Regulation of membrane band 3 Tyr-phosphorylation by proteolysis of p72^syk and possible involvement in senescence process

Erythrocyte senescence is characterized by exposure of cell surface epitopes on cell membrane proteins leading to immune mediated removal of red blood cells. One mechanism for antigen formation is tyrosine phosphorylation （Tyr-P） of the transmembrane protein band 3 by Syk kinase. Our aim was to test the hypothesis that proteolytic activation of Syk kinase by conversion from 72 kDa （p72^syk） to the 36 kDa （p36^syk） isoform enhances its phosphorylating activity independently of the association of Syk kinase with the cytoskeleton. Tyr-P assay was conducted using quantification of 32p uptake into the cytoplasmic domain of band 3 after addition of p72^syk or p36syk. Effect of prephosphorylation of erythrocyte membrane band 3 protein by p36^syk on p72^syk-mediated phosphorylation and the effect of addition of a protease inhibitor （leupeptin） on p72s^yk-mediated phosphorylation were studied by autoradiographic visualization of 32p uptake. Tyr-P by Syk isoforms of membrane skeletal and soluble fractions of band 3 was visualized by immunoblotting. It was found that p36^Syk had a higher band 3 tyrosine phosphorylating activity compared with p72^syk. Pre-phosphorylation with p36^syk or p72^syk increased band 3 phosphorylating activity. Protease inhibition treatment reduced p72^syk but not p36^syk band 3 tyrosine phosphorylating activity significantly. Both soluble and membrane skeletal fractions of band 3 protein were equally tyrosine phosphorylated by each Syk isoform. In conclusion, we confirmed the hypothesis that proteolytic cleavage of p72^syk is an important regulatory step for band 3 Tyr-P and its independence of the association of band 3 with the cytoskeleton.     

Role of the bilayer in the shape of the isolated erythrocyte membrane

Summary The determinants of cell shape were explored in a study of the crenation (spiculation) of the isolated erythrocyte membrane. Standard ghosts prepared in 5mm NaP_i (pH 8) were plump, dimpled disks even when prepared from echinocytic (spiculated) red cells. These ghosts became crenated in the presence of isotonic saline, millimolar levels of divalent cations, 1mm 2,4-dinitrophenol or 0.1mm lysolecithin. Crenation was suppressed in ghosts generated under conditions of minimal osmotic stress, in ghosts from red cells partially depleted of cholesterol, and, paradoxically, in ghosts from red cells crenated by lysolecithin. The susceptibility of ghosts to crenation was lost with time; this process was potentiated by elevated temperature, low ionic strength, and traces of detergents or chlorpromazine.In that ghost shape was influenced by a variety of amphipaths, our results favor the premise that the bilayer and not the subjacent protein reticulum drives ghost crenation. The data also suggest that vigorous osmotic hemolysis induces a redistribution of lipids between the two leaflets of the bilayer which affects membrane contour through a bilayer couple mechanism. Subsequent relaxation of that metastable distribution could account for the observed loss of crenatability.     

Crystallization and preliminary X-ray analysis of the cytoplasmic domain of human erythrocyte band 3

A cytoplasmic domain of the human erythrocyte membrane protein band 3 (M_r = 42,500), residues 1–379, expressed in and purified from E. coli, has been crystallized by the method of vapor diffusion in sitting drops with subsequent streak-seeding at room temperature. Initial crystals were grown from solutions containing 65–68% saturated ammonium sulfate at pH 4.9 and 2 mg/ml protein. Subsequent streak-seeding into solutions of 50–53% ammonium sulfate at pH 4.9 and 7 mg/ml protein produced single crystals suitable fur X-ray analysis, which contained pure protein as revealed by gel electrophoresis. The crystals belong to the monoclinic space group C2 with cell dimensions of a = 178.8 Å, b = 90.5 Å, c = 122.1 Å, and β = 131.3° and diffract at least to 2.7 Å resolution (at 100 K). A self-rotation function shows the presence of approximate 222 local symmetry. © 1995 Wiley-Liss, Inc.     

大强度训练及恢复后大鼠红细胞变形能力和红细胞膜蛋白的变化

目的 :探讨运动对红细胞变形性和红细胞膜蛋白的影响及其相互关系。方法 :设计不同强度的训练方案 ,用激光衍射法测定红细胞变形能力 ,用SDS PAGE方法测定一定体积大鼠红细胞膜中的重要蛋白带 3蛋白 (band 3)和肌动蛋白 (actin)的含量 ,研究运动即刻和恢复后红细胞变形性及膜蛋白的变化。结果 :长期的运动训练会促进大鼠红细胞变形能力的改善和红细胞膜band 3蛋白和actin的良好发展 ,一次大强度训练会引起红细胞膜band 3蛋白和actin含量的减少 ,大鼠红细胞变形能力降低 ,一周和二周的大强度训练会提高恢复期大鼠红细胞的变形能力和红细胞膜band 3蛋白和actin含量。结论 :运动训练造成的红细胞膜蛋白含量的变化 ,导致了红细胞膜结构的改变 ,从而影响红细胞变形能力 ,可能是训练对红细胞变形能力的作用机制之一。     

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) interacts with the band 3 receptor to promote erythrocyte invasion by malaria parasites

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) is an erythrocyte binding protein known to be involved in malarial parasite invasion. Although anti-GAMA antibodies have been shown to block GAMA attachment to the erythrocyte surface and subsequently inhibit parasite invasion, little is known about the molecular mechanisms by which GAMA promotes the invasion process. In this study, LC-MS analysis was performed on the erythrocyte membrane to identify the specific receptor that interacts with GAMA. We found that ankyrin 1 and the band 3 membrane protein showed affinity for GAMA, and characterization of their binding specificity indicated that both Plasmodium falciparum and Plasmodium vivax GAMA bound to the same extracellular loop of band 3 (loop 5). In addition, we show the interaction between GAMA and band 3 was sensitive to chymotrypsin. Furthermore, antibodies against band 3 loop 5 were able to reduce the binding activity of GAMA to erythrocytes and inhibit the invasion of P. falciparum merozoites into human erythrocytes, whereas antibodies against P. falciparum GAMA (PfGAMA)-Tr3 only slightly reduced P. falciparum invasion. The identification and characterization of the erythrocyte GAMA receptor is a novel finding that identifies an essential mechanism of parasite invasion of host erythrocytes.     

A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Summary The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H₂DIDS and flufenamate (approx.K _l-values at 30°C: 0.1 and 0.7 m, respectively), and right-side-out orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H₂DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.     

Decreased rotational diffusion of band 3 in melanesian ovalocytes from Papua,New Guinea

Summary Melanesian ovalocytes from Papua New Guinea have an N-terminal extension of the band 3 polypeptide (Jones, G.L., Edmunson. H.M., Wesche, D., Saul, A. 1990.Biochim. Biophys. Acta 1096:33–40). The ovalocytes showed a threefold increase in shear elastic modulus as determined by micropipette aspiration measurements of membrane rigidity. Time-resolved phosphorescence anisotropy has been used to study the rotational freedom of band 3 in membranes prepared from ovalocytes. The ovalocytic polymorphism was found to be associated with a marked decrease in the rotational mobility of band 3. This may indicate participation of band 3 in large homoaggregates or in complexes with other proteins at the cytoplasmic surface. There was no morphological clustering of band 3 detectable by immunofluorescence microscopy.     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Insulin control of a transplasma membrane NADH dehydrogenase in erythrocyte membranes

A study of NADH ferricyanide reductase activity in oriented vesicles or open ghosts of human and porcine erythrocytes shows that the dehydrogenase activity can have three types of orientation in the membrane. There is activity which responds only to acceptors and NADH exclusively on the inside face, or exclusively on the outer surface. There is also activity which requires exposure of both sides of the membrane and thus is transmembranous. The transmembrane activity is inhibited by insulin, whereas the internal and external enzymes do not respond to insulin. The transmembrane dehydrogenase can be a basis for proton transport in the plasma membrane.     

The basis of echinocytosis of the erythrocyte by glucose depletion

Echinocytosis of erythrocytes by glucose depletion is attributed to adenosine triphosphate depletion, but its process still remains unknown. A mechanism of control of the erythrocyte shape has been previously proposed in which the anion exchanger Band 3, linked to flexible membrane skeleton, has a pivotal role. Recruitments of its inward facing (Band 3(i) ) and outward facing (Band 3(o) ) conformations contract and relax the membrane skeleton, thus promoting echinocytosis and stomatocytosis, respectively. The Band 3(o) /Band 3(i) equilibrium ratio increases with the increase of the Donnan equilibrium ratio, and preferential inward and outward transport by Band 3 of substrates slowly transported are echinocytogenic and stomatocytogenic, respectively. The mechanism suggests the following process. The major organic phosphate 2,3-bisphosphoglycerate is catabolized to lactate to form inorganic phosphate, 3-phosphoglycerate, and adenosine triphosphate. The last two products can be reversibly transformed into 1,3-bisphosphoglycerate and adenosine diphosphate by the glycolytic enzyme phosphoglycerate kinase, thus allowing 2,3-bisphosphoglycerate formation by 2,3-bisphosphoglycerate synthase/phosphatase. The catabolic and cyclic processes initially oppose echinocytosis by increasing the Donnan ratio and outward transport of slowly transported inorganic phosphate by Band 3 (its basic form is transported with a hydrogen ion). Echinocytosis occurs when inward transport of this product becomes predominant. This process can rationalize direct and indirect observations.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( ? )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Interaction of molybdenum with human erythrocyte membrane proteins

This study describes the interaction of molybdenum with blood components. Molybdenum-99 was added to blood, and after four washings, 3% of the total radioactivity was found in red cells. More specifically, the radioactivity was determined to be associated with the cell membrane. Molybdenum-99 in the +VI form did not interact with the human erythrocyte membrane; however, Mo(V) forms did interact. Of five different compounds, the highes uptake was observed with a brown Mo(V)-ascorbate complex generated from Mo(VI) and ascorbic acid in the molar ratio 1∶20. A membrane suspension of Mo-ascorbate-treated human erythrocytes was prepared and the solubilized proteins were separated on a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS). Molybdenum-99 binding to spectrin was demonstrated, as well as some minor interactions with membrane hemoglobin and bands 6 and 8.     

Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100-300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( - )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

The band 3 protein of the human red cell membrane: A review

Band 3 is the predominant polypetide and the purported mediator of anion transport in the human erythrocyte membrane. Against a background of minor and apparently unrelated polypeptides of similar electrophoretic mobility, and despite apparent heterogeneity in its glycosylation, the bulk of band 3 exhibits uniform and characteristic behavior. This integral glycoprotein appears to exist as a noncovalent dimer of two ～ 93,000-dalton chains which span the membrane asymmetrically. The protein is hydrophobic in its composition and in its behaviour in aqueous solution and is best solubilized and purified in detergent. It can be cleaved while membrane-bound into large, topographically defined segments. An integral, outer-surface, 38,000-dalton fragment bears most of the band 3 carbohydrate. A 17,000-dalton, hydrophobic glycopeptide fragment spans the membrane. A ～ 40,000-dalton hydrophilic segment represents the cytoplasmic domain. In vitro, glyceraldehyde 3-P dehydrogenase and aldolase bind reversibly, in a metabolite-sensitive fashion, to this cytoplasmic segment. The cytoplasmic domain also bears the amino terminus of this polypetide, in contrast to other integral membrane proteins. Recent electron microscopic analysis suggests that the poles of the band 3 molecule can be seen by freezeetching at the two original membrane surfaces, while freeze-fracture reveals the transmembrane disposition of band 3 dimer particles. There is strong evidence that band 3 mediates 1:1 anion exchange across the membrane through a conformational cycle while remaining fixed and asymmetrical. Its cytoplasmic pole can be variously perturbed and even excised without a significant alteration of transport function. However, digestion of the outer-surface region leads to inhibition of transport, so that both this segment and the membrane-spanning piece (which is slectively labeled by covalent inhibitors of transport) may be presumed to be involved in transport. Genetic polymorphism has been observed in the structure and immunogenicity of the band 3 polypeptide but this feature has not been related to variation in anion transport or other band 3 activities.     

Binding of vanadate to human erythrocyte ghosts and subsequent events

Spectroscopic techniques were used to investigate the interaction between vanadate and human erythrocyte ghosts. Direct evidence from ⁵¹V nuclear magnetic resonance (NMR) studies suggested that the monomeric and polymeric vanadate species may bind to the anion binding sites of band 3 protein of the erythrocyte membrane. The results of ⁵¹V NMR studies and the quenching effect of vanadate on the intrinsic fluorescence of the membrane proteins indicated that in the low concentration range of vanadate (<0.6 mm), monomeric vanadate binds mostly to the anion sites of band 3 protein with the dissociation constant close to 0.23 mm. The experiments of sulfhydryl content titration by the method of Ellman and residue sulfhydryl-labeled fluorescence spectroscopies clearly displayed that vanadate reacts directly with sulfhydryl groups. The appearance of the anisotropic election spin resonance (ESR) signal of vanadyl suggests that a small (c. 3%) amount of vanadate was reduced by sulfhydryl groups of membrane proteins. The fluidity and order of intact ghost membrane were reduced by the reaction with vanadate, as shown by the ESR studies employing the protein- and lipid-specific spin labels. It was concluded that although vanadates mainly bind to band 3 protein, a minor part of vanadate may oxidize the residue sulfhydryl groups of membrane proteins, and thus decrease the fluidity of erythrocyte membrane.     

Degradation of the human erythrocyte membrane band 3 studied with monoclonal antibody directed against an epitope on the cytoplasmic fragment of band 3

The mouse hybridoma monoclonal antibody BIII.136 of the IgG2a class is specific for human erythrocyte band-3 protein. It was shown by means of immunoblotting and immunoprecipitation assays that the antibody recognized an epitope located in the cytoplasmic pole of the band-3 molecule within approximately 20 kDa from the N-terminal end. The N-terminal fragments of band-3 protein, migrating in SDS/polyacrylamide gel electrophoresis in the 60-kDa, 40-kDa and 20-kDa regions, were detected with the antibody in untreated red-cell membranes as products of autolysis of band-3 protein. A correlation was found between the amount of these fragments and erythrocyte age, which suggests that partial degradation of band 3 proceeds in vivo during senescence of erythrocytes. The further degradation of band-3 protein in vitro was not observed in intact erythrocytes stored at 4 degrees C, but progressed distinctly after hemolysis of red cells, during washing and storing the membranes.     

Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli.

The cytoplasmic domain of the human erythrocyte membrane protein, band 3 (cdb3), contains binding sites for hemoglobin, several glycolytic enzymes, band 4.1, band 4.2, and ankyrin, and constitutes the major linkage between the membrane skeleton and the membrane. Although erythrocyte cdb3 has been partially purified from proteolyzed red blood cells, further separation of the water-soluble 43-kDa and 41-kDa proteolytic fragments has never been achieved. In order to obtain pure cdb3 for crystallization and site-directed mutagenesis studies, we constructed an expression plasmid that has a tandemly linked T7 promoter placed upstream of the N-terminal 379 amino acids of the erythrocyte band 3 gene. Comparison of several Escherichia coli strains led to the selection of the BL21 (DE3) strain containing the pLysS plasmid as the best host for efficient production of cdb3. About 10 mg of recombinant cdb3 can be easily purified from 4 L of E. coli culture in two simple steps. Comparison of cdb3 released from the red blood cell by proteolysis with recombinant cdb3 reveals that both have the same N-terminal sequence, secondary structure, and pH-dependent conformational change. The purified recombinant cdb3 is also a soluble stable dimer with the same Stokes radius as erythrocyte cdb3. The affinities of the two forms of cdb3 for ankyrin are essentially identical; however, recombinant cdb3 with its unblocked N-terminus exhibits a slightly lower affinity for aldolase.     

Calcium transport by pigeon erythrocyte membrane vesicles

Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of ⁴⁵Ca²⁺. Ca²⁺ accumulation ratios greater than or approximately equal to 10⁴ are readily attained. For ATP-dependent Ca²⁺ uptake, V is 1.5 mmol · 1^?1 · min^?1 at 27°C (approx. 0.9 nmol · mg^?1 protein · min^?1), [Ca²⁺]_{$\text{1}\text{2}$} is 0.18 μM, [ATP]_{$\text{1}\text{2}$} is 30–60 μM, the Ca²⁺ uptake rate depends on [Ca²⁺]² and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 ± 1.4 kcal/mol and the pH optimum is approx. 6.9.     

A 20 kDa erythrocyte membrane phosphoprotein

A small (approximately 20 kDa) protein associated with the human erythrocyte membrane undergoes phosphorylation that is potentiated by the addition of phosphatidylinositol. It is indicated that phosphatidylinositol 4,5-bisphosphate, generated in situ during the protein phosphorylation reaction, is the effective agent. Phosphorylation of the small protein is also potentiated when isolated membranes are exposed to conditions that promote the phosphorylation of the phosphatidylinositols. It is suggested that the 20 kDa membrane protein participates in biochemical signaling in the erythrocyte.Abbreviations EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

A monomeric form of human erythrocyte membrane acetylcholinesterase

Purified detergent solubilized dimeric human erythrocyte acetylcholinesterase (6.3 S form) was converted to a stable monomeric 3.9 S species when treated with 2-mercaptoethanol and iodoacetic acid. More than 60% of the enzymatic activity were recovered after this treatment. A decreased susceptibility to reduction and alkylation was observed with purified, detergent depleted acetylcholinesterase aggregates. When erythrocyte membranes (ghosts) were subjected to the same treatment, acetylcholinesterase could subsequently be solubilized as monomeric 3.9 S form and and more than 90% of the activity were recovered. Monomeric acetylcholinesterase was less reactive towards antibodies raised against (dimeric) human erythrocyte membrane acetylcholinesterase and towards antibodies against human erythrocyte membranes. The results suggest that acetylcholinesterase is present as dimeric species in human erythrocyte membranes despite the fact that fully active monomers can be obtained.