Characterization of the linkages between the erythrocyte membrane and its spectrin-based skeleton.

摘要

The principal bridge connecting the red blood cell membrane to its underlying membrane cytoskeleton is established by the band 3-ankyrin interaction. This linkage is critical for maintaining membrane shape, elasticity and mechanical stability during transit of the cell through capillaries that are smaller than the cell's diameter. The first chapter provides a simple description of the main components of the erythrocyte membrane and the main horizontal and vertical interactions therein. In chapter two, we have undertaken to evaluate the rates of rupture and reattachment of the ankyrin-band 3 interaction under a variety of resting and mechanically stressed conditions. We conclude that there exists a reasonably facile on/off rate of the ankyrin-band 3 bridge in the human erythrocyte membrane under resting conditions. This rate appears to be moderately accelerated in the presence of shear-induced stress on the erythrocyte membrane.; Another important linkage between the membrane bilayer and its underlying spectrin skeleton has been suggested to involve the bridging of glycophorin C to the junctional complex via protein 4.1. However, recent evidence suggests that this putative major tether does not contribute to erythrocyte membrane stability and/or deformability. In chapter three, we have provided evidence of a direct interaction of adducin, a component of the junctional complex, to the integral membrane protein, band 3. Specifically, we have shown that band 3 binds predominantly to the c-terminal domain of beta-adducin. Further, we have demonstrated that band 3 knockout erythrocytes show a marked reduction in the amount of adducin retained compared to wild type. Also, band 3 levels were found to be reduced in detergent extracted membrane skeletons derived from leaky eythrocytes resealed in the presence beta-adducin fragment. Based on these and other data, we conclude that adducin constitutes a major bridge between the erythrocyte membrane bilayer and the junctional complex.; Finally, in collaboration with others, we demonstrate in chapter four that the extreme N-terminal domain of band 3 is critical to the binding and organization of the glycolytic enzyme complex. Red cells obtained from a proband (human missing the first 11 N-terminal amino acid residues of band 3) show reduced levels of aldolase and G3PDH compared to erythrocytes obtained from normal healthy patients.