The erythrocyte membrane cytoskeleton, a lattice of proteins on the inner surface of the cell membrane, determines its unique shape, its flexibility and durability. Spectrin, dimer containing alpha- and beta-subunits, is the major component of the membrane cytoskeleton and its unique molecular properties and interactions with other cytoskeletal proteins are vital to the function and survival of the red blood cell in vivo. Defects of the structural network are considered to be the cause of the fragility of red blood cells in various forms of inherited hemolytic anemias including hereditary elliptocytosis where the defect has been located in the alpha-spectrin subunit. In order to understand how erythrocyte spectrin fulfills its functions and how mutations disrupt it, the alpha-spectrin molecule was characterized by cloning and nucleotide sequencing of its mRNA. The cloned cDNAs were used to clone and determine the structure of the alpha-spectrin gene. In this proposal the investigators intend to further characterize the alpha-spectrin molecule. Based on the amino acid sequence and structure of the alpha-spectrin gene, the NH2 terminal domain, and the alphaI domain of alpha-spectrin, will be expressed in a high expression system. The recombinant alphaI domain peptide will be characterized with respect to its secondary and tertiary structure, its side to side interaction with beta-spectrin and the head interaction of dimers to form tetamers. The role of the 106 amino acid repeat unit and the highly conserved amino acids of the repeat unit will be investigated by using oligonucleotide site directed mutagenesis on the cloned alphaI domain cDNA to create modified forms of the alphaI domain peptide. The structure and interactions of the modified alphaI domain with beta-spectrin will be determined. Mutagenesis will also be used to create the mutations, observed in hereditary elliptocytosis where the alpha-spectrin molecule is deficient, in the cloned alphaI domain cDNA, so that mutated alphaI domain peptide will be produced. The effect of the mutation of the secondary tertiary structure of the alphaI domain will be determined, as well as its ability to interact with alpha-spectrin. The formation of heterodimers is essential for the functioning of erythrocyte spectrin. Therefore the beta-spectrin gene will also be cloned and characterized so that the functioning of the beta-spectrin protein can be studied in the same way as alpha-spectrin.
