly higher than that in P.przewalskii suggested that the whool blood affinity may be elevated,since the ?D-type Hbs typically have a higher oxygen affinity than ?A-type Hbs.The high-affinity HbD isoforms can ensure efficient pulmonary oxygen loading in the lung whereas low-affinity HbA isoforms can ensure the efficient unloading of oxygen to the cells of aerobically metabolizing tissues.Finally,the HbAA2 models of the P.przewalskii and P.erythrurus are distinguished by an amino acid substitution in the ?2-chain,21Gly-Ser,which introduces an additional hydroxyl group by which two additional hydrogen bonds were formed between 21 Ser and 61/65 Lys.The ultimate effect was that a subtle conformational change in the ?2-chain distal heme pocket can reduce steric hindrance of ?2 heme ligand-binding,thereby conduciving to the oxygen loading and unloading.Under acute hypoxia,contrary to mammals and birds,there was no hypoxic ventilatory response(HVR)in both P.przewalskii and P.erythrurus indicated that the respiratory system of Phrynocephalus lizards was not sensitive to acute hypoxia.This desensitization may be a common phenotype in all Phrynocephalus lizards and it seems to not directly related to the elevation.The respiratory rate of P.erythrurus was significantly higher than that of P.przewalskii,but the heart rate was significantly lower than that of P.przewalskii.The characteristics of respiration rate and heart rate in both lizards seem to be environmental adaptation under long-term evolution which may involve the desensitization of oxygen concentration in the peripheral chemoreceptors.The elevated respiratory rate of P.erythrurus did not result in respiratory alkalosis(no significnt difference in pH between the two species),so we suggested that the elevated respiratory rate of this species was a result of long-term evolutionary adaptation in the high altitude environment.Our results are consistent with the findings of Ying Xin(2015)that the expression of Mb was significantly upregulated in the heart and skeletal muscle of P.erythrurus comparaed with P.przewalskii.In addition,we also detected the expression of the this gene in the brain and liver of the high-altitude P.erythrurus,whereas the low-altitude P.przewalskii did not express Mb.However the relatively low expression abundance rendered the oxygen supply function of Mb in both tiusses unlikely.Mb is mainly deoxygenated in the brain and liver with high aerobic metabolism,thus we speculate that the expression of Mb in the brain and liver of P.erythrurus may be involved in NO-mediated blood flow adjustment.Although the physiological function of the globin new members are not yet clear,our study suggested that Ngb and Cygb may play important roles in high altitude adaptation of Phrynocephalus lizards.The amino acid mutations at positions 11,33,37 and 131 of Ngb increased the hydrophilicity of the protein and hence facilitated the transfer of the protein from the surface of the mitochondria to the cytoplasm.This may suggested that high altitude Phrynocephalus lizards are likely to accumulate higher concentrations of Ngb in the cytoplasm,and it may perform physiological functions such as oxygen supply,NO detoxification and ROS defense in the cytoplasm.Compared with P.przewalskii,the expression of Cygb-X1 in the liver of P.erythrurus was significantly up-regulated.The aerobic metabolism of liver is active,and hypoxia will inhibit its aerobic metabolism.The up-regulation of Cygb-X1 in the liver of P.erythrurus may be beneficial to alleviate hypoxia of this tiusse.Compared with P.przewalskii,protein structure analysis showed that amino acid mutation at position 194 of Cygb-X1 of P.putjatia at high altitude could reduce the stability of Cygb-X1 dimer structure,which may be beneficial to the oxygenation-linked dissociation of dimer into ligated monomers and hence increase its oxygen affinity and oxygen supply efficiency.Consequently,Cygb-X1 of high altitude Phrynocephalus lizards have evolved different adaptative mechanisms to cope with hypoxic environment.In conclusion,the globin gene family(except GbY)of Phrynocephalus lizards showed a variety of high altitude adaptive characteristics.Our results fill the gap of vertebrate globin gene family research in squamates and the physiological and biochemical adaptation to high altitude in genus of Phrynocephalus.We hope that this work is usefull to enrich and improve the knowledge and theory about adaptations to high altitude hypoxia and the development of plateau medicine.