Marguerite M. B. Kay

Autoantibodies to band 3 during aging and disease and aging interventions.

An aging antigen, senescent cell antigen, resides on the 911-amino acid membrane protein band 3. It marks cells for removal by initiating specific IgG autoantibody binding. Band 3 is a ubiquitous membrane transport protein found in the plasma membrane of diverse cell types and tissues, and in nuclear, mitochondrial, and golgi membranes. Band 3 in tissues such as brain performs the same functions as it does in red cells. Senescent cell antigen is generated on brain menbranes. Oxidation is a mechanism for generating senescent cell antigen. Neither cross-linking nor hemoglobin appears to play a role in generating senescent cell antigen. Although storage is the only in vitro model that mimics cellular aging in situ, we have discovered three alterations/mutations of band 3 that permit insight into aging in situ. One mutation with an addition to band 3 has normal or decelerated red cell aging. In contrast, another band 3 alteration with a suspected deletion or substitution that renders band 3 more susceptible to proteolysis, shows accelerated aging. The third alteration, which is also more susceptible to proteolysis, is associated with neurologic defects. Peptide technology was used to map the aging antigenic sites and anion transport sites on band 3 using a competitive inhibition assay and immunoblotting with IgG directed against the aging antigen on old cells. Results indicate that: a) aging antigenic sites reside on human band 3 residues 538-554, and 812-830; b) a putative ankyrin binding region peptide is not involved in senescent cell antigen activity; and (c) carbohydrate moieties are not required for the antigenicity or recognition of senescent cell antigen since synthetic peptides alone abolish binding of senescent cell IgG to erythrocytes. Peptide residues 588-594 (a 7-amino acid peptide), 822-839, and 869-883 were the most active inhibitors of anion transport (p < or = 0.001 compared to control without peptide). Localization of the active antigenic and transport sites on band 3 molecule facilitates definition of the molecular changes occurring during aging that initiate molecular as well as cellular degeneration. The role of senescent cell antigen and band 3 in brain aging and Alzheimers disease is discussed. Antibodies to one component of synthetic senescent cell antigen distinguish between Alzheimers and normal tissue.

Drosophila to Bacteriophage to Erythrocyte: The Erythrocyte as a Model for Molecular and Membrane Aging of Terminally Differentiated Cells

Senescent cell antigen appears on old cells and marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes in mammals and other vertebrates. Although the initial studies are done using erythrocytes as a model, senescent cell antigen has been found on all cells examined. Oxidation generates senescent cell antigen in situ. Senescent cell antigen is generated by the modification of an important structural and transport membrane molecule, protein band 3. Band 3 is a ubiquitous protein. It is present in cell, nuclear, Golgi, and mitochondrial membranes. Band 3 is the most heavily used anion exchanger in the human body because of its crucial role in respiration and acid-base balance. Senescent cell antigen has been localized to band 3 residues 538-554 and 812-827, using competitive inhibition studies with synthetic peptides of band 3 to absorb the IgG isolated from senescent erythrocytes and immunoblotting studies. In mammalian brain, band 3 performs the same functions as that of erythroid band 3. These functions are anion transport, ankyrin binding, and generation of senescent cell antigen, an aging antigen that terminates the life of cells. Our results suggest that the transport domain of erythroid and neural band 3 are similar functionally and structural. This supports the hypothesis that the immunological mechanism of maintaining homeostasis is a general physiologic process for removing senescent and damaged cells in mammals and other vertebrates.

Molecular mapping of human band 3 aging antigenic sites and active amino acids using synthetic peptides.

An aging antigen, senescent cell antigen appears on old cells and marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes. This antigen is derived from the major anion transport protein, protein band 3, that is involved in respiration and acid base balance. We use synthetic peptides from the transmembrane, anion transport segment of band 3 to “walk” band 3 to identify potential aging antigenic sites. A competitive inhibition assay with affinity purified IgG autoantibody from senescent red cells was used. Results indicate that: aging antigenic sites reside on human band 3 residues 538–554, 593–601, and 812–830; and that the smallest residues which act as aging antigenic sites are 593–601 and 813–818. The contribution of lysine and/or arginine to antigenicity is examined by synthesizing peptide analogs in which glycines or arginines are substituted for lysines or arginines. Substitution of neutral glycine for the positively charged amino acids arginine or lysine or both arginine and lysine did not result in a significant difference in antigenicity between the analog and the native band 3 peptide. Substitution of the positively charged arginine for the positively charged lysine resulted in a significant reduction in antigenicity. The chicken sequence of band 3 peptides 538–554 and 812–827 differs from that of the human peptides at several sites. Antigenicity of these chicken “analogs” were tested and compared to the human peptides. The data suggest that the three-dimensional configuration of band 3 segments plays a dominant role in defining the antigenic determinants reactive with senescent cell IgG autoantibodies.

Degradation of proteins in the membrane-cytoskeleton complex in Alzheimer's disease. Might amyloidogenic APP processing be just the tip of the iceberg?

Many transmembrane proteins are linked to cytoskeletal proteins and form the membrane-cytoskeleton complex (FIG. 1). Membrane proteins, such as band-3, Na+/K+ ATPase, Na+ channel, and amyloid protein precursor (APP), are likely to be anchored to elements of the cytoskeleton, such as microfilaments, intermediate filaments, and microtubules via interactions with ankyrin, spectrin, and various yet unidentified proteins.’.* APP is abnormally degraded in Alzheimer’s disease (AD). Here we present evidence that other membrane and cytoskeletal proteins including band-3 and spectrin may also be abnormally proteolyzed. A single abnormal protease may be responsible for this rather general proteolysis in AD, but another possibility worth considering is that the altered membrane structure in AD3 may affect the interaction of membranecytoskeleton complex proteins with the membrane, thereby exposing previously hidden sites on these proteins, and making them susceptible to proteolytic activity. For example, recent studies show APP mutations near or within the transmembrane regions of the m ~ l e c u l e , ~ ~ ~ which might alter the interaction of APP with the membrane and increase its vulnerability to amyloidogenic proteolysis. However, mutations in APP are rare and only segregate with certain families that are susceptible to early-onset AD.6 Thus, in the majority of AD cases, the aberrant amyloidogenic degradation of APP might be a result ofaltered membrane properties rather than mutations within the APP molecule. The altered membrane properties could be a result of mutated cytoskeletal proteins. A recent study in a dystrophin-deficient mouse genetic model of Duchenne

Cells, Signals, and Receptors: The Role of Physiological Autoantibodies in Maintaining Homeostasis

Macrophages can distinguish mature “self” from senescent “self” cells. This is reflected by their ability to phagocytize cells which have reached the end of their functional lifespan, while sparing the mature cells. For example, mononuclear phagocytes of the liver and spleen remove syngeneic lymphocytes as well as antibody-coated red blood cells (RBC) (1,2,3). Erythrophagocytosis also occurs in lymph nodes (4). Studies on the fate of aged RBC indicate that they are eliminated intracellularly by mononuclear phagocytes rather than by osmotic lysis both in vitro and in situ (5,6,7,8,9). In this way, mononuclear phagocytes may perform an essential homeostatic role by permitting the more efficient mature cells to carry out their vital functions without hindrance from the less efficient senescent cells, or by preventing pathological reactions which could arise as a consequence of senescent cells dying and decaying within the organism.

Band 3 and its peptides during aging, radiation exposure, and Alzheimer's disease: Alterations and self-recognition

An aging antigen, senescent cell antigen, resides on the 911 amino acid membrane protein band 3. It marks cells for removal by initiating specific IgG autoantibody binding. Band 3 is a ubiquitous membrane transport protein found in the plasma membrane of diverse cell types and tissues, and in nuclear, mitochondrial, and golgi membranes. Band 3 in tissues such as brain performs the same functions as it does in red blood cells forming senescent cell antigen. Oxidation is a mechanism for generating senescent cell antigen. The aging antigenic sites reside on human band 3 map residues 538-554, and 812-830. Carbohydrate moieties are not required for the antigenicity or recognition of senescent cell antigen. Anion transport site were mapped to residues 588-594, 822-839, and 869-883. The aging vulnerable site which triggers the antigenic site and the transport sites of band 3 were mapped using overlapping synthetic peptides along the molecule. Naturally occurring autoantibodies to regions of band 3 comprising both senescent cell antigen and B cells producing these antibodies were demonstrated in the sera of normal, healthy individuals. The presence of these antibodies tend to increase with age. Individuals with autoimmune diseases (rheumatoid arthritis and systemic lupus erythematosus) have increased antibodies to senescent cell antigen peptides. Radiation exposure results in an increase in antibodies to peptides 588-602 which lies in a transport region containing the aging vulnerable site. Band 3 ages as cells and tissues age. Our studies, to date, indicate, that the anion transport ability of band 3 decreases in brains and lymphocytes from old mice. This decreased transport ability precedes obvious structural changes such as band 3 degradation and generation of SCA, and is the earliest change thus far detected in band 3 function. Other changes include a decreased efficiency of anion transport (decreased Vmax) in spite of an increase in number of anion binding sites (increased Km), decreased glucose transport, increased phosphorylation, increased degradation to smaller fragments as detected by quantitative binding of antibodies to band 3 breakdown products and residue 812-830, and binding of physiologic IgG autoantibodies in situ. The latter 3 findings indicate that post-translational changes occur. In Alzheimers Disease (AD), our results indicate that post-translational changes occur in band 3. These include decreased band 3 phosphorylation of a 25-28kD segment, increased degradation of band 3, alterations in band 3 recognized by antibodies, and decreased anion and glucose transport by blood cells. Serum autoantibodies were increased in AD patients compared to controls to band 3 peptide 822-839. This band 3 residue lies in an anion transport/binding region.

Regulatory Autoantibody and Cellular Aging and Removal

Senescent cell antigen (SCA), an aging antigen, was discovered in 1975 (Kay, 1975). It is a protein that appears on old cells and marks them for death. It acts as a specific signal for cellular termination by initiating the binding of IgG autoantibody and subsequent removal by phagocytes (Kay, 1975, 1978,1981, 1984, 1983, 1986, 1988a, 1988b, 1988c, 1990; Kay et al., 1986, 1989, 1982, 1983a, 1988a, 1988b,1991, 1990a, 1990b, 1990c, 1990d; Kay and Lin, 1990; Bennett and Kay, 1981; Singer et al., 1986; Glass et al., 1983, 1985; Bartosz et al., 1982a, 1982b; Khansari et al., 1983; Khansari and Fedenberg, 1983; Walker et al., 1984; Lutz et al., 1984; Petz et al., 1984; Hebbel and Miller, 1984). This appears to be a general physiologic process for removing senescent and damaged cells in mammals and other vertebrates (Kay, 1981). Although the initial studies is done using human erythrocytes as a model, senescent cell antigen was discovered on cells besides erythrocytes in 1981 (Kay, 1981). It occurs on all cells examined (Kay, 1981). The aging antigen itself is generated by the degradation of an important structural and transport membrane molecule, protein band 3 (Kay, 1984). Besides its role in the removal of senescent and damaged cells, senescent cell antigen also appears to be involved in the removal of erythrocytes in clinical hemolytic anemias (Kay et al., 1989, 1990a), sickle cell anemia (Petz et al., 1984; Hebbel and Miller, 1984) and the removal of malaria-infected erythrocytes (Okoye and Bennett, 1985; Friedman et al., 1985). Oxidation generates senescent cell antigen in situ (Kay et al., 1986).

Hypothesis: synthetic aging antigen can be used to manipulate cellular lifespan.

Physiologic removal of old and damaged erythrocytes, platelets, and other terminally differentiated cells is initiated by the appearance of an aging antigen that marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes. We have developed a synthetic aging antigen peptide that blocks binding of IgG to senescent cells in vitro. We hypothesize that the synthetic antigen can be used to prevent cell destruction in diseases such as autoimmune hemolytic anemias and idiopathic thrombocytopenia purpura, and that the antigen itself can be used to manipulate cellular lifespan in vivo.

Molecular Mapping of the Active Site of an Aging Antigen

An aging antigen, senescent cell antigen, resides on the 911 amino acid membrane protein band 3. It marks cells for removal by initiating specific IgG autoantibody binding (1–22). This appears to be a general physiologic process for removing senescent and damaged cells in mammals and other vertebrates (4). Although the initial studies were done using human erythrocytes as a model, senescent cell antigen occurs on all cells examined (4). The aging antigen itself is generated by the degradation of an important structural and transport membrane molecule, protein band 3 (5). Besides its role in the removal of senescent and damaged cells, senescent cell antigen also appears to be involved in the removal of erythrocytes in clinical hemolytic anemias (7,8), and the removal of malaria-infected erythrocytes (23,24). Oxidation generates senescent cell antigen in situ (6). Neither cross-linking nor hemoglobin appear to play a role. Although storage is the only in vitro model that mimics cellular aging in situ, we have discovered three alterations/mutations of band 3 that permit insight into aging in situ. One mutation with an addition to band 3 has normal or decelerated red cell aging. In contrast, another band 3 alteration with a suspected deletion or substitution that renders band 3 more susceptible to proteolysis, shows accelerated aging. The third alteration which is also more susceptible to proteolysis is associated with neurologic defects.