Missense, nonsense, and frameshift mutations in the human anion exchanger 1 (AE1) have been associated with inherited distal renal tubular acidosis and hereditary spherocytosis. These two disorders are almost always mutually exclusive. However, we have recently found an unusual exception, i.e, a patient with complete distal renal tubular acidosis and severe hereditary spherocytosis. DNA sequencing revealed a novel mutation AE1 E522K (Band 3 Kaohsiung) combined with AE1 G701D mutation in this patient. We hypothesize these AE1 mutations cause these two disorders because of trafficking defect. To elucidate this hypothesis, we analyzed protein trafficking and subcellular location of AE1 and these mutants transfected into MDCK cells. Our results showed that they formed homodimers or heterodimers with each other. Homodimers of the wild-type and E522K mutant were localized at the plasma membrane, whereas the G701D mutant largely remained in the cytoplasm. On the other hand, heterodimers of either E522K or G701D and the wild-type AE1 were located in the plasma membrane, whereas E522K/G701D heterodimers remained in the cytoplasm. As for erythroid isoform of anion exchanger 1, analysis of protein trafficking and subcellular localization of the wild-type erythroid isoform of human anion exchanger 1 and these mutants transfected into k562 cells also showed that they can form homodimers or heterodimers with each other. Erythroid AE1 E522K/G701D cell-surface expression was significantly lower compared with WT homodimer expression. This result coincided with that erythroid AE1 of the patient¡¦s red cell membrane can be detected 28% that of normal control in immunoblotting. Our study shows that the compound E522K/G701D mutation of human anion exchanger 1 causes trafficking defects in kidney and red blood cell lines, and these may explain the complete distal renal tubular acidosis and hereditary spherocytosis of the patient.

No description available.

Abstract Glycans, as a part of glycoproteins, glycolipids and other glycoconjugates, are involved in many vital intra- and inter-cellular tasks, such as protein folding and sorting, protein quality control, vesicular trafficking, cell signalling, immunological defence, cell motility and adhesion. Therefore, their correct construction is crucial for the normal functioning of eukaryotic cells and organisms they form. Most cellular glycans are constructed in the Golgi, and abnormalities in their structure may derive, for instance, from alkalinization of the Golgi lumen. In this work we show that Golgi pH is generally higher and more variable in abnormally glycosylating, i.e. strongly T-antigen (Gal-β1,3-GalNAc-ser/thr) expressing cancer cells, than in non-T-antigen expressing cells. We also confirmed that the Golgi pH alterations detected in cancer cells have the potential to induce glycosylation changes. A mere 0.2 pH unit increase in Golgi pH is able to induce T-antigen expression and inhibit terminal N-glycosylation in normally glycosylating cells. The mechanism of inhibition involves mislocalization of the corresponding glycosyltransferases. We also studied potential factors that can promote Golgi pH misregulation in health and disease, and found that cultured cancer cells, despite variation and elevation in Golgi pH, are fully capable of acidifying the Golgi lumen under the normal Golgi pH. Moreover, we introduce a Golgi localized Cl-/HCO3- exchanger, AE2a, that participates in Golgi pH regulation by altering luminal bicarbonate concentration and thus also buffering capacity. Participation of AE2a in Golgi pH regulation is especially intriguing, because it also provides a novel mechanism for expelling protons from the Golgi lumen.

Introduction: Microbial fermentation of carbohydrates in forestomach of ruminants produces large amounts of short-chain fatty acids (SCFA, mainly acetic acid, propionic acid, and n-butyric acid). The majority of these substrates is taken up directly across the ruminal wall. After luminal uptake into the epithelial cells, SCFA mainly occur in the dissociated form due to the intracellular pH of ~7.4. Moreover, a big portion of SCFA is metabolised within the cytosol. Main end products of epithelial SCFA metabolism are ketone bodies (D-3-hydroxybutyric acid and acetoacetic acid) and lactic acid. Both intact SCFA and ketone bodies and lactate need to be efficiently extruded from the ruminal epithelial cells to prevent a lethal drop of intracellular pH and counteract osmotic load of the cytosol. All these substances are less lipophilic in comparison to the undissociated form of SCFA. Thus, dissociated SCFA (SCFA-) and their metabolites need Protein mediated mechanisms for the extrusion across the basolateral side of ruminal epithelium. One mechanism suggested to be involved in the extrusion of SCFA- across basolateral membrane of the ruminal epithelium is the monocarboxylate transporter 1 (MCT1). Functionally, MCT1 was first assumed to operate as proton-coupled transporter for monocarboxylates including SCFA. Nonetheless, a recent study found a bicarbonate dependent anion exchange mechanism which turned out to be sensitive to MCT1 Inhibitors at the basolateral side of the ruminal epithelium pointing to the ability of MCT1 to act as an anion exchanger. However, in these experiments the inhibition of MCT1 abolished bicarbonate dependent transport only by half. This suggests the involvement of further anion exchanger(s) in the transport of SCFA across the basolateral membrane of ruminal epithelium. Promising candidates to underlie this exchange are the putative Anion exchanger 1 (PAT1) and a transport protein designated „down-regulated in adenoma“ (DRA). Materials and Methods: Sheep rumen epithelium was mounted in Ussing Chambers under short-circuit conditions. Radioactively labelled acetate (ac) was added to the serosal side. Serosal to mucosal flux of ac (Jsm ac) was measured with or without anion Exchange inhibitors (50 mM NO3- or 1 mM DIDS) or the MCT1 inhibitor p-hydroxy mercuribenzoic acid (pHMB; 1.5 mM) in the serosal buffer solution. The inhibitors were added alone or in combination with each other. Furthermore, mucosal to serosal flux of radioactivelly labelled ac or butyrate (bu) (Jms ac, bu) was measured in the presence or absence of SO42-, Cl- or NO3- (50 mM respectively) as exchange substrate in the serosal buffer solution. Immunohistochemical staining was conducted to locate PAT1 and DRA by use of commercially available antibodies. Results: NO3- and pHMB significantly reduced Jsm ac by 57 % and 51 %, respectively. When pHMB was applied after pre-incubation with NO3- an additional inhibition of Jsm ac was observed. Vice versa, NO3- further inhibited Jsm ac when epithelia were pre-incubated with pHMB before. DIDS had no inhibitory effect on SCFA flux. Serosal presence of SO42- or Cl- enhanced Jms ac significantly. Regarding bu, Cl- or SO4 2- also enhanced Jms bu significantly. The different anions available in the serosal buffer solution numerically enhanced Jms in the order of SO4 2- > Cl- for both ac and bu, which corresponds to the known affinity sequence of PAT1 and DRA. Immunohistochemistry revealed localization of PAT 1 in the stratum basale, whereas DRA was not detectable using this method. Conclusions: Basically, this study supports the suggestion that MCT1 works as an Anion exchanger in ruminal epithelium. In addition, it clearly shows that there is at least one further anion exchanger involved in the basolateral extrusion of SCFA and their metabolites. The functional and immunohistochemical findings suggest that PAT1 holds a significant role in this respect.

Introduction: Microbial fermentation of carbohydrates in forestomach of ruminants produces large amounts of short-chain fatty acids (SCFA, mainly acetic acid, propionic acid, and n-butyric acid). The majority of these substrates is taken up directly across the ruminal wall. After luminal uptake into the epithelial cells, SCFA mainly occur in the dissociated form due to the intracellular pH of ~7.4. Moreover, a big portion of SCFA is metabolised within the cytosol. Main end products of epithelial SCFA metabolism are ketone bodies (D-3-hydroxybutyric acid and acetoacetic acid) and lactic acid. Both intact SCFA and ketone bodies and lactate need to be efficiently extruded from the ruminal epithelial cells to prevent a lethal drop of intracellular pH and counteract osmotic load of the cytosol. All these substances are less lipophilic in comparison to the undissociated form of SCFA. Thus, dissociated SCFA (SCFA-) and their metabolites need Protein mediated mechanisms for the extrusion across the basolateral side of ruminal epithelium. One mechanism suggested to be involved in the extrusion of SCFA- across basolateral membrane of the ruminal epithelium is the monocarboxylate transporter 1 (MCT1). Functionally, MCT1 was first assumed to operate as proton-coupled transporter for monocarboxylates including SCFA. Nonetheless, a recent study found a bicarbonate dependent anion exchange mechanism which turned out to be sensitive to MCT1 Inhibitors at the basolateral side of the ruminal epithelium pointing to the ability of MCT1 to act as an anion exchanger. However, in these experiments the inhibition of MCT1 abolished bicarbonate dependent transport only by half. This suggests the involvement of further anion exchanger(s) in the transport of SCFA across the basolateral membrane of ruminal epithelium. Promising candidates to underlie this exchange are the putative Anion exchanger 1 (PAT1) and a transport protein designated „down-regulated in adenoma“ (DRA). Materials and Methods: Sheep rumen epithelium was mounted in Ussing Chambers under short-circuit conditions. Radioactively labelled acetate (ac) was added to the serosal side. Serosal to mucosal flux of ac (Jsm ac) was measured with or without anion Exchange inhibitors (50 mM NO3- or 1 mM DIDS) or the MCT1 inhibitor p-hydroxy mercuribenzoic acid (pHMB; 1.5 mM) in the serosal buffer solution. The inhibitors were added alone or in combination with each other. Furthermore, mucosal to serosal flux of radioactivelly labelled ac or butyrate (bu) (Jms ac, bu) was measured in the presence or absence of SO42-, Cl- or NO3- (50 mM respectively) as exchange substrate in the serosal buffer solution. Immunohistochemical staining was conducted to locate PAT1 and DRA by use of commercially available antibodies. Results: NO3- and pHMB significantly reduced Jsm ac by 57 % and 51 %, respectively. When pHMB was applied after pre-incubation with NO3- an additional inhibition of Jsm ac was observed. Vice versa, NO3- further inhibited Jsm ac when epithelia were pre-incubated with pHMB before. DIDS had no inhibitory effect on SCFA flux. Serosal presence of SO42- or Cl- enhanced Jms ac significantly. Regarding bu, Cl- or SO4 2- also enhanced Jms bu significantly. The different anions available in the serosal buffer solution numerically enhanced Jms in the order of SO4 2- > Cl- for both ac and bu, which corresponds to the known affinity sequence of PAT1 and DRA. Immunohistochemistry revealed localization of PAT 1 in the stratum basale, whereas DRA was not detectable using this method. Conclusions: Basically, this study supports the suggestion that MCT1 works as an Anion exchanger in ruminal epithelium. In addition, it clearly shows that there is at least one further anion exchanger involved in the basolateral extrusion of SCFA and their metabolites. The functional and immunohistochemical findings suggest that PAT1 holds a significant role in this respect.:1 Introduction 1 2 Literature Review 3 2.1 Importance of short-chain fatty acid production of ruminants 3 2.2 Apical uptake of short-chain fatty acids from the rumen 5 2.2.1 Apical uptake of undissociated SCFA from the rumen 6 2.2.2 Apical uptake of dissociated fatty acids from the rumen 8 2.3 Intraepithelial metabolism of short-chain fatty acids 9 2.4 Mechanisms for the basolateral discharge of the short-chain fatty acids 11 2.4.1 Basolateral extrusion of short-chain fatty acids in other gastrointestinal tract epithelia 12 2.4.2 Basolateral extrusion of short-chain fatty acids in ruminal epithelium 14 2.4.3 Further candidate proteins for extrusion of SCFA- in exchange for HCO3 - 19 2.4.3.1 Putative Anion transporter 1 (PAT1 = SLC26A6) 19 2.4.3.2 Down-regulated in adenoma (DRA = SLC26A3) 21 2.4.3.3 Anion exchanger 2 (AE2 = SLC4A2) 22 2.5 Literature implications for this study 23 3 Materials and Methods 24 3.1 Animals 24 3.2 Ussing chamber studies 24 3.2.1 Buffer solutions 24 3.2.2 Preparation of ruminal epithelium 25 3.2.3 Incubation 25 3.2.4 Electrophysiological parameters 26 3.3 Experimental procedure 27 3.3.1 Determination of the unidirectional SCFA flux rate 29 3.4 Experimental Setups 30 3.4.1 Sensitivity of Jsm ac to inhibitors 30 3.4.1.1 Effect of nitrate and pHMB on Jsm ac 30 3.4.1.2 Effect of DIDS, NO3 - and pHMB on Jsm ac 31 3.4.2 Effect of the basolateral replacement of the anions on the extrusion of SCFA 32 3.4.2.1 Effect of Cl- and NO3 - on Jms of acetate and butyrate 32 3.4.2.2 Effect of SO4 2- on Jms of acetate and butyrate 32 3.4.3 Effect of different anions available in the serosal solution on Jms of acetate and butyrate 33 3.5 Immunohistochemistry 34 3.5.1 Preparation of the samples. 34 3.5.2 Fixation and staining of the samples. 34 3.5.3 Evaluation 35 3.6 Statistical analysis 36 4 Results 37 4.1 Inhibitors sensitivity 37 4.1.1 Effect of nitrate and pHMB on Jsm ac 37 4.1.2 Effect of DIDS, pHMB and NO3 - on Jsm ac 41 4.2 Effect of Cl- and NO3 - on Jms of acetate and butyrate 43 4.2.1 Effect of SO4 2- on Jms of acetate and butyrate 44 4.3 Effect of Cl-, NO3 - or SO4 2- when present in the serosal solution for 150 min 49 4.4 Immunohistochemistry 52 5 Discussion 54 5.1Ussing chamber experiments 56 5.1.1 Effect of Cl- and NO3 - on Jms of acetate 56 5.1.2 Effect of nitrate and pHMB on Jsm of acetate 57 5.1.3 Effect of DIDS, pHMB or NO3 - on Jsm of acetat 58 5.1.4 Effect of SO4 2- on Jms of acetate 59 5.1.5 Comparison between different anions as exchange substrate for the basolateral extrusion of acetate 60 5.2 Immunohistochemistry 62 5.3 Comparison between basolateral extrusion of butyrate and acetate 62 5.4 Conclusions 64 6 Summary 66 7 Zusammenfassung 68 8 References 70 Ac Aknowledgements

Mutations in the SLC4A1 gene result in misfolding and trafficking defects of the human erythroid (AE1) and kidney (kAE1) forms of the anion exchanger 1 glycoprotein. This affects the amount of functional protein at the cell surface, resulting in hematological and renal diseases. In this thesis, the role of the quality control system of molecular chaperones (cytosolic and ER) was examined during the biosynthesis of wild type and mutant AE1 in different cellular models. The hypothesis to be tested is that molecular chaperones are responsible for the intracellular retention of AE1 mutants. Chaperones were found to interact with AE1 and kAE1 in vitro and in vivo (HEK-293, K562, MDCK cells). Disruption of the calnexin-AE1 interaction in K562 cells did not affect the cell surface levels of wild type or mutant erythroid AE1. AE1 also trafficked to the cell surface in mouse embryonic fibroblasts completely deficient in calnexin or calreticulin. In contrast, in MDCK cells, disruption of the calnexin-kAE1 interaction allowed functional dominant (R589H, R901stop), but not misfolded kAE1 mutants (kSAO, G701D), to escape the ER and traffic to the cell surface. Calnexin is therefore not required for the cell surface expression of erythroid AE1, but can be responsible for the intracellular retention of certain kAE1 mutants in cells with the complete complement of molecular chaperones. Components involved in membrane glycoprotein folding and quality control (calnexin, ERp57, Hsc70, Hsp70), were lost at later stages during the differentiation of CD34+ erythroid progenitor cells. This suggests that the loss of molecular chaperones may facilitate the massive production of red cell glycoproteins, allowing erythroid AE1 mutants to escape quality control, traffic to the plasma membrane, and be present in mature red blood cells. These studies demonstrate that the role chaperones play varies, depending on cellular context. By understanding the cellular context and factors involved, therapeutic strategies may be tailored to deal with protein misfolding diseases, and in the case of kAE1, rescue the cell surface trafficking of misfolded, but functional, transport protein using pharmacological modulators.

Mutations in the SLC4A1 gene result in misfolding and trafficking defects of the human erythroid (AE1) and kidney (kAE1) forms of the anion exchanger 1 glycoprotein. This affects the amount of functional protein at the cell surface, resulting in hematological and renal diseases. In this thesis, the role of the quality control system of molecular chaperones (cytosolic and ER) was examined during the biosynthesis of wild type and mutant AE1 in different cellular models. The hypothesis to be tested is that molecular chaperones are responsible for the intracellular retention of AE1 mutants. Chaperones were found to interact with AE1 and kAE1 in vitro and in vivo (HEK-293, K562, MDCK cells). Disruption of the calnexin-AE1 interaction in K562 cells did not affect the cell surface levels of wild type or mutant erythroid AE1. AE1 also trafficked to the cell surface in mouse embryonic fibroblasts completely deficient in calnexin or calreticulin. In contrast, in MDCK cells, disruption of the calnexin-kAE1 interaction allowed functional dominant (R589H, R901stop), but not misfolded kAE1 mutants (kSAO, G701D), to escape the ER and traffic to the cell surface. Calnexin is therefore not required for the cell surface expression of erythroid AE1, but can be responsible for the intracellular retention of certain kAE1 mutants in cells with the complete complement of molecular chaperones. Components involved in membrane glycoprotein folding and quality control (calnexin, ERp57, Hsc70, Hsp70), were lost at later stages during the differentiation of CD34+ erythroid progenitor cells. This suggests that the loss of molecular chaperones may facilitate the massive production of red cell glycoproteins, allowing erythroid AE1 mutants to escape quality control, traffic to the plasma membrane, and be present in mature red blood cells. These studies demonstrate that the role chaperones play varies, depending on cellular context. By understanding the cellular context and factors involved, therapeutic strategies may be tailored to deal with protein misfolding diseases, and in the case of kAE1, rescue the cell surface trafficking of misfolded, but functional, transport protein using pharmacological modulators.

Anion exchanger 1 (AE1) is a 95 kDa glycoprotein that facilitates Cl-/HCO3- exchange across the erythrocyte plasma membrane. Seven conserved tryptophan (Trp) residues are in the AE1 membrane domain; at the membrane interface (Trp648, Trp662, and Trp723), in transmembrane segment (TM) 4 (Trp492 and Trp496), and in hydrophilic loops (Trp831, and Trp848). All 7 Trp residues were individually mutated into alanine (Ala) and phenylalanine (Phe) and transiently expressed in human embryonic kidney (HEK)-293 cells. The 7 Trp residues could be grouped into three classes according to the impact of the mutations on the functional expression of AE1: class 1, normal expression, class 2, expression decreased, and class 3, expression decreased by Ala substitution. These results indicate that Trp residues play differential roles in AE1 expression depending on their location in the protein and suggest that Trp mutants with a low expression are misfolded and retained in the ER.

Anion exchanger 1 (AE1) is a red cell membrane glycoprotein that associates with cytoskeletal protein 4.2 in a complex bridging the cell membrane to the cytoskeleton. Disruption of this linkage results in unstable erythrocytes and hereditary spherocytosis (HS). Three HS mutations (E40K, G130R and P327R) in the cytoplasmic domain of AE1 (cdAE1) result in a decreased level of protein 4.2 in the red cell yet maintain normal amounts of AE1. Biophysical analyses showed the HS mutations had little effect on the structure and conformational stability of the isolated domain. However, the conformation of the cytoplasmic domain of the kidney anion exchanger, lacking the first 65 amino acids including a central -strand, was thermally destabilized relative to cdAE1 and had a more open structure. In transfected human embryonic kidney (HEK)-293 cells the HS mutants had similar expression levels as wild-type AE1, and protein 4.2 expression level was not dependent on the presence of AE1. Protein 4.2 localized to the plasma membrane with wild-type AE1, the HS mutants of AE1, the membrane domain of AE1 and kidney AE1, and to the ER with Southeast Asian ovalocytosis AE1. A fatty acylation mutant of protein 4.2, G2A/C173A, could not localize to the plasma membrane in the absence of AE1. Subcellular fractionation showed wild-type and G2A/C173A protein 4.2 were mostly associated with the cytoskeleton. Co-immunoprecipitation and Ni-NTA pull-down assays revealed impaired binding of protein 4.2 to HS mutants compared to AE1, while the membrane domain of AE1 was unable to bind protein 4.2. These studies show that HS mutations in cdAE1 cause impaired binding of protein 4.2, without causing gross structural changes in the domain. The mutations change the binding surface on cdAE1 by the introduction of positive charges into an otherwise acidic domain. This binding impairment may render protein 4.2 more susceptible to degradation or loss during red cell development.

Anion exchanger 1 (AE1) is a 95 kDa glycoprotein that facilitates Cl-/HCO3- exchange across the erythrocyte plasma membrane. Seven conserved tryptophan (Trp) residues are in the AE1 membrane domain; at the membrane interface (Trp648, Trp662, and Trp723), in transmembrane segment (TM) 4 (Trp492 and Trp496), and in hydrophilic loops (Trp831, and Trp848). All 7 Trp residues were individually mutated into alanine (Ala) and phenylalanine (Phe) and transiently expressed in human embryonic kidney (HEK)-293 cells. The 7 Trp residues could be grouped into three classes according to the impact of the mutations on the functional expression of AE1: class 1, normal expression, class 2, expression decreased, and class 3, expression decreased by Ala substitution. These results indicate that Trp residues play differential roles in AE1 expression depending on their location in the protein and suggest that Trp mutants with a low expression are misfolded and retained in the ER.