生命科学学院知识库

高原鼢鼠(Myospalax baileyi)是青藏高原特有的一种地下鼠，隶属啮齿目鼹形鼠科(Spalacidae)，鼢鼠属(Myospalax)，几乎终生生活在低氧高二氧化碳的地下封闭洞道之中。已有很多研究从形态学、生理生化及分子生物学等方面来探究高原鼢鼠对其低氧高二氧化碳环境的适应机制。血红蛋白是脊椎动物血液中运输氧气和二氧化碳的重要工具，它的结构和功能在动物对极端环境的适应中起着重要作用。为了研究高原鼢鼠血红蛋白对低氧高二氧化碳环境的适应机制，本实验根据“改良扩散室法”自制了氧解离曲线测定装置，测定纯化后的高原鼢鼠血红蛋白在不同温度、pH及CO₂浓度下以及在变构效应物Cl^-和2,3-二磷酸甘油酸（DPG）不存在（stripped）和存在时的氧解离曲线，以小鼠血红蛋白为对照，对比分析高原鼢鼠血红蛋白的氧结合特性，包括对两种变构效应物Cl^-和DPG的敏感度、对高浓度二氧化碳的敏感度、对温度的敏感度以及其波尔效应，此外还以小鼠脱氧血红蛋白的晶体结构(3HRW.pdb)为模板，通过同源建模的方法构建了高原鼢鼠脱氧血红蛋白的结构模型，并对模板和模型进行分子动力学模拟以研究高原鼢鼠血红蛋白的结构特性，与高原鼢鼠血红蛋白的氧结合特性相联系，研究高原鼢鼠血红蛋白氧结合特性对低氧高二氧化碳环境适应的分子机制。

结果表明，高原鼢鼠的血红蛋白拥有显著高的固有氧亲和力，并且在所有相同实验条件下高原鼢鼠血红蛋白的P50均显著小于小鼠；在接近正常体温和pH的条件下，即温度为37℃、pH为7.6时，高原鼢鼠血红蛋白对Cl^-和DPG的敏感度低于相同条件下小鼠，并且5%CO₂的存在使得两种血红蛋白对Cl^-与DPG的敏感度进一步降低，而在低温25℃和低pH条件下高原鼢鼠血红蛋白有着和小鼠血红蛋白相似的高变构阴离子敏感度；变构效应物Cl^-和DPG不存在时（stripped），高原鼢鼠血红蛋白对5%CO₂的敏感度略低于小鼠，但在接近生理条件下（Cl^-与DPG都存在），高原鼢鼠血红蛋白对5%CO₂的敏感度高于小鼠；高原鼢鼠和小鼠血红蛋白在变构效应物不存在时（stripped）和存在时的波尔效应都小于相似实验条件下其它哺乳动物血红蛋白的波尔效应，且二者之间无显著差异，因此高原鼢鼠血红蛋白对pH的变化不敏感；虽然高原鼢鼠血红蛋白的固有温度敏感性高于小鼠，但仍与低温度敏感性的动物相似，并且在接近生理条件下（Cl^-和DPG都存在）高原鼢鼠的血红蛋白具有很显著的低温度敏感性。

根据以上高原鼢鼠血红蛋白氧结合特性的研究结果，我们认为高原鼢鼠血红蛋白与氧气的高亲和力最主要归因于其显著高的固有血氧亲和力，另外正常温度、正常pH及高CO₂条件下相对较低的阴离子变构效应敏感度也有助于提高血红蛋白与氧气的亲和力；在低温、低pH条件下较高的阴离子变构效应敏感度有助于氧气在身体末梢组织中释放；在接近生理条件（Cl^-和DPG都存在）下相对较高的CO₂敏感度能够提高在CO₂含量较高的代谢组织中高原鼢鼠血红蛋白的O₂释放能力和CO₂携带能力；高原鼢鼠血红蛋白的低温度敏感性有助于确保氧气运输过程顺利进行，血红蛋白能够将氧气平稳地运输至温度较低的身体末梢，同时减少身体的热量损失；尽管高原鼢鼠的低波尔效应不利于氧气在需氧组织中释放，但是上述其它氧结合特性的适应性改变及代谢组织如骨骼肌中微血管密度的增加有可能补偿这种不利影响。

分子动力学模拟结果的分析表明，高原鼢鼠血红蛋白显著高的固有氧亲和力主要归因于高原鼢鼠血红蛋白T态时（脱氧）两个半刚性二聚体（α1β1/α2β2）之间氢键的减少，尤其是α2链上131位Ser→Asn的氨基酸突变使得α1链N末端141位Arg与β2链35位Tyr之间的两个氢键丢失，这两个因素共同作用使两个半刚性二聚体间的相互作用变弱，减小了血红蛋白由T态向R态过渡时两个半刚性二聚体相对滑移的空间位阻，进而提高了高原鼢鼠血红蛋白的固有氧亲和力。此外α亚基的131位也是形成Cl^-结合位点（131Ser与1Val之间）的关键部位，该位点的突变可能是正常温度、pH条件下对Cl^-的敏感度低于小鼠的原因，同时该突变能够促进CO₂与其结合位点N端Val的结合，进而造成高原鼢鼠血红蛋白对CO₂的高敏感度；而对DPG的相对低敏感性可能归功于靠近β链N-末端DPG结合位点的β4Thr→Ser和β5Asp→Gly突变。α链111位Ala→Asn的氨基酸替换和β链的115位Gly→Ala的氨基酸替换，使这两个位点之间产生一个额外的氢键并且使半刚性二聚体内α与β之间的氢键增多，从而使高原鼢鼠血红蛋白的正常四聚体状态更加稳定，一定程度上也能提高血红蛋白与氧气的亲和力。最后，与小鼠血红蛋白相比，高原鼢鼠血红蛋白α1、α2及β2链的血红素口袋开口处肽段上的氨基酸残基具有高灵活性，有利于氧气进出血红素口袋，这可能是高原鼢鼠血红蛋白高固有氧亲和力的另一个分子机制。

Myospalax baileyi (The plateau zokor) is a one of subterranean rodents native to Qinghai-Tibet Plateau. Belonging to genus Myospalax family of Spalacidae, they spend nearly entire life underground in sealed burrows with hypoxia-hypercapnia. Many studies have been done to explore the adaptation mechanism of the plateau zokor to hypoxia-hypercapnia environment from the aspects of morphology, physiology, biochemistry and molecular biology, etc. Hemoglobin transports oxygen and carbon dioxide in the vertebrate blood and plays an important role in the adaptation of animals to extreme environments. To explore the adaptation mechanism of the plateau zokor hemoglobin to hypoxia-hypercapnia environment, an oxygen dissociation curve measuring apparatus has been developed according to the “modified diffusion chamber” method. Then the O₂ equilibrium of the purified hemoglobin were performed at different pH, temperature and CO₂ content in the absence (stripped) and present of Cl^- and DPG (two major anion allosteric effectors of Hb-O₂ affinity in the red cells) with mouse hemoglobin as control. The Hb-O₂ binding properties of the plateau zokor were compared with mouse hemoglobin, including its sensitivities to Cl^-, DPG, high CO₂, temperature and their Bohr effects. In addition, the structural model of deoxyhemoglobin in the plateau zokor was constructed by homology modeling using the crystal structure of mouse deoxyhemoglobin (3HRW.pdb) as template. Then molecular dynamics simulations were performed on templates and models to investigate the structural characteristics of the plateau zokor hemoglobin. Combined with O₂ binding properties of the plateau zokor hemoglobin, the molecular underpinnings of the adaptation of Hb-O₂ binding properties to hypoxia-hypercapnia were studied.

The results of Hb-O₂ binding properties showed that the plateau zokor hemoglobin has remarkably high intrinsic oxygen affinity, and the P50 of the plateau zokor hemoglobin are smaller than mice under all the same experimental conditions. Under conditions close to normal body temperature and pH, ie 37°C and pH 7.6, the plateau zokor hemoglobin is less sensitive to Cl^- and DPG than mouse under the same conditions. The presence of 5%CO₂ further reduced the sensitivity of these two kinds of hemoglobins to Cl^- and DPG. However, at low temperature (25℃) and low pH, the plateau zokor hemoglobin has a high anion sensitivity similar to that of mouse hemoglobin. The stripped Hb of the plateau zokor is slightly less sensitive to 5%CO₂ than mouse, while the plateau zokor hemoglobin is more sensitive to 5%CO₂ than mouse hemoglobin in near-physiological conditions (both Cl^- and DPG were presented). The Bohr effects of hemoglobin in the plateau zokor and mouse are less than that in other mammal hemoglobin under similar experimental conditions, and there are no significant differences between them. Therefore, the plateau zokor hemoglobin is insensitive to pH changes. Although the intrinsic temperature sensitivity of the plateau zokor hemoglobin is higher than that of mouse, it’s still similar to that of animals with low temperature sensitivity, and the hemoglobin of the plateau zokor has significant low temperature sensitivity when both Cl^- and DPG were presented.

Based on the results of Hb-O₂ binding properties of the plateau zokor, we suggested that the elevated Hb-O₂ affinities are mainly attributed to its remarkably high intrinsic Hb-O₂ affinity combined with a relative suppressed anion sensitivities (under normal temperature, pH, and high CO₂ conditions). High sensitivities to anion allosteric effectors at low temperature and low pH accelerate the releasing of O₂ in peripheral tissues of the body. The relatively high CO₂ effects (both Cl^- and DPG were present) elevate the O₂ releasing capacity and CO₂ carrying capacity of the plateau zokor hemoglobin in metabolic tissues where CO₂ content is higher. The low temperature sensitivity of the plateau zokor hemoglobin ensures steadily delivering O₂ to the cold peripheral tissues, while reducing the loss of heat in the body. Although the low Bohr effect of the plateau zokor hemoglobin is not conducive to O₂ releasing in aerobic tissues, the adaptive changes in other O₂-binding properties mentioned above and the increased microvessel density in metabolic tissues are act as the trade off of high intrinsic Hb-O₂ affinity to accelerate O₂ unloading.

The main molecular underpinnings of the intrinsic high Hb-O₂ affinity in the plateau zokor is the decrease of hydrogen bonds between α1β1 and α2β2 semirigid dimers interface. Specifically, amino acid substitution of the 131Ser→Asn on the α2 chain weakens the connection between the two semirigid dimers, thus reducing the space resistance of T/R quaternary structural transition. These two effects work together making the plateau zokor has remarkably high intrinsic Hb-O₂ affinity. Furthermore, 131 postion of α subunit is the key Cl^- binding site (between 131Ser and 1Val), substitution at this site may be the reason of lower Cl^- sensitivity of the plateau zokor hemoglobin compared with mouse hemoglobin at normal temperature and pH. Simultaneously, this mutation can promote the binding of CO₂ to N-terminal Val, thus leads to the high sensitivity of plateau zokor hemoglobin to CO₂. With regard to the suppressing sensitivity to DPG, it may be attributed to the β4Thr→Ser and β5Asp→Gly substitutions near the DPG binding site at β chains. Another mechanism of the high Hb-O₂ affinity of the plateau zokor may be the stabilization of dimer internal structure caused by the substitution of nonpolar Ala to polarity Asn at α111 and the substitution of Gly→Ala at β115, which generate an additional hydrogen bond in interdimer (α1β1 and α2β2) and increase the hydrogen bonds between α and β chain in each dimer. Lastly, compared with mouse hemoglobin, the amino acids line the opening of heme pockets of the α1, α2 and β2 chains of plateau zokor hemoglobin have higher flexibility, which may be beneficial to O₂ entering and leaving heme pockets. This may be another molecular mechanism of high intrinsic Hb-O₂ affinity of the plateau zokor.