Isolation and partial characterization of antibody- and globin-enriched complexes from membranes of dense human erythrocytes.
In previous studies we have described a process whereby an erythrocyte in biochemical distress can initiate its own removal by macrophages of the reticuloendothelial system. This process involves the clustering of the integral membrane protein band 3 by denatured haemoglobin and the subsequent recognition of the exofacial poles of clustered band 3 and associated proteins by autologous antibodies. To determine whether this clearance pathway might mediate normal cell turnover, the fraction of normal erythrocytes containing the 0.5% densest cells, which are known to be destined for immediate removal, was isolated and characterized biochemically. This densest fraction was found to contain 6 times more membrane-bound globin (haemichromes) and 10 times more surface-bound autologous IgG than the other fractions containing cells of lower density. To determine whether the autologous IgG was physically associated with the haemichrome-stabilized membrane protein clusters, a procedure was developed for isolation and characterization of the microscopic aggregates. The isolated aggregates were found to contain a disulphide-cross-linked mixture of several membrane proteins, predominantly haemichromes, spectrin and band 3. Although the aggregates constituted only 0.09% of the total membrane protein, they still contained approximately 55% of the total cell-surface IgG. Since in control studies anti-(blood group A) antibodies, which are distributed randomly over the surface of type A cells, could not be recovered in the aggregate, we conclude that the autologous cell-surface IgGs were physically associated with the membrane protein clusters when they were co-isolated with them in our procedure. Thus the 640-fold enrichment of autologous IgG in the aggregates compared with regions of the membrane devoid of tightly clustered protein suggests that sites of integral protein clustering either are non-specifically sticky to IgG or are viewed as foreign or 'non-self' by the immune system and aggressively opsonized with IgG.
Kannan
R
R
Department of Chemistry, Purdue University, W. Lafayette, IN 47907.
Yuan
J
J
Low
P S
PS
D001457:Anion Exchange Protein 1, Erythrocyte / Q000302:isolation & purification; D000936:Antigen-Antibody Complex / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006420:Hemeproteins / Q000302:isolation & purification*; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000097:blood*; D013049:Spectrin / Q000302:isolation & purification
D001457:Anion Exchange Protein 1, Erythrocyte; D000936:Antigen-Antibody Complex; D006420:Hemeproteins; D007074:Immunoglobulin G; D008565:Membrane Proteins; C001332:hemichrome; D013049:Spectrin