POLYMERIZATION OF RECOMBINANT SICKLE HEMOGLOBIN MUTANTS: TRANSGENIC MOUSE MODEL

为了提供有关这些地点的定量信息， 促进镰状血红蛋白形成后的聚合， 涉及瓦尔-6（p）（E6 V（p））初始疏水键，还涉及 提供具有增强的聚合作用的血红蛋白 在镰状细胞性贫血小鼠模型中，我们表达了重组 双、三和四极HbS突变体， α-和0-链，E6 V（P）/EI 2 IR（P），D 75 Y（α）/E6 V（P）/EI 21 R（P）和 D6A（a）/D75Y（a）/E6V（0）/EI2IR（P）。 这些重组血红蛋白是 通过HPLC分析、SDS-PAGE、等电 聚焦、氨基酸分析和质谱。 他们保留 血红蛋白四聚体和血红蛋白四聚体的功能特性， Hb浓度逐渐降低时的线性方式， 取代度，表明这些远端位点（aD 6A， aD 75 Y和PE 121 R）在α-和0-链上表现出加和、增强的 聚合性能 四极突变体具有聚合作用 浓度接近于来自 由HbS、Hb A ntilles和Hb D组成的转基因小鼠红细胞 - 旁遮普。 正常小鼠Hb增加聚合浓度 每一个变种人 因此，使用重组的一般方法 这里描述的血红蛋白应该证明有助于阐明 HbS聚合机制的定量方面， 确定各个站点对总体 过程 本文所述的战略证明了以下做法的可行性： 获得未来重组HbS突变体的系统方法， 可以提供新一代的转基因小鼠模型， 镰状细胞性贫血 已经提交了一份介绍这项工作的文件。

In order to provide quantitative information on the sites that promote polymerization of sickle hemoglobin after formation of the initial hydrophobic bond involving Val-6(p) (E6V(p)) and also to provide hemoglobins with an enhanced polymerization that could be used in a mouse model for sickle cell anemia, we have expressed recombinant double, triple and quadrupole HbS mutants with substitutions on both the a- and 0- chains, E6V(P)/EI2IR(P), D75Y(a)/E6V(P)/EI21R(p) and D6A(a)/D75Y(a)/E6V(0)/EI2IR(P). These recombinant hemoglobins were extensively characterized by HPLCanalysis, SDS-PAGE, isoelectric focusing, amino acid analysis and mass spectroscopy. They retained the functional properties of the Hb tetramer and polymerize in a linear manner at progressively lower Hb concentration as a function of the degree of substitution, suggesting that these remote sites (aD6A, aD75Y and PE121R) on the a- and 0-chains exhibit additive, enhanced polymerization properties. The quadrupole mutant has a polymerization concentration close to that of the purified SAD hemoglobin from transgenic mouse red cells consisting of HbS , Hb A ntilles and Hb D -Punjab. Normal mouse Hb increases the polymerization concentration of each mutant. Thus, the general approach of using recombinant hemoglobins as described here should prove useful in elucidating the quantitative aspects of the mechanism of HbS polymerization and in identifying the contribution of individual sites to the overall process. The strategy described here demonstrates the feasibility of a systematic approach to achieve future recombinant HbS mutants which could provide a new generation of the transgenic mouse model for sickle cell anemia. A paper describing this work has been submitted.