Trifillis P, Adachi K, Yamaguchi T, Schwartz E, Surrey S
Division of Hematology, Abramson Pediatric Research Center, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1996 Oct 25;271(43):26931-8. doi: 10.1074/jbc.271.43.26931.
We previously identified five delta-globin gene alleles associated with reduced hemoglobin (Hb) A2 (Trifillis, P., Ioannou, P., Schwartz, E., and Surrey, S. (1991) Blood 78, 3298-3305). We have now evaluated functional consequences of the changes after expression in COS-1 cells to monitor effects on RNA splicing. In addition, variant Hb A2 tetramers were expressed in yeast to assess effects of amino acid changes on oxygen binding and stability to heat and mechanical agitation. The G --> T change at codon 27 and the A --> G change in IVS-2 both affect RNA splicing, whereas the C --> T change at codon 97 and the AT deletion in IVS-2 have no effect. Oxygen equilibrium curves of the Hb A2 variants expressed in yeast were similar to that of wild type Hb A2. None of the three variant Hb A2 tetramers (Thr --> Ile at codon 4 (Hb deltaT4I), Ala --> Ser at codon 27 (Hb deltaA27S), and Arg --> Cys at codon 116 (Hb deltaR116C)) showed decreased heat stability compared with Hb A2, whereas the Hb deltaT4I variant showed highest instability to mechanical agitation. Co-expression in yeast of alpha-globin chain and the delta-chain variant containing a Leu --> Pro change at codon 141 yielded no identifiable tetramers, suggesting lack of assembly or severe tetramer instability. These studies show the probable cause for decreased Hb A2 for two alleles is due to defective splicing, whereas decreased protein stability, increased tetramer association with red cell membranes, increased interdisulfide bond formation of delta-chains, which inhibits assembly with alpha-chains, and/or reduced assembly is suggested for the other three alleles.
我们之前鉴定出了五个与血红蛋白（Hb）A2降低相关的δ-珠蛋白基因等位基因（Trifillis, P., Ioannou, P., Schwartz, E., and Surrey, S. (1991) Blood 78, 3298 - 3305）。我们现在评估了在COS - 1细胞中表达后这些变化的功能后果，以监测对RNA剪接的影响。此外，在酵母中表达了变异的Hb A2四聚体，以评估氨基酸变化对氧结合以及对热和机械搅拌稳定性的影响。密码子27处的G→T变化和IVS - 2中的A→G变化均影响RNA剪接，而密码子97处的C→T变化和IVS - 2中的AT缺失则无影响。在酵母中表达的Hb A2变体的氧平衡曲线与野生型Hb A2相似。与Hb A2相比，三个变异的Hb A2四聚体（密码子4处的Thr→Ile（Hb δT4I）、密码子27处的Ala→Ser（Hb δA27S）和密码子116处的Arg→Cys（Hb δR116C））均未表现出热稳定性降低，而Hb δT4I变体对机械搅拌表现出最高的不稳定性。α-珠蛋白链与密码子141处含有Leu→Pro变化的δ-链变体在酵母中共表达未产生可识别的四聚体，这表明缺乏组装或四聚体严重不稳定。这些研究表明，两个等位基因导致Hb A2降低的可能原因是剪接缺陷，而另外三个等位基因则提示蛋白质稳定性降低、四聚体与红细胞膜的结合增加、δ-链间二硫键形成增加（这抑制了与α-链的组装）和/或组装减少。
