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.     

红细胞膜蛋白与膜骨架

近10多年红细胞膜领域的研究取得了可观的进展,在红细胞膜蛋白的结构-功能相关和相互作用以及红细胞膜障碍多方面都有新的发现和开拓．现主要就国内外有关报道作一扼要综述,涉及红细胞膜蛋白的组成、功能及其相互作用,红细胞膜骨架和红细胞膜蛋白疾病等研究进展．     

Interaction of molybdenum with human erythrocyte membrane proteins

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

Structural basis of membrane skeleton organization in red blood cells

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Quantitative composition and characterization of the proteins in membrane vesicles released from erythrocytes by dimyristoylphosphatidylcholine. A membrane system without cytoskeleton

Membrane vesicles were prepared by incubation of human erythrocytes with dimyristoylphosphatidylcholine [3] and isolated by isopycnic centrifugation on Dextran density gradients. Protein analyses were carried out with crossed immunoelectrophoresis and dodecylsulfate polyacrylamide gel electrophoresis. The right-side-out-oriented membrane vesicles contained membrane and cytoplasmic proteins of the erythrocyte but lacked cytoskeletal components. Comparison of proteins in vesicles and erythrocyte membranes showed that acetylcholinesterase was enriched two to six times in the vesicles relative to both membrane-spanning proteins, band 3, and glycophorin. Two further, hitherto unidentified, sialic acid-containing membrane antigens were found in the vesicles. Both faced the outside of the membranes and were enriched two to seven times. Ankyrin was not present in the membrane vesicles and spectrin could not be detected by dodecylsulfate polyacrylamide gel electrophoresis. We suggest that the redistribution of proteins in the vesicles reflects differences in their interactions with other membrane components and their relative mobility within the erythrocyte membrane.     

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.     

Regulation Mechanism of the Lateral Diffusion of Band 3 in Erythrocyte Membranes by the Membrane Skeleton

Mechanisms that regulate the movement of a membrane spanning protein band 3 in erythrocyte ghosts were investigated at the level of a single or small groups of molecules using single particle tracking with an enhanced time resolution (0.22 ms). Two-thirds of band 3 undergo macroscopic diffusion: a band 3 molecule is temporarily corralled in a mesh of 110 nm in diameter, and hops to an adjacent mesh an average of every 350 ms. The rest (one-third) of band 3 exhibited oscillatory motion similar to that of spectrin, suggesting that these band 3 molecules are bound to spectrin. When the membrane skeletal network was dragged and deformed/translated using optical tweezers, band 3 molecules that were undergoing hop diffusion were displaced toward the same direction as the skeleton. Mild trypsin treatment of ghosts, which cleaves off the cytoplasmic portion of band 3 without affecting spectrin, actin, and protein 4.1, increased the intercompartmental hop rate of band 3 by a factor of 6, whereas it did not change the corral size and the microscopic diffusion rate within a corral. These results indicate that the cytoplasmic portion of band 3 collides with the membrane skeleton, which causes temporal confinement of band 3 inside a mesh of the membrane skeleton.     

Structure prediction of membrane proteins

There is a large gap between the number of membrane protein (MP) sequences and that of their decoded 3D structures, especially high-resolution structures, due to difficulties in crystal preparation of MPs. However, detailed knowledge of the 3D structure is required for the fundamental understanding of the function of an MP and the interactions between the protein and its inhibitors or activators. In this paper, some computational approaches that have been used to predict MP structures are discussed and compared.     

Iron-regulated membrane proteins and bacterial virulence

The amount of iron that might be readily available to bacteria in body fluids is extremely small. This iron restricted environment induces phenotypic changes in the metabolism and in the composition of the membrane proteins of bacteria growingin vivo. These changes are now providing a fresh insight into the capabilities of bacteria to multiply in host tissues and are suggesting new possibilities for targetting therapeutic and prophylactic measures.     

Interrelationships among the plasma membrane,the membrane skeleton and surface form in a unicellular flagellate

Summary Surface isolates or membrane skeletons from surface isolates can maintain the cell and surface form characteristic of euglenoids. We now report that the plasma membrane alone obtained by trypsin or urea digestion of surface isolates can also maintain surface form, but the membrane skeleton is able to produce striking changes in membrane organization. Trypsin digests microtubules, the membrane skeleton and partially digests the major integral membrane protein from surface isolates but does not alter the paracrystalline plasma membrane interior. Extraction of surface isolates with 4M urea leaves an insoluble plasma membrane and a subset of proteins arranged perpendicularly to the membrane surface. To resolve further the relationship between the plasma membrane and the membrane skeleton we have perturbed membrane organization by extraction of surface isolates with NaOH and find that readdition of the extract followed by neutralization restored important features of the membrane skeleton and caused patching of the membrane interior. Biochemically, the reassembled membrane skeleton consisted of 80 and 86 kD polypeptides and other less abundant proteins, and structurally the reassembled membrane skeleton was about the same thickness as the native membrane skeleton. Reassembly of the membrane skeleton appeared to be saturatable in that addition of an excess of extract had no effect on the thickness of the membrane skeletal layer. When the 80 kD protein was depleted from the reassembly mixture by affinity chromatography using Sepharose-bound monoclonal antibodies, the amount of 86 kD protein bound was significantly reduced, suggesting a dependance of 86 kD protein on 80 kD binding. A urea soluble fraction enriched in the 80 and 86 kD proteins was added to alkali-stripped membranes and 170 Å filaments were formed perpendicularly to the membrane surface. From the sum of these experiments we suggest that a) the native amorphous membrane skeleton ofEuglena may consist of a framework of 80 and 86 kD filaments arranged in a brush-like layer, b) the framework can direct plasma membrane organization, but once determined, membrane form remains stable to urea and trypsin but not to alkali, and c) new surface growth can in theory occur as an expansion of the brush-like layer by direct intercalation of filaments enriched in or consisting wholly of 80 and 86 kD proteins.Abbreviations BSA bovine serum albumin - ELISA enzyme linked immunosorbant assay - EF ectoplasmic fracture face - IMPs intramembrane particles - PF protoplasmic fracture face This work was supported by a University of Illinois Fellowship to RRD and NSF grant DCB-8602793 to GBB.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Structural changes in the erythrocyte membrane of the trout Salmo irideus at seasonal acclimatization

Differences of thermostability were studied in red blood cells of the trout Salmo irideus differing in sex and age, as well as structural-dynamic characteristics of erythrocyte membrane proteins at seasonal acclimatization in the interval of reservoir water temperature of 0–19°C. An increase of resistance of erythrocytes to temperature lysis with elevation of the environmental temperature was revealed to be accompanied by a rise of the proteins segmental mobility and a decrease of intermolecular interactions in spectrin-actin cytoskeleton from the data of the ESR spin labeling method. Regulation of erythrocyte stability during acclimation was concluded to occur both changes of the fatty acids chain package at the variations of lipid composition and by changes of the cytoskeleton structural lability. Thereby this provides an increase of the bilayer firmness, on the one hand, while, on the other hand, a rise of elasticity and expansibility of the membrane on the whole, which increases resistance of cells to colloidal-osmotic hemolysis. Changes of concentration of oxygen dissolved in water, which are caused by temperature fluctuations, do not deem to be of crucial importance for structural stability of erythrocytes, as it can be compensated by another mechanism, specifically by changes of affinity of hemoglobin to oxygen.     

红细胞膜带4.2蛋白的结构与功能

带4.2蛋白是一种重要的红细胞膜蛋白,与红细胞的形态、可变形性及携氧功能有至关重要的联系。它通过与带3蛋白(阴离子通道蛋白)、锚蛋白结合,稳定的连接在细胞膜的内表面,连接着膜骨架网架结构与细胞膜,是膜骨架与脂质双分子层连接的重要纽带。带4.2蛋白的缺失会引起球形或椭圆形红细胞增多症及不同程度的溶血性贫血,严重的情况需要摘除脾脏来进行治疗。近年来研究认为,带4.2蛋白在维持细胞膜骨架的完整性和稳定性方面扮演了重要角色。现对带4.2蛋白结构及功能的研究状况进行综述。     

Statistical approach for lysosomal membrane proteins (LMPs) identification

Discrimination of Lysosomal membrane proteins (LMP’s) from folding types of globular (GPs) and other membrane proteins (OtMPs) is an important task both for identifying LMPs from genomic sequences and for the successful prediction of their secondary and tertiary structures. We have systematically analyzed the amino acid frequencies as well as dipeptide count of GPs, LMPs and OtMPs. Based on the above calculated single amino acid frequency combined with dipeptide count information, we statistically discriminated LMPs from GPs and OtMPs. This approach correctly classified the LMPs with an accuracy of 95 %. On the other hand, the amino acid frequency alone can discriminate LMPs with an accuracy of only 79 %. Similarly dipeptide count alone has an accuracy of 87 % for the discrimination of LMPs. Thus the combined information of both amino acid frequencies and dipeptide composition gives us significant high accurate results.

Electronic supplementary material

The online version of this article (doi:10.1007/s11693-014-9153-7) contains supplementary material, which is available to authorized users.     

红细胞4．1蛋白与血型糖蛋白C／D结合位点研究进展

红细胞膜骨架与脂双层间存在着相互作用,其中带4.1蛋白与血型糖蛋白C/D间的相互作用对维持正常红细胞的形态和机械稳定性起着重要作用,研究表明,带4.1蛋白在血型糖蛋白C、D上的结合位点分别位于血型糖蛋白C的第82～98位氨基酸残基和血型糖蛋白D的第61～77位氨基酸残基．     

Computational modeling of membrane proteins

The determination of membrane protein (MP) structures has always trailed that of soluble proteins due to difficulties in their overexpression, reconstitution into membrane mimetics, and subsequent structure determination. The percentage of MP structures in the protein databank (PDB) has been at a constant 1–2% for the last decade. In contrast, over half of all drugs target MPs, only highlighting how little we understand about drug‐specific effects in the human body. To reduce this gap, researchers have attempted to predict structural features of MPs even before the first structure was experimentally elucidated. In this review, we present current computational methods to predict MP structure, starting with secondary structure prediction, prediction of trans‐membrane spans, and topology. Even though these methods generate reliable predictions, challenges such as predicting kinks or precise beginnings and ends of secondary structure elements are still waiting to be addressed. We describe recent developments in the prediction of 3D structures of both α‐helical MPs as well as β‐barrels using comparative modeling techniques, de novo methods, and molecular dynamics (MD) simulations. The increase of MP structures has (1) facilitated comparative modeling due to availability of more and better templates, and (2) improved the statistics for knowledge‐based scoring functions. Moreover, de novo methods have benefited from the use of correlated mutations as restraints. Finally, we outline current advances that will likely shape the field in the forthcoming decade. Proteins 2015; 83:1–24. © 2014 Wiley Periodicals, Inc.     

Analysis and separation of synaptosomal membrane proteins

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with twodimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondorial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.     

Effects of the calcium-mediated enzymatic cross-linking of membrane proteins on cellular deformability

Summary Excess calcium binding affects the shape and dynamics of cellular deformation of human erythrocytes. It may be hypothesized that incorporation of calcium may modify cellular deformability by processes which include specific cross-linking of membrane proteins with resultant changes in cell shape and deformability. Since previous studies indicate that accumulation of calcium ions causes development of -glutamyl--lysine bridges in membrane proteins, under control of a membrane transamidating enzyme which specifically requires calcium ions for activation, experiments were devised to examine the relationship between cross-linking and deformability and to determine the effects of specific inhibitor of membrane protein cross-linking on the calcium-dependent modification of erythrocyte to the echinocytic shape. The elastic shear modulus of the membrane was not significantly affected by calcium-induced cross-linking, indicating that induced shape change, not altered elasticity, causes the observed reduction in cellular deformability. These findings support the interpretation that Ca⁺⁺-induced and transamidase-catalyzed cross-linking of membrane proteins contributes to fixation of altered cellular shape and decreased cellular deformability.