Molecular defect in the sickle erythrocyte skeleton. Abnormal spectrin binding to sickle inside-our vesicles

Although functional abnormalities of the sickle erythrocyte membrane skeleton have been described, there is little quantitative data on the function of the proteins that compose the skeleton. We have examined the association of spectrin, the major skeletal protein, with ankyrin, its high-affinity membrane binding site, and found sickle erythrocytes to have markedly reduced binding. Binding is assayed by incubation of purified 125I-spectrin with spectrin-depleted inside-out vesicles (IOVs) and measurement of the label bound to IOVs. Sickle IOVs bind approximately 50% less ankyrin than do controls IOVs (P less than 0.001). Control experiments show that this reduced binding is not a function of faulty composition or orientation of sickle IOVs, or of reticulocytosis per se. Our least symptomatic patient has the highest binding capacity, suggesting that this abnormality may be related to clinical severity. This trend is supported by experiments showing that asymptomatic subjects with sickle trait, sickle cell anemia and high fetal hemoglobin, and sickle beta +-thalassemia have normal binding, whereas a symptomatic patient with sickle beta zero-thalassemia has abnormal binding. In contrast to what we see with ankyrin in situ on the IOV, when isolated and studied in solution, sickle ankyrin binds normally to spectrin. This discrepancy may be related to preferential purification of the normal ankyrin species or to an abnormal topography of the membrane near the spectrin attachment site. We hypothesize that sickle hemoglobin or perhaps the metabolic consequences of sickling damage the protein skeleton. This damage may alter the surface of the erythrocyte and result in abnormal cell-cell interactions which may be related to clinical severity.