BAND 3 PROTEIN IN SOUTHEAST ASIAN OVALOCYTOSIS

东南亚卵圆细胞增多症（SAO）是一种独特的遗传性 椭圆形红细胞增多症，在东南亚某些地区非常普遍。 卵红细胞非常坚硬，对疟疾的侵袭有抵抗力。 最近，我们发现卵母细胞含有一个功能上和 结构异常带3蛋白，主要的跨膜蛋白 的红细胞。 在功能上，胞质结构域（氨基酸1-403）的 来自SAO的带3（cdb 3）比正常的cdb 3结合得更紧密， 锚蛋白 卵母细胞带3与下面的锚蛋白的紧密结合- 包含膜骨架与显著减少的外侧 卵母细胞带3在膜上的移动性。 在结构上，我们发现 CDB 3的胰蛋白酶肽的异常。 cDNA序列分析 对应于氨基酸1-200的区域揭示了 赖氨酸-56被谷氨酸取代。 目前还不完全清楚 这种结构和cDNA缺陷是否是上述原因 功能异常或仅仅是连锁多态性， 没有功能性后果。 后一种可能性意味着， SAO的潜在分子缺陷存在于cdb 3内的其他地方 主要结构。 因为这一缺陷可能会提供重要的见解， 通过带3调节膜可变形性的机制 蛋白质，我们计划扩大这些研究，重点放在以下领域： (1)确定SAO带3在一级结构上的分子缺陷 通过对cDNA或对应于以下的18 kD胰蛋白酶片段进行测序， 该区域衍生自条带3的氨基酸201-403。 (2)以验证 患有SAO的个体，无论其种族来源如何， （3）检测带3-孟菲斯或其他带型的个体是否存在突变， 使用狭缝印迹杂交技术， 用适当的寡核苷酸探针。 (4)研究突变体的作用 带3在红细胞膜刚性增加的引入 突变体带3进入正常红细胞并检测膜的变化 通过核孔过滤器抽吸的变形性， 棘细胞刺激和带3横向移动性的测量。 (5)到 阐明导致膜增加的分子机制 刚性，包括骨骼蛋白的长距离稳定性， 带3的蛋白质缔合。 (6)为了研究SAO带3在脑缺血中的作用， 通过将突变带3引入抗疟疾入侵的基因， 正常红细胞和随后测量其对 疟疾入侵，并探讨是否以及如何形成一个过程， 寄生虫和宿主膜之间的连接， 膜空泡，宿主细胞膜内陷和细胞膜的 在SAO红的疟疾入侵期间，条带3的重排受损 免疫组化、负染和冷冻断裂电镜观察 技术. 总之，这些研究应该提供重要的线索， 关于异常带3在膜调节中的作用， 刚性和疟疾抗性以及疟疾的分子机制 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.