Role of sulfhydryl groups in band 3 in the inhibition of phosphate transport across erythrocyte membrane in visceral leishmaniasis

Membrane destabilization in erythrocytes plays an important role in the premature hemolysis and development of anemia during visceral leishmaniasis (VL). Marked degradation of the anion channel protein band 3 is likely to allow modulation of anion flux across the red cell membrane in infected animals. The present study describes the effect of structural modification of band 3 on phosphate transport in VL using (31)P NMR. The result showed progressive decrease in the rate and extent of phosphate transport during the post-infection period. Interdependence between the intracellular ionic levels seems to be a determining factor in the regulation of anion transport across the erythrocyte membrane in control and infected conditions. Infection-induced alteration in band 3 made the active sites of transport more susceptible to binding with amino reactive agents. Inhibition of transport by oxidation of band 3 and subsequent reversal by reduction using dithiothreitol suggests the contribution of sulfhydryl group in the regulation of anion exchange across the membrane. Quantitation of sulfhydryl groups in the anion channel protein showed the inhibition to be closely related to the decrease of sulfhydryl groups in the infected hamsters. Downregulation of phosphate transport during leishmanial infection may be ascribed to the sulfhydryl modification of band 3 resulting in the impaired functioning of this protein under the diseased condition.     

Identification of immunoreactive forms of human erythrocyte band 3 in nonerythroid cells

Antibodies directed to the cytoplasmic domain of human erythrocyte band 3, the major integral protein of the erythrocyte membrane which is thought to be the main anchoring site of the membrane cytoskeleton, were demonstrated in the present study to react with the membrane of various nonerythroid cells, such as human leucocytes, fibroblasts or human umbilical mesenchyme cells, amniotic epithelium and vascular smooth muscle. In cultured fibroblasts staining was confined to small dots and streaks associated with both the dorsal and ventral cell membrane. In human lymphocytes band 3 antigen accompanied capping of concanavalin A binding surface receptors. The immunoreactive form of band 3 in fibroblasts was shown by immunoblotting studies to be a polypeptide of approximately 60 000 dalton. This polypeptide is immunologically and electrophoretically related to a major immunoreactive form of band 3 naturally occurring in the red blood cell membrane. Considering the recent identification in nonerythroid cells of immunoreactive forms of other major components of the erythrocyte membrane cytoskeleton, the present observation in nucleated cells of a polypeptide related to erythrocyte band 3 may indicate some of the features of erythrocyte membrane architecture are also present in nonerythroid cells.     

Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti‐phosphotyrosine and anti‐phosphoserine antibodies following 2‐DE in conjunction with double channel laser‐induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans‐membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.     

Band 3 tyr-phosphorylation in human erythrocytes from non-pregnant and pregnant women

Pregnancy is associated with changes in circulating red blood cells, mainly involving band 3 protein and membrane lipid peroxidation. Membrane band 3 is a multifunctional protein containing four Tyr-phosphorylatable residues which modulate the physiological status of erythrocytes by regulating glycolysis, cell shape and membrane transport. Erythrocytes from nine pregnant and 12 age-matched non-pregnant healthy women were subjected to oxidative and hyperosmotic stress conditions and the extent of band 3 Tyr-phosphorylation and membrane Syk recruitment as a membrane marker were evaluated. Results indicated that, in pregnancy, red blood cells show a decrease in band 3 Tyr-phosphorylation and a clear-cut rearrangement of band 3 protein within the membrane. In fact, band 3 shows a decrease in high molecular weight aggregates (HMWA), with different subdivision between Triton-soluble and -insoluble compartments, and an increase in proteolytic fragments. In conclusion, it is demonstrated that pregnancy is associated with membrane adjustments which reduce the sensitivity of erythrocytes to both oxidative and osmotic stress. Band 3 Tyr-phosphorylation is proposed as a new parameter in the evaluation of erythrocyte membrane arrangement.     

Band 3 tyr-phosphorylation in human erythrocytes from non-pregnant and pregnant women

Pregnancy is associated with changes in circulating red blood cells, mainly involving band 3 protein and membrane lipid peroxidation. Membrane band 3 is a multifunctional protein containing four Tyr-phosphorylatable residues which modulate the physiological status of erythrocytes by regulating glycolysis, cell shape and membrane transport. Erythrocytes from nine pregnant and 12 age-matched non-pregnant healthy women were subjected to oxidative and hyperosmotic stress conditions and the extent of band 3 Tyr-phosphorylation and membrane Syk recruitment as a membrane marker were evaluated. Results indicated that, in pregnancy, red blood cells show a decrease in band 3 Tyr-phosphorylation and a clear-cut rearrangement of band 3 protein within the membrane. In fact, band 3 shows a decrease in high molecular weight aggregates (HMWA), with different subdivision between Triton-soluble and -insoluble compartments, and an increase in proteolytic fragments. In conclusion, it is demonstrated that pregnancy is associated with membrane adjustments which reduce the sensitivity of erythrocytes to both oxidative and osmotic stress. Band 3 Tyr-phosphorylation is proposed as a new parameter in the evaluation of erythrocyte membrane arrangement.     

Band 3 and ankyrin homologues are present in eye lens: evidence for all major erythrocyte membrane components in same non-erythroid cell

Although immunological homologues of erythrocyte membrane proteins have been individually discovered in a wide variety of tissues and cultured cells, the major structural components of the membrane have not yet been demonstrated simultaneously in the same cell type. Thus, considerable uncertainty continues to exist concerning whether the red cell homologues form elements of a structure which is similar to or unique from the framework which supports the erythrocyte membrane. Because the red cell cytoskeletal proteins, spectrin, actin and band 4.1, have been previously found in the superficial cortex of the lens, we decided to determine whether the corresponding membrane anchoring components of band 3 and ankyrin also occur in this cell type. Using antiserum specific for band 3 and ankyrin, we report the existence of immunologically cross-reactive proteins of similar molecular weight. Because these anchoring proteins appear and disappear coordinately with the aforementioned cytoskeletal proteins during the intermediate stages of lens cell maturation, it is conceivable that an erythrocyte-like membrane structural organization may occur transiently in the eye lens.     

The plasma membrane of erythrocytes plays a fundamental role in the transport of oxygen, carbon dioxide and nitric oxide and in the maintenance of the reduced state of the heme iron

Here we review new insights into the role of the erythrocyte membrane and the implications of its architecture on the several functions accomplished by the red blood cells. The picture which emerges highlights the capability of Hb and band 3 to modulate erythrocyte metabolism and to meet the needs of the cell.     

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.     

Glycophorin A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes.

The effects of human red cell glycophorin A (GPA) on the expression of the human erythrocyte anion transporter (band 3, AE1) has been examined in Xenopus oocytes. The coexpression of GPA with band 3 increased stilbene disulfonate-sensitive chloride transport into the oocytes. The effect of GPA was particularly noticeable at low band 3 concentrations and less marked at high band 3 cRNA concentrations. The enhancement of chloride transport was specific to GPA and was not observed when either glycophorin B or glycophorin C was coexpressed with band 3. Immunoprecipitations of whole oocyte homogenates showed the amount of band 3 synthesized was not affected by GPA at subsaturating cRNA concentrations. More band 3 was detected at the oocyte surface by immunoprecipitation when GPA was also expressed. Chymotrypsin treatment of intact oocytes was also used to assess surface band 3 and greater cleavage of band 3 by chymotrypsin was observed when GPA was present. Band 3 synthesis and assembly into canine pancreatic microsomes in the reticulocyte cell-free translation system was not altered by cotranslation of GPA. We suggest that GPA facilitates the translocation of band 3 to the plasma membrane at some point during band 3 biosynthesis in Xenopus oocytes. However, GPA is not essential for the expression of band 3 in red cells, since GPA-deficient individuals have apparently normal levels of band 3. Other GPA-independent mechanisms must also allow translocation of band 3 to the surface membrane in erythroid cells and oocytes. GPA may affect the rate of accumulation of band 3 at the cell surface, rather than the final level in the plasma membrane.     

Proteolytic activity of human erythrocyte membrane during ghosts preparation

1. Proteins in human erythrocyte membranes after red blood cells hemolysis revealed relatively high rate of self-digestion. 2. This indicates hemolysis as a critical moment for membrane proteases activation. 3. The detailed pattern of band 3 protein and spectrin degradation during ghosts preparation was more complicated and reflected both the changes in proteolytic susceptibility and extraction of some proteases. 4. Further extraction of membrane proteins by alkali stripping resulted in an increase in the self-digestion rate and decrease in the degradation rate of an exogenous substrate.     

Combination of Ascorbate and α-Tocopherol as a Preventive Therapy against Structural and Functional Defects of Erythrocytes in Visceral Leishmaniasis

The redox unbalance in erythrocytes has been found to contribute significantly in the development of anemia in visceral leishmaniasis (VL). The present study revealed enhanced production of reactive oxygen species (ROS) and gradual depletion of α-tocopherol and ascorbate in the erythrocytes of infected animals. The response of erythrocytes to chronic treatment with antioxidants was studied in hamsters during leishmanial infection. Treatment with a combination of α-tocopherol and ascorbate proved to be the most effective preventive for the proteolytic degradation of erythrocyte membrane. Erythrocytes from infected animals were thermally more sensitive compared to the control ones. Combination of both antioxidants was most successful in resisting heat induced structural defects in the cells. Cross-linking of membrane proteins subsequent to oxidative damage in the red cells was accompanied by the formation of high molecular weight protein band at the top of the resolving gel in the presence of the cross-linking agent dimethyladepimidate (DMA). Marked inhibition of cross-linking was observed with combination of both antioxidants. Treatment with α-tocopherol and ascorbate together could withstand osmotic lysis of erythrocytes in the infected animals very efficiently. Decreased hemoglobin (Hb) level was successfully replenished and was coupled with significant increase in the life span of red cells after treating the animals with both antioxidants. Results indicate better efficacy of the combination therapy with α-tocopherol and ascorbate in protecting the erythrocytes from structural and functional damages during leishmanial infection.     

Combination of ascorbate and alpha-tocopherol as a preventive therapy against structural and functional defects of erythrocytes in visceral leishmaniasis

The redox unbalance in erythrocytes has been found to contribute significantly in the development of anemia in visceral leishmaniasis (VL). The present study revealed enhanced production of reactive oxygen species (ROS) and gradual depletion of alpha-tocopherol and ascorbate in the erythrocytes of infected animals. The response of erythrocytes to chronic treatment with antioxidants was studied in hamsters during leishmanial infection. Treatment with a combination of alpha-tocopherol and ascorbate proved to be the most effective preventive for the proteolytic degradation of erythrocyte membrane. Erythrocytes from infected animals were thermally more sensitive compared to the control ones. Combination of both antioxidants was most successful in resisting heat induced structural defects in the cells. Cross-linking of membrane proteins subsequent to oxidative damage in the red cells was accompanied by the formation of high molecular weight protein band at the top of the resolving gel in the presence of the cross-linking agent dimethyladepimidate (DMA). Marked inhibition of cross-linking was observed with combination of both antioxidants. Treatment with alpha-tocopherol and ascorbate together could withstand osmotic lysis of erythrocytes in the infected animals very efficiently. Decreased hemoglobin (Hb) level was successfully replenished and was coupled with significant increase in the life span of red cells after treating the animals with both antioxidants. Results indicate better efficacy of the combination therapy with alpha-tocopherol and ascorbate in protecting the erythrocytes from structural and functional damages during leishmanial infection.     

Vesicle-to-cell protein transfer: insertion of band 3, the erythrocyte anion transporter, into lymphoid cells

Band 3, the erythrocyte anion transporter, transfers spontaneously between human red cells and model membranes. During incubation of intact erythrocytes with sonicated dimyristoylphosphatidylcholine vesicles, the transporter inserts in functional form and native orientation into the liposome bilayer, with the cytoplasmic segment of the protein contacting the lumen of the vesicle [Newton, A. C., Cook, S. L., & Huestis, W. H. (1983) Biochemistry 22, 6110-6117; Huestis, W. H., & Newton, A. C. (1986) J. Biol. Chem. 261, 16274-16278]. When band 3-vesicle complexes are incubated with erythrocytes whose native band 3 has been inhibited irreversibly, reverse transfer of the protein restores anion transport capacity to the cells [Newton, A. C., Cook, S. L., & Huestis, W. H. (1983) Biochemistry 22, 6110-6117]. Here we report the vesicle-mediated transfer of band 3 to human peripheral blood lymphocytes and to cultured murine lymphoma cells (BL/VL3). Subsequent to incubation with protein-vesicle complexes, both lymphoid cell types exhibit a 2-4-fold increase in the rate of chloride uptake. This enhanced permeability is inhibited greater than or equal to 98% by the exofacial band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, consistent with right-side-out insertion of functional band 3 into the lymphoid cell membrane.     

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.     

Glyceraldehyde-3-phosphate dehydrogenase of rat erythrocytes has no membrane component

In the course of studying mammalian erythrocytes we noted prominent differences in the red cells of the rat. Analysis of ghosts by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed that membranes of rat red cells were devoid of band 6 or the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). Direct measurements of this enzyme showed that glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was about 25% of that in human cells; all of the glyceraldehyde-3-phosphate dehydrogenase activity in rat erythrocytes was within the cytoplasm and none was membrane bound; and in the human red cell, about 1/3 of the enzyme activity was within the cytoplasm and 2/3 membrane bound. The release of glyceraldehyde-3-phosphate dehydrogenase from fresh rat erythrocytes immediately following saponin lysis was also determined using the rapid filtration technique recently described. The extrapolated zero-time intercepts of these reactions confirmed that, in the rat erythrocyte, none of the cellular glyceraldehyde-3-phosphate dehydrogenase was membrane bound. Failure of rat glyceraldehyde-3-phosphate dehydrogenase to bind to the membranes of the intact rat erythrocyte seems to be due to cytoplasmic metabolites which interact with the enzyme and render it incapable of binding to the membrane.     

The kinetics of haemolysis of spherocytic erythrocytes

Spherocytosis is a hereditary disease. It results from mutations in genes that encode proteins participating in the attachment of the membrane skeleton to the plasma membrane bilayer of the erythrocyte. In affected cells, interaction between the spectrin-actin meshwork and integral membrane proteins is altered. This results in the weakening of plasma membrane mechanical resistance and diminishing its elasticity. Since defective cells are prone to mechanical destruction and phagocytosis in the spleen, the fraction of morphologically-altered erythrocytes is rather small; this in turn means such an examination is prone to errors. In this paper, we describe a simple method which could be useful in the identification of red blood cells with altered osmotic properties. The method is based on the measurement of the amount of light scattered by a suspension of the red blood cells, during which cells are exposed to osmotic stress in the stopped-flow regime. The obtained plots are fitted to a mathematical formula, the parameters of which can be used as quantitative indicators of the changes in red blood cells' osmotic features. Two types of spherocytotic samples were examined: those with a proven deficiency in ankyrin and those with a decrease in the band 3 anion transporting protein. The presented data show that this method gives a reliable indication of altered osmotic properties of the spherocytic cells.     

Immunoelectron microscopic detection of band 3 protein during erythroid cell differentiation by a monoclonal antibody

We created a monoclonal antibody, designated EB1 (IgM, kappa), that reacts with erythroblasts by fusion of P3-X63-Ag8.653 with splenocytes of rats immunized with erythroblastic islands isolated from mice spleens. Western blotting revealed that EB1 reacted with the band 3 protein of the erythrocytic membrane. It stained erythrocytes and erythroblasts, forming clusters in the bone marrow, splenic red pulp, and fetal liver, but did not stain other tissues in the cryostat sections. The EB1 antigen was detected during dimethyl sulfoxide-induced differentiation of murine erythroleukemia cells. Immunoelectron microscopy revealed that the EB1 antigen was expressed from the basophilic erythroblasts during normal erythroid differentiation. Preferential segregation of the EB1 antigen on the cell membrane of the nucleating erythroblasts was not observed. These results suggest that EB1 is specific for erythrocyte band 3 protein and may be useful for studying erythroid cell differentiation.     

The band 3-rich membrane of llama erythrocytes: Studies on cell shape and the organization of membrane proteins

The erythrocyte membrane of the llama was characterized in comparison to that of the human. The llama erythrocyte was an elliptical disk that resisted shape alterations in hyperosmotic buffers and following metabolic depletion, both of which induce speculation of the human red cell. Lysophosphatidylcholine incorporation produced minor serrations of the edge of the llama disk but no spicules, whereas human red cells became sphero-echinocytes. The polypeptide profiles in the membranes of the two species were similar, except for several noteworthy differences: a marked elevation in the relative content of band 3; the absence of membrane-bound band 6; and simpler glycoprotein pattern in the llama. The concentration of band 3 in llama was about two and a half to three times that in the human and intramembrane particles in the protoplasmic leaflet of freeze-fractured llama membrane were correspondingly increased. The selective solubilization of bands 1, 2 and 5 in low ionic strength buffer, and all of the peripheral proteins in high alkaline buffer were similar except for increased retention of ankyrin by the llama membrane. These data suggest a similar disposition of membrane proteins. The llama membrane was markedly resistant to the solubilization of integral proteins by the nonionic detergent, Triton X-100. This property and the general resistance to shape changes may be related to the high concentration of band 3.     

Caffeine inhibits erythrocyte membrane derangement by antioxidant activity and by blocking caspase 3 activation

The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation–deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine–haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO₂ thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane.     

Plasmodium falciparum histidine-rich protein 1 associates with the band 3 binding domain of ankyrin in the infected red cell membrane

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm. Changes occur in the infected erythrocyte due to altered phosphorylation of proteins and to novel interactions between host and parasite proteins, particularly at the membrane skeleton. In erythrocytes, the spectrin based red cell membrane skeleton is linked to the erythrocyte plasma membrane through interactions of ankyrin with spectrin and band 3. Here we report an association between the P. falciparum histidine-rich protein (PfHRP1) and phosphorylated proteolytic fragments of red cell ankyrin. Immunochemical, biochemical and biophysical studies indicate that the 89 kDa band 3 binding domain and the 62 kDa spectrin-binding domain of ankyrin are co-precipitated by mAb 89 against PfHRP1, and that native and recombinant ankyrin fragments bind to the 5' repeat region of PfHRP1. PfHRP1 is responsible for anchoring the parasite cytoadherence ligand to the erythrocyte membrane skeleton, and this additional interaction with ankyrin would strengthen the ability of PfEMP1 to resist shear stress.