Alteration in membrane protein band 3 associated with accelerated erythrocyte aging.

Abstract

We report a human band 3 alteration that is associated with anemia as determined by a reticulocyte count of 20%. Erythrocyte defects included increased IgG binding, increased breakdown products of band 3, and altered anionand glucose-transport activity in middle-aged cells. These changes were observed during normal erythrocyte aging in situ. Binding of ankyrin to band 3 was normal. Serum/cell crossover studies indicated that a neoantigen appears on the propositus' erythrocytes to which IgG from both propositus and control serum binds as measured with a protein A binding assay. IgG eluted from the propositus' erythrocytes appeared to have a specificity for senescent cell antigen as determined by a phagocytosis inhibition assay. Immunoelectron microscopy showed that antibodies to band 3, which do not normally bind to intact erythrocytes, bound to the propositus' erythrocytes. Antibody 980 binds to normal old cells but not young or middle-aged cells. It also binds to a distinct region of band 3 in immunoblots of membranes from the propositus' middle-aged cells. Cells from both of the propositus' parents exhibited increased IgG binding and altered anion and glucose transport. The results of these studies suggest that (i) band 3 is aging prematurely in erythrocytes from the propositus, (it) senescent cell antigen appears on the propositus' middle-aged red cells, and (Ui) band 3 alterations observed in the propositus may have a genetic component.As part of our ongoing studies on mechanisms of cellular aging, we searched for models of accelerated and decelerated cellular aging. We anticipated that such models would allow us to dissect molecular aging and provide insight into mechanisms. Initially, we investigated models for aging in vitro (1).We subjected intact human erythrocytes to treatments that have been reported to result in changes in band 3 (the major anion-transport polypeptide) and/or to mimic aging in vitro.

Band 3 and glycophorin are progressively aggregated in density-fractionated sickle and normal red blood cells. Evidence from rotational and lateral mobility studies.

“…Others have estimated that, at 37°C, 25-35% of band 3 molecules rotate with correlation times < 150 ,us, 30-50% rotate with correlation times -3 ms, and 25-35% are rotationally immobile on the time scale ofthe experiment (67,69). Recent measurements with improved instrumentation indicate that about one-half of the rapidly rotating fraction of band 3 molecules rotate with correlation times of 25 to 30 As (67) (81,82), abnormally exposed galactosyl residues (46,83,84), and clustered band 3 molecules (27,48,50). Our data support the hypothesis that band 3 aggregation provides an important surface marker for RBC removal from the circulation.…”

Definition of a physiologic aging autoantigen by using synthetic peptides of membrane protein band 3: localization of the active antigenic sites.

“…Peptides were analyzed on 6-25% or 12-25% linear gels (6,7,(27)(28)(29). Immunoautoradiography was performed by immunoblotting as described (6,7,(28)(29)(30). Immunoblots were exposed to Kodak Ortho G (OG-1) film at -700C by our standard procedures (29,30).…”

“…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 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). SCA occurs on all cells examined (4).…”

“…The aging antigen itself is generated by the degradation ofan important structural and transport membrane molecule, protein band 3 (5). Besides its role in the removal of senescent and damaged cells, SCA also appears to be involved in the removal of erythrocytes in clinical hemolytic anemias (7,8) and the removal of malariainfected erythrocytes (15). Oxidation generates SCA in situ (6).…”

Erythrocyte: Programmed Cell Death

“…Because release of band 3 from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties, the authors suggested that tyrosine phosphorylation of band 3 may produce changes in erythrocyte biology that allow the cell to respond to initial stress (Ferru et al, 2011). Another marker of red blood cell apoptosis is band 3 clustering, which generates a cell surface epitope identified by autologous IgG antibodies and may act as a signal for the removal of erythrocytes from circulation (Kay et al, 1989). The nitration of tyrosine residues in proteins occurs through the action of reactive oxygen and nitrogen species such as peroxynitrite, the product of the reaction between nitric oxide and superoxide anion.…”