title: Binding of anti-band 3 autoantibody to sialylated poly-N-acetyllactosaminyl sugar chains of band 3 glycoprotein on polyvinylidene difluoride membrane and sepharose gel: further evidence for anti-band 3 autoantibody binding to the sugar chains of oxidized and senescent erythrocytes.
abstract: Binding specifically of naturally occurring anti-band 3 IgG antibody isolated from human plasma was investigated in a cell-free binding system. 125I-labeled human anti-band 3 IgG specifically bound to band 3 glycoprotein and lactoferrin, a glycoprotein that has poly-N-acetyllactosamine-type sugar chains like band 3, on the polyvinylidene difluoride blotting membrane. Binding was decreased by 50-70% when band 3 and lactoferrin were pretreated with N-glycosidase F, endo-beta-galactosidase, or neuraminidase. Binding of 125I-anti-band 3 IgG to band 3-Sepharose gel was partially inhibited by band 3 oligosaccharides or lactoferrin, but was less inhibited by them after they had been treated with N-glycosidase F or endo-beta-galactosidase. A significant part of 125I-anti-band 3 IgG that bound to the band 3-Sepharose gel was released upon treatment of the gel with N-glycosidase F or endo-beta-galactosidase. IgG that binds to lactoferrin (anti-lactoferrin IgG) was isolated from normal human plasma. 125I-Anti-lactoferrin IgG bound to the band 3-Sepharose gel as effectively as to the lactoferrin-Sepharose. The antibody specifically bound to the band 3- and lactoferrin-blotted membrane depending on the poly-N-acetyllactosaminyl sugar chains of the blotted glycoproteins. The results indicate that a major part (about 70%) of anti-band 3 IgG recognizes the sialylated poly-N-acetyllactosaminyl sugar chains of band 3 and lactoferrin, and the remaining part (about 30%) of the antibody may recognize the polypeptide portion of band 3. This was supported by the observation that anti-band 3 IgG effectively bound to lactoferrin-Sepharose but 33% of the antibody did not. Anti-band 3 IgG with the carbohydrate-binding property was equally obtained whether fully denatured or barely denatured band 3 was used for isolation of anti-band 3 IgG by affinity chromatography. These results provide further evidence for our proposal that the binding sites of anti-band 3 IgG to oxidized and senescent erythrocytes reside on the locally condensed sialylated poly-N-acetyllactosaminyl sugar chains of band 3 on the cell surface.
authors: School of Pharmacy, Tokyo University of Pharmacy and Life Science.
authors: K
authors: K
authors: Ando
authors: K
authors: K
authors: Kikugawa
authors: M
authors: M
authors: Beppu
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000276:immunology* / Q000302:isolation & purification; D001667:Binding, Competitive; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D000939:Epitopes / Q000276:immunology; D004905:Erythrocyte Aging; D004910:Erythrocyte Membrane / Q000737:chemistry; D004912:Erythrocytes / Q000276:immunology*; D006026:Glycoside Hydrolases; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism; D007781:Lactoferrin / Q000276:immunology; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000276:immunology*; D010084:Oxidation-Reduction; D011134:Polysaccharides / Q000276:immunology*; D011145:Polyvinyls; D011485:Protein Binding; D012685:Sepharose; D005465:Fluorocarbon Polymers
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D001323:Autoantibodies; D000939:Epitopes; D006026:Glycoside Hydrolases; D007074:Immunoglobulin G; D007781:Lactoferrin; D008567:Membranes, Artificial; D009844:Oligosaccharides; D011134:Polysaccharides; D011145:Polyvinyls; D012685:Sepharose; C016735:sialooligosaccharides; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C037199:poly-N-acetyllactosamine