Proteomic identification of erythrocyte membrane protein deficiency in hereditary spherocytosis

Hereditary spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The molecular defect in one of the erythrocytes (RBC) membrane proteins underlying HS like; spectrin-α, spectrin-β, ankyrin, band 3 and protein 4.2 that lead to membrane destabilization and vesiculation, may change the RBCs into denser and more rigid cells (spherocytes), which are removed by the spleen, leading to the development of hemolytic anemia. It is classified as mild, moderate and severe, according to the degree of the hemolytic anemia and the associated symptoms. Two-dimensional gel electrophoresis (2-DE) is potentially valuable method for studying heritable disorders as HS that involve membrane proteins. This separation technique of proteins based upon two biophysically unrelated parameters; molecular weight and charge, is a good option in clinical proteomics in terms of ability to separate complex mixtures, display post-translational modifications and changes after phosphorylation. In this study, we have used contemporary methods with some modifications for the solubilisation, separation and identification of erythrocyte membrane proteins in normal and in HS RBCs. Spectrin alpha and beta chain, ankyrin and band 3 proteins expression differences were found with PDQuest software 8.0.1. and peptide mass fingerprinting (PMF) analysis performed for identification of proteins in this study.     

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.     

Hereditary erythrocyte pyruvate-kinase (PK) deficiency and chronic hemolytic anemia: Clinical,genetic and molecular studies in six new Spanish patients

Summary Clinical, familial and biochemical studies from six unrelated Spanish patients with hereditary hemolytic anemia and erythrocyte pyruvate-kinase (PK) deficiency are described. A remarkable molecular heterogeneity of mutant PK variants involving kinetic properties, molecular stability or electrophoretic mobility is demonstrated. In two patients whose PK variants showed abnormal electrophoretic pattern, and strongly aberrant kinetic properties, a chronic hemolytic anemia assciated with several other clinical manifestations of chronic hemolysis occurred. In patients whose PK variants showed less abnormal kinetic and electrophoretic characteristics, there was only moderate or mild hemolytic anemia. One patient's PK variant with no obvious kinetic or electrophoretic alterations, showed a markedly decreased heat stability with severe diminution of antigenic concentration of the enzyme. This patient presented a spectacular clinical and hematological improvement after splenectomy.The purpose of the present study is to describe six new PK variants of Spanish origin. In addition, an attempt is made to find relationships between molecular abnormalities of mutant PK variants and the severity of hemolytic anemia, in these patients. The possible role of some kinetic alterations, such as fructose diphosphate (FDP) activation or ATP inhibition of PK variants, in the clinical manifestations of chronic hemolysis is also suggested.     

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。     

Differences in erythrocyte membrane proteins and glycoproteins in sickle cell disease

Erythrocyte membrane proteins obtained from individuals with sickle cell anemia show an SDS polyacrylamide gel pattern that differs in five regions from the normal pattern. These membranes when compared with membranes from normal individuals also show a marked decrease in sialic acid content which correlates with a marked reduction of the periodic acid-Schiff staining of the three major glycoprotein components. The observed membrane protein and glycoprotein changes are a characteristic of all the red cells in sickle cell anemia and do not correlate with the proportion of irreversibly sickled cells.     

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using γ-³²P ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.     

Localization of cytochromes in the outer membrane of Desulfovibrio vulgaris (Hildenborough) and their role in anaerobic biocorrosion

A protocol was developed whereby the outer and cytoplasmic membranes of the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) were isolated and partially characterized. The isolated outer membrane fractions from cultures grown under high (100 ppm) and low (5 ppm) Fe2+ conditions were compared by SDS-PAGE electrophoresis, and showed that several protein bands were derepressed under the low iron conditions, most notably at 50 kDa, and 77.5 kDa. Outer membrane isolated from low iron cultured cells was found to contain two proteins, 77.5 kDa and 62.5 kDa in size, that reacted with a heme-specific stain and were referred to as high molecular weight cytochromes. Studies conducted on the low iron isolated outer membrane by a phosphate/mild steel hydrogen evolution system showed that addition of the membrane fraction caused an immediate acceleration in H2 production. A new model for the anaerobic biocorrosion of mild steel is proposed.     

Molecular characterization of the hemolysin determinant of Serratia marcescens.

The nucleotide sequence of a 7.3-kilobase-pair fragment of DNA encoding a hemolytic activity from Serratia marcescens was determined. Two large open reading frames were identified, designated shlA (Serratia hemolysin) and shlB, capable of encoding polypeptides of 165, 056 and 61,897 molecular weight, respectively. Both reading frames were expressed in vivo. The shlB gene product was localized to the outer membrane of Escherichia coli cells harboring the S. marcescens hemolysin determinant. Consistent with this location, a signallike sequence was identified at the N terminus of the polypeptide predicted from the nucleotide sequence of the shlB gene. Hyperexpression of the shlB locus permitted the identification of two shlB-encoded polypeptides of 65,000 and 62,000 molecular weight, respectively. Determination of the N-terminal amino acid sequence of the purified 62,000-molecular-weight protein confirmed that it was the mature form of the ShlB protein initially synthesized as a precursor (65,000-molecular-weight protein). By using polyclonal antisera raised against the purified proteins, ShlA and ShlB were identified in the outer membrane of S. marcescens. The shlA gene product was shown to interact with erythrocyte membranes, confirming it as the hemolysin proper. Both hemolysis and the interaction of ShlA with erythrocyte membranes did, however, require the ShlB function. Progressive deletion of the C terminus of the ShlA protein gradually reduced hemolytic activity until 37% of the amino acids had been removed. Elimination of 54% of the amino acids produced a nonhemolytic protein which, however, was still capable of associating with erythrocyte membranes.     

Assessment of the red cell proteome of young patients with unexplained hemolytic anemia by two-dimensional differential in-gel electrophoresis (DIGE)

Erythrocyte cytosolic protein expression profiles of children with unexplained hemolytic anemia were compared with profiles of close relatives and controls by two-dimensional differential in-gel electrophoresis (2D-DIGE). The severity of anemia in the patients varied from compensated (i.e., no medical intervention required) to chronic transfusion dependence. Common characteristics of all patients included chronic elevation of reticulocyte count and a negative workup for anemia focusing on hemoglobinopathies, morphologic abnormalities that would suggest a membrane defect, immune-mediated red cell destruction, and evaluation of the most common red cell enzyme defects, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency. Based upon this initial workup and presentation during infancy or early childhood, four patients classified as hereditary nonspherocytic hemolytic anemia (HNSHA) of unknown etiology were selected for proteomic analysis. DIGE analysis of red cell cytosolic proteins clearly discriminated each anemic patient from both familial and unrelated controls, revealing both patient-specific and shared patterns of differential protein expression. Changes in expression pattern shared among the four patients were identified in several protein classes including chaperons, cytoskeletal and proteasome proteins. Elevated expression in patient samples of some proteins correlated with high reticulocyte count, likely identifying a subset of proteins that are normally lost during erythroid maturation, including proteins involved in mitochondrial metabolism and protein synthesis. Proteins identified with patient-specific decreased expression included components of the glutathione synthetic pathway, antioxidant pathways, and proteins involved in signal transduction and nucleotide metabolism. Among the more than 200 proteins identified in this study are 21 proteins not previously described as part of the erythrocyte proteome. These results demonstrate the feasibility of applying a global proteomic approach to aid characterization of red cells from patients with hereditary anemia of unknown cause, including the identification of differentially expressed proteins as potential candidates with a role in disease pathogenesis.     

Phosphorylation of component a of the human erythrocyte membrane in myotonic muscular dystrophy.

Endogenous membrane protein kinase activity in fresh erythrocyte ghosts is altered in myotonic muscular dystrophy. Phosphorylation of erythrocyte Component a, which migrates with an apparent molecular weight of 90,000 to 100,000, is significantly reduced compared to age- and sex-matched controls. The difference in endogenous membrane protein kinase activity in fresh RBC membranes lends confirmation to the suggestion that myotonic dystrophy is a disease of widespread membrane alterations.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia.

The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl.     

Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging

The addition of malondialdehyde to erythrocytes in vitro causes a decrease in bands 1 and 2 of spectrin and an increase in high molecular weight protein polymers. Additionally, this agent causes the formation of fluorscent chromolipids characteristic of those produced during the peroxidation of endogenous membrane phospholipids. These same alterations in proteins and lipids are observed in the membranes of older cells fractionated from freshly drawn blood and in the membranes of reticulocytes induced by treatment of animals with phenylhydrazine, but not in reticulocytes induced by bleeding. The former reticulocytes have a much shorter half-life in the circulation than do either normal erythrocytes or reticulocytes produced consequent to bleeding. These experiments and the apparent paradox of "young" reticulocytes with short half-lives suggest that the in vivo polymerization of membrane proteins consequent to radical-induced peroxidation of membrane lipids may contribute to the altered rheological behavior and hence to the splenic sequestration of cells. They also suggest that increases in intrinsic membrane rigidity due to lipid peroxidation, malondialdehyde, and protein polymerization may be a common feature of both aging in normal erythrocytes and in the accelerated aging that accompanies the administration of radical-generating, hemolytic agents. However, it is cautioned that other polymerization reactions involving disulfides, calcium, or direct radical attack on protein monomers may also be important determinants of the visco-elastic properties of erythrocyte membranes.     

Dysferlin and Other Non-Red Cell Proteins Accumulate in the Red Cell Membrane of Diamond-Blackfan Anemia Patients

Diamond Blackfan Anemia (DBA) is a congenital anemia usually caused by diverse mutations in ribosomal proteins. Although the genetics of DBA are well characterized, the mechanisms that lead to macrocytic anemia remain unclear. We systematically analyzed the proteomes of red blood cell membranes from multiple DBA patients to determine whether abnormalities in protein translation or erythropoiesis contribute to the observed macrocytosis or alterations in the mature red blood cell membrane. In depth proteome analysis of red cell membranes enabled highly reproducible identification and quantitative comparisons of 1100 or more proteins. These comparisons revealed clear differences between red cell membrane proteomes in DBA patients and healthy controls that were consistent across DBA patients with different ribosomal gene mutations. Proteins exhibiting changes in abundance included those known to be increased in DBA such as fetal hemoglobin and a number of proteins not normally found in mature red cell membranes, including proteins involved in the major histocompatibility complex class I pathway. Most striking was the presence of dysferlin in the red blood cell membranes of DBA patients but absent in healthy controls. Immunoblot validation using red cell membranes isolated from additional DBA patients and healthy controls confirmed a distinct membrane protein signature specific to patients with DBA.     

Mitogenic proteins of the 3T3 plasma membrane

A mitogenic factor from 3T3 plasma membranes has been identified and partially characterized. The factor appears to be a peripheral membrane protein that can be released by mild trypsin, chymotrypsin, or plasmin treatment. This component is sensitive to heat and acid, and has a molecular weight in the range of 150,000–200,000 daltons as determined by gel filtration. A similar mitogenic activity has also been found on the membranes of both SV-40-transformed 3T3 cells and human fibroblasts. The factor appears to be distinct from all previously described mitogenic components.     

The activity of the red blood cell Ca pump is decreased in hemolytic anemia of the beagle dog

A mild hereditary nonspherocytic anemia in Beagle dogs was studied. Compared to RBCs from normal dogs, RBCs from hemolytic Beagles were larger on average, contained more potassium, and exhibited an approximately 50% decrease in rate of loss of ATP induced by Ca and the ionophore, A23187. Under certain conditions, this rate of ATP loss can be taken as a measure of the Ca pump ATPase activity of intact RBCs. From RBC fractionation studies it appeared that the defective Ca pump ATPase was acquired during the relatively short life-span of the hemolytic RBC. Significant loss of Ca pump ATPase may be causally related to the hemolytic anemia. The mechanism(s) by which Ca pump ATPase activity is lost in this hemolytic anemia remain(s) to be determined.     

The oligomeric state of spectrin in the rat erythrocyte membrane skeleton

The oligomeric state of spectrin in the erythrocyte membrane skeleton of the rat was investigated following extraction in a low ionic strength buffer for 24 and 96 h. All analyses were quantitatively compared with preparations from human erythrocyte membranes. After nondenaturing agarose-polyacrylamide gel electrophoresis, the human samples revealed their characteristic spectrin oligomer pattern; there were high molecular weight complexes near the origin of the gel, followed by several high order oligomers, tetramers, and dimers. The pattern in the rat membrane skeleton also included tetramers and a high molecular weight complex band, but had only one oligomer and no dimers. With time the high molecular weight complex diminished and oligomers accumulated in both the rat and human, while dimers accumulated only in the human and tetramers accumulated only in the rat. Tetramers decreased with time in the human. Extraction of spectrin increased with time and was greater from rat than the human red cell membrane at both time points. The percentage of spectrin and actin in the low ionic strength extract was similar between species, as analyzed by SDS-polyacrylamide electrophoresis, staining, and densitometry. Proteins 4.1 and 4.9 were present in greater percentages in the human. The only temporal effect on monomeric protein composition was an increase of protein A in the rat. There was no species difference in protein A percentage at 24 h, but at 96 h the rat was greater than the human. The results suggest that there are significant differences in the structural arrangement of the rat and human erythrocyte membrane skeleton.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Distribution of calmodulin and calmodulin-binding proteins in membranes from bovine epididymal spermatozoa

Reproducible concentrations of calmodulin representing approximately 0.1% of the membrane protein were detected in purified plasma membranes from bovine epididymal spermatozoa. When membranes were isolated in the presence of 1 mM EGTA, the amount of calmodulin associated with the plasma membranes was not reduced. Calmodulin-binding proteins were detected in both purified plasma membranes and in a mixed membrane fraction containing both plasma membranes and cytoplasmic droplet membranes. A calcium-dependent, calmodulin-binding protein of apparent molecular weight 123,000 was detected in both fractions. In the presence of 1 mM EDTA, putative calcium-independent calmodulin-binding proteins of apparent molecular weights 93,000, 32,000, 18,000, and 15,000 were detected in the plasma membrane fraction. The 15,000 Mr polypeptide was also present in the mixed membrane fraction but the three proteins of higher molecular weight were reduced or absent in this fraction.     

Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes

The assembly polypeptides are an integral component of coated vesicles and may mediate the linkage of clathrin to the vesicle membrane. We have purified assembly polypeptides in milligram quantities from bovine brain by an improved procedure. Hydrodynamic and chemical crosslinking studies indicate that the protein is an asymmetric heterotetramer with a molecular weight of 252,000, containing two subunits of Mr 98,000-115,000, one subunit of 52,000, and one subunit of 16,000. Two-dimensional peptide maps of the subunits show that the 16- and 52-kD polypeptides are not derived from the higher molecular weight species, and that the group of bands at 98-115 kD are related. Electron microscopic visualization shows an essentially globular protein with one or two knob-like tails. We demonstrate a specific membrane protein binding site for 125I-labeled assembly polypeptides in 0.1 N sodium hydroxide-extracted bovine brain membranes based on the following criteria: (a) binding is displaceable by unlabeled ligand, (b) the binding site is destroyed by protease treatment of the membranes, and (c) the distribution of binding between vesicle-depleted membranes and coated vesicle membranes parallels the in vivo localization of assembly polypeptides and clathrin. This binding site is likely to be an integral membrane protein because (a) it is enriched in the sodium hydroxide-extracted membranes stripped of most of their peripheral membrane proteins, and (b) the binding site is partially extracted by 0.5% Triton X-100. A similar binding site appears to be present in coated vesicles. Clathrin binds to the hydroxide-stripped membranes in an assembly polypeptides dependent manner, and this binding is diminished by Triton extraction of the membranes. This assay may aid in identification of the membrane receptor for the assembly polypeptides.