Examination of the protein interaction between ankyrin and the anion exchanger in the erythrocyte membrane

Ankyrin-band 3 bridge is believed to play a critical role in maintaining membrane stability and red blood cell morphology. This work demonstrates that D3-D4 repeats of ankyrin interact with cdb3 with a stoichiometry of 1:1 monomer to monomer, in solution. In a separate assay using his-tagged cdb3 dimer along with a 125I-labeled cdb3 dimer we showed that a D3-D4 ankyrin fragment is capable of bridging two cdb3 dimers suggesting an existence of tetramer species of band 3 maintained in a solution by only two of the four subdomains of ankyrin involved in the membrane binding. Oxygenation state of the red blood cell has been shown to modulate critical functions in the erythrocytes such as membrane transport, glycolysis and glycolitic enzyme binding to membrane. Little is known about the effect of oxygenation on membrane-cytoskeleton interactions. We report here that deoxygenation of RBCs leads to the reduction of ankyrin binding to stripped inside-out-vesicles. Furthermore, ankyrin retains its membrane localization while displaying increased accessibility in deoxygenated erythrocytes. A beta-hairpin loop comprising residues 175-185 of the cytoplasmic domain of band 3 was shown in vitro to be a major ankyrin binding site on the erythrocyte membrane. We showed that a surgical deletion of this region of band 3 in transgenic mice was enough to ablate ankyrin binding to murine erythrocyte membranes while retaining a biconcave cellular morphology. Clearly, the ankyrin-band 3 linkage is not a requirement for maintenance of membrane stability and red cell morphology