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ABSTRACT: Malaria parasites interact with the host cell membrane inserting new proteins and inducing oxidative and phosphorylative changes of erythrocyte proteins. In the present report we monitored the time dependent oxidative and phosphorylative modifications induced by parasites in heterozygous beta thalassemia (Het-βThal). Het-βThal causes mild anemia and is known to determine a pro-oxidant milieu and a protective effect against severe malaria. In malaria cultures Het-βThal has been reported to induce accumulation of hemoglobin denaturation products. At early parasite development stages (rings), tyrosine hyper-phosphorylation of band 3 was the most notable modification, and at later development stages (trophozoites), additional membrane proteins displayed significant hyper-phosphorylation of their serine and tyrosine residues (adducins, ankyrin, catalase). Het-βThal also caused membrane destabilization. Free radical scavengers effectively inhibited the phosphorylative response and membrane destabilization. Kinase inhibitors exerted similar effects suggesting a causal relationship between oxidative stress, membrane protein hyper-phosphorylation and increased membrane damage exacerbated by Het-βThal. In conclusion, different lines of evidence suggest that Het-βThal enhances the redox stress caused by malaria parasites inducing its protective effect destabilizing the host cell membrane. This article is part of a Special Issue entitled: Integrated omics.
Journal of proteomics 09/2012; · 5.07 Impact Factor
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ABSTRACT: A constantly increasing number of mABs are required for the validation of a large proportion of proteomic and protein-protein interaction data. The development of new robotic platforms has greatly enhanced the throughput of monoclonal antibody production; however, the availability of highly purified proteins to use as antigens currently represents the major bottleneck of the process. In this article, we describe a new 2DE approach to purify hundreds of proteins from cellular extracts in a very cost-effective and time-efficient way. The accuracy of the new purification method is shown to be comparable to high-resolution analytical 2DE. The effectiveness and the throughput of the method to purify proteins suitable for the development of mAbs are then assessed. Using this methodology, we were able to separate 447 proteins starting from 50 mg of proteins extracted from HT29 cells. Fractions containing more than 30 μg of protein constantly induced immunization in mice. Using a high-throughput process for monoclonal antibody production, we obtained an average of 3.5 mAbs for each protein. According to pilot experiments, we can predict that starting from an unfractionated cellular extract it is possible to obtain approximately 200 proteins usable for monoclonal antibody development. Our results indicate that the number of antigens available for monoclonal antibody production can be further increased by running parallel separations. The proposed methodology will then facilitate the high-throughput monoclonal antibody process providing a vast array of high quality antigens at very low cost.
Electrophoresis 08/2012; 33(16):2546-52. · 3.30 Impact Factor
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ABSTRACT: During blood bank storage, red blood cells (RBCs) undergo a number of biological and biochemical alterations collectively referred to as "storage lesions". These injuries include loss and oxidative cross-linking of band 3, the major integral protein of RBC membranes. Denaturation of hemoglobin (Hb) and damage to the amino-terminal of band 3 are recognised as the starting events for immunological recognition mechanisms and phagocytic removal of senescent or impaired RBCs from circulation. Consequently, studies focusing on the Hb-association and oxidative status of the cytoskeleton of stored RBCs intended for transfusion are of extreme interest. In this work, two storage-related fragments of band 3 were documented and biochemically characterised.
Four RBC units were collected from normal volunteers and stored for 21 days under (i) standard blood bank conditions, (ii) anaerobic conditions, or (iii) in the presence of caspase 3-inhibitor. Degradation products of band 3 were followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis coupled with western blot and mass spectrometry analyses.
Two different degradation products of the cytoplasmic domain of the erythrocyte band 3 (CDB3) were detected in RBC membranes during storage in saline-adenine-glucosemannitol (SAGM) preservation medium. One of these fragments showed an apparent molecular weight of 34 kDa and was demonstrated to be the product of a free-radical attack on the protein main chain, whereas another fragment of 24 kDa was the result of a caspase 3-mediated cleavage.
Although to different extent, anaerobic conditions reduced the formation of both truncated products indicating an enhanced activity of the pro-apoptotic caspase 3 enzyme following oxidative stress. Interestingly, both CDB3 fragments were tightly associated to the erythrocyte membrane supporting the involvement of Cys-201 and/or Cys-317 in clustering different band 3 monomers.
Blood transfusion = Trasfusione del sangue 05/2012; 10 Suppl 2:s55-62. · 2.10 Impact Factor
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ABSTRACT: We propose a new method for the selective labeling, isolation and electrophoretic analysis of the Plasmodium falciparum protein exposed on the erythrocyte cell surface. Historically, membrane surface proteins have been isolated using a surface biotinylation followed by capture of biotin-conjugated protein via an avidin/streptavidin-coated solid support. The major drawback of the standard methods has been the labeling of internal proteins due to fast internalization of biotin.
To solve this problem, we used a biotin label that does not permeate through the membrane. As a further precaution to avoid the purification of non surface exposed proteins, we directly challenged whole labeled cells with avidin coated beads and then solubilized them using non ionic detergents.
A marked enrichment of most of the RBC membrane proteins known to face the external surface of the membrane validated the specificity of the method; furthermore, only small amounts of haemoglobin and cytoskeletal proteins were detected. A wide range of P. falciparum proteins were additionally described to be exposed on the erythrocyte surface. Some of them have been previously observed and used as vaccine candidates while a number of newly described antigens have been presently identified. Those antigens require further characterization and validation with additional methods.
Surface proteins preparations were very reproducible and identification of proteins by mass spectrometry has been demonstrated to be feasible and effective.
The Journal of Infection in Developing Countries 01/2012; 6(6):536-41. · 1.19 Impact Factor
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Antonella Pantaleo, Emanuela Ferru,
Rosa Vono,
Giuliana Giribaldi,
Omar Lobina,
Françoise Nepveu,
Hany Ibrahim,
Jean-Pierre Nallet,
Franco Carta,
Franca Mannu,
Proto Pippia,
Estela Campanella,
Philip S Low,
Francesco Turrini
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ABSTRACT: Although indolone-N-oxide (INODs) genereting long-lived radicals possess antiplasmodial activity in the low-nanomolar range, little is known about their mechanism of action. To explore the molecular basis of INOD activity, we screened for changes in INOD-treated malaria-infected erythrocytes (Pf-RBCs) using a proteomics approach. At early parasite maturation stages, treatment with INODs at their IC(50) concentrations induced a marked tyrosine phosphorylation of the erythrocyte membrane protein band 3, whereas no effect was observed in control RBCs. After INOD treatment of Pf-RBCs we also observed: (i) accelerated formation of membrane aggregates containing hyperphosphorylated band 3, Syk kinase, and denatured hemoglobin; (ii) dose-dependent release of microvesicles containing the membrane aggregates; (iii) reduction in band 3 phosphorylation, Pf-RBC vesiculation, and antimalarial effect of INODs upon addition of Syk kinase inhibitors; and (iv) correlation between the IC(50) and the INOD concentrations required to induce band 3 phosphorylation and vesiculation. Together with previous data demonstrating that tyrosine phosphorylation of oxidized band 3 promotes its dissociation from the cytoskeleton, these results suggest that INODs cause a profound destabilization of the Pf-RBC membrane through a mechanism apparently triggered by the activation of a redox signaling pathway rather than direct oxidative damage.
Free radical biology & medicine 11/2011; 52(2):527-36. · 5.42 Impact Factor
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Lucia De Franceschi,
Carlo Tomelleri,
Alessandro Matte,
Anna Maria Brunati,
Petra H Bovee-Geurts,
Mariarita Bertoldi,
Edwin Lasonder,
Elena Tibaldi,
Adrian Danek,
Ruth H Walker,
Hans H Jung,
Benedikt Bader,
Angela Siciliano, Emanuela Ferru,
Narla Mohandas,
Giel J C G M Bosman
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ABSTRACT: Acanthocytic RBCs are a peculiar diagnostic feature of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder. Although recent years have witnessed some progress in the molecular characterization of ChAc, the mechanism(s) responsible for generation of acanthocytes in ChAc is largely unknown. As the membrane protein composition of ChAc RBCs is similar to that of normal RBCs, we evaluated the tyrosine (Tyr)-phosphorylation profile of RBCs using comparative proteomics. Increased Tyr phosphorylation state of several membrane proteins, including band 3, β-spectrin, and adducin, was noted in ChAc RBCs. In particular, band 3 was highly phosphorylated on the Tyr-904 residue, a functional target of Lyn, but not on Tyr-8, a functional target of Syk. In ChAc RBCs, band 3 Tyr phosphorylation by Lyn was independent of the canonical Syk-mediated pathway. The ChAc-associated alterations in RBC membrane protein organization appear to be the result of increased Tyr phosphorylation leading to altered linkage of band 3 to the junctional complexes involved in anchoring the membrane to the cytoskeleton as supported by coimmunoprecipitation of β-adducin with band 3 only in ChAc RBC-membrane treated with the Lyn-inhibitor PP2. We propose this altered association between membrane skeleton and membrane proteins as novel mechanism in the generation of acanthocytes in ChAc.
Blood 09/2011; 118(20):5652-63. · 9.90 Impact Factor
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ABSTRACT: The cytoplasmic domain of band 3 serves as a center of erythrocyte membrane organization and constitutes the major substrate of erythrocyte tyrosine kinases. Tyrosine phosphorylation of band 3 is induced by several physiologic stimuli, including malaria parasite invasion, cell shrinkage, normal cell aging, and oxidant stress (thalassemias, sickle cell disease, glucose-6-phosphate dehydrogenase deficiency, etc). In an effort to characterize the biologic sequelae of band 3 tyrosine phosphorylation, we looked for changes in the polypeptide's function that accompany its phosphorylation. We report that tyrosine phosphorylation promotes dissociation of band 3 from the spectrin-actin skeleton as evidenced by: (1) a decrease in ankyrin affinity in direct binding studies, (2) an increase in detergent extractability of band 3 from ghosts, (3) a rise in band 3 cross-linkability by bis-sulfosuccinimidyl-suberate, (4) significant changes in erythrocyte morphology, and (5) elevation of the rate of band 3 diffusion in intact cells. Because release of band 3 from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties, tyrosine phosphorylation of band 3 is argued to enable adaptive changes in erythrocyte biology that permit the cell to respond to the above stresses.
Blood 06/2011; 117(22):5998-6006. · 9.90 Impact Factor
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PROTEOMICS - CLINICAL APPLICATIONS 04/2011; 5(3-4):190. · 1.81 Impact Factor
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ABSTRACT: While G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs.
Our results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation.
Collectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products.
PLoS ONE 01/2011; 6(1):e15847. · 4.09 Impact Factor
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ABSTRACT: Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti-phosphotyrosine and anti-phosphoserine antibodies following 2-DE in conjunction with double channel laser-induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans-membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.
Proteomics 08/2010; 10(19):3469 - 3479. · 4.43 Impact Factor
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Blood Cells Molecules and Diseases 03/2010; 45(1):65-6. · 2.35 Impact Factor
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ABSTRACT: With the advent of proteomic techniques the number of known post-translational modifications (PTMs) affecting red cell membrane proteins is rapidly growing but the understanding of their role under physiological and pathological conditions is incompletely established. The wide range of hereditary diseases affecting different red cell membrane functions and the membrane modifications induced by malaria parasite intracellular growth represent a unique opportunity to study PTMs in response to variable cellular stresses. In the present review, some of the major areas of interest in red cell membrane research have been considered as modifications of erythrocyte deformability and maintenance of the surface area, membrane transport alterations, and removal of diseased and senescent red cells. In all mentioned research areas the functional roles of PTMs are prevalently restricted to the phosphorylative changes of the more abundant membrane proteins. The insufficient information about the PTMs occurring in a large majority of the red membrane proteins and the general lack of mass spectrometry data evidence the need of new comprehensive, proteomic approaches to improve the understanding of the red cell membrane physiology.
Journal of proteomics 09/2009; 73(3):445-55. · 5.07 Impact Factor
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ABSTRACT: Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS(3) (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.
Biochemical Journal 11/2008; 418(2):359-67. · 4.90 Impact Factor
