Induction of Band 3 Aggregation in Erythrocytes Results in Anti-band 3 Autoantibody Binding to the Carbohydrate Epitopes of Band 3

Involvement of band 3 aggregation in the mechanism of anti-band 3 autoantibody binding to the cell surface carbohydrate epitopes of band 3 was investigated. When erythrocytes were treated nonoxidatively with a known protein-aggregating agent acridine orange, protein aggregates of the cell membrane which are insoluble in a nonionic detergent C12E8solution were remarkably increased. Analysis of the protein aggregates by SDS–PAGE indicated that they were composed of several species of noncovalently associated membrane proteins including band 3.125I-labeled anti-band 3 bound to the acridine orange-treated cells, and the binding increased depending on the concentrations of acridine orange used. The binding was inhibited by band 3 and its oligosaccharides but not by the oligosaccharides pretreated with endo-β-galactosidase, an enzyme specifically cleaves poly-N-acetyllactosaminyl saccharide chains of band 3. When erythrocytes were pretreated with endo-β-galactosidase to remove poly-N-acetyllactosaminyl saccharide chains from cell surface prior to acridine orange treatment, the cells did not become susceptible to anti-band 3 binding. The results indicate that induction of band 3 aggregation in erythrocyte membrane leads to anti-band 3 binding to the poly-N-acetyllactosaminyl saccharide chains of band 3. Consistently, membrane proteins including band 3 were found to be aggregated when erythrocytes were oxidized with ADP-chelated Fe3+under the conditions that induce anti-band 3 binding to the cells. Similar band 3 aggregation was observed on senescent erythrocytes whose carbohydrate epitopes of band 3 had been occupied with anti band 3. These results indicate that anti-band 3 binds to the carbohydrate epitopes of band 3 on erythrocytes when band 3 is aggregated by oxidative and nonoxidative mechanisms.