STRUCTURE & FUNCTION OF HUMAN RBC MEMBRANE PROTEIN 7.2B

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The band 7 proteins (MW 26-30 kda) of the human erythrocyte constitute about 3.4% of the membrane protein mass. One of the proteins (protein 7.2b) is missing from the erythrocytes of all Caucasian cases of hereditary stomatocytosis, a disorder of monovalent cation permeability that gives rise to chronic hemolytic anemia. Although this finding suggests that protein 7.2b may be part of an erythrocyte ion channel, its actual function is currently unknown. The objective and specific aims of this proposal are 1) to understand the structure of protein 7.2b and its topography within the erythrocyte membrane, 2) to evaluate its putative function as a component of an ion channel, and 3) to explore the timing and mechanics of its biosynthesis in erythrocyte progenitors and of its insertion into the membrane lipid bilayer. To achieve these goals, protein 7.2b c DNA from erythroid tissues will be identified immunologically or by oligonucleotides using a bacteriophage expression library in E. Coli. Positive clones will be sequenced (Sanger) to determine the nucleotide sequence (and from this the amino acid sequence) of the entire coding region of protein 7.2b. The amino acid sequence will be verified by chemical methods and the sites of known structural modification (phosphorylation, palmitylation) determined. Parallel protein and nucleic acid studies in hereditary stomatocytes are expected to yield an explanation for the absence of protein 7.2b in these cells. The possible role of protein 7.2b in monovalent cation transport will be evaluated by 1) comparison of the structure of 7.2b to that of known transport proteins, 2) modification of protein 7.2b function by incorporation of polyvalent anti-7.2b antibody into intact red cells using the technique of osmotic lysis and resealing with subsequent evaluation of 86Rb transport, and 3) introduction of protein 7.2b into liposomes followed by evaluation of effects on cation transport. Protein 7.2b function will also be evaluated in non-erythroid cells. The time of appearance of protein 7.2b during erythroid maturation as well as its insertion into the plasma membrane will be determined by immunofluorescent microscopy and by Northern blot analysis, searching for the presence of 7.2b mRNA. The significance of this work lies in its potential for defining the molecular basis of a cation transport system in the erythrocyte and of a disease associated with hemolytic anemia in man.