Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components

Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane perturbants. Fluorescence photobleaching recovery was used to measure the lateral mobility of two integral membrane proteins, glycophorin and band 3, and two lipid analogues, fluorescein phosphatidylethanolamine (Fl-PE) and carbocyanine dyes, in RBCs and ghosts adherent to schistosomula. Adherent ghosts manifested 95-100% immobilization of both membrane proteins and 45-55% immobilization of both lipid probes. In separate experiments, diamide-induced cross-linking of RBC cytoskeletal proteins slowed transmembrane protein diffusion by 30-40%, without affecting either transmembrane protein fractional mobility or lipid probe lateral mobility. Wheat germ agglutinin- and polylysine-induced cross-linking of glycophorin at the extracellular surface caused 80-95% immobilization of the transmembrane proteins, without affecting the fractional mobility of the lipid probe. Egg lysophosphatidylcholine (lysoPC) induced both lysis of RBCs and a concentration-dependent decrease in the lateral mobility of glycophorin, band 3, and Fl-PE in ghost membranes. At a concentration of 8.4 micrograms/ml, lysoPC caused a pattern of protein and lipid immobilization in RBC ghosts identical to that in ghosts adherent to schistosomula. Schistosomula incubated with labeled palmitate released lysoPC into the culture medium at a rate of 1.5 fmol/h per 10(3) organisms. These data suggest that lysoPC is transferred from schistosomula to adherent RBCs, causing their lysis.     

Challenges for red blood cell biomarker discovery through proteomics

Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Molecular basis of red cell membrane rheology. Part 1

The biorheological properties and behavior of red blood cells (RBCs), as other types of cells, have a biochemical and molecular basis. The shape maintenance and deformability of RBCs depend on the structural and functional integrity of the membrane proteins. These proteins are composed of transmembrane proteins inserted in the lipid bilayer, the skeletal proteins forming a network lining the membrane endoface, and the linking proteins which link together the other two types of proteins to form a three-dimensional protein structure to effect the complex and intricate biorheological functions of the RBC. The application of molecular biological techniques has led to the establishment of the molecular structures of all major RBC membrane proteins and generated insights into the nature and energy of protein interactions in the membrane. Abnormalities or deficiencies of these proteins in hereditary disorders in humans and animals have offered opportunities to assess the rheological significance of each of these proteins and their interactions. Parallel molecular biological and biorheological studies on RBC membranes under a variety of conditions can provide the fundamental information required for theoretical modeling of RBC membrane rheology at the molecular level. Such interdisciplinary research will contribute to not only the elucidation of normal rheology of RBCs and other types of cells, but also the understanding of pathorheology of their disorders and the development of new methods of diagnosis and treatment.     

The inactivation of the π gene in chicken erythroblasts of adult lineage is not mediated by packaging of the embryonic part of the α-globin gene domain into a repressive heterochromatin-like structure

The developmental switch of globin gene expression is a characteristic feature of vertebrate organisms. The switch of β-globin expression is believed to depend on reconfiguration of the active chromatin hub, which contains transcribed genes and regulatory elements. Mechanisms controlling the switch of α-globin gene expression are less clear. Here, we studied the mode of chromatin packaging of the chicken α-globin gene domain in red blood cells (RBCs) of primitive and definite lineages and the spatial configuration of this domain in RBCs of primitive lineage. It has been demonstrated that RBCs of primitive lineage already contain the adult-type active chromatin hub but the embryonal α-type globin π gene is not recruited to this hub. Distribution of active and repressive histone modifications over the α-globin gene domain in RBCs of definite and primitive lineages does not corroborate the hypothesis that inactivation of the π gene in RBCs of adult lineage is mediated via formation of a local repressed chromatin domain. This conclusion is supported by the demonstration that in chicken erythroblasts of adult lineage, the embryonal and adult segments of the α-globin gene domain show similar elevated sensitivities to DNase I.     

The inactivation of the π gene in chicken erythroblasts of adult lineage is not mediated by packaging of the embryonic part of the α-globin gene domain into a repressive heterochromatin-like structure

The developmental switch of globin gene expression is a characteristic feature of vertebrate organisms. The switch of β-globin expression is believed to depend on reconfiguration of the active chromatin hub, which contains transcribed genes and regulatory elements. Mechanisms controlling the switch of α-globin gene expression are less clear. Here, we studied the mode of chromatin packaging of the chicken α-globin gene domain in red blood cells (RBCs) of primitive and definite lineages and the spatial configuration of this domain in RBCs of primitive lineage. It has been demonstrated that RBCs of primitive lineage already contain the adult-type active chromatin hub but the embryonal α-type globin π gene is not recruited to this hub. Distribution of active and repressive histone modifications over the α-globin gene domain in RBCs of definite and primitive lineages does not corroborate the hypothesis that inactivation of the π gene in RBCs of adult lineage is mediated via formation of a local repressed chromatin domain. This conclusion is supported by the demonstration that in chicken erythroblasts of adult lineage, the embryonal and adult segments of the α-globin gene domain show similar elevated sensitivities to DNase I.     

Fusion Activity of Transmembrane and Cytoplasmic Domain Chimeras of the Influenza Virus Glycoprotein Hemagglutinin

The role of the sequence of transmembrane and cytoplasmic/intraviral domains of influenza virus hemagglutinin (HA, subtype H7) for HA-mediated membrane fusion was explored. To analyze the influence of the two domains on the fusogenic properties of HA, we designed HA-chimeras in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. These chimeras, as well as constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type1 (NF1) or c-Raf-1, NF78 (residues 1441 to 1518), and Raf81 (residues 51 to 131), respectively, were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Wild-type and chimeric HA were cleaved properly into two subunits and expressed as trimers. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins was studied by a lipid-mixing assay with the lipid-like fluorophore octadecyl rhodamine B chloride (R18). No profound differences in either extent or kinetics could be observed. After the pH was lowered, the above proteins also induced a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-1 cells, indicating that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore. We conclude that neither HA-specific sequences in the transmembrane and cytoplasmic domains nor their length is crucial for HA-induced membrane fusion activity.     

The accumulation of basic nonhemoglobin proteins during the differentiation of primitive (embryonic) red cells of amphibia

The preferential accumulation of hemoglobin is a characteristic of the differentiation of definitive (adult) red blood cells. Since primitive (embryonic or larval) red blood cells have many properties which contrast with definitive red cells, the accumulation of red cell proteins was analyzed during the differentiation of primtive red cells to determine whether or not hemoglobin was the only protein which showed a substantial increase in amount. Primitive red cells of amphibia were used because the mature circulating cell retains large amounts (13%) of specific, well characterized, nonhemoglobin proteins (CP). Preparations of primitive red cells enriched in immature cells were obtained from the circulation of bullfrog tadpoles recovering from phenylhydrazine-induced anemia. The amount of CP, determined by electrophoresis, imunodiffusion, and ion-exchange chromatography, was compared for red cells from normal animals and anemic animals. Mature cells contained three to six times the amount of CP and three times the amount of hemoglobin found in the population of red cells enriched in immature cells. The accumulation of CP during maturation of primitive red cells indicates that differentiation of primitive red cells is less restrictive than differentiation of definitive red cells. Since the primitive red cell is less specialized than the definitive red cell, it is possible that primitive red cells have several roles in the developing animal, in contrast to the single role of synthesizing and maintaining hemoglobin in the adult animal.     

Global proteome discovery using an online three-dimensional LC-MS/MS

We have developed a proteomics technology featuring on-line three-dimensional liquid chromatography coupled to tandem mass spectrometry (3D LC-MS/MS). Using 3D LC-MS/MS, the yeast-soluble, urea-solubilized peripheral membrane and SDS-solubilized membrane protein samples collectively yielded 3019 unique yeast protein identifications with an average of 5.5 peptides per protein from the 6300-gene Saccharomyces Genome Database searched with SEQUEST. A single run of the urea-solubilized sample yielded 2255 unique protein identifications, suggesting high peak capacity and resolving power of 3D LC-MS/MS. After precipitation of SDS from the digested membrane protein sample, 3D LC-MS/MS allowed the analysis of membrane proteins. Among 1221 proteins containing two or more predicted transmembrane domains, 495 such proteins were identified. The improved yeast proteome data allowed the mapping of many metabolic pathways and functional categories. The 3D LC-MS/MS technology provides a suitable tool for global proteome discovery.     

Identification of a role for the PfEMP1 semi‐conserved head structure in protein trafficking to the surface of Plasmodium falciparum infected red blood cells

Transport of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) variants to the red blood cell (RBC) surface enables malarial parasite evasion of host immunity by modifying the antigenic and adhesive properties of infected RBCs. In this study, we applied the Bxb1 integrase system to integrate transgenes encoding truncated PfEMP1‐GFP fusions into cytoadherent A4 parasites and characterize their surface transport requirements. Our studies revealed that the semi‐conserved head structure of PfEMP1 proteins, in combination with the predicted transmembrane region and cytoplasmic tail, encodes sufficient information for RBC surface display. In contrast, miniPfEMP1 proteins with truncated head structures were exported to the RBC cytoplasm but were not detected at the RBC surface by flow cytometry or immuno‐electron microscopy. We demonstrated the absence of a mechanistic barrier to having native and miniPfEMP1 proteins displayed simultaneously at the RBC surface. However, surface‐exposed miniPfEMP1 proteins did not convey cytoadherence properties to their host cells, implicating potential steric considerations in host‐receptor interactions or the need for multiple domains to mediate cell binding. This study establishes a new system to investigate PfEMP1 transport and demonstrates that the PfEMP1 semi‐conserved head structure is under selection for protein transport, in addition to its known roles in adhesion.     

Cathodal proteins from primitive (embryonic) red cells of amphibia. Isolation and characterization.

A group of abundant (15% of the soluble protein) nonhemoglobin proteins was isolated from the primitive (embryonic) red cells found in tadpoles, using the cationic properties of the proteins at pH 8.6 to separate them from hemoglobin and other red cell proteins. The cathodal proteins (CP) were resolved into five components, and the two most predominant proteins were separated and characterized. Purified CP-1b and CP-2 had an amino acid composition similar to that of unfractionated cathodal proteins and to each other, except for small variations in the lysine and half-cystine content. The molecular weight of the purified CP-1b and CP-2 was 13 to 14,000, determined by gel filtration chromatography and electrophoresis in the presence of sodium dodecyl sulfate. Cathodal proteins were immunologically related although there were quantitative differences in reactivity. The concentration of cathodal proteins in primitive (embryonic) red cells was 100 times that in definitive (adult) red cells coincided with the replacement of primitive red cells. The synthesis of the cathodal proteins appeared to continue throughout the life of the primitive red cells; when hemoglobin synthesis declined in primitive red cells, approximately half of the protein synthesized by the cells was cathodal protein. Although the function of the cathodal proteins is as yet unknown, the data suggest that the cathodal proteins are a unique characteristic of erythroid differentiation in early development.     

Bicarbonate permeability and immunological evidence for an anion exchanger-like protein in the red blood cells of the sea lamprey,Petromyzon marinus

Physiological and immuno-blotting experiments were used to determine whether the red blood cell membrane of a primitive vertebrate, the sea lamprey Petromyzon marinus, contained a counterpart similar to the vertebrate anion exchange protein known as AE1 or band 3. Results of the physiological experiments which measured CO₂ production after adding H¹⁴CO ₃ ^- to the extracellular saline, indicated significant transmembrane bicarbonate movement in lamprey blood which unlike that in most vertebrates, was insensitive to inhibition by 4,4 diisothiocyanatostilbene-2,2 disulfonic acid. The present study also showed that lamprey red blood cells possess acetazolamide-sensitive carbonic anhydrase which is an important component of CO₂ production by vertebrate red blood cells. Polyclonal immunoglobulins against a 12 amino acid domain in the C-terminus of the mouse AE1 recognized a trout red blood cell membrane protein with a relative molecular mass of 97 kDa, but failed to immunoreact with any membrane proteins from the red blood cells of lamprey. Antibodies against trout AE1 immunoreacted with trout red blood cell membrane proteins of approximately 97 kDa, 200 kDa and >200 kDa. Interestingly, only a 200-kDa membrane protein from the red blood cells of the primitive lamprey immunoreacted with the trout anti-AE1 immunoglobulin proteins. Therefore, lamprey red blood cells appear to possess an AE1-like protein that may be physiologically different than that in most other vertebrates.     

Effects of r—HuEPO on the biophysical characteristics of erythrocyte membrane in patients with anemia of chronic renal failure

Using electron spin resonance (ESR) spin labeling technique,we have studied the conformation of sulfhydryl groups(-SH) binding sites in membrane proteins and mem brane fluidity of red blood cells(RBCs) from two groups of patients with anemia of chronic renal failure(ACRF).One of the groups is composed of patients who were untreated with recombinant human erythropoietin(r-HuEPO),and the other is composed of patients who were treated with r-HuEPO.The results indicated:1)the conformation of SH group binding site in RBC membrane proteins from former group was different from those of healty people.2)the fluidity in the region near the surface of RBC membrane from former group was lower than those of healthy people.3)However,the above biophysical properties of RBC membrane from later group were normal.We concluded that RBC membrane in patients with ACRF was abnormal,and the treatment of r-HuEPO may promote the production of normal RBCs,thus ameliorate the biophysical properties of RBCs from the patients with ACRF.     

Poly(2-dimethylamino ethylmethacrylate)-based polymers to camouflage red blood cell antigens

Poly(2-dimethylamino-ethylmethacrylate) (PDMAEMA) is a cationic polymer when dissolved in a 7.4 pH fluid. Owing to its ionic nature, this polycation interacts with the negatively charged cell membrane surface of red blood cells (RBCs). The electrostatic self-assembly of PDMAEMA on RBCs membrane can be employed for inducing the formation of a polymeric shield camouflaging blood group antigens on RBCs as a valuable strategy for developing "universal RBCs" for blood transfusion. The purpose of this research was to evaluate the camouflaging ability of PDMAEMA homopolymers and PDMAEMA-co-poly(ethylene glycol) copolymers differing in molecular weight and architecture. Surprisingly, the PDMAEMAs caused a partially masking, no masking, and sensitization of the same RBCs population. The MW and architecture of the polymers as well as temperature of PDMAEMA-RBCs treatment influenced the results observed. Herein, the very particular reactivity of PDMAEMAs and RBCs is analyzed and discussed.     

Membrane-bound hemoglobin as a marker of oxidative injury in adult and neonatal red blood cells

A comparative study of the effect of hydrogen peroxide on adult and neonatal red blood cell (RBC) membrane protein composition has been carried out. The results indicate that (a) the native neonatal RBC membranes contain higher levels of membrane-bound hemoglobin (MBHb) than the adult RBC membranes. (b) The content of MBHb increases when RBCs are incubated with increasing concentrations of hydrogen peroxide (H2O2), more so in neonatal than in adult RBCs; however, neonatal RBC membrane proteins are less susceptible to H2O2 oxidation than adult ones. This could be attributed to the fact that Hb F, which is more susceptible to oxidation than Hb A, adds to the reduction potential of neonatal RBC (in which it is present in large amounts) and partially protects neonatal membrane proteins against oxidant stress compared to Hb A in adult RBC. (c) In both neonatal and adult RBCs, Spectrin 1 is relatively more susceptible to oxidant stress than spectrin 2, and spectrins in adult RBC are more labile for peroxidation than the spectrins in neonatal RBC. (d) Based on electrophoretic studies with and without reduction of membranes with mercaptoethanol, we have classified two types of MBHb: Type I is adsorbed to membrane by noncovalent interactions and Type II MBHb is chemically crosslinked to membrane components by disulfide bridges; the content of both these types increases when RBCs are incubated with increasing concentrations of H2O2. (e) Band 6 protein is present in higher amounts in neonatal than in adult RBC membranes. (f) Since the total content of MBHb increases linearly with the level of oxidant stress, we suggest that it could be used as a marker for oxygen radical-induced injury to tissues.     

Surface properties of crosslinked erythrocytes as studied by counter-current distribution in aqueous polymer two-phase systems

The bifunctional imidoester dimethyl suberimidate hydrochloride can stabilize rat red blood cells (RBCs) by membrane protein crosslinking, and in that way they can be used as carrier systems for exogenous substances. Counter-current distribution fractionation in charge-sensitive dextran-polyethyleneglycol two-phase systems has been used to detect slight changes in surface charge in stabilized cells. A decrease in the surface charge of crosslinked RBCs and an apparent masking of the age-related cell surface properties have been found to result from the protein crosslinking. Digitonin treatment used to permeabilize crosslinked RBCs produces a significant decrease of the cell surface charge while the age-related surface properties do not seem to be modified by the treatment.     

Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol

Cell membrane proteome analysis is limited by inherent membrane hydrophobicity. Conventional membrane protein extraction techniques use detergents, chaotropes and organic acids that require sample clean-up or pH adjustment, and are associated with significant sample loss. We extracted membrane proteins from red blood cells (RBCs) using methanol (MeOH), trifluoroethanol (TFE) and urea, and identified membrane proteins using 2-D LC coupled with MALDI-TOF/TOF-MS. We show that organic solvents MeOH- and TFE-based methods have membrane protein analysis efficiencies comparable to urea, and are complementary for the recovery of both hydrophilic and hydrophobic peptides. The mean grand average of hydropathicity (GRAVY) value of identified peptides from the TFE-based method (-0.107) was significantly higher than that of the MeOH-based method (-0.465) (p<0.001). Sequential and adjunctive use of the organic solvents MeOH and TFE increases the number of proteins identified, and the confidence of their identification. We show that this strategy is effective for shotgun membrane proteome analysis.     

Identification and function of transmembrane glycoproteins--the red cell model

Transmembrane glycoproteins in the red cell membrane traverse the plasma membrane, have their carbohydrate moieties on the extracellular surface, are sialyated (except for band 3) and are tethered to the membrane cytoskeleton proteins on the cytoplasmic surface. This linkage between the transmembrane proteins and the skeletal membrane proteins provides a two-way communication between the extracellular surface and the interior of the red cell; i.e., a transmembrane effect can be initiated from either side. These interactions are discussed in this review, including the example of sickle cell anemia in which the membrane bound hemoglobin may exert a transmembrane effect to change the conformation or distribution of transmembrane glycoproteins and and hence the extracellular surface receptors. This, in turn, may explain why sickle cells adhere to endothelium in vitro. Although the RBC transmembrane sialoglycoproteins may function in communication, regulation of cell shape, and adhesion, uncertainties exist regarding many of their functions. To study these sialoglycoproteins, we have developed a double staining technique (Dzandu et al., 1984) that differentially stains human RBC membrane sialoglycoproteins and asialoproteins in SDS-polyacrylamide gels. This should aid in elucidating the conformational structure and function of transmembrane glycoproteins.     

Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM

The surface topography of red blood cells (RBCs) was investigated under near-physiological conditions using atomic force microscopy (AFM). An immobilization protocol was established where RBCs are coupled via molecular bonds of the membrane glycoproteins to wheat germ agglutinin (WGA), which is covalently and flexibly tethered to the support. This results in a tight but non-invasive attachment of the cells. Using tapping-mode AFM, which is known as gentle imaging mode and therefore most appropriate for soft biological samples like erythrocytes, it was possible to resolve membrane skeleton structures without major distortions or deformations of the cell surface. Significant differences in the morphology of RBCs from healthy humans and patients with systemic lupus erythematosus (SLE) were observed on topographical images. The surface of RBCs from SLE patients showed characteristic circular-shaped holes with approx. 200 nm in diameter under physiological conditions, a possible morphological correlate to previously published changes in the SLE erythrocyte membrane.     

Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is an important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: one-dimensional gel liquid chromatography-tandem MS analysis (one-dimensional gel electrophoresis LC-MS/MS), biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer "sandwich" gel electrophoresis with multidimensional protein identification technology. A total of 2241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T. gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially suitable targets for the development of therapeutic strategies.     

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.