Rettig M P, Low P S, Gimm J A, Mohandas N, Wang J, Christian J A
Departments of Chemistry and Veterinary Pathobiology, Purdue University, West Lafayette, IN, USA.
Blood. 1999 Jan 1;93(1):376-84.
One hypothesis to explain the age-dependent clearance of red blood cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC's life span induces clustering of the integral membrane protein, band 3. In turn, band 3 clustering generates an epitope on the senescent cell surface leading to autologous IgG binding and consequent phagocytosis. Because dog RBCs have survival characteristics that closely resemble those of human RBCs (ie, low random RBC loss, approximately 115-day life span), we decided to test several aspects of the above hypothesis in the canine model, where in vivo aged cells of defined age could be evaluated for biochemical changes. For this purpose, dog RBCs were biotinylated in vivo and retrieved for biochemical analysis at various later dates using avidin-coated magnetic beads. Consistent with the above hypothesis, senescent dog RBCs were found to contain measurably elevated membrane-bound (denatured) globin and a sevenfold enhancement of surface-associated autologous IgG. Interestingly, dog RBCs that were allowed to senesce for 115 days in vivo also suffered from compromised intracellular reducing power, containing only 30% of the reduced glutathione found in unfractionated cells. Although the small quantity of cells of age >/=110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster in response to hemichrome binding. Importantly, band 3 in RBCs >/=112 days old was found to be 25% less restrained by skeletal interactions than band 3 in control cells, indicating that the normal linkages between band 3 and the membrane skeleton had been substantially disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly assumed to reflect RBC age, was found to be maximal in RBCs isolated only 58 days after labeling, implying that while this marker is useful for identifying very young populations of RBCs, it is not a very sensitive marker for canine senescent RBCs. Taken together, these data argue that several of the readily testable elements of the above hypothesis implicating band 3 in human RBC senescence can be validated in an appropriate canine model.
一种解释红细胞（RBC）从循环中按年龄依赖性清除的假说提出，在RBC寿命后期产生的变性/氧化血红蛋白（高铁血红素）会诱导整合膜蛋白带3发生聚集。反过来，带3聚集会在衰老细胞表面产生一个表位，导致自体IgG结合并随之发生吞噬作用。由于犬RBC的存活特征与人类RBC非常相似（即随机RBC损失低，寿命约115天），我们决定在犬模型中测试上述假说的几个方面，在该模型中可以评估特定年龄的体内衰老细胞的生化变化。为此，犬RBC在体内进行生物素化，并在之后的不同时间使用抗生物素蛋白包被的磁珠取回进行生化分析。与上述假说一致，发现衰老的犬RBC含有可测量到的膜结合（变性）球蛋白升高以及表面相关自体IgG增强了7倍。有趣的是，在体内衰老115天的犬RBC也存在细胞内还原能力受损的情况，其还原型谷胱甘肽含量仅为未分级细胞中的30%。尽管年龄≥110天的细胞数量较少，无法直接定量带3聚集，但仍然可以利用单细胞微变形方法来评估失去正常骨架连接并因高铁血红素结合而自由聚集的带3分子的比例。重要的是，发现年龄≥112天的RBC中的带3受骨架相互作用的限制比对照细胞中的带3少25%，这表明带3与膜骨架之间的正常连接已被严重破坏。有趣的是，通常认为反映RBC年龄的蛋白4.1a/蛋白4.1b比值在标记后仅58天分离的RBC中最高，这意味着虽然该标志物对于识别非常年轻的RBC群体有用，但它不是犬衰老RBC的非常敏感的标志物。综上所述，这些数据表明，上述涉及带3在人类RBC衰老中的几个易于测试的要素可以在合适的犬模型中得到验证。
