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.
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