Red cell shape regulation by band 3–ankyrin–spectrin linkage: implications for clinical severity of bovine hereditary spherocytosis

• Band 3–ankyrin–spectrin link promotes spectrin tetramerization to maintain mechanical stability and deformability of the red cell membrane. • The E91K substitution in α-spectrin destabilizes spectrin tetramerization and exacerbates spherocytic phenotype due to band 3 deficiency. The mechanical stability and shear elasticity of the red cell membrane are regulated by the proteins of the membrane skeleton consisting of rod-like α 2 β 2 spectrin tetramers attached at their distal ends to the junctional complexes, whereas the stability of the lipid bilayer is maintained by the linkage between transmembrane proteins, such as band 3, and the underlying spectrin-based skeleton. Although it has long been suggested that the lipid bilayer–membrane skeleton linkage is also involved in stabilizing the spectrin-based skeleton, no naturally occurring cases exist to substantiate this hypothesis. Here, we report that a novel substitution mutation in bovine α-spectrin E91K, located in the middle of the first spectrin repeat α1, causes disruption of the stable triple-helical bundle of this domain and impairs the spectrin dimer-dimer self-association, resulting in decreased spectrin tetramer formation leading to pronounced decrease in red cell mechanical stability. The E91K substitution markedly exacerbated the loss of membrane surface areas in hereditary spherocytosis due to band 3 deficiency, presumably through the increased membrane fragmentation. Notably, the red cells carrying E91K substitution and normal band 3 contents had only mild spectrin deficiency without significant hematological abnormalities, but on perturbation of band 3–ankyrin association, they exhibited disruption and fragmentation of the spectrin network. Taken together, these findings demonstrate that the band 3–ankyrin–spectrin linkage plays a key role in promoting and reforming the spectrin tetramer to maintain mechanical stability and deformability of the membrane skeleton, in addition to stabilizing the lipid bilayer in red cells.