3-related Protein Research Articles

Starting DNA Synthesis: Initiation Processes during the Replication of Chromosomal DNA in Humans.

The initiation reactions of DNA synthesis are central processes during human chromosomal DNA replication. They are separated into two main processes: the initiation events at replication origins, the start of the leading strand synthesis for each replicon, and the numerous initiation events taking place during lagging strand DNA synthesis. In addition, a third mechanism is the re-initiation of DNA synthesis after replication fork stalling, which takes place when DNA lesions hinder the progression of DNA synthesis. The initiation of leading strand synthesis at replication origins is regulated at multiple levels, from the origin recognition to the assembly and activation of replicative helicase, the Cdc45-MCM2-7-GINS (CMG) complex. In addition, the multiple interactions of the CMG complex with the eukaryotic replicative DNA polymerases, DNA polymerase α-primase, DNA polymerase δ and ε, at replication forks play pivotal roles in the mechanism of the initiation reactions of leading and lagging strand DNA synthesis. These interactions are also important for the initiation of signalling at unperturbed and stalled replication forks, "replication stress" events, via ATR (ATM-Rad 3-related protein kinase). These processes are essential for the accurate transfer of the cells' genetic information to their daughters. Thus, failures and dysfunctions in these processes give rise to genome instability causing genetic diseases, including cancer. In their influential review "Hallmarks of Cancer: New Dimensions", Hanahan and Weinberg (2022) therefore call genome instability a fundamental function in the development process of cancer cells. In recent years, the understanding of the initiation processes and mechanisms of human DNA replication has made substantial progress at all levels, which will be discussed in the review.

A phase I trial of elimusertib in combination with cisplatin or with cisplatin plus gemcitabine in advanced solid tumors with an emphasis on urothelial carcinoma.

TPS3174 Background: Cisplatin, a well-established backbone of combination therapy of various advanced solid tumors, inhibits DNA synthesis by forming DNA cross-links and adducts. Despite the activity of cisplatin, tumor cells can either be refractory or develop resistance to treatment. Cisplatin has been demonstrated to cause cell cycle G2/M arrest, which may allow for DNA damage response (DDR) and repair. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad 3-related (ATR) protein kinases are key regulators of DDR, and contribute to maintaining genomic integrity in response to various exogenous and endogenous genotoxic insults like cytotoxic chemotherapy. In fact, cisplatin has been shown to transiently increase ATR expression. Inhibitors of ATR have been studied in combination with cisplatin both in vitro and in vivo, demonstrating enhanced activity. The oral small molecule ATR inhibitor elimusertib, which has been studied as a single agent in a Phase I study, has also demonstrated enhanced activity with cisplatin in vitro in lung cancer and bladder cancer cell lines. We sought to conduct a Phase I study evaluating the combination of elimusertib with cisplatin or with cisplatin and gemcitabine. Methods: In the first cohort, patients with histologically confirmed advanced solid tumors for which cisplatin-based therapy would be considered appropriate, who exhibit adequate organ function, and have received < 300 mg/m2 of cisplatin previously are treated with cisplatin on Day (D) 1 and with elimusertib on D2 & 9 of each 21-day cycle. The study follows a phase I queue (IQ) 3+3 dose escalation design, following standard practices for dose-limiting toxicity (DLT) impact on escalation, and when the maximum tolerated dose (MTD) is established with cisplatin alone, this will inform the first dose level of the next cohort, in which patients will be treated with cisplatin on D1, gemcitabine on D1 & 8, and elimusertib on D2 & 9 of each 21-day cycle. An expansion cohort will enroll urothelial carcinoma patients when the second MTD is established. The primary objective of the study is to evaluate the safety and MTD of elimusertib in combination with cisplatin, as well as in combination with cisplatin and gemcitabine. Secondary study objectives include evaluation of pharmacokinetics of elimusertib in these combinations, preliminary efficacy, and evaluating the association between ATM expression and responses to therapy. Currently, 6 patients have been enrolled to the first cohort of the study. Clinical trial information: NCT04491942.

Activity of anti-CR3-RP polyclonal antibody against biofilms formed by Candida auris, a multidrug-resistant emerging fungal pathogen

Fungal biofilm has remained a serious medical problem that complicates treatment of mycoses. In particular, once biofilms are formed, they display high levels of resistance against most common antifungals. Candida auris is currently considered as a serious emerging fungal pathogen frequently exhibiting high levels of resistance to antifungals. Recent studies have confirmed that C. auris shares similarity with Candida albicans in regards to virulence-associated proteins involved in adherence and biofilm development. Complement receptor 3-related protein (CR3-RP) is one of the key surface antigens expressed by Candida species during biofilm formation. Here, we have investigated the presence of this cell surface moiety on the surface of C. auris, as well as the potential of anti-CR3-RP polyclonal antibody (Ab) to inhibit biofilm formation by this emerging fungal pathogen. Using indirect immunofluorescence and ELISA, we were able to confirm the presence of CR3-RP in C. auris cells within biofilms. Further, not only anti-CR3-RP Ab was able to inhibit biofilm formation by multiple C. auris strains when added during the adherence phase, but it also demonstrated activity against C. auris 24-h pre-formed biofilms, which compared favorably to levels of inhibition achieved by treatment with current conventional antifungals fluconazole, amphotericin B, and caspofungin. Overall, our data demonstrate the presence of this antigen on the surface of C. auris and points to the potential of anti-CR3-RP Ab in eradication of biofilms formed by this novel fungal pathogen.

Two Opsin 3-Related Proteins in the Chicken Retina and Brain: A TMT-Type Opsin 3 Is a Blue-Light Sensor in Retinal Horizontal Cells, Hypothalamus, and Cerebellum.

Opsin family genes encode G protein-coupled seven-transmembrane proteins that bind a retinaldehyde chromophore in photoreception. Here, we sought potential as yet undescribed avian retinal photoreceptors, focusing on Opsin 3 homologs in the chicken. We found two Opsin 3-related genes in the chicken genome: one corresponding to encephalopsin/panopsin (Opn3) in mammals, and the other belonging to the teleost multiple tissue opsin (TMT) 2 group. Bioluminescence imaging and G protein activation assays demonstrated that the chicken TMT opsin (cTMT) functions as a blue light sensor when forced-expressed in mammalian cultured cells. We did not detect evidence of light sensitivity for the chicken Opn3 (cOpn3). In situ hybridization demonstrated expression of cTMT in subsets of differentiating cells in the inner retina and, as development progressed, predominant localization to retinal horizontal cells (HCs). Immunohistochemistry (IHC) revealed cTMT in HCs as well as in small numbers of cells in the ganglion and inner nuclear layers of the post-hatch chicken retina. In contrast, cOpn3-IR cells were found in distinct subsets of cells in the inner nuclear layer. cTMT-IR cells were also found in subsets of cells in the hypothalamus. Finally, we found differential distribution of cOpn3 and cTMT proteins in specific cells of the cerebellum. The present results suggest that a novel TMT-type opsin 3 may function as a photoreceptor in the chicken retina and brain.

Humoral immune responses to Candida albicans complement receptor 3-related protein in the atopic subjects with vulvovaginal candidiasis. Novel sensitive marker for Candida infection.

In vitro evaluation of specific anti-Candida albicans sera antibodies based on synthetically prepared complement receptor 3-related protein (CR3-RP) mimicking the structure of native complement receptor 3 in a cohort of 72 patients with atopy and recurrent Candida vulvovaginitis (RVC) revealed effective humoral response against Candida CR3-RP. The most significant have been IgM and IgA isotype antibodies (33 and 47% positive cases, respectively). The quantitative evaluation of anti-CR3RP isotype antibodies was confronted with results of commercial ELISA anti-C. albicans antibodies diagnostics based on C. albicans cell wall mannan and β-glucan antigens, the most significant correlation being observed with anti-CR3-RP IgM and anti-β-D-glucan IgM (r(2) = 0.624) followed by isotype IgA (r(2) = 0.381). The immunogenicity and immunoreactivity of CR3RP antigen in RVC patients' sera had been evaluated with regard to the results reached by counterimmunoelectrophoresis and heterogeneous enzyme immunoassay. Obviously, synthetically prepared CR3-RP mimicking the Candida cell-wall-derived structure moiety represents a promising immunological tool not only for Candida serodiagnostics, but also prospectively for follow-up of targeted antifungal therapy and as promising Candida vaccine candidate.

Dissecting cellular responses to irradiation via targeted disruptions of the ATM-CHK1-PP2A circuit

Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (ATM), ATM and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, ATM or both for dissecting cross-talk and compensatory effects on G₂/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G₂/M checkpoint arrest. ATM phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G₂/M checkpoint response compared with the individual knockdowns. ATM inhibition and 90% CHK1 depletion abrogated the early G₂/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments.

Meiotic Checkpoints: Repair or Removal?

Defects in meiosis can produce different checkpoint responses in female and male animals, suggesting that meiotic checkpoints exhibit sexual dimorphism. A recent study in Caenorhabditis elegans indicates that meiotic checkpoint activation is similar between the sexes and the primary difference lies in the downstream consequences.

Participation of theCandida albicanssurface antigen in adhesion, the first phase of biofilm development

The lack of work dealing with possible ways of reducing biofilm production via inhibiting Candida albicans adherence in the first stage of biofilm formation was a motivation for this study. The study was focused on two questions: (1) can a decrease in adherence affect the quantity of mature biofilm? and (2) can blocking the surface C. albicans complement receptor 3-related protein (CR3-RP) with polyclonal anti-C3-RP antibody or monoclonal antibody OKM1 significantly contribute to a reduction in adherence during biofilm formation? The presence and quantity the CR3-RP expressed in the biofilm was confirmed by immunofluorescence, immunocytometry and enzyme-linked immunosorbent assay. To determine the changes in adherence of C. albicans CCY 29-3-162 and C. albicans catheter isolate, 30-, 60-, 90- and 120-min time points were selected and viability was determined by XTT assay. The strains were preincubated with both antibodies to block CR3-RP, which proved to be effective at reducing adhesion and the formation of a mature biofilm (64.1-74.6%). The duration of adhesion, between 30 and 120 min, seems to have a significant effect on the mature biofilm. The blocking of CR3-PR by antibodies before adherence affected the fitness of biofilm, which was not able to revitalize in the later stages.

Antibody response to the 45 kDa Candida albicans antigen in an animal model and potential role of the antigen in adherence

The Candida antigen CR3-RP (complement receptor 3-related protein) is supposed to be a 'mimicry' protein because of its ability to bind antibody directed against the alpha subunit of the mammalian CR3 (CD11b/CD18). This study aimed to (i) investigate the specific humoral isotypic response to immunization with CR3-RP in vivo in a rabbit animal model, and (ii) determine the role of CR3-RP in the adherence of Candida albicans in vitro using the model systems of buccal epithelial cells (BECs) and biofilm formation. The synthetic C. albicans peptide DINGGGATLPQ corresponding to 11 amino-acids of the CR3-RP sequence DINGGGATLPQALXQITGVIT, determined by N-terminal sequencing, was used for immunization of rabbits to obtain polyclonal anti-CR3-PR serum and for subsequent characterization of the humoral isotypic response of rabbits. A significant increase of IgG, IgA and IgM anti-CR3-RP specific antibodies was observed after the third (P<0.01) and the fourth (P<0.001) immunization doses. The elevation of IgA levels suggested peptide immunomodulation of the IgA1 subclass, presumably in coincidence with Candida epithelial adherence. Blocking CR3-RP with polyclonal anti-CR3-RP serum reduced the ability of Candida to adhere to BECs, in comparison with the control, by up to 35 % (P<0.001), and reduced biofilm formation by 28 % (P<0.001), including changes in biofilm thickness and integrity detected by confocal laser scanning microscopy. These properties of CR3-RP suggest that it has potential for future vaccine development.

BCR/ABL translocates to the nucleus and disrupts an ATR-dependent intra-S phase checkpoint

Chronic myelogeneous leukemia (CML) is a two-stage disease associated with expression of the BCR/ABL tyrosine kinase protein. However, whether BCR/ABL expression directly causes blast crisis, and if so by what mechanism, is unknown. We have found that BCR/ABL translocates from the cytoplasm to the nucleus after genotoxic stress. Furthermore, BCR/ABL increases DNA double-strand damage after etoposide treatment and leads to a defect in an intra-S phase checkpoint, causing a radioresistant DNA synthesis (RDS) phenotype. In the nucleus, BCR/ABL associates with the ataxia-telangiectasia and rad 3-related protein (ATR) and disrupts ATR-dependent signal transduction. Overexpression of ATR in a BCR/ABL-expressing cell line corrects the DNA damage phenotype. These results demonstrate a nuclear role for BCR/ABL in altering the cellular response to DNA damage.

Vitamin E prevents oxidative modification of brain and lymphocyte band 3 proteins during aging.

Antioxidants may play an important role in preventing free radical damage associated with aging by interfering directly in the generation of radicals or by scavenging them. We investigated the effects of a high vitamin E and/or a high beta-carotene diet on aging of the anion transporter, band 3, in lymphocytes and brain. The band 3 proteins function as anion transporters, acid base regulators, C02 transporters, and structural proteins that provide a framework for membrane lipids and that link the plasma membrane to the cytoskeleton. Senescent cell antigen (SCA), which terminates the life of cells, is a degradation product of band 3. This study was conducted as a double-blind study in which eight groups of middle-aged or old mice received either high levels of beta-carotene and/or vitamin E or standard levels of these supplements in their diets. Anion transport kinetic assays were performed on isolated splenic lymphocytes. Immunoreactivity of an antibody that recognizes aging changes in old band 3 preceding generation of SCA was used to quantitate aged band 3 in brain tissue. Results indicate that vitamin E prevented the observed age-related decline in anion transport by lymphocytes and the generation of aged band 3 leading to SCA formation. beta-Carotene had no significant effect on the results of either assay. Since increased aged band 3 and decreased anion transport are initial steps in band 3 aging, which culminates in the generation of SCA and cellular removal, vitamin E prevents or delays aging of band 3-related proteins in lymphocytes and brain.

Functional characterization and regulation by pH of murine AE2 anion exchanger expressed in Xenopus oocytes

cRNA encoding the murine band 3-related protein AE2 was expressed in Xenopus oocytes. AE2-mediated transport function and regulation were analyzed by unidirectional 36Cl- influx and efflux studies. AE2 cRNA-injected oocytes took up 36Cl- as much as 40-fold faster than did water-injected oocytes. AE2-mediated 36Cl- uptake increased as a function of increasing uptake time, number of days after cRNA injection, and amount of injected cRNA. Among the functional properties of AE2 evaluated were transport mechanism and substrate specificity, inhibitor pharmacology, and regulation by pH. The apparent Km for external Cl- was 5.6 mM. AE2 was defined as a Cl-/anion exchanger by two criteria: 1) 36Cl- efflux from AE2-expressing oocytes was maximally stimulated by extracellular Cl- or nitrate; AE2-associated 36Cl- efflux was supported by substitution of extracellular Cl- with other anions in the rank order bromide > isethionate > or = gluconate > iodide and 2) prolonged preincubation of AE2 cRNA-injected oocytes in Cl(-)-free media containing isethionate, gluconate, or glutamate decreased subsequent AE2-associated 36Cl- uptake from Cl- media in rough proportion to the degree of intracellular Cl- depletion, whereas preincubation in nitrate medium had no effect. AE2-associated 36Cl- uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid at half-maximally inhibitory concentrations between 0.5 and 19 microM, depending on extracellular Cl- concentration, and progressed to irreversibility at 20 degrees C with a half-time of 20-30 min. Many additional inhibitors showed lower potency for AE2 than previously reported for AE1. Although AE2 expression did not change oocyte resting intracellular pH, AE2-associated 36Cl- influx and efflux were each decreased in acid incubation medium and increased in alkaline medium.

Characterization of the "hepatic" asialoglycoprotein receptor in rat late-stage spermatids and epididymal sperm.

Northern blot analysis of rat testicular (Te) poly(A)+RNA reveals that a transcript homologous to the major form of the asialoglycoprotein receptor (ASGP-R), designated RHL-1, is expressed as early as one week postnatally and that steady-state levels are approx. 8-times higher in the Te of an 8-week-old rat (sexually mature) as compared to an 84-week-old rat (aged). Partial cDNAs encoding RHL-1 and the minor form of the ASGP-R, designated RHL-2/3, have been cloned from two rat Te/epididymal (Ep) cDNA libraries and rat Te poly(A)+RNA. Sequence analysis of the Te/Ep RHL-1 cDNA and the Te/Ep RHL-2/3 cDNA indicates that these cDNAs are identical to the forms expressed in rat liver. Western blot analysis demonstrates the presence of a 49-kDa Te/Ep RHL-1-related protein band and a 54-kDa Te/Ep RHL-2/3-related protein band in both rat Te membrane fractions (MF) and rat Ep sperm MF. The RHL-1-related protein has been localized to late-stage Te spermatids at the time of release from the seminiferous tubules and to Ep sperm in the region of the sperm tail, referred to as the middle piece. Taken collectively, these data indicate that the authentic RHL-1 and RHL-2/3 genes of the ASGP-R are expressed in late-stage spermatids; however, the Te/Ep RHL-1-related protein differs in size from the hepatic RHL-1 polypeptide, possibly indicating a specific function of the RHL-1-related protein in spermatogenesis.

Subtypes of Madin-Darby canine kidney (MDCK) cells defined by immunocytochemistry: Further evidence for properties of renal collecting duct cells

The Madin-Darby canine kidney (MDCK) cell line has been proposed as a model for studying intercalated (IC) cells of the renal cortical collecting duct. The IC cells are characterized by peanut lectin (PNA) binding capacity, carbonic anhydrase (CA) activity and Cl(-)-HCO3- exchange mediated by a band 3-related protein. It has been suggested that these properties are also expressed in MDCK cells. So far however, the nature of the specific protein involved in Cl(-)-HCO3- exchange, the type of CA isozyme and the relationship between these two characteristics and PNA binding, have not been investigated in MDCK cells by immunocytochemical methods. Using two antibodies raised against human erythrocyte band 3 protein and two against human erythrocyte CA I and II isozymes, our study provides evidence that a protein related to band 3 is expressed in about 5% of cultured MDCK cells; these band 3-positive cells do not bind PNA and are not reactive for CAI or CAII. About 30% of the MDCK cells bind PNA, two-thirds of which are also CAII-positive. A majority (about 65%) of MDCK cells is not reactive for the three markers used; their density is increased after incubation with aldosterone. These data indicate (i) that the Cl(-)-HCO3- exchange of the MDCK cells could be related to human erythrocyte band 3, (ii) that the CA activity of the MDCK cell line bears antigenic identity with the erythrocyte CA II isozyme and (iii) that the latter is always co-localized with PNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Polarized Expression of a Band 3-Related Protein in Mammalian Sperm Cells

Extracellular bicarbonate is known to be essential for sperm motility. This finding indirectly suggests that sperm cells possess a specific carrier protein, allowing this impermeant anion to cross the cell's plasma membrane. In this study, we have identified a protein in both human and rat sperm cells that is immunologically related to erythrocyte bicarbonate/Cl exchanger (AE1) and its close homolog (AE2). We used antibodies raised against synthetic C-terminal peptides of either AE1 (band 3) or a related protein (AE2) expressed mainly in the stomach. Indirect immunofluorescence experiments revealed that both antibodies recognized a protein that is expressed in a highly polarized fashion in the sperm cells, being present only in the equatorial segment of the sperm head. Confocal laser scanning microscopy further showed that the human protein is arranged circularly close to or at the plasma membrane and that it forms a ring-like structure around the sperm head. In the human testis, the seminiferous tubules were also stained with the anti-AE2 antiserum, indicating that the protein is also expressed in developing sperm cells. This observation was also supported by Northern blot analysis, which confirmed the presence of a 4.5-kb AE2 mRNA in the rat testis tissue. We suggest that the sperm band 3-related protein could be the transport protein that mediates the effects of extracellular bicarbonate on sperm motility.

Expression cloning of human and rat renal cortex Na/Pi cotransport.

We have isolated two cDNA clones, NaPi-2 and NaPi-3, by screening rat kidney cortex and human kidney cortex cDNA libraries, respectively, for expression of sodium-dependent phosphate transport in Xenopus laevis oocytes. Substrate specificity and a detailed kinetic analysis (Na, Pi, H+ concentrations) suggested that expressed uptake activities relate to proximal tubular brush border membrane Na/Pi cotransport. NaPi-2 cDNA contains 2464 bp encoding a protein of 637 aa; NaPi-3 cDNA contains 2573 bp encoding a protein of 639 aa. NaPi-2- and NaPi-3-deduced protein sequences show high homology to each other but are different from the protein sequence deduced from the previously cloned NaPi-1 cDNA (from rabbit proximal tubules). Hydropathy profile predictions suggest at least eight membrane-spanning regions in NaPi-2/3-related proteins. In vitro translation results in proteins of the expected size and suggests glycosylation. Northern blot analysis shows corresponding mRNA species (approximately 2.7 kb) in kidney cortex of various species but no hybridization with RNAs isolated from a variety of other tissues (including intestinal segments); a hybridization signal (approximately 4.8 kb) was observed only in the lung (human). We conclude that we have structurally identified two closely related proteins most likely involved in human and rat renal brush border Na/Pi cotransport.

Localization of a Band 3-related protein in the mitochondria-rich cells of amphibian skin epithelium

Based on immunoblotting procedure, the isolated epithelium of amphibian skin was found to contain a 180 kDa protein which cross-reacts with a polyclonal antiserum raised against human erythrocyte Band 3. Immunoperoxidase and immunofluorescence staining techniques indicated that the Band 3-related protein was localized in the mitochondria-rich cells (MRC) of this epithelium, with characteristic apical labelling pattern. Our findings show that the putative apical anion exchanger of the MRC is immunologically related to the band 3 multigenic family, which catalyzes Cl -HCO 3 − transmembranous exchange. It thus suggests a molecular basis for the role played by these cells in the transepithelial Cl pathway and acid-base regulation.

Band 3 Memphis: a widespread polymorphism with abnormal electrophoretic mobility of erythrocyte band 3 protein caused by substitution AAG---- GAG (Lys----Glu) in codon 56

Band 3 Memphis (b3M) is a variant of the erythrocyte band 3 protein detected in individuals of virtually all ethnic groups and characterized by a reduced mobility of proteolytic fragments derived from the N-terminus of the cytoplasmic domain of band 3 (cdb3). We have sequenced band 3 cDNA corresponding to cdb3 in 12 heterozygotes for the b3M polymorphism including one white, one black, one Chinese, one Philippino, one Malay, and seven Melanesian subjects. In all individuals, we found a single-base substitution in codon 56 of one band 3 allele changing lysine to glutamic acid (AAG----GAG) which, in some of them, was linked with an additional mutation in cdb3. Since the change of codon 56 from AAG to GAG was the only mutation in the studied individuals found within the cDNA segment coding for the abnormally migrating fragment of cdb3, we conclude that it represents the underlying molecular basis of the b3M polymorphism. We further support this conclusion by showing that electrophoresis in the presence of 4 mol/L urea abolished the difference in migration between proteolytic products of b3M and normal band 3, and that a fusion protein prepared from cDNA coding for the b3M allele again exhibits reduced electrophoretic mobility compared with the normal fusion protein. Finally, since most of the previously cloned mouse, rat, and chicken band 3 and band 3-related proteins contain glutamic acid in the position corresponding to amino acid 56 in the human band 3, we propose that the Memphis variant is the evolutionarily older form of band 3.

Band 3 Memphis: A Widespread Polymorphism With Abnormal Electrophoretic Mobility of Erythrocyte Band 3 Protein Caused by Substitution AAG → GAG (Lys → Glu) in Codon 56

Band 3 Memphis (b3M) is a variant of the erythrocyte band 3 protein detected in individuals of virtually all ethnic groups and characterized by a reduced mobility of proteolytic fragments derived from the N-terminus of the cytoplasmic domain of band 3 (cdb3). We have sequenced band 3 cDNA corresponding to cdb3 in 12 heterozygotes for the b3M polymorphism including one white, one black, one Chinese, one Philippino, one Malay, and seven Melanesian subjects. In all individuals, we found a single-base substitution in codon 56 of one band 3 allele changing lysine to glutamic acid (AAG----GAG) which, in some of them, was linked with an additional mutation in cdb3. Since the change of codon 56 from AAG to GAG was the only mutation in the studied individuals found within the cDNA segment coding for the abnormally migrating fragment of cdb3, we conclude that it represents the underlying molecular basis of the b3M polymorphism. We further support this conclusion by showing that electrophoresis in the presence of 4 mol/L urea abolished the difference in migration between proteolytic products of b3M and normal band 3, and that a fusion protein prepared from cDNA coding for the b3M allele again exhibits reduced electrophoretic mobility compared with the normal fusion protein. Finally, since most of the previously cloned mouse, rat, and chicken band 3 and band 3-related proteins contain glutamic acid in the position corresponding to amino acid 56 in the human band 3, we propose that the Memphis variant is the evolutionarily older form of band 3.

CDNA cloning and localization of a band 3-related protein from ileum

A Cl(-)-HCO3- exchanger in the brush-border membrane mediates active Cl- absorption and regulates intracellular pH in rabbit ileum. The molecular identity of the ileal Cl(-)-HCO3- exchanger has not been established. The best-characterized plasma membrane Cl(-)-HCO3- exchanger is erythroid band 3. Structurally related proteins in nonerythroid tissues comprise an anion exchanger (AE) family. We used the polymerase chain reaction to amplify and clone a cDNA encoding an ileal band 3-related protein (B3RP) from rabbit ileal enterocytes. The composite sequence is 3,909 bp and is predicted to encode a protein of 136 kDa. The deduced amino acid sequence is 95% identical to murine renal AE2, indicating that ileal B3RP is rabbit AE2. Antisera generated against a cytoplasmic fragment of ileal B3RP recognized a 160- to 170-kDa polypeptide in the brush-border membrane, but not the basolateral membrane, of ileal crypt and villus enterocytes. This correlates with previous studies indicating that a Cl(-)-HCO3- exchange is present in brush-border but not basolateral membrane vesicles from rabbit ileal enterocytes. We conclude that ileal B3RP is a product of the AE gene family, and is present in the brush-border of ileal enterocytes, where it may mediate Cl(-)-HCO3- exchange.