The objective of the proposed studies is to elucidate the molecular basis of inherited elliptocytic and spherocytic anemias. The work will be designed to aid neonates, so that they may be treated quickly and appropriately. Red blood cells of subjects with hereditary spherocytosis, elliptocytosis and variant forms of these hemolytic anemias are initially screened for abnormal membrane components on SDS-polyacrylamide gels stained for protein or immunoblotted with site specific antibodies to spectrin, protein 4.1, and ankyrin. Membrane components suspected of being abnormal are extracted, purified, and subjected to restricted proteolysis; the resulting peptides are resolved on one or two dimensional gels which are stained and immunoblotted with antibodies directed toward specific domains within the protein. The ultimate goal of these studies is to identify mutant sites (point mutations, deletions, or insertions), responsible for the abnormal shape and survival of the red cell. In some cases, this is achieved by direct amino acid sequence of mutant peptides, followed by confirmation at the DNA level. In other cases, protein studies identify only the general location of the mutant site and are followed by amplification and sequence of DNA in that region. Identification of mutant sites within cytoskeletal proteins will be in some cases initiated, or directed by, studies of patient DNA extracted from peripheral blood leukocytes or spleen, using alpha and beta subunits, protein 4.1, and ankyrin. To evaluate the significance of altered cytoskeletal components, we propose to measure their capacity to modify the stability of the membrane skeleton. Spectrin mutants will be assayed for their ability to form stable dimers and undergo dimer-tretramer-oligomer transitions. Mutants of both spectrin and protein 4.1 will be assayed for their capacity to form ternary complexes of spectrin, 4.1 and actin. We will also search for mutant forms that destabilize the links between the membrane skeleton and its transmembrane protein anchoring sites. We believe that the characterization of molecular defects in the erythrocyte cytoskeleton responsible for hemolytic disorders will not only explain their altered shape, deformability and life span, but will help elucidate the functions and interactions of normal cytoskeletal components.
| Marchesi, S L; Conboy, J; Agre, P et al. (1990) Molecular analysis of insertion/deletion mutations in protein 4.1 in elliptocytosis. I. Biochemical identification of rearrangements in the spectrin/actin binding domain and functional characterizations. J Clin Invest 86:516-23 |
