-
[show abstract]
[hide abstract]
ABSTRACT: Erythrocyte lipid rafts are anchored to the underlying spectrin membrane skeleton [A. Ciana, C. Achilli, C. Balduini, G. Minetti, On the association of lipid rafts to the spectrin skeleton in human erythrocytes, Biochim. Biophys. Acta 1808 (2011) 183-190]. The nature of this linkage and the molecules involved are poorly understood. The interaction is sensitive to the increase in pH and ionic strength induced by carbonate. Given the role of palmitoylation in modulating the partitioning of certain proteins between various sub-cellular compartments and the plasma membrane, we asked whether palmitoylation of p55, a peripheral protein located at the junctional complex between spectrin-actin-protein 4.1 that anchors the membrane skeleton to the lipid bilayer via the transmembrane protein glycophorin C, could contribute to the anchoring of lipid rafts to the membrane skeleton. We adopted a new, non-radioactive method for studying protein palmitoylation, based on bio-orthogonal chemical analogues of fatty acids, containing an omega-alkynyl group, to metabolically label cell proteins, which are then revealed by a "click chemistry" reaction of the alkynyl moiety with an azide-containing reporter tag. We show that the membrane localization and palmitoylation levels of p55 did not change after carbonate treatment. 2-bromopalmitate and cerulenin, two known palmitoylation inhibitors, completely inhibited p55 palmitoylation, and protein palmitoyl thioesterase-1 (PPT1) reduced it, without affecting the association between lipid rafts and membrane-skeleton, indicating, on the one hand, that p55 palmitoylation is enzymatic, and, on the other, that it is not involved in the modulation of the linkage of lipid rafts to the membrane-skeleton.
Biochimica et Biophysica Acta 12/2012; · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Lipid rafts are local inhomogeneities in the composition of the plasma membrane of living cells, that are enriched in sphingolipids and cholesterol in a liquid-ordered state, and proteins involved in receptor-mediated signalling. Interactions between lipid rafts and the cytoskeleton have been observed in various cell types. They are isolated as a fraction of the plasma membrane that resists solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs). We have previously described that DRMs are anchored to the spectrin-based membrane skeleton in human erythrocytes and can be released by increasing the pH and ionic strength of the solubilization medium with sodium carbonate. It was unexplained why this carbonate treatment was necessary and why this requirement was not reported by other workers in this area. We show here that when contaminating leukocytes are present in erythrocyte preparations that are subjected to detergent treatment, the isolation of DRMs can occur without the requirement for carbonate treatment. This is due to the uncontrolled breakdown of erythrocyte membrane components by hydrolases that are released from contaminating neutrophils that lead to proteolytic disruption of the supramolecular assembly of the membrane skeleton. Results presented here corroborate the concept that DRMs are anchored to the membrane skeleton through electrostatic interactions that most likely involve the spectrin molecule.
Biochimica et Biophysica Acta 01/2011; 1808(1):183-90. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Supposedly "homogeneous" red blood cell (RBC) samples are commonly obtained by "washing" whole blood free of plasma, platelets, and white cells with physiological solutions, a procedure that does not result, however, in sufficient removal of polymorphonuclear neutrophils (PMNs), leading to possible artifactual results. Pure RBC samples can be obtained only by leukodepletion procedures. Proposed here is a version of gelatin zymography adapted to detect matrix metalloproteinase 9 (MMP-9), selectively expressed by PMNs, in heterogeneous mixtures of RBCs and PMNs that can reveal contamination at levels as low as 1 PMN/10⁶ RBCs.
Analytical Biochemistry 10/2010; 409(2):296-7. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C(12)E(8)) at 37 degrees C, and by treatment at 4 degrees C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37 degrees C, or from cholesterol-depleted cells at 4 degrees C, for both detergents. For 5-SASL only, increased S values were measured in 4 degrees C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C(12)E(8) DRMs prepared at 4 degrees C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C(12)E(8) DRMs. However, contrary to the 4 degrees C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C(12)E(8) treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C(12)E(8) to the study of DRMs.
Journal of Membrane Biology 03/2010; 234(3):195-205. · 1.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: ZO-1 is a peripheral protein that plays a central role in the macromolecular assembly of tight junctions by interacting with integral proteins (occludin, claudins, JAMs) of the membrane of adjoining cells, with the actin cytoskeleton, and with nuclear factors. Human ZO-1 is expressed in all epithelia and some specialized endothelia as variable amounts of two related isoforms, which originate from the alternatively spliced mRNA transcripts alpha(+) and alpha(-) and whose specific differential role is still unknown. Moreover, little is known about the timing of expression of ZO-1 isoforms at the protein and mRNA level. This study shows that during growth of freshly plated Caco-2 cells, the alpha(+)/alpha(-) ratio increased as a result of simultaneous increase of alpha(+) and decrease of alpha(-). Differences in the isoform ratio also correlated with differences in epithelium differentiation. This was determined by aminopeptidase N measurements of cells grown on conventional substrates and on modified, micro/nano-patterned surfaces. A comparable shift of ZO-1 isoforms was not observed in other tumour cell lines of non-intestinal origin (A549, Calu-3). Pancreatic stem cells, propagated without exogenous differentiation stimuli, displayed a slight, stable prevalence of the alpha(-) isoform. Of the intestinal cell lines examined (Caco-2 and T84), only Caco-2 cells displayed a dramatic shift in isoform expression. This suggests that this tumour cell line retains to a higher degree a developmental programme related to the dynamic of enterocytic differentiation in vivo.
Cell Biology International 03/2010; 34(6):669-78. · 1.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4 degrees C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C12E8. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C12E8, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C12E8 in comparison to intact cells, while the difference in the S values between Triton X-100 and C12E8 DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C12E8 detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.
Journal of Membrane Biology 01/2009; 227(1):39-48. · 1.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: L-Methionine (Met), in its free form or when inserted in proteins, is sensitive to oxidation of its thioether group by reactive oxygen species from exogenous or endogenous sources. Two stable diastereomers of Met sulfoxide [Met-(O)] may be formed [Met-S-(O) and Met-R-(O)], but these can be reduced by two classes of Methionine-sulfoxide-reductase (Msr) enzymes: MsrA, which reduces the S, and MsrB, which reduces the R sulfoxide. In this study, we have examined the levels of expression of Msr in human blood cells by enzymatic activity assay, Western blotting, and RT-PCR of purified populations of polymorphonuclear neutrophils and eosinophils, mononuclear cells, platelets, and erythrocytes. Our data indicate that of the blood cells analyzed, neutrophils expressed the highest activity, which was mainly of MsrB type. During degranulation of activated neutrophils, Msr activity was not released but remained confined within the cell, indicating a non-granular localization. Immunoprecipitation and RT-PCR studies indicated the almost complete lack of mitochondrial forms of Msrs in granulocytes. It is thus likely that Msrs are important as antioxidant/repair systems for neutrophils, cells with enormous capacity for the generation of reactive oxidants and hence, susceptible to oxidative damage.
Journal of Leukocyte Biology 02/2008; 83(1):181-9. · 4.99 Impact Factor
-
Arduino Arduini,
Giampaolo Minetti, Annarita Ciana,
Claudio Seppi,
Augusta Brovelli,
Antonella Profumo,
Cristina Vercellati,
Manuela Zappa,
Alberto Zanella,
Secondo Dottori,
Mario Bonomini
[show abstract]
[hide abstract]
ABSTRACT: L-Carnitine (LC) in the preservation medium during storage of red blood cells (RBC) can improve the mean 24-hr percent recovery in vivo and increase RBC life-span after reinfusion. The purpose of the study was to investigate the differences in the biochemical properties of RBCs stored in the presence or absence of LC, and the cell-age related responses to storage conditions and to LC. RBC concentrates in saline-adenine-glucose-mannitol (SAG-M) were stored in the presence or absence of 5 mM LC at 4 degrees C for up to 8 weeks. RBC subpopulations of different densities were prepared by centrifugation on Stractan density gradient. Cells were sampled at 0, 3, 6, and 8 weeks, and hematological and cellular properties analyzed (MCV, MCHC, 4.1a/4.1b ratio as a cell age parameter, intracellular Na(+) and K(+)). After 6 weeks, MCV of RBC stored in the presence of LC was lower than that of controls (6 weeks MCV: controls 95.4 +/- 1.8 fl; LC 91.5 +/- 2.0 fl; n = 6; P < 0.005). This was due to swelling of control cells, and affected mainly older RBCs. LC appeared to reduce or retard cell swelling. Among the osmotically active substances whose changes during storage could contribute to cell swelling, only intracellular Na(+) and K(+) differed between stored control RBCs and LC-treated cells. LC reduces the swelling of older cells during storage at 4 degrees C in SAG-M, possibly by acting on the permeability of cell membrane to monovalent cations.
American Journal of Hematology 01/2007; 82(1):31-40. · 4.67 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recent studies have suggested that Rap1 and Rap2 small GTP-binding proteins are both expressed in human red blood cells (RBCs). In this work, we carefully examined the expression of Rap proteins in leukocytes- and platelets-depleted RBCs, whose purity was established on the basis of the selective expression of the beta2 subunit of the Na+/K+ -ATPase, as verified according to the recently proposed "beta-profiling test" [J.F. Hoffman, A. Wickrema, O. Potapova, M. Milanick, D.R. Yingst, Na pump isoforms in human erythroid progenitor cells and mature erythrocytes, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14572-14577]. In pure RBCs preparations, Rap2, but not Rap1 was detected immunologically. RT-PCR analysis of mRNA extracted from highly purified reticulocytes confirmed the expression of Rap2b, but not Rap2a, Rap2c, Rap1a or Rap1b. In RBCs, Rap2 was membrane-associated and was rapidly activated upon treatment with Ca2+/Ca2+ -ionophore. In addition, Rap2 segregated and was selectively enriched into microvesicles released by Ca2+ -activated RBCs, suggesting a possible role for this GTPase in membrane shedding.
Biochimica et Biophysica Acta 04/2006; 1763(3):330-5. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In cell membranes, local inhomogeneity in the lateral distribution of lipids and proteins is thought to exist in vivo in the form of lipid 'rafts', microdomains enriched in cholesterol and sphingolipids, and in specific classes of proteins, that appear to play specialized roles for signal transduction, cell-cell recognition, parasite or virus infection, and vesicular trafficking. These structures are operationally defined as membranes resistant to solubilization by nonionic detergents at 4 degree C (detergent-resistant membranes, DRMs). This definition appears to be necessary and sufficient, although additional manoeuvres, not always described with sufficient detail, may be needed to ensure isolation of DRMs, like mechanical homogenization, and changes in the pH and/or ionic strength of the solubilization medium. We show here for the human erythrocyte that the different conditions adopted may lead to the isolation of qualitatively and quantitatively different DRM fractions, thus contributing to the complexity of the notion itself of lipid raft. A significant portion of erythrocyte DRMs enriched in reported lipid raft markers, such as flotillin-1, flotillin-2 and GM1, is anchored to the spectrin membrane-skeleton via electrostatic interactions that can be disrupted by the simultaneous increase in pH and ionic strength of the solubilization medium.
Journal of Biosciences 07/2005; 30(3):317-28. · 1.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The phosphorylation of tyrosine residues of human red blood cell (RBC) band 3 is regulated in vivo by constitutively active tyrosine-kinases (PTKs) and phosphotyrosine-phosphatases (PTPs), identified so far as, respectively, p72(syk) and p56/53(lyn), and PTP1B and SHPTP-2. Tyr-phosphorylation of band 3 increases upon reduction of cell volume as in hypertonic media or during Ca(2+)-induced membrane vesiculation. We show here that old RBCs display higher Tyr-phosphorylation levels of band 3 than younger cells under hypertonic conditions, at least in part due to the reduced cell volume of old RBCs, a condition of lowered threshold for activation of volume-sensitive PTKs. We have also analysed the membrane-bound PTP activity and the relative abundance of PTP1B (as the main membrane-associated PTP) in RBCs of different age. Immunodetection of PTP1B in purified ghost membranes revealed that the catalytic, N-terminal domain of the PTP is partially cleaved, in an age-dependent manner, from the membrane-bound domain, and it is lost during the preparation of ghost membranes. This suggests that erythrocytes may undergo in vivo activation of the Ca(2+)-dependent calpain system that proteolytically regulates PTP1B activity, as already documented for other cell types. On the other hand, the assay of the PTP activity that remains associated with the membranes of RBCs of different age indicated that the PTP undergoes oxidative inactivation that can be further differentiated into reversible and irreversible components.
Bioelectrochemistry 06/2004; 62(2):169-73. · 3.76 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: One of the most intensively studied post-translational modifications of erythrocyte proteins is the phosphorylation of tyrosine residues of band 3, which is strictly regulated in vivo by PTKs (protein-tyrosine kinases) and PTPs (protein-phosphotyrosine phosphatases). Two PTKs (p72(syk) and p56/53(lyn)) and two PTP activities (PTP1B and SHPTP-2) have been immunologically identified so far in mature human erythrocytes. We have shown previously that band 3 undergoes tyrosine phosphorylation upon a decrease in cell volume, as occurs when erythrocytes treated with Ca(2+)/Ca(2+) ionophore (A23187) lose KCl and release microvesicles. Similar levels of band 3 tyrosine phosphorylation in vesicles and in the parent cells are induced by this treatment. However, we have found that tyrosine phosphorylation of band 3 in vesicles is more stable than in whole erythrocytes. Examination of how the identified PTPs and PTKs are partitioned between the vesicles and the remnant cells during vesiculation reveals that PTP1B, unlike the PTKs, is retained entirely in the parent cell compartment. Since a tight association between PTP1B and band 3 has been documented previously, we have investigated the partitioning of PTP1B and band 3 between the membrane and the membrane-skeletal fractions prepared from resting or Ca(2+)/A23187-treated cells. Our results rule out the possibility that the preferential retention of PTP1B within the cell was due to an increase in the amount of membrane-skeleton-associated band 3 (and of PTP1B) during the release of spectrin-free vesicles, suggesting a more complex modality of interaction of PTP1B with band 3 in the erythrocyte membrane. Analysis of erythrocytes of different cell ages revealed that PTP1B, unlike the other enzymes examined, was quantitatively conserved during erythrocyte aging. This suggests important roles for the down-regulation of tyrosine phosphorylation of band 3 in erythrocyte physiology, and for vesiculation as a mechanism of human erythrocyte senescence.
Biochemical Journal 02/2004; 377(Pt 2):489-97. · 4.90 Impact Factor
-
Blood 06/2003; 101(9):3751; author reply 3751-3. · 9.90 Impact Factor
