Carboxyl methylation of human erythrocyte band 3 in intact cells. Relation to anion transport activity.

The anion transport protein of the human erythrocyte membrane, band 3, is reversibly methylated by an endogenous protein carboxyl methyltransferase. The physiological consequence of this modification was studied by measuring the rate of phosphate transport by intact erythrocytes incubated under conditions where protein methylation reactions are inhibited. No change in phosphate transport was detected when cells were treated with either methionine-free media or cycloleucine, whereas cells incubated with adenosine and homocysteine thiolactone displayed a marginally slower rate of transport, which was not reversed by subsequent remethylation of the membrane proteins. These results suggest that erythrocyte protein carboxyl methylation does not directly regulate this activity of band 3.     

Labeling of human erythrocyte band 3 with maltosylisothiocyanate. Interaction with the anion transporter

Maltosylisothiocyanate (MITC), synthesized as an affinity label for the hexose carrier, has been reported to label a Band 3 or Mr = 100,000 protein in human erythrocytes, in contradistinction to many studies showing the carrier as a Band 4.5 or Mr = 45,000-66,000 protein on gel electrophoresis. In this work the possibility that MITC interacts with the Band 3 anion transporter was studied. In intact human erythrocytes, MITC labeling was largely confined to Band 3 and was decreased by several competitive inhibitors of hexose transport. However, MITC also appeared to react with the anion transport protein, since MITC labeling of Band 3 was irreversibly decreased by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and since MITC also irreversibly inhibited both tritiated dihydro-DIDS labeling of Band 3 and sulfate uptake in intact cells. Although 20 microM DIDS had little effect on hexose transport, the labeling of erythrocyte Band 3 by the dihydro analog was significantly diminished by competitive inhibitors of hexose transport. These data suggest that MITC labels in part the anion transporter as well as other DIDS-reactive sites on Band 3 which appear to be sensitive to competitive inhibitors of hexose transport.     

Flexibility of the cytoplasmic domain of the anion exchange protein, band 3, in human erythrocytes.

The rotational flexibility of the cytoplasmic domain of band 3, in the region that is proximal to the inner membrane surface, has been investigated using a combination of time-resolved optical anisotropy (TOA) and saturation-transfer electron paramagnetic resonance (ST-EPR) spectroscopies. TOA studies of rotational diffusion of the transmembrane domain of band 3 show a dramatic decrease in residual anisotropy following cleavage of the link with the cytoplasmic domain by trypsin (E. A. Nigg and R. J. Cherry, 1980, Proc. Natl. Acad. Sci. U.S.A. 77:4702-4706). This result is compatible with two independent hypotheses: 1) trypsin cleavage leads to dissociation of large clusters of band 3 that are immobile on the millisecond time scale, or 2) trypsin cleavage leads to release of a constraint to uniaxial rotational diffusion of the transmembrane domain. ST-EPR studies at X- and Q-band microwave frequencies detect rotational diffusion of the transmembrane domain of band 3 about the membrane normal axis of reasonably large amplitude that does not change upon cleavage with trypsin. These ST-EPR results are not consistent with dissociation of clusters of band 3 as a result of cleavage with trypsin. Global analyses of the ST-EPR data using a newly developed algorithm indicate that any constraint to rotational diffusion of the transmembrane domain of band 3 via interactions of the cytoplasmic domain with the membrane skeleton must be sufficiently weak to allow rotational excursions in excess of 32 degrees full-width for a square-well potential. In support of this result, analyses of the TOA data in terms of restricted amplitude uniaxial rotational diffusion models suggest that the membrane-spanning domain of that population of band 3 that is linked to the membrane skeleton is constrained to diffuse in a square-well of approximately 73 degrees full-width. This degree of flexibility may be necessary for providing the unique mechanical properties of the erythrocyte membrane.     

The functional role of arginine 901 at the C-terminus of the human anion transporter band 3 protein

To determine which arginine residues are responsible for band 3-mediated anion transport, we analyzed hydroxyphenylglyoxal (HPG)-modified band 3 protein in native erythrocyte membranes. HPG-modification leads to inhibition of the transport of phosphoenolpyruvate, a substrate for band 3-mediated transport. We analyzed the HPG-modified membranes by reverse phase-HPLC, and determined that arginine 901 was modified by HPG. To determine the role of Arg 901 in the conformational change induced by anion exchange, we analyzed HPG-modification of the membranes when 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethypyrocarbonate (DEPC) was present. DNDS and DEPC fix band 3 in the outward and inward conformations, respectively. HPG-modification was unaffected in the presence of DEPC but decreased in the presence of DNDS. In addition to that, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which specifically reacts with the outward conformation of band 3, did not react with HPG-modified membranes. Furthermore, we expressed a band 3 mutant in which Arg 901 was replaced by alanine (R901A) on yeast membranes. The kinetic parameters indicated that the R901A mutation affected the rate of conformational change of the band 3 protein. From these results, we conclude that the most C-terminal arginine, Arg 901, has a functional role in the conformational change that is necessary for anion transport.     

Internal motions of band 3 of human erythrocytes

Band 3 was labeled with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate either exofacially in the intact washed erythrocytes or endofacially by treating inside-out vesicles. Exo labeling resulted in the labeling of several other proteins, besides band 3, which could not be removed from the membrane. Therefore, the exo-labeled band 3 was extracted and purified by chromatography on DEAE-cellulose in Triton X-100. The endo labeling also resulted in the labeling of several other proteins. In this case, washing with NaOH removed all labeled material except band 3 from the vesicles. The lifetime of bound N-[(acetylamino)ethyl]-5-naphthylamine-1-sulfonate was heterogeneous, suggesting the positioning of the label in different environments either because different sites were labeled or because of positional freedom of the label at the same point of attachment. The main fraction of emission intensity had a lifetime near 20 ns, as expected for a hydrophobic environment. The rest showed a lifetime of about 3 ns in the exo-labeled band 3 and 9 ns in the endo-labeled band 3. Both lifetimes appeared to be independent of temperature between 5 and 25 degrees C, suggesting shielding of the probe from the solvent. Quenching phenomena must be responsible for both the 3- and 9-ns lifetimes, not due to residual heme, as proven by the persistence of such quenching in the Triton X-100 extracted protein. The correlation times indicated the presence of a short component, between 2 and 4 ns in the different systems, probably due to the presence of a flexible portion in the structure of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrodynamic examination of the dimeric cytoplasmic domain of the human erythrocyte anion transporter, band 3.

Solution studies of the cytoplasmic domain (molecular mass approximately 40kDa) of band 3, the anion exchanger from human erythrocyte membranes, previously suggested a dimeric molecule on the basis of the relative techniques of calibrated gel filtration and calibrated preparative ultracentrifugation. This dimeric behavior is firmly established on an absolute basis by a combination of calibrated gel chromatography and absolute ultracentrifugation techniques. Sedimentation velocity in the analytical ultracentrifuge combined with calibrated gel chromatography give a molecular mass M of (77 +/- 4) kDa, a value confirmed by low-speed sedimentation equilibrium. Velocity sedimentation in the analytical ultracentrifuge gave a single sedimenting species with an s o 20,w of (3.74 +/- 0.07)S. Sedimentation equilibrium analysis was also used to establish the strength of the binding via the dissociation constant Kd, with a value from direct fitting of the concentration distribution curves of (2.8 +/- 0.5) microM, confirmed by a value of approximately 3 microM obtained from fitting a plot of molecular weight Mw,app versus cell loading concentration. Hydrodynamic calculations based on the classical translational frictional ratio showed that the protein was highly asymmetric, with an axial ratio of approximately 10:1, consistent with observations from electron microscopy.     

Efflux of dipicolinic acid from human erythrocytes, sealed membrane fragments, and band 3-liposome complexes: a fluorescence probe for the erythrocyte anion transporter

The greatly enhanced fluorescence of Tb3+ when complexed with dipicolinic acid affords a simple and highly sensitive method for monitoring continuous anion flux through the erythrocyte anion transporter, band 3. Dipicolinic acid (pyridine-2,6-dicarboxylic acid) is entrapped in human erythrocytes and other band 3-membrane preparations. Efflux of dipicolinic acid from preequilibrated systems into Tb3+-containing medium is monitored fluorometrically. Dipicolinic acid efflux is demonstrated in intact erythrocytes, sealed red cell membrane fragments, and band 3-liposome complexes. In each system, dipicolinic acid efflux is blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid. The technique is highly sensitive and reproducible, and is applicable to hemoglobin-containing and hemoglobin-free systems of widely varying surface area to volume ratios.     

Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites.

Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes compared to control cells. In contrast, the lateral mobility of neither glycophorins nor a fluorescent phospholipid analog was altered in band 4.2-deficient cells. Compared to controls, band 4.2-deficient erythrocytes manifested a decreased ratio of band 3 to spectrin, and band 4.2-deficient membrane skeletons had decreased extractability of band 3 under low-salt conditions. Normal band 4.2 was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. We conclude that band 4.2 provides low-affinity binding sites for both band 3 oligomers and spectrin dimers on the human erythrocyte membrane. Band 4.2 may serve as an accessory linking protein between the membrane skeleton and the overlying lipid bilayer.     

Effect of glycyrrhetinic acid on membrane band 3 in human erythrocytes

Glycyrrhetinic acid (GA) is a hydrolytic product of the triterpene glycoside of glycyrrhizic acid, one of the main constituents of licorice root, which has long been studied, due to its several biological and endocrine properties. In this paper, GA was tested on human erythrocytes, and GA-induced alterations were compared with those caused by diamide, a mild oxidant inducing well-characterized cell/membrane alterations, and n-ethylmaleimide (NEM), as alkylating agent. In order to verify the biochemical steps underlying the action of GA, band 3 Tyr-phosphorylation level, enzyme recruitment and band 3 clustering in cells pre-incubated with GA before diamide treatment were all examined. Results show that GA, in a dose-dependent manner, prevents both diamide and NEM-induced band 3 Tyr-phosphorylation, but not GSH decrease caused by both compounds. In addition, diamide-induced band 3 clustering and IgG binding to altered cells were also completely reversed by GA pre-treatment. Also, when membrane sensitivity toward proteolytic digestion was tested, GA-treated cells showed high resistance to proteolysis. In conclusion, in human erythrocytes, GA is proposed to strengthen membrane integrity against both oxidative and proteolytic damage.     

Metabolism of S-adenosyl-L-methionine in intact human erythrocytes

Freshly isolated human erythrocytes contain S-adenosyl-L-methionine (AdoMet) at a concentration of about 3.5 mumol/l cells. When such cells are incubated in a medium containing 30 microM L-methionine, 18 mM D-glucose and 118 mM sodium phosphate (pH 7.4), intracellular AdoMet levels continuously decrease to a value of about 0.1 microM after 24 h. This occurs in spite of the fact that the cellular concentrations of the substrates for the AdoMet synthetase reaction, ATP and L-methionine, remain relatively constant. In a search for incubation conditions that lead to stable levels of AdoMet in incubated cells, we have developed a sodium-Hepes-buffered medium which includes 1 mM adenine and a stoichiometric excess of MgCl2 over its ligand, phosphate. The inclusion of magnesium ion (and a reduction in phosphate) appears to increase intracellular free Mg2+, which is required for full activity of the erythrocyte AdoMet synthetase. Even in the presence of MgCl2, however, the AdoMet pool level can drop 4-6-fold within the first 2 h of incubation. We present evidence that suggests that this initial fall in the cellular AdoMet level may be due to the activation of AdoMet-dependent protein carboxyl methyltransferase, an enzyme which accounts for a large fraction of the total cellular AdoMet utilization. Adenine, or related compounds in the medium may prevent this activation, although the mechanism of this action is not clear at present.     

Cross-linking and chymotryptic digestion of the extractoplasmic domain of the anion exchange channel in intact human erythrocytes

We have applied our new high yield, membrane-impermeant, protein cross-linking reagents (J.V. Staros, 1982. Biochemistry 21:3950-3955) together with chymotryptic digestion of the surface of intact erythrocytes (T.L. Steck, B. Ramos, and E. Strapazon, 1976. Biochemistry 15:1154-1161) in an investigation of the topology of the extracytoplasmic domain of the anion exchange channel of intact human erythrocytes. In intact erythrocytes, these cross-linking reagents have been shown to cross-link subunits of the anion exchange channel to dimers in the extracytoplasmic domain of the protein. Chymotryptic treatment of intact erythrocytes has been shown to cleave subunits of the anion exchange channel into two fragments of distinct Mr. Sequential treatment of intact erythrocytes with either of two membrane-impermeant cross-linkers, followed by digestion with chymotrypsin, yields chymotryptic fragments of the anion exchange channel cross-linked to one another. The cross-linked products observed appear to arise by cross-linking of unlike chymotryptic fragments, whether the cross-links are intersubunit or intrasubunit. These results are consistent with a model of the anion exchange channel in which the subunits form a head-to-head dimer with a twofold center of symmetry perpendicular to the plane of the membrane.     

Transfer of band 3, the erythrocyte anion transporter, between phospholipid vesicles and cells

Band 3, the anion transport protein of human erythrocyte membranes, can be transferred from cells to liposomes and from liposomes back to cell membranes, retaining function and native orientation. After incubation with cells, sonicated phosphatidylcholine vesicles bind a transmembrane protein that comigrates with band 3 on sodium dodecyl sulfate-polyacrylamide gels. Like native red cell band 3, the vesicle-bound protein is cleaved by chymotrypsin into 65- and 30-kdalton fragments and is not cleaved by trypsin. The protein can be cross-linked by copper-phenanthroline oxidation either before or after transfer to vesicles; in either case, the vesicle fractions contain high molecular weight material that is dissociated into 95-kdalton species by mercaptoethanol. Band 3-vesicle complexes contain no detectable cell lipid and are specifically permeable to anions. Greater than 99% of their anion uptake can be blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Red cells whose band 3 function has been blocked irreversibly by DIDS or eosin maleimide regain part of their anion permeability upon incubation with band 3-vesicle complexes. Under the conditions employed, an average of one copy of functional band 3 is delivered to half of the cells, increasing by 2.3-fold the number of cells containing functional anion transporters. Incubation of pure lipid vesicles or red cell membrane buds with either normal red cells or eosin maleimide inhibited cells has no detectable effect on the cells' anion permeability.     

The effects of glycophorin A on the expression of the human red cell anion transporter (band 3) in Xenopus oocytes

The effects of human red cell glycophorin A (GPA) on the translocation to the plasma membrane and anion transport activity of the human erythrocyte anion transporter (band 3; AE1) have been examined using the Xenopus oocyte expression system. We show that band 3 accumulates steadily at the oocyte surface with time in the presence or absence of GPA, but this occurs more quickly when GPA is coexpressed. The amount of band 3 at the surface is determined by the concentrations of band 3 and GPA cRNA that are injected, with a higher proportion of total band 3 being translocated to the surface in the presence of GPA cRNA. The increased expression of DNDS-sensitive chloride transport is highly specific to GPA, and is not observed when the cRNA to the putative glycophorin E or a very high concentration of the cRNA to glycophorin C are coexpressed with band 3 in oocytes.We thank Dr. Kay Ridgwell and Charlotte Ratcliffe for supplying plasmids and Dr. David Anstee for antibodies. This work was supported by grants from the Medical Research Council.     

Enzymatic basis for the Ca2+-induced cross-linking of membrane proteins in intact human erythrocytes.

The accumulation of Ca2+ ions in intact human erythrocytes leads to the production of membrane protein polymers larger than spectrin. The polymer has a heterogeneous size distribution and is rich in gamma-glutamyl-epsilon-lysine cross-links. Isolation of this isodipeptide, in amounts as high as 6 mol/10(5) g of protein, confirms the idea [Lorand L., Weissmann, L.B., Epel, D.L., and Bruner-Lorand, J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 4479] that the Ca2+-induced membrane protein polymerization is mediated by transglutaminase. Formation of the polymer in the intact cells is inhibited by the addition of small, water-soluble primary amines. Inasmuch as these amines are known to prevent the Ca2+-dependent loss of deformability of the membrane, it is suggested that transglutaminase-catalyzed cross-linking may be a biochemical cause of irreversible membrane stiffening.     

Hypoxanthine-guanine exchange by intact human erythrocytes

The uptake and release of [14C]hypoxanthine by human erythrocytes, suspended in a tris(hydroxymethyl)aminomethane (Tris)-glucose-NaCl isotonic medium (pH 7.4), have been studied at 37 degrees C. The uptake of hypoxanthine, mediated by its incorporation into inosine 5'-monophosphate (IMP), was markedly stimulated by preincubating the cells in phosphate-buffered saline. After a lag time, [14C]IMP-enriched erythrocytes released [14C]hypoxanthine in the medium. Formycin B, at concentrations known to inhibit purine nucleoside phosphorylase in intact erythrocytes, affected hypoxanthine uptake and release and led to an increase in the intracellular concentration of inosine, suggesting that the main catabolic path of IMP is the sequential degradation of the nucleotide to inosine and hypoxanthine. The addition of guanine to a suspension of [14C]IMP-enriched erythrocytes led to an increase in the rate of [14C]hypoxanthine release, which was unaffected by the presence of formycin B. During the guanine-induced hypoxanthine release, guanine was taken up by the cells as GMP. These results suggest that the presence of guanine in the incubation medium activates a catabolic path in human erythrocytes leading to IMP degradation without formation of inosine.     

Phosphorylation in membranes of intact human erythrocytes.

Studies of phosphorylation in membranes of intact human erythrocytes were performed by incubating erythrocytes in inorganic [32P]phosphate. Analysis of membrane proteins by polyacrylamide gel electrophoresis showed a pattern of phosphorylation similar to that observed when ghost membranes were incubated with [gamma-32P]ATP. Membrane lipid phosphorylation was also similar in intact cells and ghosts. The most heavily phosphorylated lipid, polyphosphoinositide, was closely associated with glycophorin A, the major erythrocyte membrane sialoglycoprotein obtained when the sialoglycoprotein fraction was isolated by the lithium diiodosalicylate-phenol partition procedure. Only 1 molecule of glycophorin A out of every 100 was found to be phosphorylated, and the phosphate exchange occurred specifically in the COOH-terminal intracellular portion of glycophorin A. These studies show that the human erythrocyte can be used as a model for membrane phosphorylation in an intact cell system.     

Molecular mapping of human band 3 anion transport regions using synthetic peptides

Band 3 is a ubiquitous membrane transport protein found in Golgi, mitochondrial, nuclear, and cell membranes. It is the most heavily used anion transport system in the body because it is responsible for CO2 exchange in all tissues and organs and for acid-base balance. The anion transport regions are mapped along the band 3 molecule using synthetic peptides (pep) from extracellular regions of band 3 and/or suspected anion transport regions. Assays include anion transport/inhibition and immunoblotting with anti-idiotypic antibodies to a transport inhibitor. Results indicate that anion binding/transport regions of band 3 reside within residues 549-594, (588-594 being the most active) and 804-839 (822-839 being the most active), and 869-883. Pep-COOH (residues 812-827), which is part of senescent cell antigen, is an anion binding site with most of the activity localized to residues 813-818 (the six amino acids on the amino side of pep-COOH). The stilbene disulfonate inhibitors of transport bind to peptide 812-830, and possibly peptides 788-805 and 800-818, as determined with anti-idiotypic antibodies. Residues 538-554, which have been reported to be a transport segment of band 3, do not bind sulfate. Band 3 external loops containing residues 539-553 and 812-830, and internal segments containing residues 588-594 and 869-883, are in close spacial proximity in the membrane. The contribution of lysine and/or arginine to anion transport is examined by synthesizing peptides in which glycines or arginines are substituted for lysines or arginines. Lysines can contribute to anion binding but are not required.