Electrical hemolysis of human and bovine red blood cells

Summary The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50% hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50% hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Electrical hemolysis of human and bovine red blood cells.

The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50 per cent hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50 per cent hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be approximately 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.     

Low-pH association of proteins with the membranes of intact red blood cells. I. Exogenous glycophorin and the CD4 molecule

Glycophorin and CD4 proteins are tightly associated with intact human erythrocyte membranes after a short-time incubation at low pH (1-2 min, pH lower than 5, 37 degrees C). Flow cytometry and epifluorescence microscope observations showed that after incubation of red cells with fluorescein isothiocyanate (FITC) labeled glycophorin at pH values lower than 5, the erythrocyte membrane and subsequently formed ghost membranes were fluorescent. Unlabeled glycophorin was reacted with mouse erythrocytes using the same low-pH conditions. Flow cytometry and fluorescence microscopy showed that anti-glycophorin monoclonal antibodies were able to recognize the epitopes of glycophorin associated with the mouse erythrocytes. Kinetic experiments showed that the interaction of FITC-glycophorin with red cell membranes can be monitored by a decrease in the fluorescence intensity. Erythrocyte associated glycophorin was not removed from the membranes after 24 h incubation in human plasma (in vitro, 39 degrees C). A glycoprotein extract containing CD4 was isolated from a T4-lymphoma cell line (CEM). This protein extract was incubated with erythrocytes using the same low-pH conditions. Fluorescently labeled monoclonal antibodies against CD4 stained the red cells after association of CD4 with the membranes. Electron microscopy showed 10 nm immunoglobulin G-coated gold beads associated with CD4-bearing erythrocyte membranes after incubation with anti-CD4 antibodies and then with the gold beads. The potential use of the CD4-erythrocyte complex as a therapeutical agent against acquired immune deficiency syndrome (AIDS) is suggested.     

Lipopolysaccharide from Proteus mirabilis O29 induces changes in red blood cell membrane lipids and proteins

Alterations in red blood cell (RBC) plasma membranes, i.e. in lipids and proteins, and osmotic fragility of these cells after treatment with Proteus mirabilis O29 endotoxin (lipolysaccharide (LPS)) were examined using a spin labelling method. At the highest concentration of LPS, insignificantly decreased fluidity of membrane lipids was observed. Changes in conformation of membrane proteins were determined by two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (MSL) and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The analysis of spectra of MSL and ISL showed modifications in membrane proteins in red blood cells treated with the highest concentration of lipopolysaccharide. On the other hand, in the case of isolated membranes, disturbances in membrane were observed for all concentrations of LPS. The alterations in membrane lipids and proteins are paralleled in a significant rise in osmotic fragility of RBCs upon endotoxin treatment. These results provide experimental evidence that P. mirabilis O29 LPS causes deleterious changes in membranes of human red blood cells. They show that action of lipopolysaccharide mainly concerns the membrane cytoskeleton.     

Cystic fibrosis transmembrane conductance regulator in human and mouse red blood cell membranes and its interaction with ecto-apyrase

Elevated blood ATP and increased red blood cell (RBC) ATP transport is associated with cystic fibrosis (CF). In this report, we demonstrate the presence of the wild-type and the DeltaF508 mutant form of the CF transmembrane conductance regulator protein in RBC membranes and its putative interaction with ecto-apyrase, an ATP hydrolyzing enzyme also present in the RBC membrane. RBC membranes of control and DeltaF508 individuals and of wild-type and CF transmembrane conductance regulator-knockout mice were examined by immunoblot using several antibodies directed against different epitopes of this protein. These experiments indicated that human RBC membranes contain comparable amounts of the wild-type CF transmembrane conductance regulator protein and the DeltaF508 mutant form of the protein, respectively. CF transmembrane conductance regulator protein was also detected in wild-type mouse RBC membranes but not in the gene knockout mouse RBC membranes. Antibodies directed against ecto-apyrase co-immunoprecipitated CF transmembrane conductance regulator protein of human RBC membranes indicating a physical interaction between these two membrane proteins consistent with ATP transport and extracellular hydrolysis. We conclude that RBCs are a significant repository of CF transmembrane conductance regulator protein and should provide a novel system for evaluating its expression and function.     

The effects of cholesterol oxidation products in sickle and normal red blood cell membranes.

The oxysterol content in normal and sickle red blood cell (RBC) membranes was assessed using thin-layer chromatography and capillary gas chromatography/mass spectrometry. Several more oxysterols were present in sickle RBCs compared to normal RBCs. Sickle RBC membranes had a higher concentration of 5 alpha,6 alpha-epoxycholesterol, 5 alpha-cholestane-3 beta,5,6 beta-triol, 7-ketocholesterol and 19-hydroxycholesterol than normal RBC membranes. The increased oxysterols in sickle RBC may be an effect of the increased oxidative stress which occurs in sickle RBC membranes. Physical characteristics of normal and sickle RBC membrane ghosts with and without inserted oxysterols were examined by Fourier transform infrared spectroscopy. The data are consistent with a greater sterol content in sickle cells compared to normal RBC membranes, and a possible oxysterol-cholesterol synergism.     

Reversible deformation-dependent erythrocyte cation leak. Extreme sensitivity conferred by minimal peroxidation.

To determine the threshold at which red blood cells (RBC) begin to manifest deformation-dependent leakiness to monovalent cations, we examined net passive potassium leak during elliptical deformation. Normal RBC did not begin to leak appreciable amounts of potassium until shear stress reached 204 dyn/cm2, at which point they had attained greater than 96% of their maximal deformation. In striking contrast, RBC that had undergone minimal, physiologically relevant degrees of peroxidative damage induced by t-butylhydroperoxide began to leak potassium at only 59 dyn/cm2 when they had reached only 63% of their maximal deformation. The cation leak identified in this manner is not prelytic, and it is fully reversible. Therefore, these data may be relevant to abnormal cation leaks that develop in sickle red cells that have membranes damaged by auto-oxidative stress and that manifest an exuberant but reversible leakiness to monovalent cation during sickling-induced deformation of the cell membrane.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane.

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10-3-10-4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strength it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yeilds a mean value of about 1.6 V. for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 degrees C. as shown by incorporation of 131I-labeled albumin and repeated dielctric breakdown.     

Effect of cholesterol on the stability of human erythrocyte membranes to electric breakdown

Electrical stability of human erythrocyte membranes with different cholesterol content was studied. Breakdown in the cell membranes was generated by application of electric pulses with field strengths of 1.4-3.2 kV/cm. The share of perforated cells was registered by measuring hemolysis level. The red blood cells from patients with psoriasis and normal erythrocytes after incubation in the presence of liposomes were used as a model of cells with cholesterol-rich membranes. It was discovered that an increase of cholesterol content in the membranes moved the field-dependent curves to a higher field range. The obtained effect is attributed to the increase of the breakdown membrane potential. Application of high-pulse-electric-field technique for investigating the properties of cell membranes is discussed.     

Kinetics of pH-dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red blood cells. Measurement by dequenching of fluorescence

Fusion between membranes of 3T3 fibroblasts expressing hemagglutinin (HA) from the Japan strain of influenza virus and human red blood cells (RBC) was measured using an assay for lipid mixing based on the relief of self-quenching (dequenching) of fluorescence of the lipid probe octadecylrhodamine (R18). The probe was incorporated into the membrane of intact RBC at self-quenching concentrations, and the RBCs were bound to the 3T3 cells. Fusion, which allowed movement of R18 into 3T3 cell membranes, was monitored by spectrofluorometry as an increase in fluorescence. Upon lowering the pH below 5.4, the fluorescence increased after a delay of about 30 s at 37 degrees C, and leveled off within 2 min. In control experiments where R18 RBCs bound to 3T3 cells expressing the uncleaved precursor hemagglutinin (HA0) were incubated at 37 degrees C and low pH, no fluorescence increase was observed. This indicated that the R18 dequenching occurred as a result of HA-induced fusion of plasma membranes. Fusion showed a very steep pH dependence with a threshold at pH 5.4 and a maximum at pH 5.0, similar to HA-induced fusion seen previously using cell biological techniques. The fusion rate increased and the delay for the onset of fusion decreased as the temperature was raised above 20 degrees C. Low pH activation of the fusion process at 37 degrees C could be partially arrested by raising the pH after 2-10 s, but not after 15 s, indicating that the irreversible pH-activated conformational change of HA necessary for fusion was complete within about 15 s. Analysis of the data indicates that the pH-induced membrane fusion activity of HA is a highly cooperative event.     

Influence of the membrane undercoat on filipin perturbation of the red blood cell membrane

Filipin, a polyene antibiotic, interacts with beta-hydroxy sterols such as cholesterol in most cell membranes, forming bumps and pits that are visible by electron microscopy of freeze-fracture replicas. The markedly reduced perturbability of the red blood cell (RBC) membrane, compared to other cells, has been attributed to the constraining influence of the red cell membrane skeleton, the undercoat composed of spectrin, actin, and protein 4.1. To test the influence of the membrane skeleton on filipin-induced perturbation of the RBC membrane, we studied the interaction of filipin with red cells that were inherently devoid of spectrin and RBC in which spectrin had been crosslinked or denatured. These spectrin-deficient, crosslinked, and denatured cells have a fivefold increase in the number of filipin-induced perturbations as compared to control cells, despite equivalent membrane cholesterol content. These findings confirm that the spectrin-based membrane skeleton strongly influences the organization of the membrane so as to limit perturbation by filipin:cholesterol interaction and that for membranes in which the cholesterol content is known, filipin is a useful probe for testing the avidity of spectrin-based cytoskeletal attachment.     

A novel pathway of human T lymphocyte activation. Identification by a monoclonal antibody generated against a rheumatoid synovial T cell line

Substantial evidence indicates that compartmentalized infiltrates of T lymphocytes are central to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, but the mechanisms by which such cells become activated remain unknown. To define surface components of activation pathways important in the function of these cells, we have generated mAb against a rheumatoid synovial T cell line. One such antibody, termed anti-UM4D4, reacts with an Ag, termed UM4D4, which is strongly expressed on most rheumatoid synovial T cell lines and clones, and on a subset of peripheral blood T cells, resting or activated. Anti-UM4D4 is mitogenic in soluble form for PBMC and certain T cell clones, and is comitogenic with the phorbol ester PMA for purified resting T lymphocytes. These functional effects are similar to those previously observed with antibodies to epitopes of CD2 and CD3, surface Ag involved in two well defined pathways of human T cell activation. Binding of anti-UM4D4 to T cells is not, however, blocked by antibodies directed at various epitopes of CD2 and CD3. Moreover, UM4D4 does not comodulate with CD3, and is expressed on a T cell line that lacks CD2, CD3, and CD28. The data, therefore, indicate that anti-UM4D4 identifies a T cell activation pathway, distinct from those previously described, that could play a role in the pathogenesis of T cell-mediated autoimmune diseases.     

Lateral mobility of a lipid analog in the membrane of irreversible sickle erythrocytes

The major feature of sickle cell anemia is the tendency of erythrocytes to sickle when exposed to decreased oxygen tension and to unsickle when reoxygenated. Irreversible sickle cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation. ISCs are believed to owe their biophysical abnormalities to acquired membrane alterations which decrease membrane deformability. While increased membrane surface viscosity has been measured in ISCs, the lateral dynamics of membrane lipids in these cells have not heretofore been examined. We have measured the lateral diffusion of the lipid analog 3,3'-dioctadecylindocyanine iodide (DiI) in the plasma membrane of intact normal erythrocytes, reversible sickle cells (RSCs), and irreversible sickle cells by fluorescence photobleaching recovery (FPR). The diffusion coefficients +/- standard errors of the mean of DiI in intact normal red blood cells (RBCs), RSCs, and ISCs at 37 degrees C are (8.06 +/- 0.29) X 10(-9) cm2 X s-1, (7.74 +/- 0.22) X 10(-9) cm2 X s-1, and (7.29 +/- 0.24) X 10(-9) cm2 X s-1, respectively. A similar decrease in the diffusion coefficient of DiI in the plasma membranes of the three cell types was observed at 4, 10, 17, 23, and 30 degrees C. ANOVA analysis of the changes in DiI diffusion showed significant differences between the RBC and ISC membranes at all temperatures examined. The characteristic breaks in Arrhenius plots of the diffusion coefficients for the RBCs, RSCs, and ISCs occurred at 20, 19, and 18.6 degrees C, respectively. Photobleaching recovery data were used to estimate (Boullier, J.A., Melnykovich, G. and Barisas, B.G. (1982) Biochim. Biophys. Acta 692, 278-286) the microviscosities of the plasma membranes of the three cell types at 25 degrees C. We find significant differences between our microviscosity values and those obtained in previous fluorescence depolarization studies. However, both methods indicate qualitatively similar differences in membrane microviscosity among the various cell types.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10³–10⁴ V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strenght it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yields a mean value of about 1.6 V for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 °C as shown by incorporation of ¹³¹I-labeled albumin and repeated dielectric breakdown.     

Effect of procaine hydrochloride on the electrophoretic mobility of human red blood cells

The interaction of cationic anesthetics with biological membranes and the resulting alterations of membrane electrokinetic properties continue to be of current interest. The present study was designed to examine the effects of procaine hydrochloride (PRHCL) on the mobility of human red blood cells (RBC); electrophoretic measurements were made on RBC suspended in phosphate-buffered saline (PBS, pH = 5.0, 7.4, or 9.2), autologous plasma or 3 g% dextran T70/PBS (pH = 7.4), with PRHCL concentrations from 8 x 10(-6) to 8 x 10(-2) M. Low concentrations of PRHCL (8 x 10(-5)-8 x 10(-3) M) significantly (p less than 0.001) increased RBC mobility, with a maximal increase of 8.2% at 8 x 10(-4) M. Conversely, a higher PRHCL concentration (8 x 10(-2) M significantly (p less than 0.001) decreased RBC mobility. Both glutaraldehyde fixation and lipid extraction abolished any PRHCL-induced increase in RBC mobility; the observed increases in mobility for normal cells are, thus, consistent with a mechanism based on expansion of the RBC membrane glycocalyx. Microelectrophoretic methods were also used to study the effect of PRHCL (8 x 10(-4) and 8 x 10(-2) M) on RBC membrane calcium binding, with the results indicating that PRHCL competes with calcium for neuraminate binding sites. We conclude that the observed changes in RBC electrokinetic properties reflect incorporation of PRHCL into the RBC membrane; such changes may be of importance in modulating cell-cell interactions.     

T细胞亚群CD4测定方法的研究

为比较外周血T淋巴细胞亚群CD4不同测定方法的差别,以流式细胞术为定量手段,测定了猴外周血中三种不同方法处理后CD4的表达.结果表明：先标后溶法——先用异硫氰荧光素标记的单克隆抗体（FITC-CD4 McAb）标记后,再加入红细胞溶解液溶掉红细胞的处理方法,结果基本等同于传统的淋巴细胞分离法,但样本用量仅为传统方法的1/5,且操作简单.激光共焦显微术的形态学研究也证实：先标后溶法与淋巴细胞分离法相似,其细胞膜表面荧光标记清晰,优于先溶后标法.     

The Effects of Ethanol on the Morphological and Biochemical Properties of Individual Human Red Blood Cells

Here, we report the results of a study on the effects of ethanol exposure on human red blood cells (RBCs) using quantitative phase imaging techniques at the level of individual cells. Three-dimensional refractive index tomograms and dynamic membrane fluctuations of RBCs were measured using common-path diffraction optical tomography, from which morphological (volume, surface area, and sphericity); biochemical (hemoglobin (Hb) concentration and Hb content); and biomechanical (membrane fluctuation) parameters were retrieved at various concentrations of ethanol. RBCs exposed to the ethanol concentration of 0.1 and 0.3% v/v exhibited cell sphericities higher than those of normal cells. However, mean surface area and sphericity of RBCs in a lethal alcoholic condition (0.5% v/v) are not statistically different with those of healthy RBCs. Meanwhile, significant decreases of Hb content and concentration in RBC cytoplasm at the lethal condition were observed. Furthermore, dynamic fluctuation of RBC membranes increased significantly upon ethanol treatments, indicating ethanol-induced membrane fluidization.     

Plasmodium lophurae: membrane proteins of erythrocyte-free plasmodia and malaria-infected red cells

Plasma membranes of normal duckling erythrocytes were prepared by blender homogenization and nitro-en decompression. Surface membrane vesicles of red cells infected with the avian malaria Plasmodium lophurae were produced by nitrogen decompression. Membranes of erythrocyte-free malaria parasites were removed from cytoplasmic constituents by Dounce homogenization. These membranes were collected by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Red cell membranes had a buoyant density of 1.159 g/cm3, whereas plasmodial membranes banded at 2 densities: 1.110 g/cm3 and 1.158 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the isolated red cell membranes revealed 7 major protein bands with molecular weights (MW) ranging from 230, 000 to 22,000, and 3 glycoprotein bands with MW of 160,000, 88,000 and 37,000. Parasite membranes also had 7 major bands with MW ranging from 100,000 to 22,000. No glycoproteins were identifiable in these membranes. The proteins of the surface membranes from infected red cells had MW similar to those from normal red cells; however, there was some evidence of a reduction in the amount of the high MW polypeptides. The red cell membrane contained 79 nmoles sialic acid/mg membrane protein, whereas plasmodial membranes had 8 nmoles sialic acid/mg membrane protein. The sialic acid content of the surface membranes of infected red cells was significantly smaller than that of normal cells. Lactoperoxidase-glucose oxidase-catalyzed iodination of intact normal and malaria-infected erythrocytes labeled 7 surface components. Although no observable differences in iodinatable proteins were seen in these preparations, there was a striking reduction in the iodinatability of erythrocytic membranes obtained from P. lophurae-infected cells. Erythrocyte-free plasmodia bound very little radioactive iodine; the small amount of radioactivity was distributed among 3 major bands with MW of 42,000, 32,000 and 28,000. It is suggested that the alterations of the surface of the P. lophurae-infected erythrocyte do not occur by a wholesale insertion of plasmodial membrane proteins into the red cell plasma membrane, but rather that there are parasite-mediated modifications of existing membrane polypeptides.     

A spectrin-like protein in retinal rod outer segments

Biochemical and immunochemical studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the alpha-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghosts and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labeled the alpha-chain of RBC spectrin having an apparent molecular weight of 240,000. Polyclonal anti-spectrin antibody that bound to both the alpha- and beta-chain of RBC spectrin predominantly labeled a Mr 240,000 polypeptide of ROS membranes. Two faintly labeled bands in the molecular weight range of 210,000-220,000 were also observed. These components may represent variants of the beta-chain of spectrin that are weakly cross-reacting or present in smaller quantities than the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

T and B cell epitope mapping of SM23, an integral membrane protein of Schistosoma mansoni.

SM23 is an integral membrane protein of the blood-vessel dwelling parasitic worm Schistosoma mansoni. This protein has been detected with antibodies in all stages of the parasite found in the human host, notably the lung stage, and therefore is of interest as a vaccine candidate. In addition SM23 has been shown to be a member of a proposed new superfamily of membrane proteins whose structures do not conform to the previously known classifications. To date there are 13 members including ME491 (CD63, Pltgp40), CD9 (p23), TAPA-1, CD37, CD53, MRC OX-44, CO-029, MRP-1, L6, the gene product of TI-1, the target of mAb AD-1, SM23, and SJ23 (the Schistosoma japonicum homologue). Most of these molecules except for those in the two blood vessel-dwelling parasites are found in membranes of hemopoietic and/or malignant cells and all have unknown function. In this study we used recombinantly expressed full-length and partial molecules as well as synthesized peptides to map T cell and B cell epitopes of SM23. The two predicted external hydrophilic domains were found to be highly immunogenic and contained several B cell epitopes. There were at least four T cell epitopes in the large hydrophilic domain. One segment of 23 amino acids contained both a T cell and B cell epitope as well as the putative glycosylation site. This particular segment was recognized by immune sera and cells of every mouse strain tested. The elucidation of these epitopes demonstrates the immunogenic nature of this molecule and raises questions as to the role of SM23 in the host/parasite relationship.