Senescent cell antigen (SCA), an aging antigen, is a protein that appears on old cells and acts as a specific signal for the termination of that cell by initiating the binding of IgG autoantibody and subsequent removal by phagocytes. This appears to be a general physiologic process for removing senescent and damaged cells in mammals and other vertebrates. SCA is generated by degradation of the major anion transport protein of vertebrates, band 3. Because of its central role in respiration of CO2, band 3 is the most heavily used ion transport system in vertebrate animals. We have used synthetic peptides to identify antigenic sites on membrane protein band 3 recognized by the IgG that binds to old cells. Results indicate that crucial anion transport segments of the band 3 molecule carry the aging determinants and suggest that these may be the aging sites of the molecule. The purpose of this proposal is to further define the antigenic sites in senescent cell antigen and the aging vulnerable site(s) of the band 3 molecule which are involved in its demise. We will use purified IgG antibodies from old cells (SCIgG) to identify the antigenic site(s) on old cells. Three distinct assays will be used. These are a phagocytosis inhibition assay, a protein A/IgG binding assay, and immunoblotting. In the first aim, we will purify the specific IgG autoantibody to SCA from aged red blood cells. We will use this autoantibody to determine the amino acids critical to antigenicity using synthetic peptides. Peptides with single amino acid changes will be used to locate amino acid residues that are crucial to aging antigens. Results obtained in this manner will be tested using site directed mutagenesis. This information will be used to develop """"""""designer"""""""" peptides for which SCIgG has a greater affinity and/or that are more resistant to degradation than the native aging antigen. Then, on the basis of this information, site-directed mutagenesis will be used to construct """"""""mutant"""""""" band 3 proteins that contain amino acid substitutions at these positions. This allows assessment of the functional importance of these altered residues. Functional assays for anion transport will be added to the other assays for this part of the study. Molecular changes occurring during aging that initiate changes in the band 3 molecule will be defined using synthetic peptides and 980 antibodies which bind to aged band 3 before degradation to SCA. Site directed mutagenesis will be used as an independent approach to identifying the aging vulnerable site. These studies will allow us to identify and define the aging antigenic and aging vulnerable site, and to determine the critical amino acid residues for each site. We anticipate that the results of these studies will facilitate regulation of cellular lifespan and the development of peptides to alleviate cell destruction in hematologic diseases.
