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.

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.

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.