Crystallization of Human Erythrocyte Band 3, the anion exchanger, at the International Space Station "KIBO".

Band 3 mediates the Cl- and HCO3- exchange across the red blood cell membrane and plays a pivotal role for delivering oxygen appropriately to metabolically active tissues. For understanding molecular mechanisms, it is essential to know the structure and function relationship. In terrestrial environments, however, nobody could make good quality crystals of Band 3 for the X-ray crystallographic study. In this study, we purified the transmembrane domain of Band 3 from human red blood cells and crystallized the purified Band 3 without the Fab fragment at the International Space Station "KIBO" under microgravity environments.

[Structure of human erythrocyte band 3: two-dimensional crystallographic analysis of the membrane domain].

Band 3 (also known as anion exchanger 1, AE1) is one of the most abundant membrane proteins in human erythrocytes. Band 3 has 911 amino acids and consists of two structurally and functionally distinct domains. One is a 40-kDa N-terminal cytoplasmic domain and the other is a 55-kDa C-terminal membrane domain. The cytoplasmic domain maintains red cell shape through interactions with cytoskeletal proteins, such as protein 4.1, protein 4.2, ankyrin, and spectrin. On the other hand, the membrane domain mediates electroneutral exchange of anions, such as bicarbonate and chloride across the erythrocyte membrane. We reported the three-dimensional structure of the outward-open membrane domain of band 3, which was cross-linked between K539 and K851 with H2DIDS, at 7.5 Å resolution using cryo-electron crystallography. Although the results showed significantly improved resolution as compared with previous structural analyses, we could not assign all α-helices because of low resolution and uncertainty persists regarding the fold of band 3. However, we recognized that band 3 has internal repeats, because the structure exhibited distinctive anti-parallel V-shaped motifs, which protrude from the membrane bilayer on both sides. One of the helices in the motif is very long and highly tilted with respect to the normal structure of the bilayer.

Characterization of band 3-ankyrin-Protein 4.2 complex by biochemical and mass spectrometry approaches.

The elastic property of red blood cell is supported by interaction between red cell membrane and the intricate cytoskeleton network underlying the membrane bilayer cytoplasmic face. One of the major scaffold protein linkers is band 3-ankyrin complex. Defects occurring in this complex have been found in many inherited diseases, causing red blood cell abnormalities. Here we combined the power of mass spectrometry with conventional biochemical purification methods in order to study the native interactions among band 3, ankyrin and Protein 4.2. This approach provided in vivo evidence for the association between band 3 and N-terminal ankyrin purified directly from the cell membrane. The C-terminal regions of ankyrin were not found to be a stable partner of the band 3 complex. Protein 4.2 was shown here to be an integral part of the complex. Its association to the band 3-ankyrin complex could withstand harsh purification conditions. Our findings lend additional support to the interaction between band 3 and ankyrin N-terminal domain previously shown by in vitro binding assays and provide evidence for a band 3 core complex comprising of band 3, ankyrin and Protein 4.2.

Purification of functional human Cl(-)/HCO(3)(-) 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.

Automation of RNA-based biomarker extraction from dried blood spots for the detection of blood doping.

Aim: Transcriptomic biomarkers originating from reticulocytes measured in dried blood spots (DBSs) may be reliable indicators of blood doping. Methods/results: Here, we examined changes in the expression levels of the erythropoiesis-related ALAS2, CA1 and SLC4A1 genes in DBS samples from elite athletes and volunteers of clinical study with recombinant erythropoietin dose. Conclusion: By comparing the mean intraday coefficients of variation for ALAS2L, ALASLC, CA1 and SLC4A1 between manual and automated RNA extractions, an average improvement was observed, whereas the assessment of interday variability provided comparable results for both manual and automated approaches. Our results confirmed that RNA biomarkers on DBS support are efficient to detect blood doping.

Defective anion transport and marked spherocytosis with membrane instability caused by hereditary total deficiency of red cell band 3 in cattle due to a nonsense mutation.

We studied bovine subjects that exhibited a moderate uncompensated anemia with hereditary spherocytosis inherited in an autosomal incompletely dominant mode and retarded growth. Based on the results of SDS-PAGE, immunoblotting, and electron microscopic analysis by the freeze fracture method, we show here that the proband red cells lacked the band 3 protein completely. Sequence analysis of the proband band 3 cDNA and genomic DNA showed a C --> T substitution resulting in a nonsense mutation (CGA --> TGA; Arg --> Stop) at the position corresponding to codon 646 in human red cell band 3 cDNA. The proband red cells were deficient in spectrin, ankyrin, actin, and protein 4.2, resulting in a distorted and disrupted membrane skeletal network with decreased density. Therefore, the proband red cell membranes were extremely unstable and showed the loss of surface area in several distinct ways such as invagination, vesiculation, and extrusion of microvesicles, leading to the formation of spherocytes. Total deficiency of band 3 also resulted in defective Cl-/HCO3- exchange, causing mild acidosis with decreases in the HCO3- concentration and total CO2 in the proband blood. Our results demonstrate that band 3 indeed contributes to red cell membrane stability, CO2 transport, and acid-base homeostasis, but is not always essential to the survival of this mammal.

Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE).

We describe a duplication of 10 nucleotides (2,455-2,464) in the band 3 gene in a kindred with autosomal dominant hereditary spherocytosis and a partial deficiency of the band 3 protein that is reflected by decreased rate of transmembrane sulfate flux and decreased density of intramembrane particles. The mutant allele potentially encodes an abnormal band 3 protein with a 3.5-kD COOH-terminal truncation; however, we did not detect the mutant protein in the membrane of mature red blood cells. Since the mRNA levels for the mutant and normal alleles are similar and since the band 3 content is the same in the light and dense red cell fractions, we conclude that the mutant band 3 is either not inserted into the plasma membrane or lost from the membrane prior to the release of red blood cells into circulation. We further show that the decrease in band 3 content principally involves the dimeric laterally and rotationally mobile fraction of the band 3 protein, while the laterally immobile and rotationally restricted band 3 fraction is left essentially intact. We propose that the decreased density of intramembrane particles decreases the stability of the membrane lipid bilayer and causes release of lipid microvesicles that leads to surface area deficiency and spherocytosis.

Identification of sheep red blood cell (SRBC) surface immune-responsive peptides detected by antisera from SRBC-immunized rats.

To identify the sheep red blood cell (SRBC) surface immune-responsive peptides, immuno-reactive fraction of SRBC was detected by SDS-PAGE and western blot analysis with antisera from SRBC-immunized rats. Then the most intense immuno-reactive band on SDS-PAGE was subjected to nanoLC-ESI-MS/MS analysis, and 17 proteins were identified including membrane proteins of erythrocytes such as band 3 anion transport protein isoform 1 (Anion exchange protein 1; AE-1, CD233), Ammonium transporter Rh type A (Rh type A glycoprotein, CD241) and Ankyrin-1 (ANK-1), Spectrin beta chain. Among them, plasma protein AE-1 (CD233) and Rh type A glycoprotein (CD241) have transmembrane domain and correspond to extracellular region in their sequences. These extracellular regions of the plasma membrane proteins are supposed to be major immune-responsive peptides of SRBC in rats. These peptides are promising for the construction of an ELISA system which does not require the processing of SRBC membrane ghosts.

The state of association of Band 3 of the human erythrocyte membrane: evidence of a hexamer.

Band 3 of the human erythrocyte membrane was isolated from 32P-labelled erythrocytes in non-ethyleneglycol n-dodecyl ether (C12E9), Triton X-100, and Brij 58 solutions, and its states of association were studied by sucrose gradient sedimentation and by Sepharose 4B column chromatography. The sedimentation and elution profiles indicated that Band 3 exists under two stable forms in each detergent solution, a slow form and a fast form. The fraction of the fast form in a Brij 58 solution was 2-3-times higher than those of the fast forms in C12E9 and Triton X-100 solutions. Moreover, depending of the conditions of isolation, only the slow form or the fast form was present in a Brij 58 solution. The apparent values of sedimentation coefficients, Stokes radii, and effective masses of the slow and fast forms in C12E9, Triton X-100, and Brij 58 solutions were determined. On the basis of these values, we have concluded that the slow and fast forms of Band 3 were dimer and tetramer in C12E9 and Triton X-100 solutions but were dimer and hexamer in a Brij 58 solution.

Determination of polypeptide chain molecular weights of human and bovine band 3 protein from erythrocyte membranes by low-angle laser light scattering combined with high-performance gel chromatography in the presence of sodium dodecyl sulfate.

Polypeptide chain molecular weights of human and bovine band 3 proteins which are glycoproteins of the erythrocyte membrane were determined as 101,000 +/- 2000 for the former and 107,000 +/- 2000 for the latter by using the low-angle laser light scattering technique combined with a high-performance gel chromatography column, an ultraviolet spectrophotometer and a differential refractometer in the presence of sodium dodecyl sulfate. The advantage of this method is that, unlike the sedimentation equilibrium technique, neither information on the binding to proteins of all ligands present nor the partial specific volume is required to evaluate the polypeptide chain molecular weight of proteins in a multicomponent system.

An interspecies approach to the investigation of the red cell membrane glucose transporter.

Glucose transport differs in red cells of various species. The following sequence of transport velocities was found: man greater than newborn pig greater than rat, dog greater than cattle greater than pig. No correlation was found between the amount of protein in band 3 and glucose transport activity. By contrast, a very clear peak in the band 4.5 region was found for newborn pigs, whereas adult pigs did not exhibit a corresponding peak in the electrophoresis. Thus further evidence is provided by our investigations in favour of band 4.5 region for glucose transport activity in red cells.

Binding of naturally occurring antibodies to oxidatively and nonoxidatively modified erythrocyte band 3.

Both oxidative clustering (elicited by diamide treatment) and nonoxidative clustering (elicited by zinc/BS3 (bis[sulfosuccinimidyl]suberate) treatment) of erythrocyte integral membrane proteins induce binding of autologous antibodies with anti-band 3 specificity, followed by complement deposition and phagocytosis. Autologous antibodies eluted from nonoxidatively clustered erythrocytes bind to and stimulate phagocytosis of oxidatively damaged erythrocytes. Those eluted antibodies bind specifically to disulfide-crosslinked band 3 dimers generated by diamide treatment. Band 3 dimerization and antibody binding are abrogated by cleavage of band 3 cytoplasmic domain. Thus, disulfide-crosslinked band 3 dimers are the minimal band 3 aggregate with enhanced affinity for anti-band 3 antibodies. The eluted antibodies do not bind to band 3 dimers generated nonoxidatively by BS3 treatment but bind avidly to larger band 3 clusters generated nonoxidatively by zinc/BS3 treatment. Possibly, disulfide crosslinking of cytoplasmic domain cysteines induces reorientation of intramembrane domains as to expose putative anti-band 3 epitopes and allow bivalent binding of anti-band 3 antibodies. Extensive nonoxidative band 3 clustering appears to disrupt the native band 3 conformation and generate reoriented dimers which expose putative anti-band 3 epitopes in the proper distance and orientation as to allow bivalent antibody binding.

Effect of detergent on protein structure. Action of detergents on secondary and oligomeric structures of band 3 from bovine erythrocyte membranes.

With special interest in the mode of action of zwitterionic detergents on proteins, a variety of detergents were examined for their ability to disrupt the secondary and quaternary structures of an anion transport protein, band 3, and its cytoplasmic 38 kDa fragment from bovine erythrocyte membranes and for their effect on the binding of an anion transport inhibitor to band 3. Nonionic detergents and Chaps also acted as a nondenaturant in these instances, as well accepted for other proteins. Though deoxycholate and cholate inhibited the binding of an anion transport inhibitor to band 3, these detergents did not show any effect on the native structure of band 3. Zwitterionic detergents (Zwittergent 3-10, Zwittergent 3-12 and N, N-dimethyl-N-dodecyl glycine) were suggested to denature the water-soluble 38 kDa fragment at concentrations above the critical micelle concentration, but to be weak in disrupting interacting forces between hydrophobic membrane-bound domains of band 3. The results indicated that these zwitterionic detergents are similar in the mode of denaturing action to dodecyltrimethylammonium bromide rather than sodium dodecyl sulfate.

Major proteolytic fragments of the murine band 3 protein as obtained after in situ proteolysis.

Proteolytic fragments of murine band 3 were produced by exposure to extracellular chymotrypsin and intracellular trypsin. The ensuing proteolytic fragments were isolated, their N-terminal sequences were determined and their locations in the known amino acid sequence of murine band 3 established. Equivalents of the human 60, 35 and 17 kDa fragments were obtained through the cleavage sites were situated at locations that are not strictly homologous to the corresponding cleavage sites in human band 3, although all of them were near such sites. Exposure of the intact murine red cell to chymotrypsin leads to the formation of two fragments of 67 kDa and 41 kDa, which are equivalent to the 60 kDa and the 35 kDa fragments of the human band 3. Internal trypsin cleaves the chymotryptic 67 kDa fragment while the 41 kDa fragment appears essentially unaffected. The 67 kDa fragment is first degraded to 64 kDa, then further to 22 kDa and finally to 19 kDa. The anion transport inhibitor H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) combines with murine band 3 protein as it does with human band 3. Anion transport is maximally inhibited when 5.10(5) H2DIDS molecules per cell are bound to band 3. As in the human red cell, after exposure to high pH (9.0-9.5) of the H2DIDS-labeled, chymotryptically cleaved band 3 intramolecular cross-linking takes place. This joins the 67 and 41 kDa chymotryptic pieces together to form a peptide of the original molecular mass of band 3 of 108 kDa. If cross-linking is performed after additional tryptic cleavage, the 19 and 22 kDa pieces join together with 41 kDa pieces to form overlapping bands that cover the molecular weight range from 60 to 63 kDa.

Human erythrocyte band 3. Solubilization and reconstitution into two-dimensional crystals.

Various polyoxyethylene alkylethers were used to extract integral proteins from human erythrocyte membranes. The solubilization power of these detergents and the oligomerization of solubilized band 3 were studied. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that short-chain detergents induced oligomers larger than the band 3 dimer. In contrast, after solubilization with long-chain detergents, the predominant band on SDS-containing gels was the monomeric band 3. Oligomerization in short-chain detergents occurred preferentially at room temperature whereas monomeric band 3 prevailed at 4 degrees C. Consistent with these results, negative stain electron microscopy of solubilized isolated band 3 showed larger complexes with short-chain detergents than with long-chain detergents. Cu2+/o-phenanthroline-induced crosslinking had no effect on size or shape of band 3 particles. Despite their rather heterogeneous dimensions, octylpolyoxyethylene-solubilized band 3 complexes assembled into two-dimensional trigonal lattices (a = b = 11 (+/- 0.5) nm) in the presence of dimyristoyl phosphatidylcholine. The unit cell exhibited a pronounced stain-filled region surrounded by three elongated morphological subunits. Each subunit most likely represents a band 3 dimer. Freeze-drying/metal-shadowing of reconstituted lattices revealed one large elevation per unit cell protruding from an otherwise smooth surface.

Local structural difference between human and bovine band 3 in the anion transport inhibitor-binding region.

We have examined molecular properties of inhibitor-complexed human and bovine band 3, an anion transport protein of erythrocyte membrane, in order to demonstrate the structural characteristics of the inhibitor binding region. Band 3 modified with DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonate), a potent anion transport inhibitor, generated a positive circular dichroic band at a wavelength of 345 nm, corresponding to a DIDS chromophore. The dichroic spectra of human band 3-DIDS complex and its bovine counterpart differed markedly in their ellipticity. Under the conditions that H2DIDS (the dihydro-derivative of DIDS) cross-linked two chymotryptic fragments of human band 3, the reagent failed to cross-link the equivalent bovine fragments. The inhibitory effect of PLP (pyridoxal 5'-phosphate), a substrate and affinity label, on phosphate influx into red blood cells was more pronounced for human band 3 than for bovine band 3. The residue Lys-562 of human band 3 was found to be modified with PLP, while the corresponding residue of bovine band 3 was devoid of reactivity with PLP.

The nature of the stable noncovalent dimers of band 3 protein from erythrocyte membranes in solutions of Triton X-100.

Stable noncovalent dimers of band 3 protein from human erythrocyte membranes, in which state the protein is thought to exist after solubilization by the nonionic detergent Triton X-100, do not occur when purified batches of the detergent are used. Instead, the protein is in a monomer/dimer/tetramer association equilibrium. The stable dimers do appear, however, when the detergent has been 'aged'. They thus seem to be artifacts.

Band 3-hemoglobin associations. The band 3 tetramer is the oxyhemoglobin binding site.

The associations between the band 3 protein of the human erythrocyte membrane and oxyhemoglobin, in solutions of a nonionic detergent, were studied by sedimentation equilibrium experiments in the analytical ultracentrifuge. The following results were obtained: (i) hemoglobin is bound virtually exclusively to the band 3 tetramer, but not to the monomer or dimer; (ii) the band 3 tetramer can bind up to four hemoglobin tetramers; (iii) unlike the unstable dimers of unmodified band 3, stable dimers crosslinked via S-S-bridges also represent hemoglobin binding sites.

Microcalorimetry and electrophoresis of the erythrocyte membrane of spontaneously hypertensive rats.

Abnormalities of both structure and function have been described in the erythrocyte membrane of spontaneously hypertensive rats (SHR). In order to elucidate the molecular basis of these abnormalities we have carried out differential scanning microcalorimetry of the erythrocyte membrane and gel electrophoresis of membrane polypeptides. The partial enthalpy of so-called 'C-transition' (at 63 degrees C) was found to be increased. This may be explained by increased content of band 3 protein in SHR erythrocyte membrane.

Isolation of a 5,300-dalton peptide containing a pyridoxal phosphate binding site from the 38,000-dalton domain of band 3 of human erythrocyte membranes.

A hydrophobic 5,300-dalton peptide was isolated from the 38,000-dalton domain of Band 3 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography. The peptide was affinity labeled with pyridoxal phosphate and sodium [3H]borohydride when erythrocytes were incubated in vitro. The peptide was not labeled with these agents when cells were incubated in the presence of a specific inhibitor of anion transport, suggesting that the peptide contains at least a part of the active center for the anion transport system in the cell membrane. The peptide was eluted from a reversed-phase high-performance liquid chromatography column with a high concentration of acetonitrile (more than 65%), although the elution pattern of the hydrophobic peptide was not as sharp as that of the soluble peptides. However, a satisfactory separation was achieved when this procedure was employed in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis.