Molecular mechanism for the assembly of red cell membrane skeletons based on pathobiology of congenital hemolytic anemia in cattle

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Stable transfectants of normal bovine band 3 (bebWT) or the mutant band 3 with R664X mutation (bebRX) were established in K562 cells and HEK293 cells using retoriviral vectors. Transfected cells expressing EGFP-bebWT and EGFP-bebRX, and N-terminal domain of ankyrin (AnkN90) in combination with band 3 proteins were also prepared.The bebWT and EGFP-bebWT showed stable expression on the plasma membrane of the transfected cells, whereas the mutant proteins, bebRX and EGFP-bebRX were degraded by Ub-proteasome system soon after synthesis on the ER or after retrograde transported from the Golgi apparatus to the ER. Stability of the bebWT was extremely reduced when bebRX was co-transfected. AnkN90 showed membrane localization within the cells and was destabilized in the cells that had the mutant band 3.These findings indicate that the mutant band 3 (bebRX) plays a dominant-negative role on the expression of normal band 3 and a partner in the membrane skeleton, ankyrin, and the interaction of band 3 with ankyrin occurs on the ER membrane soon after band 3 synthesis is started during erythroid development.