Expression Of Anion Exchanger 1 Research Articles

A Balance between Transmembrane-Mediated ER/Golgi Retention and Forward Trafficking Signals in Glycophorin-Anion Exchanger-1 Interaction.

Anion exchanger-1 (AE1) is the main erythroid Cl-/HCO3- transporter that supports CO2 transport. Glycophorin A (GPA), a component of the AE1 complexes, facilitates AE1 expression and anion transport, but Glycophorin B (GPB) does not. Here, we dissected the structural components of GPA/GPB involved in glycophorin-AE1 trafficking by comparing them with three GPB variants-GPBhead (lacking the transmembrane domain [TMD]), GPBtail (mainly the TMD), and GP.Mur (glycophorin B-A-B hybrid). GPB-derived GP.Mur bears an O-glycopeptide that encompasses the R18 epitope, which is present in GPA but not GPB. By flow cytometry, AE1 expression in the control erythrocytes increased with the GPA-R18 expression; GYP.Mur+/+ erythrocytes bearing both GP.Mur and GPA expressed more R18 epitopes and more AE1 proteins. In contrast, heterologously expressed GPBtail and GPB were predominantly localized in the Golgi apparatus of HEK-293 cells, whereas GBhead was diffuse throughout the cytosol, suggesting that glycophorin transmembrane encoded an ER/Golgi retention signal. AE1 coexpression could reduce the ER/Golgi retention of GPB, but not of GPBtail or GPBhead. Thus, there are forward-trafficking and transmembrane-driven ER/Golgi retention signals encoded in the glycophorin sequences. How the balance between these opposite trafficking signals could affect glycophorin sorting into AE1 complexes and influence erythroid anion transport remains to be explored.

Stronger Association between Hemoglobin and Blood Pressure in People Bearing GP.Mur Blood Type

GP.Mur, commonly known as Miltenberger subtype III, is a clinically-important red blood cell (RBC) phenotype unique among Southeast Asians (SEA). On the GP.Mur+ erythrocyte membrane, glycophorin B-A-B hybrid protein is expressed, instead of glycophorin B (GPB). Different from GPB that does not have any known function, GP.Mur hybrid protein promotes expression of anion exchanger-1 (AE1) on the RBC membrane and support AE1-relevant physiological functions in GP.Mur+ carriers. In our previous human studies, we unexpectedly uncovered that GP.Mur+ carriers have slightly higher blood pressure (BP) than GP.Mur-negative people. Since AE1 has been implicated a role in modulation of NO bioavailability and hemoglobin (Hb) is a major NO scavenger, we hypothesized that the slightly but significantly higher BP in GP.Mur carriers might be related to Hb-mediated NO processing. Methods With the approval of hospital Institutional Review Board, healthy, non-smoker subjects aged between 20 and 55 were recruited at Taitung MacKay Memorial Hospital (TT-MMH). Besides general physical assessment, recruited subjects were tested for CBC and RBC phenotypes, including GP.Mur. Results 345 male and 515 female non-smoker adults were recruited from Taitung County, Taiwan, and 22.1% of them bear GP.Mur blood type. For male and female cohorts, GP.Mur erythrocyte expression was significantly associated with slightly higher systolic BP (SBP) (122.2±12.9 mmHg [non-GP.Mur male] versus 127.8±14.4 mmHg [GP.Mur male]; 115.0±13.0 mmHg [non-GP.Mur female] versus 118.0±13.4 mmHg [GP.Mur female]). Hb levels were not affected by GP.Mur. Hb is known to be positively associated with SBP. We indeed found a Pearson correlation of 0.164 between Hb (g/dL) and SBP (mmHg) in non-GP.Mur male subjects (*p~0.007). However, Hb-SBP associations were not observed in our female cohorts with or without GP.Mur. Intriguingly, the Pearson correlation between Hb and SBP from the GP.Mur male group was doubled, compared to that from the GP.Mur-negative male group (Pearson correlation: 0.299 [GP.Mur male] versus 0.164 [non-GP.Mur male]). Conclusion From the differences in Hb-SBP correlations in male, the effects of NO-scavenging by Hb were likely more substantial in the presence of GP.Mur and/or higher AE1 expression. We did not observe any positive associations in our SEA female subjects, presumably due to menstrual cycle and that SEA women were generally more anemic.

Stomatin modulates the activity of the Anion Exchanger 1 (AE1, SLC4A1)

Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO3− and Cl− exchange. Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl−/HCO3− exchange was evaluated using stopped-flow methods. In HEK293 cells expressing recombinant AE1 and stomatin, the permeabilities associated with AE1 activity were 30% higher in cells overexpressing stomatin, compared to cells with only endogenous stomatin expression. Ghosts from stomatin-deficient RBCs and controls were resealed in the presence of pH- or chloride-sensitive fluorescent probes and submitted to inward HCO3− and outward Cl− gradients. From alkalinization rate constants, we deduced a 47% decreased permeability to HCO3− for stomatin-deficient patients. Similarly, kinetics of Cl− efflux, followed by the probe dequenching, revealed a significant 42% decrease in patients. In situ Proximity Ligation Assays confirmed an interaction of AE1 with stomatin, in both HEK recombinant cells and RBCs. Here we show that stomatin modulates the transport activity of AE1 through a direct protein-protein interaction.

Renal peroxiredoxin 6 interacts with anion exchanger 1 and plays a novel role in pH homeostasis.

Peroxiredoxin 6 (PRDX6) is one of six members of the PRDX family, which have peroxidase and antioxidant activity. PRDX6 is unique, containing only one conserved cysteine residue (C47) rather than the two found in other PRDXs. A yeast two-hybrid screen found PRDX6 to be a potential binding partner of the C-terminal tail of anion exchanger 1 (AE1), a Cl−/HCO3− exchanger basolaterally expressed in renal α-intercalated cells. PRDX6 immunostaining in human kidney was both cytoplasmic and peripheral and co-localized with AE1. Analysis of native protein showed it was largely monomeric, whereas expressed tagged protein was more dimeric. Two methionine oxidation sites were identified. In vitro and ex vivo pulldowns and immunoprecipitation assays confirmed interaction with AE1, but mutation of the conserved cysteine resulted in loss of interaction. Prdx6 knockout mice had a baseline acidosis with a major respiratory component and greater AE1 expression than wild type animals. After an oral acid challenge, PRDX6 expression increased in wild type mice, with preservation of AE1. However, AE1 expression was significantly decreased in knockout animals. Kidneys from acidified mice showed widespread proximal tubular vacuolation in wild type but not knockout animals. Knockdown of PRDX6 by siRNA in mammalian cells reduced both total and cell membrane AE1 levels. Thus, PRDX6-AE1 interaction contributes to maintenance of AE1 during cellular stress such as during metabolic acidosis.

Differential roles of tryptophan residues in the functional expression of human anion exchanger 1 (AE1, Band 3, SLC4A1).

Anion exchanger 1 (AE1) is a 95 kDa glycoprotein that facilitates Cl(-)=HCO(-)(3) exchange across the erythrocyte plasma membrane. This transport activity resides in the 52 kDa C-terminal membrane domain (Gly(361)-Val(911)) predicted to span the membrane 14 times. To explore the role of tryptophan (Trp) residues in AE1 function, the seven endogenous Trp residues in the membrane domain were mutated individually to alanine (Ala) and phenylalanine (Phe). Expression levels, cell surface abundance, inhibitor binding and transport activities of the mutants were measured upon expression in HEK-293 cells. The seven Trp residues divided into three classes according the impact of mutations on the functional expression of AE1: Class 1, dramatically decreased expression (Trp(492) and Trp(496)); Class 2, decreased expression by Ala substitution but not Phe (Trp(648), Trp(662) and Trp(723)); and Class 3, normal expression (Trp(831) and Trp(848)). The results indicate that Trp residues play differential roles in AE1 expression and function depending on their location in the protein and that Trp mutants with low expression are misfolded and retained in the endoplasmic reticulum.

Rapid Cl−/HCO3− exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells

Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.

Anti‐tumour effects of small interfering RNA targeting anion exchanger 1 in experimental gastric cancer

Anion exchanger 1 (AE1) is an integral membrane protein found in erythrocytes. Our previous studies have demonstrated that AE1 is expressed in human gastric cancer cells and may be involved in the carcinogenesis of cancer. In this study, we further investigated the role of AE1 in gastric carcinogenesis and the anti-tumour effects of AE1-targeted small interfering RNAs (siRNAs) in two experimental models of gastric cancer. Molecular and cellular experiments were performed to elucidate the role of AE1 in the malignant transformation of gastric epithelium and the effects of AE1-targeted siRNAs on gastric cancer cells. The anti-tumour effect of the siRNA was evaluated in vivo in two mouse models, nude mice implanted with human gastric cancer xenografts (Model I) and mice with gastric cancer induced by N-methyl-N-nitrosourea (MNU) and Helicobacter pylori (Model II). AE1 was found to increase gastric carcinogenesis by promoting cell proliferation. AE1-targeted siRNA significantly suppressed AE1 expression and hindered tumour growth. Furthermore, the siRNA markedly decreased the detection rate of gastric cancer, in parallel with an increase in atypical hyperplasia at the end of the experiment in Model II. Knockdown of AE1 expression in gastric mucosa by administration of synthetic siRNAs significantly inhibits the growth of gastric cancer and decreases the detection rate of this tumour in experimental mice. These results suggest that AE1 is potentially a key therapeutic target and the silencing of AE1 expression in gastric mucosa could provide a new therapeutic approach for treating gastric cancer.

Induction of anion exchanger-1 translation and its opposite roles in the carcinogenesis of gastric cancer cells and differentiation of K562 cells

Anion exchanger-1 (AE1), an erythroid-specific membrane protein, mediates the Cl(-)/HCO(-)(3) exchange across the plasma membrane and regulates intracellular pH. We have found that AE1 was unexpectedly expressed in gastric cancer cells and participated in the tumorigenesis of the cancer. Here, we focus on the induction of AE1 expression and its role in gastric carcinogenesis as well as in the differentiation of K562 cells. The results show that expression of AE1 is not related to genetic mutation or the mRNA level, but rather, that it is modulated by miR-24. miR-24 decreases the expression of AE1 through binding to the 3'UTR of AE1 mRNA. Transfection of an miR-24 into gastric cancer cells reduced the elevation of the AE1 protein, which resulted in return of AE1-sequestrated p16 to the nucleus, thereby inhibiting proliferation of the cells. Furthermore, the miR-24 inhibitor cooperated with hemin to induce the expression of AE1 in K562 cells and differentiation of the cells, which is consistent with results obtained from the cells cultured at pH 7.6 or from forced stable expression of AE1. These findings establish a novel regulation of miR-24-related AE1 expression in gastric carcinogenesis and erythropoiesis.

Gastrin suppresses the interdependent expression of p16 and anion exchanger 1 favoring growth inhibition of gastric cancer cells

Our previous studies demonstrated that expression and interaction of p16 with anion exchanger 1 (AE1) in gastric cancer cells is correlated with progression and shorter survival of the cancer. In this article, the effects of gastrin on p16 and AE1 and its implication in prevention and treatment of gastric cancer were studied by molecular biology techniques, animal experiment and clinical analysis. The results showed that expression of p16 in human gastric body carcinoma was downregulated along with the progression of the cancer, suggesting the reverse correlations between gastrin and p16 in vivo. Further experiments indicated that gastrin suppressed the expression of p16 via the p16 promoter and thereafter resulted in the degradation of AE1 in gastric cancer cells. Silencing of AE1 or p16 significantly inhibited the proliferation of the cancer cells. Using a xenograft tumor model in nude mice, we showed that experimental systemic hypergastrinemia induced by the administration of omeprazole led to decreased expression of AE1 and p16 as well as to a marked growth inhibition of SGC7901 tumors. It is concluded that a moderate plasma gastrin level is beneficial to the growth inhibition of gastric cancer by suppressing the expression of AE1 and p16. This finding may have an important implication for the prevention and treatment of cancers arise in the gastric antrum.

Miltenberger blood group antigen type III (Mi.III) enhances the expression of band 3

The special blood group antigen Mi.III exhibits a characteristic hybrid structure of glycophorin A (GPA) and glycophorin B, termed Gp.Mur. This phenotype has exceptionally high occurrence rates in several indigenous tribes in Taiwan ( approximately 21.2%-88.4%). Because glycophorin/Miltenberger begins interaction with anion exchanger-1 (AE1) in the endoplasmic reticulum, we hypothesized that the AE1-based macrocomplexes on erythrocyte membranes obtained from Mi.III(+) people could be differentiated from those obtained from non-Miltenberger people. Quantitative mass spectrometric comparison of the AE1-based complexes by iTRAQ (Applied Biosystems) revealed 25% to 67% higher expression of AE1 in Mi.III(+) erythrocytes. In accordance with the higher AE1 level, the Mi.III(+) erythrocytes exhibited superior HCO(3)(-) capacities, pH homeostasis, and osmotic resistance. Cotransfection experiments in HEK293 cells showed that Gp.Mur, like GPA, enhanced trafficking of AE1 to the plasma membrane. In summary, the increased surface expression of AE1 in Mi.III(+) erythrocytes could be attributed to the additive effect of GPA and Gp.Mur coexpression.

Expression of anion exchanger 1 is associated with tumor progress in human gastric cancer

Anion exchanger 1 (AE1) is a transmembrane glycoprotein which is abundantly expressed in erythrocyte plasma membrane and mediates the electroneutral exchange of Cl(-) and HCO(3) (-). We previously reported that the AE1 protein was unexpectedly expressed in the gastric and colonic cancer and take part in the carcinogenesis of the cancer cells. The aim of the present study is to determine the potential clinical implications of AE1 expression in gastric carcinoma. Immunohistochemistry assay was used to determine the expression of AE1 protein. The expression of AE1 in normal and malignant tissues from 286 patients with early and advanced gastric carcinoma was examined. The correlations of AE1 expression with clinicopathological parameters, including age, tumor size, location and subtypes, expression frequency, survival period and lymph metastasis were assessed by Chi-squared test and t test analysis. AE1 immunoreactivity was negative in normal gastric tissue. Positive immunostaining of AE1 was detected in gastric carcinoma regardless of the location. AE1 was most frequently expressed in the gastric antrum carcinoma compared with gastric body cancer (P = 0.034). Expression of AE1 was significantly associated with bigger tumor size, deeper invasion, shorter survival period, and non-lymph metastasis. In para-cancer tissues of intestinal-type gastric cancer, the expression frequency of AE1 was higher than that in diffuse-type (P = 0.011). The results showed a strong association of AE1 expression with the onset and progression of the gastric cancer and that may be helpful for improving the tumor classification and the treatment of cancer.

Expression of cytoplasmic p16 and anion exchanger 1 is associated with the invasion and absence of lymph metastasis in gastric carcinoma

The tumor suppressor p16 is a negative regulator of the cell cycle, commonly believed to act in the nucleus. We recently found that p16 protein is expressed in the cytoplasm of gastric cancer cells, concomitantly with anion exchanger 1 (AE1). The aim of this study was to analyze the significance of cytoplasmic p16 and its relationship to AE1 in the progression of gastric cancer. Expression of p16 and AE1 was examined by immunohistochemical analysis in 196 patients; 98 with early gastric cancer and 98 with advanced gastric cancer. The relationship between cytoplasmic p16 and clinicopathological features, and the relationship between cytoplasmic p16 and AE1, were analyzed statistically. Expression of p16 was observed in the nucleus in early stage gastric cancer, but was located mainly in the cytoplasm in advanced cancer cells. Furthermore, cytoplasmic expression of p16 was correlated with AE1 expression, and both were associated with the absence of lymph metastasis in gastric cancer. In conclusion, cytoplasmic immunoreactivity of p16 appears to be a good prognostic indicator in advanced gastric cancer. Co-localization of p16 and AE1 predicts a lack of metastasis in gastric cancer. The role of cytoplasmic p16 and AE1, and the mechanisms involved in the progression of gastric cancer, warrant further investigation.

Effect of block deletions in the C-terminus on the functional expression of human anion exchanger 1 (AE1)

The human anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells and is responsible for the exchange of Cl(-) for HCO(3)(-). However, the detailed role played by the AE1 C-terminal region in the anion translocation and membrane trafficking process remains unclear. In this paper, we created four mutants in the human AE1 C-terminus by deletion of the residues Ala(891)-Phe(895), Asp(896)-Glu(899), Asp(902)-Glu(906) and Val(907)-Val(911), to investigate the role of these sequences in functional expression of AE1. WT AE1 and its deletion mutant constructs were expressed in HEK 293 cells. Western blotting showed that deletions of Ala(891)-Phe(895), Asp(896)-Glu(899), and Val(907)-Val(911) induced high expression of AE1, whereas loss of Asp(902)-Glu(906) results in stable low expression. Pulse chase assays of WT AE1 and its mutants showed that the stability of protein is unaffected by the levels of expression of the AE1 and its mutants. Ala(891)-Phe(895), Asp(902)-Glu(906) and Val(907)-Val(911) mutants exhibited lower levels of trafficking to the plasma membrane compared with WT AE1, while the Asp(896)-Glu(899) mutant was more highly expressed at the plasma membrane. The decreased ability of the mutants to mediate Cl(-)/HCO(3)(-) exchange in transfected cells revealed that the deletion sequences have an important role in transport activity. These results demonstrate that the studied residues in the AE1 C-terminus differently affect the expression, membrane trafficking and functional folding of AE1.

Ubiquitylation-independent ER-associated degradation of an AE1 mutant associated with dominant hereditary spherocytosis in cattle

Various mutations in the AE1 (anion exchanger 1, band 3) gene cause dominant hereditary spherocytosis, a common congenital hemolytic anemia associated with deficiencies of AE1 of different degrees and loss of mutant protein from red blood cell membranes. To determine the mechanisms underlying decreases in AE1 protein levels, we employed K562 and HEK293 cell lines and Xenopus oocytes together with bovine wild-type AE1 and an R664X nonsense mutant responsible for dominant hereditary spherocytosis to analyze protein expression, turnover, and intracellular localization. R664X-mutant protein underwent rapid degradation and caused specifically increased turnover and impaired trafficking to the plasma membrane of the wild-type protein through hetero-oligomer formation in K562 cells. Consistent with those observations, co-expression of mutant and wild-type AE1 reduced anion transport by the wild-type protein in oocytes. Transfection studies in K562 and HEK293 cells revealed that the major pathway mediating degradation of both R664X and wild-type AE1 employed endoplasmic reticulum (ER)-associated degradation through the proteasomal pathway. Proteasomal degradation of R664X protein appeared to be independent of both ubiquitylation and N-glycosylation, and aggresome formation was not observed following proteasome inhibition. These findings indicate that AE1 R664X protein, which is associated with dominant hereditary spherocytosis, has a dominant-negative effect on the expression of wild-type AE1.

Differential expression of AE1 in renal HCO3-secreting and -reabsorbing intercalated cells.

The cortical collecting duct of the kidney contains two types of intercalated cells that transport HCO3 in opposite directions. HCO3 reabsorption takes place in the alpha-type intercalated cells, which express a Cl/HCO3 exchanger on the basolateral membrane. This exchanger is the product of the anion exchanger 1 (AE1) or band 3 gene. HCO3 secretion occurs in the beta-intercalated cells, which have a Cl/HCO3 exchanger on the apical membrane. Based on studies in an immortalized cell line, recently it was proposed that the apical anion exchanger of beta-intercalated cells is also AE1 (van Adelsberg, J. S., Edwards, J. C., and Al-Awqati, Q. (1993) J. Biol. Chem. 268, 11283-11289). In the present study we reinvestigated this issue by determining the distribution of AE1 mRNA and protein in the two intercalated cell types using cells freshly isolated from the native epithelium. Using quantitative reverse transcriptase polymerase chain reaction, we found that alpha-intercalated cells, isolated from rabbit kidney by fluorescence-activated cell sorting, have high levels of AE1 mRNA, whereas beta-intercalated cells express very low levels. The ratio of AE1 mRNA levels in alpha- versus beta-intercalated cells averaged 10.1 +/- 2.6. In addition, metabolic acidosis increased the levels of AE1 mRNA by 3-5-fold in cortical collecting duct cells. This difference was confirmed by Northern blotting. Western blotting using an antibody against rabbit AE1 revealed a major immunoreactive product with a molecular weight of approximately 110 kDa in cortical collecting duct cells. Deglycosylation reduced the size of the immunoreactive product to approximately 90 kDa, which is compatible with the presence of a truncated form of AE1. Metabolic acidosis increased the intensity of the AE1 immunoreactive band. The level of AE1-immunoreactive protein was significantly higher in alpha-intercalated cells than in beta-intercalated cells. In aggregate, these data provide evidence for the differential expression of AE1 in HCO3-reabsorbing versus HCO3-secreting renal intercalated cells both at the mRNA and at the protein level. These results give no support to the concept that AE1 functions both as a basolateral and an apical anion exchanger in cortical collecting duct cells.