Altered Assembly of Spectrin in Red Cell Membranes in Hereditary Pyropoikilocytosis

Abstract

Red cells from hereditary pyropoikilocytosis (HPP) are unstable and more susceptible than normal to thermal damage in vitro. We have investigated whether or not the HPP instability results from alterations in spectrin assembly in the membrane. We identified the nearest membrane protein neighbors employing oxidative crosslinking by intermolecular disulfide couplings. We subsequently separated the crosslinked complexes by polyacrylamide gel electrophoresis (PAGE), subjected them to reductive cleavage and identified the crosslinked proteins by PAGE in the 2nd dimension. To prevent changes in membrane protein organization due to decrease of ATP and GSH levels during incubation, we used anaerobic incubation with adenine, inosine, and glucose. When incubated at 46°C (15 min) or 37°C (> 16 hr), HPP red cells were fragmented and converted to poikilocytes and spherocytes. Subsequent oxidative crosslinking of HPP red cells with diamide or of their ghosts with CuSO4, o-phenanthroline produced a high mol wt (>1 × 106) protein complex selectively enriched in spectrin. A similar complex was produced by crosslinking of HPP ghost amino groups with glutaraldehyde, suggesting rearrangement of spectrin to closer contacts. Normal red cells formed such aggregates only at temp >49°C. In both normal and HPP cells, aggregate formation paralleled red cell fragmentation and poikilocyte formation. In fresh HPP red cells, the crosslinking failed to produce the spectrin enriched complex. However, fresh HPP membrane protein PAGE revealed decrease in spectrin, presumably due to its aggregation in vivo followed by removal of the aggregate by the spleen. Spectrin-actin skeletons of heat treated HPP ghosts were poikilocytic, unstable and exhibited irregularities in electron density, suggesting abnormalities in cytoskeletal protein assembly. These data demonstrate that spectrin from HPP red cells is more susceptible than normal to aggregation in the membrane. Abnormal spectrin assembly may be responsible for membrane instability and shape of alterations of HPP erythrocytes.