BAND 3 PROTEIN IN SOUTHEAST ASIAN OVALOCYTOSIS

东南亚卵胞增多症是一种独特的遗传性疾病。 在东南亚某些地区非常普遍的椭圆形红细胞增多症。 卵圆细胞红细胞非常坚硬，能抵抗疟疾的侵袭。 最近，我们发现卵圆细胞含有一种功能性的和 结构异常的带3蛋白，主要的跨膜蛋白 红血球。从功能上讲，它的细胞质结构域(氨基酸1-403) 来自SAO的带3(Cd3)比正常的cdb3结合得更紧密 锚定蛋白。卵圆细胞带3与潜在的锚蛋白的紧密结合- 含有膜骨架的患者侧方显著减少 卵母细胞带3在膜上的迁移率。从结构上讲，我们发现 Cdb3的胰蛋白酶多肽的异常。该基因的克隆及序列分析 对应于氨基酸1-200的区域显示了 赖氨酸-56由谷氨酸合成。目前，还不完全清楚 这种结构和cdna缺陷是否是上述的原因 功能异常或仅仅是一种连锁多态本身 不会产生任何功能后果。后一种可能性意味着 Sao的潜在分子缺陷存在于cdb3的其他地方。 主要结构。因为这一缺陷可能会为 带3对膜变形性的调节机制 关于蛋白质，我们计划扩大这些研究，重点放在以下领域： (1)在一级结构上鉴定SAO带3的分子缺陷 水平通过测序对应的cDNAs或18kD的胰酶片段 来自带3的氨基酸201-403的区域。(2)验证 患有SAO的人，无论他们的种族血统，都有相同的 突变，(3)检查带3的个体-孟菲斯或其他 用缝隙杂交技术检测多态有这种特殊的突变 用适当的寡核苷酸探针。(4)研究突变体的作用 条带3在红细胞膜刚性增加中的作用 突变条带3进入正常红细胞及其细胞膜变化的检测 核孔过滤吸入的变形性，形状响应 棘细胞刺激和带3横向迁移率的测量。(5)至 阐明膜增厚的分子机制 刚性，包括骨骼蛋白的长期稳定性- 条带3与蛋白质的结合。(6)研究SAO带3在蛋白质结合中的作用。 通过导入突变带3对疟疾入侵的抗性 正常红细胞及其抗病能力的后续测定 疟疾的入侵，并探索是否以及如何形成一个 寄生虫与寄主细胞膜之间的连接，发育 膜液泡，寄主细胞膜的内陷和 条带3的重排在疟疾入侵期间受到损害 细胞免疫、阴性染色和冷冻断口电子显微镜观察 技巧。总而言之，这些研究应该在以下方面提供重要线索 关于异常带3在膜调节中的作用 刚性与疟疾抗性及疟疾的分子机制 寄生虫进入细胞。

Southeast Asian ovalocytosis (SAO) is a unique form of hereditary elliptocytosis that is highly prevalent in certain parts of Southeast Asia. Ovalocytic red cells are very rigid and are resistant to malaria invasion. Recently, we have found that ovalocytes contain a functionally and structurally abnormal band 3 protein, the principal transmembrane protein of red cells. Functionally, the cytoplasmic domain (amino acids 1-403) of band 3 (cdb3) from SAO bound considerably more tightly than normal cdb3 to ankyrin. This tight binding of ovalocyte band 3 to the underlying ankyrin- containing membrane skeleton is associated with a markedly reduced lateral mobility of ovalocyte band 3 in the membrane. Structurally, we found abnormalities in the tryptic peptides of the cdb3. cDNA sequencing of the region corresponding to amino acids 1-200 revealed a substitution of lysine-56 by glutamic acid. At the present time, it is not entirely clear whether this structural and cDNA defect is the cause of the above functional abnormalities or merely a linked polymorphism which in itself has no functional consequences. The latter possibility means that the underlying molecular defect of SAO resides elsewhere within the cdb3 primary structure. Because this defect may provide important insights to the mechanisms of regulation of membrane deformability by the band 3 protein, we plan to extend these studies focusing on the following areas: (1) To identify the molecular defect of SAO band 3 at the primary structure level by sequencing the cDNA or the 18 kD tryptic fragment corresponding to the region derived from amino acids 201-403 of band 3. (2) To verify that individuals with SAO, regardless of their ethnic origin have an identical mutation, (3) To examine whether individuals with band 3-Memphis or other polymorphisms have this particular mutation using slot blot hybridization with appropriate oligonucleotide probes. (4) To study the role of mutant band 3 in the increase of red cell membrane rigidity by introduction of mutant band 3 into normal red cells and testing for the change of membrane deformability by Nucleopore filter aspiration, shape responses to echinocytic stimuli and measurement of band 3 lateral mobility. (5) To elucidate the molecular mechanisms leading to the increased membrane rigidity including the long range stabilization of the skeletal protein- protein association by band 3. (6) To study the role of SAO band 3 in the resistance to malaria invasion by the introduction of mutant band 3 into normal red cells and the subsequent measurement of their resistance to malaria invasion, and to explore if and how the processes of forming a junction between the parasite and the host membrane, the development of membrane vacuoles, invagination of the host cell membrane and the rearrangement of band 3 are impaired during the malaria invasion of SAO red cells by immuno, negative staining and freeze fracture electron microscopic techniques. In summary, these studies should provide important clues in regard to the role of the abnormal band 3 in regulation of membrane rigidity and malaria resistance and the molecular mechanisms of malaria parasite entry into the cells.