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 C12E8 solution 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-beta-galactosidase, an enzyme specifically cleaves poly-N-acetyllactosaminyl saccharide chains of band 3. When erythrocytes were pretreated with endo-beta-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.
Ando
K
K
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan.
Kikugawa
K
K
Beppu
M
M
D000165:Acridine Orange / Q000737:chemistry; D001457:Anion Exchange Protein 1, Erythrocyte / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D002241:Carbohydrates / Q000276:immunology; D003432:Cross-Linking Reagents; D000939:Epitopes; D004905:Erythrocyte Aging; D004910:Erythrocyte Membrane / Q000276:immunology / Q000648:ultrastructure*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D011485:Protein Binding
D001457:Anion Exchange Protein 1, Erythrocyte; D001323:Autoantibodies; D002241:Carbohydrates; D003432:Cross-Linking Reagents; D000939:Epitopes; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D000165:Acridine Orange