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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL033884-07
Application #
3346208
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1985-05-01
Project End
1995-04-30
Budget Start
1991-05-01
Budget End
1992-04-30
Support Year
7
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Wistar Institute
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Ursitti, J A; Kotula, L; DeSilva, T M et al. (1996) Mapping the human erythrocyte beta-spectrin dimer initiation site using recombinant peptides and correlation of its phasing with the alpha-actinin dimer site. J Biol Chem 271:6636-44
Amin, K M; Scarpa, A L; Winkelmann, J C et al. (1993) The exon-intron organization of the human erythroid beta-spectrin gene. Genomics 18:118-25
Kotula, L; DeSilva, T M; Speicher, D W et al. (1993) Functional characterization of recombinant human red cell alpha-spectrin polypeptides containing the tetramer binding site. J Biol Chem 268:14788-93
Kotula, L; Laury-Kleintop, L D; Showe, L et al. (1991) The exon-intron organization of the human erythrocyte alpha-spectrin gene. Genomics 9:131-40
Kotula, L; Curtis, P (1988) Xba I polymorphisms in the 5' region of human erythrocyte alpha spectrin gene (Spna). Nucleic Acids Res 16:10950
Laurila, P; Cioe, L; Kozak, C A et al. (1987) Assignment of mouse beta-spectrin gene to chromosome 12. Somat Cell Mol Genet 13:93-7
Cioe, L; Laurila, P; Meo, P et al. (1987) Cloning and nucleotide sequence of a mouse erythrocyte beta-spectrin cDNA. Blood 70:915-20