A Bacterial Phosphatase-Like Enzyme of the Malaria Parasite Plasmodium falciparum Possesses Tyrosine Phosphatase Activity and Is Implicated in the Regulation of Band 3 Dynamics during Parasite Invasion

Eukaryotic parasites of the genus Plasmodium cause malaria by invading and developing within host erythrocytes. Here, we demonstrate that PfShelph2, a gene product of Plasmodium falciparum that belongs to the Shewanella-like phosphatase (Shelph) subfamily, selectively hydrolyzes phosphotyrosine, as shown for other previously studied Shelph family members. In the extracellular merozoite stage, PfShelph2 localizes to vesicles that appear to be distinct from those of rhoptry, dense granule, or microneme organelles. During invasion, PfShelph2 is released from these vesicles and exported to the host erythrocyte. In vitro, PfShelph2 shows tyrosine phosphatase activity against the host erythrocyte protein Band 3, which is the most abundant tyrosine-phosphorylated species of the erythrocyte. During P. falciparum invasion, Band 3 undergoes dynamic and rapid clearance from the invasion junction within 1 to 2 s of parasite attachment to the erythrocyte. Release of Pfshelph2 occurs after clearance of Band 3 from the parasite-host cell interface and when the parasite is nearly or completely enclosed in the nascent vacuole. We propose a model in which the phosphatase modifies Band 3 in time to restore its interaction with the cytoskeleton and thus reestablishes the erythrocyte cytoskeletal network at the end of the invasion process.     

Differential effects of quercetin and resveratrol on Band 3 tyrosine phosphorylation signalling of red blood cells

The protective effects of eating fruits and vegetables in the prevention of several degenerative pathologies have been attributed at least in part to the antioxidant and anti-inflammatory properties of polyphenols. In this study, we investigated the effects of two polyphenols, quercetin and resveratrol, on red blood cell Band 3 tyrosine phosphorylation signalling activated by peroxynitrite. Peroxynitrite is a physiological oxidant scavenged largely by the erythrocyte and formed by the reaction between nitrogen monoxide and superoxide anion. Quercetin and its structurally analogous (+)-catechin inhibited the peroxynitrite-dependent upregulation of Band 3 tyrosine phosphorylation. Quercetin was found to downregulate the activity of syk, which is upstream in the Band 3 tyrosine phosphorylation cascade, and partially prevented peroxynitrite-mediated phosphotyrosine phosphatase inhibition. Resveratrol and hydroxytyrosol, unexpectedly, amplified peroxynitrite-dependent upregulation of Band 3 tyrosine phosphorylation through the activation of lyn, a kinase of the src family. The present results clearly indicate that polyphenols may activate cell transduction pathways in different and sometimes opposite ways.     

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.     

Effector-induced Syk-mediated phosphorylation in human erythrocytes

Band 3 (AE1), the most prominent polypeptide of the human erythrocyte membrane, becomes heavily tyrosine phosphorylated following treatment of intact cells with protein tyrosine phosphatase inhibitors such as diamide, pervanadate, vanadate, or N-ethylmaleimide (NEM). The mechanism underlying this tyrosine phosphorylation is thought to involve the sequential action of two protein tyrosine kinases, Syk (p72syk) and Lyn (p53/56lyn). While Lyn catalysed phosphorylation appears to be strictly dependent on prior phosphorylation of Tyr8 and 21 of band 3 by Syk, little is known about the mechanism of induction of Syk phosphorylation. Data presented here show that both the fraction of Syk that associates with the membrane and the extent of phosphorylation of band 3 differ in response to the above inhibitors. While diamide and NEM stimulate syk translocation to the membrane during their induction of band 3 tyrosine phosphorylation, pervanadate and vanadate induce no change in kinase distribution. Moreover, diamide and NEM-induced Syk recruitment to the membrane are phosphotyrosine independent and involve their preferential association with Triton X-100-insoluble membrane skeletons. Together these data reveal a complex process controlling the association and catalytic activity of protein tyrosine kinases syk and lyn with the human erythrocyte membrane.     

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.     

Effects of cholesterol and PIP2 on interactions between glycophorin A and Band 3 in lipid bilayers

In the erythrocyte membrane, the interactions between glycophorin A (GPA) and Band 3 are associated strongly with the biological function of the membrane and several blood disorders. In this work, using coarse-grained molecular-dynamics simulations, we systematically investigate the effects of cholesterol and phosphatidylinositol-4,5-bisphosphate (PIP2) on the interactions of GPA with Band 3 in the model erythrocyte membranes. We examine the dynamics of the interactions of GPA with Band 3 in different lipid bilayers on the microsecond time scale and calculate the binding free energy between GPA and Band 3. The results indicate that cholesterols thermodynamically favor the binding of GPA to Band 3 by increasing the thickness of the lipid bilayer and by producing an effective attraction between the proteins due to the depletion effect. Cholesterols also slow the kinetics of the binding of GPA to Band 3 by reducing the lateral mobility of the lipids and proteins and may influence the binding sites between the proteins. The anionic PIP2 lipids prefer binding to the surface of the proteins through electrostatic attraction between the PIP2 headgroup and the positively charged residues on the protein surface. Ions in the solvent facilitate PIP2 aggregation, which promotes the binding of GPA to Band 3.     

Characterization of matrix-bound Band 3, the anion transport protein from human erythrocyte membranes

Band 3 (Mr = 95,000), the anion transport protein of human erythrocyte membranes exists primarily as a dimer in solutions of nonionic detergents such as octaethylene glycol mono-n-dodecyl ether (C12E8). The role of the oligomeric structure of Band 3 in the binding of [14C]4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), an inhibitor of anion transport (Ki = 1-2 microM), was studied by characterizing the interaction of BADS with dimers and monomers of Band 3 covalently attached to p-mercuribenzoate-Sepharose 4B. BADS bound to matrix-bound Band 3 dimers with an affinity of approximately 3 microM at a stoichiometry of 1 BADS molecule/Band 3 monomer, in agreement with the BADS binding characteristic of Band 3 in the membrane and in solutions of C12E8. Band 3 dimers could be attached to the matrix via one subunit by limiting the amount of p-chloromercuribenzoate on the Sepharose bead. Matrix-bound monomers were formed by dissociation of the dimers with dodecyl sulfate or guanidine hydrochloride. Complete removal of the denaturants allowed formation of refolded Band 3 monomers since the matrix-bound subunits could not reassociate. These refolded Band 3 monomers were unable to bind BADS. Release of the monomers from the matrix with 2-mercaptoethanol allowed reformation of dimers with recovery of the BADS binding sites. These results suggest that the dimeric structure of Band 3 is required for BADS binding and that the BADS binding sites may be at the interface between the two halves of the Band 3 dimer.     

Identification by peptide analysis of the spectrin-binding protein in human erythrocytes.

One-dimensional and two-dimensional peptide-mapping techniques are used to identify the protein which gives rise to the 72,000 dalton alpha-chymotryptic fragment previously shown to be the membrane attachment site for spectrin. Peptide maps of the 72,000 dalton fragment are very different from maps of Bands 1, 2, 2.9, 3, 3.1, 4.1, and 4.2 and very similar to maps of the apparently closely homologous polypeptides, Bands 2.1, 2.2, 2.3, and 2.6. Limited proteolysis of erythrocyte membranes is shown to generate Band 3', another polypeptide which has been associated with spectrin-binding activity. Peptide maps of Band 3' are very similar to maps of Band 2.1, suggesting that Band 3' is also a proteolytic fragment of Band 2.1. It is concluded that Band 2.1 and possibly some or all of the other, related polypeptides which electrophorese in the 2 region is (are) the spectrin-binding protein(s) of the human erythrocyte.     

The behavior of the human erythrocyte as an imperfect osmometer: a hypothesis

The human erythrocyte does not behave as a perfect osmometer that is its volume does not change as predicted with the change of the tonicity of the medium, as if there was a fraction of the cell water not participating in the osmotic exchange. A mechanism of control of the erythrocyte shape has been previously proposed in which Band 3 (AE1), the protein anion exchanger of Cl(-) and HCO(3)(-), plays a central role. Specifically, decrease and increase of the ratio of its outward-facing conformation and inward-facing conformation (Band 3(o)/Band 3(i)) contract and relax the membrane skeleton, thus favoring echinocytosis and stomatocytosis, respectively. The equilibrium Band 3(o)/Band 3(i) ratio is determined by the Donnan equilibrium ratio of anions and protons, increasing with it (r=Cl(i)(-)/Cl(o)(-)=HCO 3(i)(-)/HCO 3(o)(-)=H(o)(+)/H(i)(+)). The Donnan ratio is influenced by the erythrocyte transport and metabolic activities. The volume change of the human erythrocyte alters the skeleton conformation as it is accompanied by a change of the membrane curvature. Thus, the mechanism could be a hypothesis for explaining the behavior of the human erythrocyte as an imperfect osmometer since the Donnan ratio controls the Band 3(o)/Band 3(i) ratio which controls the volume by a control of the degree of contraction or relaxation of the skeleton. Predictions made by the hypothesis on the Ponder's coefficient R' values in the presence of sucrose or Band 3 substrates slowly transported as well as on the participation of Band 3 in the osmotic hemolysis appear to be corroborated by previous observations. If the hypothesis was valid, it would follow that there is a pressure gradient across the erythrocyte membrane. The equilibrium volume is antagonistically determined by the Donnan ratio per se and Band 3. Band 3, rather than the ratio of surface-to-volume, primarily controls the osmotic hemolysis.     

A basis of echinocytosis and stomatocytosis in the disc-sphere transformations of the erythrocyte

A mechanism of erythrocyte shape control has been previously hypothesized in which Band 3, the anion exchange protein, controls the shape. In essence, the mechanism hypothesizes that the membrane skeleton is used to generate different shapes and the alternate influx and efflux of anions mediated by Band 3, which recruit Band 3 to an inward-facing and an outward-facing conformation, contract and relax the skeleton by folding and unfolding spectrin. Spectrin is bound to Band 3 by the intermediary of ankyrin. The mechanism is shown to be consistent with rapid shape deformations of the erythrocyte in blood circulation. We have examined whether the mechanism could provide a basis of echinocytosis and stomatocytosis in disc-sphere transformations of the erythrocyte induced by a wide variety of agents. These agents were classified into four groups: lipids of the bilayer, Donnan equilibrium modifiers, Band 3 anion transport inhibitors and integral membrane protein modifiers. Evidence is presented that the lipids play a secondary function in the control of the erythrocyte shape, as indicated by the mechanism. Two possible functions of the lipids are suggested with respect to the mechanism. Without exception, echinocytogenic and stomatocytogenic Donnan equilibrium modifiers decrease and increase the equilibrium ratio of chloride (Cl-(i)/Cl-(o)), respectively, as predicted by the mechanism. Echinocytosis produced by competitive anion transport inhibitors slowly transported inward by Band 3 and by affinity labels of Band 3 is compatible with the mechanism. Evidence is presented which indicates that echinocytosis and stomatocytosis induced by amphiphilic drugs and detergents occur by inhibition of the Band 3 anion transport. Finally, echinocytosis and stomatocytosis induced by non-covalent and covalent modifiers of integral membrane proteins such as agglutinins and digestive enzymes are consistent with the mechanism.     

Reconstitution of band 3, the erythrocyte anion exchange protein

Band 3, the erythrocyte membrane protein thought to be responsible for anion transport, was purified to near homogeneity using a Concanavalin A affinity column. Band 3 was then combined with egg lecithin, erythrocyte lipid, cholesterol, and glycophorin, the major erythrocyte sialoglycoprotein, to form vesicles capable of rapid sulfate transport. The transport activity was sensitive to prior treatment of the erythrocytes with pyridoxal phosphate-NaBH₄, a potent inhibitor of anion transport in these cells.     

Carboxypeptidase Y digestion of band 3, the anion transport protein of human erythrocyte membranes

The exposure of the carboxyl-terminal of the Band 3 protein of human erythrocyte membranes in intact cells and membrane preparations to proteolytic digestion was determined. Carboxypeptidase Y digestion of purified Band 3 in the presence of non-ionic detergent released amino acids from the carboxyl-terminal of Band 3. The release of amino acids was very pH dependent, digestion being most extensive at pH 3, with limited digestion at pH 6 or above. The 55,000 dalton carboxyl-terminal fragment of Band 3, generated by mild trypsin digestion of ghost membranes, had the same carboxyl-terminal sequence as intact Band 3, based on carboxypeptidase Y digestion. Treatment of intact cells with trypsin or carboxypeptidase Y did not release any amino acids from the carboxyl-terminal of Band 3. In contrast, carboxypeptidase Y readily digested the carboxyl-terminal of Band 3 in ghosts that were stripped of extrinsic membrane proteins by alkali or high salt. This was shown by a decrease in the molecular weight of a carboxyl-terminal fragment of Band 3 after carboxypeptidase Y digestion of stripped ghost membranes. No such decrease was observed after carboxypeptidase Y treatment of intact cells. In addition, Band 3 purified from carboxypeptidase Y-treated stripped ghost membranes had a different carboxyl-terminal sequence from intact Band 3. Cleavage of the carboxyl-terminal of Band 3 was also observed when non-stripped ghosts or inside-out vesicles were treated with carboxypeptidase Y. However, the digestion was less extensive. These results suggest that the carboxyl-terminal of Band 3 may be protected from digestion by its association with extrinsic membrane proteins. We conclude, therefore, that the carboxyl-terminal of Band 3 is located on the cytoplasmic side of the red cell membrane. Since the amino-terminal of Band 3 is also located on the cytoplasmic side of the erythrocyte membrane, the Band 3 polypeptide crosses the membrane an even number of times. A model for the folding of Band 3 in the erythrocyte membrane is presented.     

A hypothesis of the disc-sphere transformation of the erythrocytes between glass surfaces and of related observations

Erythrocytes suspended at a low hematocrit in a non-buffered isotonic saline change from biconcave discs to spheres between glass surfaces of a slide and of a coverslip with the echinocyte as an intermediate. A pH increase is a major factor responsible for this disc-sphere transformation or glass effect. It is also observed between surfaces made of various polymers and of mica provided that the distance between them is controlled (0.1 mm). The glass effect is antagonized by serum, plasma, serum albumin, ammonium salts and CO2. It is not observed above a 1-2% hematocrit, but is enhanced by gamma-globulins. The sites of reappearance of the spicules are the same and the order of their disappearance is the inverse of the order of their reappearance during the repetitive cycle of the disc-sphere transformation and reversal when a small glass rod is alternatively approached near a site on the erythrocyte surface and withdrawn. A mechanism of erythrocyte shape control has been previously hypothesized in which Band 3 (AE1), the anion exchange protein, plays a central role. Specifically, decrease and increase of the ratio of its outward-facing conformation (Band 3o) and inward-facing conformation (Band 3i) contract and relax the membrane skeleton, promoting the echinocytosis and stomatocytosis, respectively. The Band 3o/Band 3i equilibrium ratio is determined by the Donnan equilibrium ratio of Cl-, HCO3- and H+ (r=Cl(i)-/Cl(o)-=HCO3i-/HCO3o-=Ho+/Hi+), increasing with it. The mechanism could explain by a change of the Donnan ratio the above observations with the assumptions that polymers are permeable to CO2 and that an unstirred layer slows the propagation of the change occurring at the site of approach of the glass rod to peripheral sites. The presence of HCO3- in serum or plasma may be the basis for the absence of the glass effect in these fluids.     

Interaction of human erythrocyte Band 3 with Ricinus communis agglutinin and other lectins

Agglutination and competition studies suggest that human erythrocyte Band 3 can interact with both mannose/glucose- and galactose-specific lectins. Purified Band 3 reconstituted into lipid vesicles binds concanavalin A, but the nonspecific binding component, measured in the presence of alpha-methylmannoside, is very high. This glycoprotein also carries binding sites for the galactose-specific lectin Ricinus communis agglutinin. Binding was inhibited poorly by lactose, but much more effectively by desialylated fetuin glycopeptides, suggesting that the lectin recognizes a complex oligosaccharide sequence on Band 3. The glycoprotein bears two separate classes of binding sites for R. communis agglutinin. High-affinity binding sites exist which show strong positive cooperativity and correspond in number to the outward-facing Band 3 molecules. A low-affinity binding mode is abolished by 40% ethyleneglycol, suggesting the involvement of hydrophobic lectin-glycoprotein interactions. Studies on binding of R. communis agglutinin to human erythrocytes indicate positively cooperative binding to 7 X 10(5) very-high-affinity sites per cell, and lectin binding is completely inhibitable by lactose. Based on its binding characteristics in vesicles, it seems likely that Band 3 forms the major receptor for this lectin in human erythrocytes. Properties such as positive cooperativity thus appear to be a common feature of the interaction of Band 3 with a variety of lectins of different specificity, both in erythrocytes and lipid bilayers.     

Water transport across red-cell membranes following reductive methylation of the major transmembrane protein, Band 3: implications to increased divalent cation membrane permeability

1H-T-NMR methods in conjunction with normally membrane-impermeable Mn2+ were used to study the effect of reductive methylation of specific lysine residues of Band 3, the major transmembrane protein, on water permeability. At 21 degrees C, the water apparent transverse relaxation time (T2) was decreased by nearly 16% (p less than .00001) for cells with modified Band 3. Atomic absorption measurements of control and methylated cells showed an increased level of Mn2+ in the erythrocyte cytosol following methylation. This increased level of this paramagnetic relaxation agent is sufficient to relax interior water protein to the values obtained. Thus, following specific methylation of band 3, increased membrane permeability to divalent cations is observed. The results are discussed with reference to possible conformation changes of Band 3 following methylation, and the findings are interpreted be mean that the conformation of Band 3 has influence on cation permeability to erythrocyte membranes.     

Reactive sulfhydryl groups of the band 3 polypeptide from human erythroycte membranes. Location in the primary structure.

Human erythrocyte membranes contain a major transmembrane protein, known as Band 3, that is involved in anion transport. This protein contains a total of five reactive sulfhydryl groups, which can be assigned to either of two classes on the basis of their susceptibility to release from the membrane by trypsin. Two of the groups are located in the region COOH-terminal to the extracellular chymotrypsin-sensitive site of the protein and remain with a membrane-bound 55,000-dalton fragment generated by trypsin treatment. The three sulfhydryl groups NH2-terminal to the extracellular chymotrypsin site are released from the cytoplasmic surface of the membrane by trypsin. All three groups are present in a 20,000-dalton tryptic fragment of Band 3. Two of these groups are located very close to the sites of trypsin cleavage that generate the 20,000-dalton fragment. The third reactve group is probably located about 15,000-daltons from the most NH2-terminal sulfhydryl group. Two other well defined fragments of the protein do not contain reactive sulfhydryl groups. They are a 23,000-dalton fragment derived from the NH2-terminal end that is also released by trypsin from the cytoplasmic surface of the membrane and a 19,000-dalton membrane-bound region of the protein that is produced by treatment with chymotrypsin in ghosts. The 20,000-dalton tryptic fragment may, therefore, constitute a sulfhydryl-containing domain of the Band 3 protein.     

Accessibility of the N-ethylmaleimide-unreactive sulfhydryl of human erythrocyte Band 3

The human erythrocyte anion exchange protein, Band 3, was reacted with N-ethylmaleimide (NEM) in cells to a stoichiometry of 5.3 mol NEM per mol Band 3, indicating that all NEM-reactive cysteines in Band 3 were labeled. Quantitatively NEM-blocked Band 3 was still able to bind to and be eluted by reducing agents from a mercurial affinity resin, [p-(chloromercuribenzamido)ethylene]amino-Sepharose. Reaction of NEM-blocked Band 3 with p-chloromercuribenzoate (pCMB) did not prevent binding to the resin due to exchange of pCMB for the immobilized mercurial. pCMB has been reported to inhibit water and urea permeation across the red cell membrane, and this has been attributed to reaction with a NEM-reactive sulfhydryl in Band 3. The interaction of Band 3 with the immobilized ligand directly demonstrates the reaction of NEM-blocked Band 3 with a mercurial and indicates that the NEM-unreactive, pCMB-reactive sulfhydryl residue is accessible to within approximately equal to 12 A (the distance from the solid support to the Hg) of the surface of the solubilized Band 3 protein.     

Reversible DIDS binding to Band 3 protein in human erythrocyte membranes

Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([³H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS-Band 3' complex at 0° C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k₊₁     

Partial puridication of a membrane protein from human erythrocytes involved in glucose transport.

In an attempt to determine which membrane proteins are essential to the stereospecific uptake of D-glucose, isolated human erythrocyte membranes were exposed to a variety of reagents capable of selectively extracting various membrane proteins. These reagents included EDTA, lithium 3,5-diiodosalicylate, sodium iodide, and 2,3-dimethylmaleic anhydride. Selective elution of spectrin and Components 2.1, 2.2, 2.3, 4.1, 4.2, 5, and 6 representing 65% of the ghost protein has no effect on the uptake of D-glucose. All of the sugar transport proteins are associated with a membrane residue consisting of the proteins of Bands 3, 4.5, and 7, the periodic acid-Schiff-sensitive glycoproteins, and ghost phospholipids. Specific cross-linking of the proteins of Band 3 of ghosts by the catalyzed oxidation of intrinsic sulfhydryl groups with the o-phenanthroline-cupric ion complex inhibits D-glucose uptake and alters the relative electrophoretic mobility of Band 3 proteins in sodium dodecyl sulfate-polyacrylamide-agarose gels. This uptake activity and the relative mobility of Band 3 proteins are recovered upon reversal of the cross-linking reaction by reduction with 2-mercaptoethanol. These results and other observations indicate that the D-glucose transport protein is an intrinsic component of the hydrophobic structure of the erythrocyte membrane and may be associated with the proteins of Band 3 which are glycoproteins spanning the membrane bilayer. It is proposed that D-glucose transport occurs through a water-filled channel formed by specific subunit aggregates of the transport proteins in the erythrocyte membrane rather than by rotation of the protein within the plane of the membrane.