A New Approach for Physical Functions of Band 3 Based on Molecular Pathobiology of Hereditary Band 3 Deficiency in Cattle

我们研究了牛受试者，表现出中度失代偿性贫血与遗传性球形红细胞增多症，遗传在一个常染色体不完全显性模式和生长迟缓。根据SDS-PAGE、免疫印迹和冷冻断裂法电镜分析的结果，我们在此表明先证者红细胞完全缺乏带3蛋白。为了阐明红细胞带3蛋白异常缺乏的分子基础，我们从正常和患病动物中克隆了红细胞带3 cDNA（~ 3.5kb）。先证者红细胞带3cDNA和基因组DNA的序列分析显示，C * T取代导致在对应于人红细胞带3cDNA中密码子646的Arg * 1位置处的无义突变（CGA * TGA ; Arg * Stop）<664>。先证者红细胞缺乏血影蛋白、锚蛋白、肌动蛋白和蛋白4.2，导致密度降低的扭曲和破坏的膜骨架网络。因此，先证者红细胞膜极不稳定，并以几种不同的方式显示表面积损失，如内陷、囊泡形成和微泡挤出，导致球形红细胞形成。带3的完全缺乏也导致CL^-/HCO_3^-交换的缺陷，引起轻度酸中毒，先证者血液中HCO_3^-浓度和总CO_2浓度降低。我们的研究结果表明，带3确实有助于红细胞膜的稳定性，CO_2的运输，酸碱平衡，但并不总是必要的，这种哺乳动物的生存。

We studied bovine subjects that exhibited a moderate uncompensated anemia with hereditary spherocytosis inherited in an autosomal incompletely dominant mode and retarded growth. Based on the results of SDS-PAGE,immunoblotting, and electron microscopic analysis by the freeze fracture method, we show here that the proband red cells lacked the band 3 protein completely. To elucidate the molecular basis of this aberrant deficiency of the band 3 protein, we cloned red cell band 3 cDNA (-3.5kb) from normal and the affected animal. Sequence analysis of the proband band 3 cDNA and genomic DNA showed a C * T substitution resulting in a nonsense mutation (CGA * TGA ; Arg * Stop) at the position of Arg^<664> that corresponded to codon 646 in human red cell band 3 cDNA.The proband red cells were deficient in spectrin, ankyrin, actin, and protein 4.2, resulting in a distorted and disrupted membrane skeletal network with decreased density. Therefore, the proband red cell membranes were extremely unstable and showed the loss of surface area in several distinct ways such as invagination, vesiculation, and extrusion of microvesicles, leading to the formation of spherocytes. Total dificiency of band 3 also resulted in defective CL^-/HCO_3^- exchange, causing mild acidosis with decreases in the HCO_3^- concentration and total CO_2 in the proband blood. Our results demonstrate that band 3 indeed contributes to red cell membrane stability, CO_2 transport, and acid-base homeostasis, but is not always essential to survival of this mammal.