Aging of human red cells

Jie Yuan, Purdue University

Abstract

Clearance of aging and damaged erythrocytes after a finite lifespan is essential for organismal survival. This process involves IgG binding, complement deposition and phagocytosis of senescent or damaged erythrocytes by macrophages. Our group has previously demonstrated that hemoglobin denaturation in the red cell triggers clustering of the major transmembrane protein, band 3, and that this process promotes the binding of senescent antibodies to the cell surface. Since these studies did not directly demonstrate that band 3 clustering is both necessary and sufficient for red cell removal, we decided to examine whether clustering of band 3 alone is an adequate signal for clearance of faltering red cells. To evaluate the above hypothesis, band 3 proteins were caused to cluster in intact erythrocytes by addition of various clustering agents, and these band 3 clusters were fixed by BS3 cross-linking. After returning the cells to their own serum for opsonization, it was found that band 3 clustering promoted autologous IgG binding, complement fixation and phagocytosis of the treated cells. Based on numerous reversibility and control studies, the sequence of events was established to be: (1) hemichrome formation, (2) band 3 clustering, (3) senescent cell autologous antibody binding, (4) complement deposition, and (5) phagocytosis by macrophages. The senescent cell antibodies were isolated and characterized and were found to be of the IgG2 subtype. In immunoblots they preferentially recognized a clustered distribution of band 3 protein, but staining of band 3 monomer was also prominent. Immune complexes from naturally occurring senescent and thalassemic cell membranes were isolated and characterized to determine whether the processes identified in the above model studies might also occur in vivo. Through gel filtration and immunoprecipitation studies, it was documented that hemichromes, band 3, IgG, and complement are present in the same cell surface immune complex. We suggest that these complexes are the major recognition sites for removal of senescent and damaged red cells. Finally, a simple, rapid method of epitope mapping was developed that avoids the arduous task of obtaining amino acid sequence information. The protein antigen is digested with a mixture of highly purified carboxypeptidases into a continuous series of polypeptides, all containing a common N-terminus. The peptides are then separated by SDS-PAGE and blotted with the antibody whose epitope is to be determined. By noting the molecular weight where the immunostain terminates, the position of the epitope relative to the N-terminus can be determined.

Degree

Ph.D.

Advisors

Low, Purdue University.

Subject Area

Biochemistry|Cellular biology

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