Characterization of the autologous antibodies that opsonize erythrocytes with clustered integral membrane proteins

In earlier studies we presented evidence that the clustering of the integral membrane protein, band 3, can serve as a signal for immune recognition and clearance of senescent or abnormal erythrocytes from circulation. In this study, we have exploited the capacity of 1 mmol/L Zn+2 to mildly and reversibly cluster band 3 in situ to characterize the nature of the autologous antibodies specific for the clustered state. We report that the autologous IgG elute almost exclusively in a high molecular weight complex with other proteins when C12E8 detergent extracts of Zn clustered membranes are chromatographed on Sepharose CL- 6B. The complex was also seen to contain complement component C3, hemoglobin, and a cross-linked oligomer of band 3. Autologous IgG and complement were virtually absent from all other fractions. When the band 3 clusters were disaggregated by removal of the Zn+2, the autologous IgG eluted from the erythrocyte surface. Collection of this IgG and use of the antibody in immunoblots of erythrocyte membranes showed that the band 3 monomer, dimer, and oligomers were the major antigenic species. Except for a minor unidentified band at approximately 78,000 d, no other proteins were significantly stained. Curiously, band 3 showed an uneven staining pattern, with oligomers and the leading edge of the monomers appearing more intensely than expected from their abundances in the Coomassie blue-stained gels. Typing of the same autologous IgG with monoclonal antibodies specific for the different subclasses of IgG showed the presence of only subtypes 2 and 3. Taken together, these data suggest that a specific population of autologous IgG recognizes sites of integral membrane protein clustering (a common lesion in senescent and abnormal red blood cells) and that the antigen within these clusters involves an aggregated state of band 3.