Human Erythrocyte Anion Exchanger Research Articles

Human erythrocyte band 3 is a host receptor for Plasmodium falciparum glutamic acid–rich protein

AbstractMalaria remains a major global threat to human health and economic development. Microvascular lesions caused by Plasmodium falciparum–infected human erythrocytes/red blood cells are hallmarks of severe pathogenesis contributing to high mortality, particularly in children from sub-Saharan Africa. In this study, we used a phage display complementary DNA library screening strategy to identify P falciparum glutamic acid–rich protein (PfGARP) as a secreted ligand that recognizes an ectodomain of human erythrocyte anion-exchanger, band 3/AE1, as a host receptor. Domain mapping of PfGARP revealed distinct nonoverlapping repeats encoding the immune response epitopes and core erythrocyte-binding activity. Synthetic peptides derived from the erythrocyte-binding repeats of PfGARP induced erythrocyte aggregation reminiscent of the rosetting phenomenon. Using peptides derived from the immunogenic repeats, a quantitative immunoassay was developed to detect a selective immune response against PfGARP in human plasma samples obtained from patients in rural Mali, suggesting the feasibility of PfGARP as a potential biomarker of disease progression. Collectively, our results suggest that PfGARP may play a functional role in enhancing the adhesive properties of human erythrocytes by engaging band 3 as a host receptor. We propose that immunological and pharmacological inhibition of PfGARP may unveil new therapeutic options for mitigating lesions in cerebral and pregnancy-associated malaria.

Topology models of anion exchanger 1 that incorporate the anti-parallel V-shaped motifs found in the EM structureThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

We recently published the three-dimensional structure of the membrane domain of human erythrocyte anion exchanger 1 (AE1) at 7.5 Å resolution, solved by electron crystallography. The structure exhibited distinctive anti-parallel V-shaped motifs, which protrude from the membrane bilayer on both sides. Similar motifs exist in the previously reported structure of a bacterial chloride channel (ClC)-type protein. Here, we propose two topology models of AE1 that reflect the anti-parallel V-shaped structural motifs. One is assumed to have structural similarity with the ClC protein and the other is only assumed to have internal repeats, as is often the case with transporters. Both models are consistent with most topological results reported thus far for AE1, each having advantages and disadvantages.

Purification of functional human [formula omitted] exchanger, AE1, over-expressed in Saccharomyces cerevisiae

There is no high-resolution structure for the membrane domain of the human erythrocyte anion exchanger, AE1 (Band 3). In this report, we have developed an expression and purification strategy for AE1 to be used in crystallization trials. Saccharomyces cerevisiae strain BJ5457 was transformed with an expression vector encoding the AE1 membrane domain (AE1MD, amino acids 388–911), fused C-terminally to an epitope tag, corresponding to the nine C-terminal amino acids of rhodopsin. The fusion protein, AE1MD-Rho, was expressed at a concentration of 0.3 mg/l of culture. Confocal immunofluorescence microscopy and sucrose gradient ultracentrifugation revealed that AE1MD-Rho did not process to the plasma membrane of S. cerevisiae, but was retained in an intracellular membrane fraction. Treatment with the endoglycosidase, PNGase F, showed that AE1MD-Rho is not N-glycosylated. AE1MD-Rho solubilized from yeast membranes, with Fos-choline detergent, was purified to 93% homogeneity in a single-step, using a 1D4 antibody affinity resin, in amounts up to 2.5 mg from 18 l of culture. The ability of purified AE1MD-Rho to transport sulfate was examined in reconstituted vesicles. The rate of sulfate efflux mediated by vesicles reconstituted with AE1MD-Rho was indistinguishable from vesicles with purified erythrocyte-source AE1. Using this purification strategy, sufficient amounts of functional, homogeneous AE1MD-Rho can be purified to enable crystallization trials.

The Drosophila Anion Exchanger (DAE) lacks a detectable interaction with the spectrin cytoskeleton

BackgroundCurrent models suggest that the spectrin cytoskeleton stabilizes interacting ion transport proteins at the plasma membrane. The human erythrocyte anion exchanger (AE1) was the first membrane transport protein found to be associated with the spectrin cytoskeleton. Here we evaluated a conserved anion exchanger from Drosophila (DAE) as a marker for studies of the downstream effects of spectrin cytoskeleton mutations.ResultsSequence comparisons established that DAE belongs to the SLC4A1-3 subfamily of anion exchangers that includes human AE1. Striking sequence conservation was observed in the C-terminal membrane transport domain and parts of the N-terminal cytoplasmic domain, but not in the proposed ankyrin-binding site. Using an antibody raised against DAE and a recombinant transgene expressed in Drosophila S2 cells DAE was shown to be a 136 kd plasma membrane protein. A major site of expression was found in the stomach acid-secreting region of the larval midgut. DAE codistributed with an infolded subcompartment of the basal plasma membrane of interstitial cells. However, spectrin did not codistribute with DAE at this site or in anterior midgut cells that abundantly expressed both spectrin and DAE. Ubiquitous knockdown of DAE with dsRNA eliminated antibody staining and was lethal, indicating that DAE is an essential gene product in Drosophila.ConclusionsBased on the lack of colocalization and the lack of sequence conservation at the ankyrin-binding site, it appears that the well-characterized interaction between AE1 and the spectrin cytoskeleton in erythrocytes is not conserved in Drosophila. The results establish a pattern in which most of the known interactions between the spectrin cytoskeleton and the plasma membrane in mammals do not appear to be conserved in Drosophila.

Structure of the Membrane Domain of Human Erythrocyte Anion Exchanger 1 Revealed by Electron Crystallography

The membrane domain of human erythrocyte anion exchanger 1 (AE1) works as a Cl −/HCO 3 − antiporter. This exchange is a key step for CO 2/O 2 circulation in the blood. In spite of their importance, structural information about AE1 and the AE (anion exchanger) family are still very limited. We used electron microscopy to solve the three-dimensional structure of the AE1 membrane domain, fixed in an outward-open conformation by cross-linking, at 7.5-Å resolution. A dimer of AE1 membrane domains packed in two-dimensional array showed a projection map similar to that of the prokaryotic homolog of the ClC chloride channel, a Cl −/H + antiporter. In a three-dimensional map, there are V-shaped densities near the center of the dimer and slightly narrower V-shaped clusters at a greater distance from the center of the dimer. These appear to be inserted into the membrane from opposite sides. The structural motifs, two homologous pairs of helices in internal repeats of the ClC transporter (helices B + C and J + K), are well fitted to those AE1 densities after simple domain movement.

Effect of Cytosolic Hereditary Spherocytosis Mutations of Human Erythrocyte Anion Exchanger 1 on Its Interaction with Cytoskeletal Protein 4.2.

Anion exchanger 1 (AE1, Band 3) is the predominant membrane protein of erythrocytes. Human AE1 has two functionally independent domains: its 52 kDa C-terminal membrane domain catalyzes the exchange of chloride for bicarbonate across the membrane while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the membrane to the cytoskeleton, giving the red cell its stability and flexibility. Several proteins bind to cdb3 including cytoskeletal protein 4.2, ankyrin, glycolytic enzymes and deoxyhemoglobin. Three mutations in cdb3 (E40K, G130R and P327R) are associated with the hemolytic anemia hereditary spherocytosis (HS) and decreased levels of erythrocyte protein 4.2 while maintaining a normal amount of AE1 at the red cell membrane. Wild-type and mutant cdb3 proteins were expressed in E. coli and purified and it was shown through a variety of biophysical methods that these three HS mutations do not cause major structural changes in this domain. Each of these mutations introduces a positive charge at the surface of the protein that is predominantly negatively charged, which may have an effect on protein interactions while maintaining its native folded structure. Full-length wild-type AE1 or HS mutants were co-expressed with protein 4.2 in HEK-293 cells in order to study their interaction in a mammalian cell line. All three HS mutant proteins were expressed at similar levels to wild-type in these cells and were shown to be present at the membrane using immunofluorescence and confocal microscopy. These proteins were also co-expressed in LLCPK-1 cells for the purpose of co-localization studies using immunofluorescence. A series of GST fusion proteins of protein 4.2 domains were designed based on a homology model of protein 4.2 and regions of the protein known to interact with AE1. These fusion proteins were expressed in E. coli and purified on glutathione-Sepharose resin and were used to study their interaction with purified wild-type and HS mutant cdb3 proteins in vitro. Blot overlay analysis showed that a protein 4.2 GST fusion protein containing a putative β-hairpin region (Asp145 to Glu203) binds specifically to wild-type cdb3 while GST does not. It is hypothesized that since these three HS mutations do not cause major structural changes in cdb3, the decreased level of protein 4.2 in the red cells of these patients is a result of impaired binding that occurs due to these mutation sites.

Substrates Induce Conformational Changes in Human Anion Exchanger 1 (hAE1) as Observed by Fluorescence Resonance Energy Transfer

The one-for-one exchange of Cl(-) and HCO(3)(-) ions is catalyzed by human erythrocyte anion exchanger 1 (hAE1) through a ping-pong mechanism whereby the protein exists in two main conformations, with the single anion-binding site exposed at either the cytoplasmic (inner) side (E(i)) or the extracellular side (E(o)), with interconversion between the two states being possible only after anion binding. Steady-state and time-resolved resonance energy transfer (FRET) techniques were used to determine the distance of the binding site for diTBA (bis-(1,3-diethylthiobarbituric acid)trimethine oxonol), a high affinity fluorescent oxonol inhibitor of hAE1, from a benchmark site (probably Lys-430) labeled by external fluorescein maleimide (FM). Using red cell ghost membranes, energy transfer distances were measured in media containing different anions between FM as the donor, covalently attached to one monomer, and diTBA as the acceptor, reversibly bound to the adjacent monomer of a hAE1 dimer. Energy transfer increased significantly in chloride or bicarbonate buffers relative to conditions where no transportable anions were present, that is, in citrate buffer. These differences in transfer efficiencies were interpreted in light of the conformational distributions of hAE1 in various buffers and the possible effects of diTBA itself on the distribution. The analysis indicates that the diTBA binding site comes closer to the FM site by approximately 7 A in chloride buffer as compared to that in citrate (or equivalent changes in diTBA orientation occur) because of the effects of anion binding. This provides the first direct physical evidence for structural changes in hAE1 induced by substrates.

Conformational Changes in the Cytoplasmic Domain of Human Anion Exchanger 1 Revealed by Luminescence Resonance Energy Transfer

The cytoplasmic domain of the human erythrocyte anion exchanger 1 (cdAE1) serves as a center of organization for the red blood cell cytoskeleton as well as several metabolic enzymes and hemoglobin. The protein is known to undergo a reversible pH-dependent conformational change characterized by a 2-fold change in the intrinsic fluorescence and an 11 A change in the Stokes radius. While the exact changes in the molecular structure are unknown, on the basis of the crystal structure of the protein at pH 4.8 and site-directed mutagenesis studies, Zhou and Low (19) have proposed that the peripheral protein binding (PPB) domain of cdAE1 moves away from the dimerization domain in response to increasing alkalinity. To test this hypothesis, we have applied luminescence resonance energy transfer (LRET) to measure the intermonomer distance between donor and acceptor probes at the Cys201 site (located in the PPB domain) within the cdAE1 dimer. This distance was found to increase as the pH is increased from 5 to 10, in recombinant forms of both the wild type and a mutant (C317S) of cdAE1. Furthermore, LRET measurements in red blood cell inside-out vesicles indicate that when cdAE1 is linked to the membrane, the intermonomer distance is larger at pH 5, compared to that of the purified cdAE1 segments, and exhibits a different pH-dependent behavior. An increase in the distance was also observed on binding of a metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, to cdAE1. These data provide the first demonstration of a defined change in the molecular structure of cdAE1, and also indicate that the structure under physiological conditions is different from the crystal structure determined at low pH.

Membrane integration and topology of the first transmembrane segment in normal and Southeast Asian ovalocytosis human erythrocyte anion exchanger 1

Anion exchanger 1 (AE1, or Band 3) is an integral membrane glycoprotein found in erythrocytes, responsible for the electroneutral exchange of chloride and bicarbonate ions across the plasma membrane. Southeast Asian ovalocytosis (SAO) results from a nine-amino acid deletion in the first transmembrane segment (TM) of the AE1 protein that abolishes its transport function. The effects of the SAO deletion on: (1) the efficiency of integration of TM1 into the membrane, and (2) the precise positioning of TM1 relative to the membrane were investigated using scanning N-glycosylation mutagenesis in a cell-free transcription/translation system and in transfected HEK293 cells. AE1 or SAO constructs containing either the endogenous N-glycosylation site at Asn642 in extracellular loop 4 (EC4) or single N-glycosylation sites engineered into an expanded extracellular loop 1 (EC1) were used. N-glycosylation efficiency of EC1 in the SAO construct was significantly lower than that of the AE1 construct, indicating that the SAO deletion impairs membrane integration of TM1 and the translocation of EC1 across the membrane. Scanning N-glycosylation mapping of EC1 in the cell-free system and in transfected cells showed that the C-terminus of both AE1 and SAO TM1 were at the same position relative to the membrane. Thus, the SAO deletion is likely to cause a pulling-in of the polar amino acid sequence immediately N-terminal to the deletion into the lipid bilayer, allowing SAO TM1 that was inserted to assume a transmembrane disposition.

The N-terminal Region of the Transmembrane Domain of Human Erythrocyte Band 3: RESIDUES CRITICAL FOR MEMBRANE INSERTION AND TRANSPORT ACTIVITY

We studied the role of the N-terminal region of the transmembrane domain of the human erythrocyte anion exchanger (band 3; residues 361-408) in the insertion, folding, and assembly of the first transmembrane span (TM1) to give rise to a transport-active molecule. We focused on the sequence around the 9-amino acid region deleted in Southeast Asian ovalocytosis (Ala-400 to Ala-408), which gives rise to nonfunctional band 3, and also on the portion of the protein N-terminal to the transmembrane domain (amino acids 361-396). We examined the effects of mutations in these regions on endoplasmic reticulum insertion (using cell-free translation), chloride transport, and cell-surface movement in Xenopus oocytes. We found that the hydrophobic length of TM1 was critical for membrane insertion and that formation of a transport-active structure also depended on the presence of specific amino acid sequences in TM1. Deletions of 2 or 3 amino acids including Pro-403 retained transport activity provided that a polar residue was located 2 or 3 amino acids on the C-terminal side of Asp-399. Finally, deletion of the cytoplasmic surface sequence G(381)LVRD abolished chloride transport, but not surface expression, indicating that this sequence makes an essential structural contribution to the anion transport site of band 3.

Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion-exchanger membrane domain

Three-dimensional crystals were obtained for the membrane domain of the human erythrocyte anion exchanger (AE1, Band 3). Protein homogeneity and stability and the delicate balance between the detergent used and the amount of phospholipids copurifying are critical to the formation of three-dimensional crystals of the AE1 membrane domain. While deglycosylation improved the protein homogeneity, its stability was significantly increased by inhibitor binding. Size-exclusion chromatography showed that the protein was monodisperse in detergents with acyl chains of 10–12 carbons over a pH range of 5.5–10.0. This pH range and the detergents that retained the protein's monodispersity were used for crystallization screening. Crystals were obtained with the protein purified in C 12E 8, dodecylmaltoside, decylthiomaltoside, and cyclohexyl-hexylmaltoside. Five to 13 lipid molecules per protein were required for the protein crystal formation. Those crystals grown in dodecylmaltoside diffracted X-rays to 14 Å. With these factors taken into consideration, ways to further improve the crystal quality are suggested.

Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface

The human erythrocyte anion-exchanger isoform 1 (AE1) is a dimeric membrane protein that exchanges chloride for bicarbonate across the erythrocyte plasma membrane. Crystallographic studies suggest that the transmembrane anion channel lies at the interface between the two monomers, whereas kinetic analysis provides evidence that each monomer contains an anion channel. We have studied the structure–function relationship of residues at the dimeric interface of AE1 by cysteine-directed cross-linking. Single cysteine mutations were introduced in 16 positions of putative loop regions throughout AE1. The ability of these residues to be chemically cross-linked to their partner within the dimeric protein complex was assessed by mobility of the protein on immunoblots. Introduced cysteine residues in extracellular loops (ECs) 1–4 and intracellular loop 1 formed disulphide cross-linked dimers. Treatment with homobifunctional maleimide cross-linkers of different lengths (6, 10 and 16 Å; 1Å≡0.1nm) also cross-linked AE1 with introduced cysteines in EC5 and close to the start of transmembrane segment (TM) 1. On the basis of these data, tentative positional constraints of TMs 1–4 and 6 relative to the dimeric interface are proposed. Neither disulphide- nor maleimide-mediated cross-linking perturbed AE1 transport function, suggesting that loop–loop contacts across the dimeric interface are not primarily responsible for allosteric interactions between monomers within the functional dimeric protein complex.

Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface.

The human erythrocyte anion-exchanger isoform 1 (AE1) is a dimeric membrane protein that exchanges chloride for bicarbonate across the erythrocyte plasma membrane. Crystallographic studies suggest that the transmembrane anion channel lies at the interface between the two monomers, whereas kinetic analysis provides evidence that each monomer contains an anion channel. We have studied the structure-function relationship of residues at the dimeric interface of AE1 by cysteine-directed cross-linking. Single cysteine mutations were introduced in 16 positions of putative loop regions throughout AE1. The ability of these residues to be chemically cross-linked to their partner within the dimeric protein complex was assessed by mobility of the protein on immunoblots. Introduced cysteine residues in extracellular loops (ECs) 1-4 and intracellular loop 1 formed disulphide cross-linked dimers. Treatment with homobifunctional maleimide cross-linkers of different lengths (6, 10 and 16 A; 1 A identical with 0.1 nm) also cross-linked AE1 with introduced cysteines in EC5 and close to the start of transmembrane segment (TM) 1. On the basis of these data, tentative positional constraints of TMs 1-4 and 6 relative to the dimeric interface are proposed. Neither disulphide- nor maleimide-mediated cross-linking perturbed AE1 transport function, suggesting that loop-loop contacts across the dimeric interface are not primarily responsible for allosteric interactions between monomers within the functional dimeric protein complex.

Effect of pH on the self-association of erythrocyte band 3 in situ

The human erythrocyte anion exchanger (band 3) contains a cytoplasmic domain (cdb3) that exists in a reversible, pH-dependent structural equilibrium among three native conformations. To understand how this conformational equilibrium might influence the association state of band 3, we have incubated stripped erythrocyte membranes in solutions ranging from pH 6.0 to pH 10.5 and have examined the oligomeric state of the protein by size exclusion high performance liquid chromatography. We demonstrate that incubation of membranes in slightly acidic conditions favors dimer formation, whereas extended incubation at higher pHs (pH>9) leads to irreversible formation of an oligomeric species larger than the tetramer. Since the pH dependence of the conformational state of the cytoplasmic domain exhibits a similar pH profile, we suggest that the conformation of the cytoplasmic domain can modulate the self-association of band 3. Importantly, this modulation would appear to require the structural interactions present within the intact protein, since the isolated membrane-spanning domain does not display any pH dependence of association. The irreversible nature of the alkali-induced aggregation further suggests that a secondary reaction subsequent to band 3 association is required to stabilize the high molecular weight aggregate. Although we were able to eliminate covalent bond formation in this irreversible aggregation process, the exact nature of the secondary reaction remains to be elucidated.

Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1, membrane glycoprotein

The human erythrocyte anion exchanger (AE)1 (Band 3) contains a single complex N-linked oligosaccharide that is attached to Asn642 in the fourth extracellular loop of this polytopic membrane protein, while other isoforms (AE2, AE3 and trout AE1) are N-glycosylated on the preceding extracellular loop. Human AE1 expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells contained a high-mannose oligosaccharide. The lack of oligosaccharide processing was not due to retention of AE1 in the endoplasmic reticulum since biotinylation assays showed that approx. 30% of the protein was expressed at the cell surface. Moving the N-glycosylation site to the preceding extracellular loop in an AE1 glycosylation mutant (N555) resulted in processing of the oligosaccharide and production of a complex form of AE1. A double N-glycosylation mutant (N555/N642) contained both a high-mannose and a complex oligosaccharide chain. The complex form of the N555 mutant could be biotinylated showing that this form of the glycoprotein was at the cell surface. Pulse-chase experiments showed that the N555 mutant was efficiently converted from a high-mannose to a complex oligosaccharide with a half-time of approx. 4 h, which reflected the time course of trafficking of AE1 from the endoplasmic reticulum to the plasma membrane. The turnover of the complex form of the N555 mutant occurred with a half-life of approx. 15 h. The results show that the oligosaccharide attached to the endogenous site in extracellular loop 4 in human AE1 is not processed in HEK-293 or COS-7 cells, while the oligosaccharide attached to the preceding loop is converted into the complex form.

Processing of N-linked oligosaccharide depends on its location in the anion exchanger, AE1, membrane glycoprotein.

The human erythrocyte anion exchanger (AE)1 (Band 3) contains a single complex N-linked oligosaccharide that is attached to Asn(642) in the fourth extracellular loop of this polytopic membrane protein, while other isoforms (AE2, AE3 and trout AE1) are N-glycosylated on the preceding extracellular loop. Human AE1 expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells contained a high-mannose oligosaccharide. The lack of oligosaccharide processing was not due to retention of AE1 in the endoplasmic reticulum since biotinylation assays showed that approx. 30% of the protein was expressed at the cell surface. Moving the N-glycosylation site to the preceding extracellular loop in an AE1 glycosylation mutant (N555) resulted in processing of the oligosaccharide and production of a complex form of AE1. A double N-glycosylation mutant (N555/N642) contained both a high-mannose and a complex oligosaccharide chain. The complex form of the N555 mutant could be biotinylated showing that this form of the glycoprotein was at the cell surface. Pulse-chase experiments showed that the N555 mutant was efficiently converted from a high-mannose to a complex oligosaccharide with a half-time of approx. 4 h, which reflected the time course of trafficking of AE1 from the endoplasmic reticulum to the plasma membrane. The turnover of the complex form of the N555 mutant occurred with a half-life of approx. 15 h. The results show that the oligosaccharide attached to the endogenous site in extracellular loop 4 in human AE1 is not processed in HEK-293 or COS-7 cells, while the oligosaccharide attached to the preceding loop is converted into the complex form.

Binding properties of the stilbene disulfonate sites on human erythrocyte AE1: kinetic, thermodynamic, and solid state deuterium NMR analyses.

A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.

Calnexin Interaction with N-Glycosylation Mutants of a Polytopic Membrane Glycoprotein, the Human Erythrocyte Anion Exchanger 1 (Band 3)

The interaction of the endoplasmic reticulum chaperone calnexin with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger (AE1), was characterized by cell-free translation and in transfected HEK293 cells, followed by co-immunoprecipitation using anti-calnexin antibody. AE1 contains 12-14 transmembrane segments and has a single site of N-glycosylation at Asn-642 in the fourth extracytosolic loop. This site was mutated (N642D) to create a nonglycosylated protein. Calnexin showed a preferential interaction with N-glycosylated AE1 relative to nonglycosylated AE1 both in vitro and in vivo. This interaction could be blocked by inhibition of glucosidases I and II with castanospermine. Calnexin had access to novel N-glycosylated sites created in other extracytosolic loops in AE1 by site-directed or insertional mutagenesis. The interaction with AE1 was enhanced when multiple sites were introduced into the same loop or into two different loops. An association of calnexin with truncated versions of N-glycosylated AE1 was detected after release of the nascent chains from ribosomes with puromycin. The results show that the interaction of calnexin with the polytopic membrane glycoprotein AE1 was dependent on the presence but not the location of the oligosaccharide. Furthermore, calnexin was associated with AE1 after release of AE1 from the translocation machinery.

Transmembrane folding of the human erythrocyte anion exchanger (AE1, Band 3) determined by scanning and insertional N-glycosylation mutagenesis

Transmembrane folding of the human erythrocyte anion exchanger (AE1, Band 3) determined by scanning and insertional N-glycosylation mutagenesis

Transmembrane folding of the human erythrocyte anion exchanger (AE1, Band 3) determined by scanning and insertional N-glycosylation mutagenesis

The human erythrocyte anion exchanger (AE1, Band 3) contains up to 14 transmembrane segments, with a single site of N-glycosylation at Asn642 in extracellular (EC) loop 4. Scanning and insertional N-glycosylation mutagenesis were used to determine the folding pattern of AE1 in the membrane. Full-length AE1, when expressed in transfected human embryonic kidney (HEK)-293 or COS-7 cells, retained a high-mannose oligosaccharide structure. Scanning N-glycosylation mutagenesis of EC loop 4 showed that N-glycosylation acceptor sites (Asn-Xaa-Ser/Thr) spaced 12 residues from the ends of adjacent transmembrane segments could be N-glycosylated. An acceptor site introduced at position 743 in intracellular (IC) loop 5 that could be N-glycosylated in a cell-free translation system was not N-glycosylated in transfected cells. Mutations designed to disrupt the folding of this loop enhanced the level of N-glycosylation at Asn743 in vitro. The results suggest that this loop might be transiently exposed to the lumen of the endoplasmic reticulum during biosynthesis but normally folds rapidly, precluding N-glycosylation. EC loop 4 insertions into positions 428, 484, 754 and 854 in EC loops 1, 2, 6 and 7 respectively were efficiently N-glycosylated, showing that these regions were extracellular. EC loop 4 insertions into positions 731 or 785 were poorly N-glycosylated, which was inconsistent with an extracellular disposition for these regions of AE1. Insertion of EC loop 4 into positions 599 and 820 in IC loops 3 and 6 respectively were not N-glycosylated in cells, which was consistent with a cytosolic disposition for these loops. Inhibitor-affinity chromatography with 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate (SITS)-Affi-Gel was used to assess whether the AE1 mutants were in a native state. Mutants with insertions at positions 428, 484, 599, 731 and 785 showed impaired inhibitor binding, whereas insertions at positions 754, 820 and 854 retained binding. The results indicate that the folding of the C-terminal region of AE1 is more complex than originally proposed and that this region of the transporter might have a dynamic aspect.