The objective of Project 3 is to study molecular defects of red cell membrane proteins in hereditary elliptocytosis (HE) and spherocytosis (HS). Our effort has four major directions: (1) We wish to continue our work involving identification, at the DNA and deduced amino acid sequence levels, of abnormal HE spectrins which we have previously characterized at the level of spectrin function. Identification of the sites of these mutations is likely to provide important insights into the molecular control of spectrin self-association. Furthermore, in four kindred, we did not detect abnormalities in the amino acid or DNA sequence in the vicinity of the cleavage site, which gives rise to the abnormal tryptic peptide. In two of those families, we found that the primary structure defect, which leads to abnormal tryptic digestion of alpha spectrin, resides on beta spectrin; thus, identification of the site of the defect can provide new information in regard to long range conformational effects and alpha beta interchain contacts. (2) We will study the nature of the primary molecular defect in HS associated with band 3 deficiency. In contrast to the majority of HS patients who have a low spectrin to band 3 ratio, the newly defined subset of patients has a high spectrin/band 3, ankyrin/band 3, 4.1/band 3, and 4.2/band 3 ratios suggesting a partial deficiency of the band 3 protein. The objective of our work is to define the underlying defect addressing the following possibilities: (a) a primary defect of the band 3 protein such as reduced synthesis, protein instability, poor binding to ankyrin or a defect involving a reduced propensity of band 3 to form dimers and tetramers thereby diminishing the fraction of band 3 molecules which is bound to ankyrin or (b) a primary defect of ankyrin, possibly involving the band 3 binding domain, a defect which may weaken the ankyrin-band 3 interaction leading to a preferential loss of band 3 from the membrane. (3) We will characterize the defect of ankyrin in HS associated with abnormally migrating ankyrin. A family has been identified where an abnormally migrating ankyrin is coinherited with HS in three generations. The defect has been tentatively mapped to the regulatory domain. The principal steps to characterize this ankyrin mutation include identification of the underlying cDNA defect employing amplification of cDNA corresponding to the regulatory domain and a functional characterization of this mutant ankyrin. (4) We will study red cells from patients with autosomal dominant HS with spectrin deficiency, but with a normal or increased ankyrin content.
Our aim i s to define a group of patients in whom the primary defect underlying the deficiency of spectrin involves a weakening binding of spectrin to ankyrin due to a functionally abnormal spectrin or ankyrin. Once the defect is assigned to one of these proteins by functional studies or RFLP studies, further approach will involve limited proteolysis, mapping the site of the mutation by RNAase protection assay or GC clamp-denaturing gradient gel electrophoresis followed by sequencing of either cDNA or the respective exons of the genomic DNA.
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