Dr. Goodman's laboratory has recently identified a molecular defect in the erythrocyte membrane skeleton of two of four hereditary spherocytosis (HS) kindreds under investigation. In these HS kindreds (Type I HS), a defective spectrin molecule leads to an altered spectrin-protein 4.1 interaction, which in turn leads to a defective spectrin-protein 4.1-actin ternary complex. As this defect does not correlate with red cell age in the circulation or splenic function, but does correlate with the defective gene dose in this autosomal dominant disorder it may be the primary defect causing HS in these kindreds. In the proposed studies the other two kindreds (Type II HS) as well as newly available HS kindreds will be screened for a defect in their membrane skeletons by functional assays performed on the purified proteins, as well as one and two dimensional peptide mapping analysis of skeletal proteins. The Type I defect will be identified by a series of experiments including [1] isolation of the defective HS gene product by affinity chromatography on a protein 4.1-sepharose column, [2] isolation of the defective 74,000 Mr Beta-4 domain from the defective HS gene product and normal spectrin, [3] protein 4.1 binding analysis and peptide mapping analysis on the 74,000 dalton tryptic fragment of the Beta subunit confirming the localization of the defect, and [4] amino acid sequence analysis on the Type I HS and normal 74,000 Mr Beta-4 domain. These studies should lead to the identification of the exact amino acid substitution (or less likely post translational modification) causing the Type I HS membrane skeletal disorder. We will also study the association of protein 4.1 with it's membrane attachment site so that this new functional assay can be used to screen HS subjects for a potential defect in this interaction, and towards our goal of identifying the high affinity protein 4.1 membrane attachment protein. These studies should lead to major advances in our understanding of the molecular basis of HS, as well as elucidating the functional interactions of protein 4.1 in the normal erythrocyte membrane skeleton.
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