Band 3 and its alterations in health and disease.

Band 3 proteins, members of the anion exchange family of proteins (AE 0-3), are involved in a number of physiological activities such as cell volume and osmotic homeostasis, HCO3-/Cl- exchange, red cell aging, IgG binding and cellular removal, and the maintenance of the structural integrity of cells. They are present in the membranes of all cells and cellular organelles examined including Golgi, mitochondria and nuclei. The first polymorphisms of band 3 discovered were the asymptomatic band 3 Memphis variants carrying the Lys --> Gly substitution at position 56 in the cytoplasmic tail, and band 3 Texas (high transport band 3 Texas) with a mutation in the critical transmembrane, anion transport domain (Pro --> Leu substitution at position 868). The rate at which band 3 mutations were discovered accelerated in the mid 1990s and there are now over 50 known. The most common polymorphisms of band 3 are the Diego blood group antigens which reside on extracellular loops of the protein. Southeast Asia ovalocytosis (SAO; a nine amino acid deletion of residues 400-408) is a band 3 mutation known only in the heterozygous state in which it does not cause disease. It is thought to confer resistance to malaria by altering red cell deformability. Band 3 mutations are responsible for a subset of the heterogeneous group of disorders known as hereditary spherocytosis (HS). HS is a relatively common congenital or inherited group of anemias characterized by chronic hemolysis and abnormal red cell morphology. Red cells in the subset of HS with band 3 mutations behave like they are band 3 deficient either because the mutant protein is not incorporated into the membrane or because it is not functional. HS can be caused by mutations in any of at least 5 proteins involved in membrane stability. Band 3 mutations are associated with diseases in cells besides erythrocytes. For example, 2 types of distal renal tubular acidosis are the result of band 3 mutations either alone or combined with SAO. Band 3 alterations are implicated in neurological diseases such as familial paroxysmal dyskinesia, idiopathic generalized epilepsies, and neuro- or choreoacanthocytosis although they have not been demonstrated to be causative. Mutations in other genes can cause changes in band 3. An example is sickle cell anemia where the increased oxidation causes accelerated aging of band 3 and increased IgG binding and cellular removal.

Immunoregulation of cellular lifespan: physiologic autoantibodies and their peptide antigens.

Physiologic autoantibodies are part of our normal immune repertoire where they function to maintain homeostasis by performing physiologic functions. The role of physiologic autoantibodies in removing senescent and damaged cells is probably the best example of a physiologic autoantibody, complete with well established function. IgG autoantibodies bind to altered band 3 anion exchanger protein on senescent cells and trigger their removal by macrophages. Band 3 isoforms are found in all cells, tissues, and membranes, and in all species examined. In this paper, we discuss the innate immune response to band 3 membrane proteins and their regulation of cellular lifespan. The role of physiologic autoantibodies and their peptide antigens in health and disease, apoptosis, and their therapeutic potential is discussed focusing on the examples of senescence and malaria.

A comparison of practice domains of clinical nurse specialists and nurse practitioners.

This qualitative study was carried out to add empirical data to the discussion of the essentials of advanced practice graduate nursing education. Study participants were 15 nurse practitioners (NPs) whose original preparation was as clinical nurse specialists (CNSs). These nurses subsequently completed post-master's preparation as NPs at a midwest US school of nursing. The nurses were interviewed regarding similarities and differences between the NP and CNS roles based on their own experiences. They stated that their 1-year post-master's NP educational program broadened their knowledge base and increased their skills of direct data collection in history taking and physical examination. They described increased NP role autonomy and clinical decision making. Comparison of study results with the six practice domains of the National Association of Nurse Practitioner Faculties (NONPF) Curriculum Guidelines appeared to verify the NONPF domains as being operant in advanced nursing practice. Each role had distinct expression of the NONPF practice domains, particularly in two areas: management of client health-illness states and the professional role. Results suggest that advanced practice graduate nursing curricular content needs to be explicit regarding CNS and NP role domains to retain the respective strengths of each. A "hybrid" CNS/NP curriculum faces the danger of lacking both the depth and specificity of knowledge that has resulted in the roles' past successes.

Brain and erythrocyte anion transporter protein, band 3, as a marker for Alzheimer's disease: structural changes detected by electron microscopy, phosphorylation, and antibodies.

Band 3, a ubiquitous membrane transport, regulatory, and structural protein, is represented in brain by at least 4 isoforms. Bands 3 in brain performs the same functions as it does in erythrocytes (RBC). It is susceptible to oxidative damage, which, ultimately, terminates its life and that of the cell. We examined the changes band 3 undergoes in Alzheimer's disease (AD) because our previous studies suggest that band 3 is a pivotal protein in neurological disease. Because we hypothesize that AD is a total body disease, we examined peripheral blood cells as well as brain tissue to determine whether the same changes occur in both. Our results indicate that posttranslational changes occur in RBC band 3 that parallel changes in brain band 3. These include decreased 32P-phosphate labeling in vitro of band 3 polypeptides in brain and RBC, increased degradation of band 3, alteration in band 3 recognized by polyclonal and monoclonal antibodies, and decreased anion and glucose transport by blood cells. Serum autoantibodies to band 3 peptides 588-602 and 822-839 were increased in AD patients compared to controls. These band 3 residues lie in anion transport/binding regions. This is consistent with alteration of this region in AD since it is recognized as antigenically different by the patients' immune system. Our data support an immunological component to AD. The finding that changes in RBC in AD reflect those in brain and can be recognized by antibodies should facilitate development of blood tests for diagnosis and monitoring, and early therapy. It is anticipated that identification of molecular sites of posttranslational modification of band 3 will enable us to design specific preventive and treatment strategies, and target drugs to crucial molecular sites.

Posttranslational modifications of brain and erythrocyte band 3 during aging and disease.

Band 3 performs the same structural and functional activities in adult brain as it does in erythrocytes. It 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. The following changes occur in lymphocytes, erythrocytes and brain band 3 with aging: 1) a decreased efficiency of anion transport (decreased Vmax) in spite of an increase in number of anion binding sites (increased K(m)), 2) a decreased glucose transport, 3) 32P labeling in vitro, 4) an increased degradation to smaller fragments as detected by quantitative binding of antibodies to band 3 breakdown products and residue 812-830, and 5) a binding of physiologic IgG autoantibodies in situ. The latter three findings indicate that posttranslational changes occur. In addition, the anion transporter, band 3, undergoes an as yet undefined change that results in binding of "980" antibodies to aged band 3. Posttranslational changes in AD include decreased brain and RBC phosphorylation of a M(r) = 135, 113 and 45 kDa band 3 polypeptides due to the phosphorylation site being already occupied, increased degradation of band 3, alterations in band 3 recognized by antibodies, and decreased anion and glucose transport by blood cells. Band 3 in erythrocytes of AD patients has a different immunological identity from normal band 3 as evidenced by the binding of antibodies described in this study. AD may be preferentially manifested in the brain because neurons accumulate damage throughout the lifetime as they do not regenerate or undergo cell division. We suspect, and our data indicate, that the same mechanism(s) of AD occurs in all cells, but that the manifestations differ due to different cell proteins and functions.

Human erythroid band 3 "anion exchanger 1" is expressed in transformed lymphocytes.

The anion transporter, band 3, is a ubiquitous protein. It is present in brain and all other tissues examined. Not only is band 3 present in cell membranes, but also in nuclear, Golgi and mitochondria membranes. There are four isoforms of band 3, the anion exchanger (AE) proteins, thus far discovered. They are products of different genes. Lymphocytes are reported to contain AE2, but not AE1. We hypothesized that induction or up-regulation of AE1 occurs when lymphocytes are transformed as an initial event in the path to malignancy. We transformed lymphocytes containing a single base mutation with Epstein Barr Virus (EBV). The mutation of band 3, high transport band 3 (HTbd3), exhibits anion transport that is 2-3 times normal in erythrocytes which contain AE1. This facilitated our identification of AE1 since the probability that 2 different gene products would have the same mutation approaches zero. Thus, we have a base mutation in addition to linear sequence to identify AE1. A 133 base pair (bp) fragment including the affected region was amplified from the mRNA of lymphocytes from the HTbd3 mutant. AE1 primers were used to amplify regions of interest. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to generate cDNA which was sequenced. The sequence of the crucial 133 base pair segment from high transport lymphocytes was 100% identical to the sequence published for the red blood cell band 3 of the same mutant. As reported previously for erythrocytes, this mutation is a C-->T base change which changes a proline to leucine in the protein sequence. Restriction enzyme digests of AE1 cDNA from normal and HTBD3 lymphocytes confirmed that the proposita was homozygous for the mutation, and showed the father to be heterozygous. Anion transport was increased in HTbd3 EBV transformed lymphocytes, as was the case with HTbd3 erythrocytes. AE2 was identified in lymphocytes by sequence. Thus, EBV transformed lymphocytes express the erythroid band 3 (AE1) in addition to AE2.

Posttranslational changes in band 3 in adult and aging brain following treatment with ergoloid mesylates, comparison to changes observed in Alzheimer's disease.

Band 3, the most heavily used anion transport system in vertebrates, ages as cells and tissues age. Posttranslational changes in band 3 in adult and aging brain were investigated following treatment with ergoloid mesylates and compared to changes observed in Alzheimer's disease (AD). The study was conducted in a double blind fashion and was decoded only after the study was completed. The following posttranslational changes in brain band 3 occur with age: increased breakdown of band 3; decreased phosphorylation; and decreased anion transport. Autoantibodies to senescent cell antigen (SCA) synthetic peptides residue 538-554 and 812-827 increase with age, but antibodies to the former peptide are significantly reduced in ergoloid mesylate treated old mice. This is a critical transport region of band 3. Results showed the aged/altered band 3 increased in Alzheimer's disease (AD) as determined by quantitative antibody binding. Ergoloid mesylates altered the age-related posttranslational changes as follows: the observed age-related decrease in brain band 3 was partially reversed and anion transport was increased. This is consistent with the data indicating decreased autoantibodies to a critical anion transport segment of band 3. Aging appears to result in damage to a critical transport region of the anion transporter which is reflected by decreased anion transport, increased breakdown, alteration of the molecule itself, and an increase in autoantibodies to the region. Ergoloid mesylates seem to protect against this damage.

Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu).

Band 3 HT (Pro-868-->Leu) is a mutant anion exchange protein which has several phenotypic characteristics, including a 2- to 3-fold larger Vmax, and reduced covalent binding of the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS). We have used fluorescence kinetic methods to study inhibitor binding to band 3 to determine if the point mutation in band 3 HT produces localized or wide-spread conformational changes within the membrane-bound domain of this transporter. Our results show that covalent binding of H2DIDS by band 3 HT is slower by a factor of 10 to 20 compared with the wild-type protein. In contrast, no such difference in the kinetics was observed for covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In addition, the kinetics of H2DIDS release from band 3 HT was abnormal, while the kinetics of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) release showed no difference when compared with the wild-type protein. We conclude that substitution of leucine for proline at position 868 does not perturb the structure of "lysine A" in the membrane-bound domain of band 3 but rather produces an apparently localized conformational change in the C-terminal subdomain of the protein which alters H2DIDS affinity. When combined with the observation of an increased Vmax, these results suggest that protein structural changes at position 868 influence a turnover step in the transport cycle.

Band 3, the anion transporter, is conserved during evolution: implications for aging and vertebrate evolution.

A cDNA fragment corresponding to a highly evolutionarily conserved region of the major anion transport protein band 3 was cloned from lamprey mRNA using PCR homology probing. This is the first report providing evidence for a band-3 like transporter in the lowest vertebrates, the agnathostomes. Semi-quantitative PCR showed expression similar to that of higher vertebrates. Lamprey serum contains antibody-like molecules that bind to synthetic peptides of band 3 comprising senescent cell antigen, an aging antigen that terminates the life of cells. The high degree of homology found in nucleic acid and derived proteins sequence and the reaction of "antibodies" in lamprey serum with senescent cell antigen peptides of band 3 suggests that lamprey band 3 plays a role comparable to that in higher vertebrates.

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 Alzheimer's 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.

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 Alzheimer's disease is discussed. Antibodies to one component of synthetic senescent cell antigen distinguish between Alzheimer's and normal tissue.

Molecular basis of human band-3 mutation associated with increased anion transport.

Band 3, the anion transporter, is a ubiquitous protein. It is present in brain and all other tissues. Not only is band 3 present in cell membranes, but also in nuclear, Golgi, and mitochondrion membranes. Band 3 is involved in respiration, acid-base balance, and is the major structural protein linking the plasma membrane to the cytoskeleton. Thus, alterations/mutations in the transport segment of the band-3 molecule might be expected to be of major importance. We discovered and sequenced a mutation of band 3, high-transport band 3 (HTbd3), that exhibits anion transport that is 2-3 time above normal. Anion transport studies of the family members revealed that red cells from the proposita, one of two siblings, and both parents had abnormally increased anion transport (increased Vmax). We used synthetic peptides of band 3 to help localize the change along the band-3 molecule. Results suggest that high-transport band 3 is altered in or near residue 869-883. This places the alteration toward the carboxyl terminal of band 3. cDNA sequencing demonstrated that the mutation was a proline to leucine at residue 868. A peptide was synthesized corresponding to residues 853-870 for testing in the anion transport inhibition assay. This peptide significantly inhibited anion transport (p < or = 0.001) indicating that it is an anion transport/binding region of band 3. Thus, DNA technology confirms the validity of the anion transport inhibition assay for localizing transport regions. Glucose transport is decreased in affected individuals. The HTbd3 mutation appears benign as determined by the red cell aging panel. IgG binding, creatinine, and glyceraldehyde-3-phosphate dehydrogenase are normal. Our studies indicate that the most rapid and sensitive techniques for detecting band-3 alterations are polyacrylamide gel electrophoresis, IgG binding, and anion transport studies. This is the only mutation of band 3 discovered to date the affects the transmembrane, anion transport region of band 3.

Distribution and function of multiple anion transporter proteins in brain tumor cell lines in relation to glucose transport.

The anion transport, "band 3," family of proteins in mammalian brain 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. The presence of 5-7 band 3 related proteins in brain tissue was suggested by the reaction of antibodies to synthetic peptides of erythroid band 3 with a number of bands in immunoblots. Since there are a number of different cell types in brain, tissue cultures of neural cell types were examined to determine whether multiple band 3 related proteins are present in each cell type or whether several band 3 related proteins are present in each cell type. The tumor cell lines exhibit anion transport and are inhibited by the anion transport inhibitors 4,4'-diisothiocyano-2,2' disulfonic acid (DIDS), phenylglyoxal, and furosemide. Glucose transport is inhibited by cytochalasin B and the anion transport inhibitor, phenylglyoxal, in these cell lines, but not by 4,4'-diisothiocyano-2,2' disulfonic acid. Furosemide gave partial inhibition of most, but not all, cell lines. Since phenylglyoxal inhibits anion transport by binding to an arginine near the transport site, inhibition of glucose transport by phenylglyoxal suggests that an arginine lies in the substrate binding site. The number of cytochalasin B and DIDS binding sites was quantitated on cell lines as an approximation of the number of glucose transporter and anion transporter sites, respectively.

Band 3 HT, a human red-cell variant associated with acanthocytosis and increased anion transport, carries the mutation Pro-868-->Leu in the membrane domain of band 3.

1. We have studied band 3 HT, a human red-cell band 3 variant with increased M(r), which is associated with abnormal red-cell shape (acanthocytosis) and increased anion-transport activity. 2. We have shown that the increased M(r) does not result from the presence of the band 3 Memphis mutation, and that the variant band 3 is covalently labelled by 4,4'-di-isothiocyanato-1,2-diphenylethane-2,2'-disulphonic acid (H2DIDS) less readily than normal. 3. cDNA cloning studies show that band 3 HT results from the mutation Pro-868-->Leu, and the possible significance of the mutation in the altered anion-transport activity and cytoskeleton binding properties of band 3 HT is discussed.

Generation of senescent cell antigen on old cells initiates IgG binding to a neoantigen.

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 membranes. Oxidation is a mechanism for generating senescent cell antigen. Neither cross-linking nor hemoglobin appear 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 the definition of 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 Alzheimer's disease is discussed.

Natural human antibodies to synthetic peptide autoantigens: correlations with age and autoimmune disease.

Clinically healthy humans as well as patients suffering from various autoimmune diseases produce natural antibodies against a variety of self-components. Such antibodies have been proposed to carry out a physiologic role in maintaining the integrity of self, as well as potentially destructive roles in the generation of autoimmune diseases. Because human autoantigens, particularly membrane proteins, are usually present in extremely small amounts, it is generally impossible to obtain enough to carry out a detailed characterization of the antibodies or the antigenic determinants recognized. To circumvent this difficulty, we developed synthetic autoantigens predicted from the gene sequence of two functionally critical membrane proteins; the band 3 anion transport protein which is found on all cells, and the T-cell receptor (beta chain) which is the antigen-specific receptor on thymus-derived lymphocytes. We have investigated the natural human IgM and IgG antibody responses to peptides selected on the basis of predicted molecular surface exposure and previously known antigenicity, and correlate levels of binding with changes in age and by comparison with autoimmune diseases. We report that the IgM response to synthetic autoantigens tends to be higher than that of IgG molecules, but significant IgG binding occurs to some peptides. This situation is particularly noticeable in comparison of rheumatoid arthritis patients with normal individuals. Distinct peptide portions of individual molecules are recognized differently by the autochthonous immune system as manifested by age dependence of the response and differential levels of IgM and IgG activity. The synthetic autoantigens that tend to generate the highest amounts of natural antibody are those that are either exposed on the surface of the cell (band 3 peptides) or are exposed in the predicted 3-dimensional folding of the molecule (T-cell receptor beta peptides). Rheumatoid arthritis patients tend to give higher IgM reactivities to both band 3 and Tcr beta peptides than do normals, with this effect being less pronounced in the distinct autoimmune disease systemic lupus erythematosus. Studies of normal humans ranging in age from 20 to 90 years suggest two major patterns for the IgM natural antibody response to synthetic peptides giving high response. The first is that the level of IgM reactivity is high early in life and remains high throughout. The second pattern is one in which the reaction is high in younger individuals, but diminishes substantially in the latter decades of life.(ABSTRACT TRUNCATED AT 400 WORDS)

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.