The Mechanism For High-altitude Adaptation In Hemoglobin Of Black-spotted Frog(Pelophylax Nigromaculatus)

High-altitude environment was characterized by hypobaric hypoxia,high ultraviolet radiation and cold.As altitude increases,both ambient oxygen partial pressure and temperature decrease gradually.The deficiency of oxygen is one of the most significant factors that impact survival and development of animals.Hemoglobin plays a pivotal role in the oxygen transport system by binding to oxygen in lung and transporting it through blood circulation to oxygen-demanding tissues and organs.Hence,hemoglobin is considered an essential functional molecule in high-altitude adaptation research.Currently,researches on high-altitude adaptation of hemoglobin are mainly focused on birds and mammals,while studies on amphibians,especially frogs,are scarce.This is mainly due to the absence of genomic sequencing data that makes it challenging to determine the subunit composition and amino acid sequence of hemoglobin.This paper focuses on the black-spotted frog（Pelophylax nigromaculatus）,a species with wide distribution,abundance,and significant ecological value in China.The study collected two populations of this species from different altitudes:one from Guide County,Qinghai Province at an altitude of 2292 m,and the other from Ningyang County,Shandong Province at an altitude of 69 m.The research is divided into three parts.In the first part,we compared the fundamental indicators,such as body weight,relative lung mass and hematological parameters between the high-and low-altitude populations.In the second part,we measured the oxygen dissociation curves of hemoglobin from two populations under different conditions of temperature（10°C,20°C）,p H（7.0,7.5）,and allosteric effectors（Cl^-,ATP）.From these measurements,we calculated the P₅₀,Hill cooperativity coefficient(n₅₀),sensitivity to allosteric effectors,temperature sensitivity and Bohr effect of the hemoglobin.In the third part,subunit composition of the hemoglobin from the high-and low-altitude populations was identified,the gene sequences of each subunit was determined,and the main reasons for the functional differences in the hemoglobin was explored from the structural perspective.In terms of basic indicators,we found that the black-spotted frogs from the high-altitude population had significantly increased lung relative weight,hemoglobin concentration,and hematocrit,which enhanced their capacity for oxygen uptake and transport.Additionally,high-altitude population had significantly lower body weight than the low-altitude population,which may be related to the larger relative surface area of skin in smaller black-spotted frogs,enabling them to absorb higher proportion of oxygen through the skin.The functional analysis of hemoglobin reveals that the high-altitude population exhibits significantly increased Hb-O₂ affinity.However,both the high-altitude and low-altitude populations show lower sensitivity to allosteric effectors,temperature,and the Bohr effect,and there is no significant difference in these indicators between the hemoglobin of the two populations.It is therefore inferred that the elevated oxygen affinity of hemoglobin in the high-altitude population is directly related to its inherent Hb-O₂ affinity variation.The structure of hemoglobin determines its function.In this study,we successfully identifiedαandβsubunits of hemoglobin in black-spotted frogs using proteomic techniques combined with liver transcriptome data.Primers were designed based on the hemoglobin gene sequences of the common frog（Rana temporaria）,and then the gene sequences of theαandβsubunits of black-spotted frog hemoglobin were amplified.After translation into amino acids,we found two amino acid substitutions of Hb onαchain（Met63Val and Gln123Glu）and one amino acid substitution onβchain（Gln76His）between high-and low-altitude black-spotted frog.The result of analysis showed differences in the theoretical isoelectric point and the count of negatively charged residue of theαsubunit in the primary structure of hemoglobin between the high-and low-altitude population.The secondary structure composition was the same,with a high proportion ofαhelices in both populations.Regarding the quaternary structure,we used the current mainstream protein structure modeling software（Modeller and Alphafold 2）to model the structure of hemoglobin in high-and low-altitude black-spotted frogs.The model was evaluated and then performed 50 ns molecular dynamics simulation（all-atom）.Molecular dynamics simulations showed that the Gln123Glu substitution onα₂ chain in high-altitude frogs could form a hydrogen bond with 127Lys onα₂ chain,resulting in the elimination of a hydrogen bond between 127Lys onα₂ chain and 141Arg onα₁ chain.This could weaken the interaction between two semirigid dimers（α₁β₁ andα₂β₂）and then lead to the high intrinsic Hb-O₂affinity in high-altitude black-spotted frogs.Therefore,the absence of this hydrogen bond in the high-altitude black-spotted frog hemoglobin makes it easier for hemoglobin to bind oxygen,which is likely the reason why it exhibits high Hb-O₂ affinity.