Erythrocyte membrane proteins and membrane skeleton

Considerable advances in the research field of erythrocyte membrane were achieved in the recent two decades.New findings in the structure-function correlation and interactions of erythrocyte membrane proteins have attracted extensive attention.Interesting progress was also made in the molecular pathogenesis of erythrocyte membrane disorders.Advances in the composition,function and interaction of erythrocyte membrane proteins,erythrocyte membrane skeleton,and relevant diseases are briefly described and summarized here on the basis of domestic and world literatures.     

Erythrocyte membrane proteins and membrane skeleton

Considerable advances in the research field of erythrocyte membrane were achieved in the recent two decades. New findings in the structure-function correlation and interactions of erythrocyte membrane proteins have attracted extensive attention. Interesting progress was also made in the molecular pathogenesis of erythrocyte membrane disorders. Advances in the composition, function and interaction of erythrocyte membrane proteins, erythrocyte membrane skeleton, and relevant diseases are briefly described and summarized here on the basis of domestic and world literatures. Translated from Life Science Research, 2005, 9(4): 283–291 [译自: 生命科学研究]     

Two-dimensional map of membrane proteins from human erythrocytes

Human erythrocyte membrane proteins were analyzed by a modified two-dimensional electrophoresis performed according to O'Farrell. This method was used to construct a two-dimensional map of human erythrocyte membrane proteins. The map plotted in the coordinates "relative molecular mass versus relative electrophoretic mobility during IEF" was used for the characterization of 189 proteins. The position of major membrane proteins in the map was determined on the basis of their Mr, pI as well as literature data. Carboanhydrase was positioned by coelectrophoresis. A comparative analysis of erythrocyte membrane and cytosol preparations by two-dimensional protein mapping revealed that some of erythrocyte proteins have dual localization.     

Malarial proteins that interact with the erythrocyte membrane and cytoskeleton

Several distinct classes of Plasmodium proteins have been proposed to interact with the submembrane skeleton of the erythrocyte based upon differential solubility and subcellular localization studies. That the parasite affects the erythrocyte membrane by interacting with the submembrane skeleton is an attractive hypothesis since the membrane skeleton likely regulates many aspects of membrane topography and function. The precise interactions between host and parasite proteins at the molecular level and how the parasite proteins are transported to the erythrocyte membrane are not completely understood. Experiments addressing these questions are under way, and such studies will provide valuable information about the host-parasite interface. In addition, the characterization of the interaction of Plasmodium proteins with the host erythrocyte membrane may also provide new insight into the structure and function of the erythrocyte membrane or membranes in general.     

Occurrence of creatine kinase activity in human erythrocyte membrane

Some evidences for creatine kinase activity in normal human erythrocyte membrane were presented. The creatine kinase was indicated to be a constituent of the integral proteins of erythrocyte membrane or to be tightly bound to the membrane, and was contrasted to the results obtained with adenylate kinase. Isoenzyme distribution of the erythrocyte creatine kinase by electrophoresis was identical to MM-creatine kinase from rabbit muscle.     

Identification of Re lipopolysaccharide-binding protein on murine erythrocyte membrane

Our recent studies have suggested that bacterial lipopolysaccharide (LPS) attaches to Pronase-sensitive proteins on the murine erythrocyte membrane. In the present study, in order to identify the LPS-binding protein on the murine erythrocyte membrane, a unique method to detect LPS-binding protein on a nitrocellulose membrane was developed. Murine erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred electrophoretically onto a nitrocellulose membrane. The membrane was incubated with LPS of Salmonella minnesota R595 (Re LPS) in phosphate-buffered saline (PBS), after the remaining sites were blocked with gelatin in PBS. We were able to obtain a non-background stain by adding the nonionic detergent octylglucoside at the low concentration of 0.1% to the Re LPS solution. The Re LPS bound to the protein on the nitrocellulose membrane was exposed to affinity purified anti-Re LPS antibodies (IgG) and then to alkaline phosphatase-conjugated anti-IgG. The alkaline phosphatase was detected on the membrane by an enzymatic reaction. This method demonstrated that Re LPS was bound to an erythrocyte protein of 96 kDa. Treatment of erythrocytes with Pronase led to disappearance of the Re LPS-binding protein on the erythrocyte membrane. There was no difference between LPS-responder and LPS-nonresponder murine erythrocyte membranes in amount and molecular weight of the Re LPS-binding protein.     

Calcium effects on human erythrocyte membrane proteins

The effects of Ca²⁺ on human erythrocyte membrane proteins were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Ca²⁺ had several effects on normal human erythrocyte membrane proteins. It affected the binding of cytoplasmic proteins to the membrane, produced a non-reversible aggregation of several membrane proteins and activated apparent proteolysis of membrane proteins. The Ca²⁺ effect could be obtained with isolated, washed membranes when the erythrocyte cytoplasm was added. These studies indicate that the Ca²⁺-induced membrane proteolysis and aggregation effects are not due simply to its presence at the time of hemolysis as previously suggested (Carraway, K.L., Triplett, R.B. and Anderson, D.R. (1975) Biochim. Biophys. Acta 379, 571–581), but are the result of more complex interactions between the erythrocyte membrane and cytoplasmic factors.     

Cholesterol ester increases the permeability of erythrocyte membrane

The effect of cholesteryl palmitate on erythrocyte membrane permeability for K+ and hemoglobin was studied. Cholesterol ether was incorporated into the erythrocyte membrane by liposomes containing cholesteryl palmitate and lecithin or by dispersion of cholesteryl palmitate. It was shown that cholesteryl palmitate considerably increases permeability of the erythrocyte membrane for K+ and hemoglobin. The leakage of K+ and hemoglobin from red blood cells is not accompanied by cell destruction.     

A possible role of cholesterol in membrane adhesion

Calcium phosphate induced membrane aggregation was studied for erythrocyte vesicles and lipid membrane vesicles. The later lipid membrane components were similar in composition to those of erythrocyte membranes. The presence of an appropriate amount of cholesterol is an important factor in the production of the calcium phosphate dependent membrane aggregation.     

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。     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Quantitative erythrocyte membrane proteome analysis with Blue-Native/SDS PAGE

The erythrocyte membrane plays a pivotal role in erythrocyte functioning. Many membrane protein aberrations are known that result in hemolytic anemia, however, the origin of numerous disorders is not known to date. To extend the current set of diagnostic tools, we used a novel proteome-wide approach to quantitatively analyze membrane proteins of healthy donor and patient erythrocytes. Blue-native PAGE has proven to be a powerful tool for separation of membrane proteins and their complexes, but has hitherto not been applied to erythrocyte membranes to find biomarkers. Using this technique, we detected almost 150 protein spots, from which more than 500 proteins could be identified by LC-MS/MS. Further, we successfully assessed the potential of using CyDye labeling to quantify the membrane proteins. Our final goal was to determine if this approach is suited to detect protein level changes in disordered erythrocyte membranes, and we could successfully confirm that erythrocyte spectrin levels were dramatically decreased for a hemolytic anemia patient.This approach provides a new tool to detect potential biomarkers and can contribute to an improved understanding of the causes of erythrocyte membrane defects in patients suffering from hemolytic anemia.     

Selective phosphorylation of erythrocyte membrane proteins by the solubilized membrane protein kinases.

This report describes the substrate and phosphoryl donor specificities of solubilized erythrocyte membrane cyclic adenosine 3',5'-monophosphate (cAMP)-independent protein kinases toward human and rabbit erythrocyte membrane proteins. Three types of substrate preparations have been utilized: heat-inactivated ghosts, isolated spectrin, and 2,3-dimethylmaleic anhydride (DMMA)-extracted membranes. A 30 000-dalton protein kinase, extracted from either human or rabbit erythrocyte membranes, catalyzes the phosphorylation of heat-inactivated membranes in the presence of ATP. The resulting phosphorylation profile is analogous to that of the autophosphorylation of membranes with ATP (in the absence of cAMP). These kinases also phosphorylate band 2 of isolated spectrin and band 3, but not glycophorin, in the DMMA-extracted ghosts. The ability of the 30 000-dalton kinases to use GTP as a phosphoryl donor appears to be related to the substrate or some other membrane factor. A second kinase, which is 100 000 daltons and derived from rabbit erythrocyte membranes, uses ATP or GTP to phosphorylate membrane proteins 2, 2.1, 2.9-3 in heat-inactivated ghosts, band 2 in isolated spectrin, glycophorin, and to a lesser extent, band 3 in the DMMA-extracted ghosts.     

Unique properties of the camel erythrocyte membrane: II. Organization of membrane proteins

Camel erythrocyte membranes are distinguished by some unique properties of stability and composition. Notable is their abundance in proteins (protein: lipid ratio of 3 : 1). Membrane proteins of camel erythrocytes were compared with those of human erythrocytes, which have been intensively investigated. Proteins were extracted with various aqueous media (EDTA, alkaline or high ionic strength) and with ionic and non-ionic detergents and were analyzed by gel electrophoresis. In membranes of camel erythrocytes, the peripheral proteins constitute, proportionally, a much smaller fraction of total proteins than in the human erythrocyte, while their distribution is identical per unit of surface area. The camel erythrocyte membrane is particularly rich in integral proteins and in intramembranous particles. The proteins in this membrane are more closely organized than in the human system, as revealed by crosslinking and freeze-etching studies. It is proposed that protein-protein interaction of integral proteins, presumably constituting an “integral skeleton”, is a dominant structural feature stabilizing the camel erythrocyte membrane.     

Atomic force microscopy of the erythrocyte membrane in obstructive jaundice

The erythrocyte intracellular pressure was calculated using a biomechanical model of the erythrocyte and atomic-force-microscopy (AFM) data. The intracellular pressure was characterized as a function of the model erythrocyte morphology. Based on numerical modeling and data of erythrocyte imaging by AFM, a method was developed to estimate the erythrocyte intracellular pressure by comparing experimental data and the results of numerical calculations. Calculations were performed for erythrocytes of dwarf domestic pigs with and without obstructive jaundice that varied in severity. The erythrocyte membrane was affected with increasing disease severity and blood bilirubin concentration, i.e., the erythrocyte volume increased on average, and substantial changes were observed for erythrocyte intracellular pressure relative to its normal value. As an example, an increase in bilirubin concentration from 5 to 96 μmol/L was associated with an increase in intracellular pressure from 0 to 2.2 kPa. An examination of the erythrocyte membrane surface by high-resolution AMF showed that the membrane is disrupted with an increase in bilirubin concentration and displays lesions and an increasing rupture length.     

The human erythrocyte membrane skeleton may be an ionic gel

Biochemical and biophysical observations indicate that the erythrocyte membrane skeleton is composed of a swollen network of long, flexible and ionizable macromolecules located at the cytoplasmic surface of the fluid membrane lipid bilayer. We have analyzed the mechanochemical properties of the erythrocyte membrane assuming that the membrane skeleton constitutes an ionic gel (swollen ionic elastomer). Using recently established statistical thermodynamic theory for such gels, our analysis yields mathematical expressions for the mechanochemical properties of erythrocyte membranes that incorporate membrane molecular parameters to an extent not achieved previously. The erythrocyte membrane elastic shear modulus and maximum elastic extension ratio predicted by our membrane model are in quantitative agreement with reported values for these parameters. The gel theory predicts further that the membrane skeleton modulus of area compression, K _G, may be small as well as large relative to the membrane elastic shear modulus, G, depending on the environmental conditions. Our analysis shows that the ratio between these two parameters affects both the geometry and the stability of the favoured cell shapes.     

Effect of the membrane potential on the fusion of the Sendai viral envelope with the membrane of chick erythrocytes and on virus-induced erythrocyte hemolysis

The ESR data on the influence of membrane potential of the fusion of Sendai virus envelope with erythrocyte membrane are presented. The hyperpolarization of cell membrane takes place at low concentration of KCl (1-5 mM) in extracellular medium in the presence of valinomycin, while at high concentration of KCl (125-150 mM) its depolarization occurs. The hyperpolarization of erythrocyte plasma membrane is accompanied by the increase of its fusion with viral envelope and virus-induced hemolysis. At the same time depolarization of erythrocyte membrane leads to the decrease of virus fusion activity. This evidence together with previously obtained by patch-clamp method data on potential-dependence of virus-induced increase of cell membrane conductivity provide us an opportunity to make a proposal that the electric field membrane damage may be the initial stage of the virus-induced membrane fusion.     

Erythrocyte membrane cholesterol levels and their effects on membrane proteins

The susceptibility of the band 3 protein of the erythrocyte membrane to proteolytic digestion at either surface of the membrane was not altered when the membrane cholesterol level was increased by 65–103%. Cross-linking of the major membrane proteins by o-phenanthroline · Cu, glutaraldehyde, dimethylsuberimidate and dimethyladipimidate was also unaffected.     

The organization of the major protein of the human erythrocyte membrane

The enzyme lactoperoxidase was used to catalyse the radioiodination of membrane proteins in intact human erythrocytes and in erythrocyte `ghosts'. Two major proteins of the erythrocyte membrane were isolated after iodination of these two preparations, and the peptide `maps' of each protein so labelled were compared. Peptides from both proteins are labelled in the intact cell. In addition, further mobile peptides derived from one of the proteins are labelled only in the `ghost' preparation. Various sealed `ghost' preparations were also iodinated, lactoperoxidase being present only at either the cytoplasmic or extra-cellular surface of the membrane. The peptide `maps' of protein E (the major membrane protein) labelled in each case were compared. Two discrete sets of labelled peptides were consistently found. One group is obtained when lactoperoxidase is present at the extra-cellular surface and the other group is found when the enzyme is accessible only to the cytoplasmic surface of the membrane. The results support the assumption that the organization of protein E in the membrane of the intact erythrocyte is unaltered on making erythrocyte `ghosts'. They also confirm previous suggestions that both the sialoglycoprotein and protein E extend through the human erythrocyte membrane.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

Abstract

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (^*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H₂O₂; ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.