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首页> 外文期刊>Journal of Molecular Biology >ROLES OF ALPHA-114 AND BETA-87 AMINO ACID RESIDUES IN THE POLYMERIZATION OF HEMOGLOBIN S - IMPLICATIONS FOR GENE THERAPY
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ROLES OF ALPHA-114 AND BETA-87 AMINO ACID RESIDUES IN THE POLYMERIZATION OF HEMOGLOBIN S - IMPLICATIONS FOR GENE THERAPY

机译：ALPHA-114和BETA-87氨基酸残基在血红蛋白S聚合中的作用-对基因治疗的意义

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Three novel recombinant mutants of sickle hemoglobin (Hb S, beta 6Glu --> Val) have been constructed to assess the role of proline at alpha 114 and threonine at beta 87 in the polymerization of deoxygenated Hb S. Using the hemoglobin expression system (pHE2) designed in our laboratory, four plasmids were expressed separately in Escherichia coli to produce the four recombinant hemoglobins: r Hb S (beta 6Glu --> Val); r Hb S-Chiapas (beta 6Glu --> Val, alpha 114Pro --> Arg); r Hb S-D-Ibadan (beta 6Glu --> Val, beta 87Thr --> Lys); and r Hb S-Chiapas-D-Ibadan (beta 6Glu --> Val, alpha 114Pro --> Arg, beta 87Thr --> Lys). The structural features of these four recombinant hemoglobins were analyzed by proton nuclear magnetic resonance spectroscopy, and were found to be similar to those of human normal adult hemoglobin (Hb A) under identical conditions. The recombinant hemoglobins were further investigated by measuring the oxygen-binding properties, which were found to be comparable to those of Hb A. Delay-time gelation studies of the three mutants of r Hb S were carried out in 1.8 M potassium phosphate (pH 7.34) by a temperature jump from 4 degrees C to 30 degrees C and an increase in delay time over that of r Hb S was observed, as well as an overall decrease in the polymerization of these three mutants of Hb S. A more detailed and quantitative investigation has also been carried out to determine the equilibrium solubility (C-sat) in 0.1 M potassium phosphate (pH 7.35) at 25 degrees C of the three Hb S mutants as well as of mixtures of these mutants with Hb S versus mixtures of fetal hemoglobin (Hb Fl and Hb A with Hb S. The inhibition of polymerization demonstrated in these experiments suggests that the interactions involving the two amino acid residues alpha 114Pro and beta 87Thr are very important to the formation of Hb S polymer, and modification of these amino acids results in an anti-sickling potential. Of particular interest is the inhibitory effect of alpha 114Pro --> Arg, which offers a novel opportunity to use an alpha-chain construct, in addition to a beta-chain construct in the same vector, in gene therapy for sickle cell anemia, with the objective of modifying a larger number of hemoglobin tetramers at a given level of expression. (C) 1996 Academic Press Limited [References: 34]
机译：已经构建了三种新型的镰状血红蛋白重组突变体（Hb S，β6Glu-> Val），以评估脯氨酸在α114和苏氨酸在β87在脱氧Hb S聚合中的作用。使用血红蛋白表达系统（pHE2 ）在我们的实验室中设计，四种质粒分别在大肠杆菌中表达，以产生四种重组血红蛋白：r Hb S（beta 6Glu-> Val）; r Hb S-Chiapas（beta 6Glu-> Val，alpha 114Pro-> Arg）; r Hb S-D-Ibadan（beta 6Glu-> Val，beta 87Thr-> Lys）;和r Hb S-Chiapas-D-Ibadan（beta 6Glu-> Val，alpha 114Pro-> Arg，beta 87Thr-> Lys）。通过质子核磁共振波谱分析了这四种重组血红蛋白的结构特征，发现它们在相同条件下与人正常成人血红蛋白（Hb A）相似。通过测量氧结合特性进一步研究了重组血红蛋白，发现其与Hb A相当。在1.8 M磷酸钾（pH 7.34）中对r Hb S的三个突变体进行了延迟时间胶凝研究。 ）导致温度从4摄氏度跃升至30摄氏度，并且观察到延迟时间超过r Hb S的延迟时间增加，而且这三个Hb S突变体的聚合反应总体下降。更详细和定量还进行了研究以确定三种Hb S突变体以及这些突变体与Hb S的混合物与胎儿的混合物在25°C下在0.1 M磷酸钾（pH 7.35）中的平衡溶解度（C-sat）血红蛋白（Hb Fl和Hb A与HbS。在这些实验中显示出的聚合抑制作用表明，涉及两个氨基酸残基alpha 114Pro和beta 87Thr的相互作用对于Hb S聚合物的形成非常重要，这些氨基酸的修饰会产生抗胶结潜力。特别令人感兴趣的是α114Pro-> Arg的抑制作用，它为在镰状细胞性贫血的基因治疗中提供了一个新的机会，除了在同一载体中使用β链构建体之外，还使用α链构建体，目的是在给定的表达水平上修饰更多的血红蛋白四聚体。 （C）1996 Academic Press Limited [参考文献：34]

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