Giampaolo Minetti

erythrocyte signal transduction pathways and their possible functions

Human erythrocytes are equipped with a diversity of receptors and effectors that mediate well‐characterized signal transduction pathways in nonerythroid cells. Some of these erythrocyte components may be vestiges of signaling pathways critical to the functions of the erythrocytes precursors but no longer needed in the mature erythrocyte. Other signaling elements, however, are likely involved in enabling the erythrocyte to detect and respond to the needs of other hematopoietic and endothelial cells with which it comes in contact.

Red cell investigations: Art and artefacts

Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology. Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards. Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy.

Detergent-resistant membranes in human erythrocytes and their connection to the membrane-skeleton.

In cell membranes, local inhomogeneity in the lateral distribution of lipids and proteins is thought to existin 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°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.

Differential sorting of tyrosine kinases and phosphotyrosine phosphatases acting on band 3 during vesiculation of human erythrocytes.

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.

Oxidation state of glutathione and membrane proteins in human red cells of different age

In this study the oxidation state of glutathione and membrane proteins was analyzed in red cells of different age in basal conditions. Red cells of different age were prepared by centrifugation and separated according to their density by two procedures: on self-forming gradients of autologous plasma (Murphys procedure) and on discontinuous Stractan gradients. The efficiency of the two procedures in the isolation of senescent cells was compared. The results indicate that, despite the evidence that total cell GSH decreases with aging, its concentration, evaluated in the cell preparations of different ages, remains constant throughout the red cell life, when correlated with cell water content. Glutathione disulfide concentration increases with aging. The oxidation state of membrane proteins does not seem to change during the red cell life span.

Evidence for membrane protein oxidation during in vivo aging of human erythrocytes.

Oxidative lesions to membrane proteins were studied in human erythrocytes of different age and were evaluated on ghost membrane preparations by assaying thiol and methionine sulphoxide groups, and in situ on intact cells, after treating erythrocytes with the fluorochrome N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM). DACM reacts with thiol groups and the amount of this reagent bound by membrane proteins was quantified after SDS-PAGE separation. Results obtained show that during aging of normal cells the oxidative state of membrane proteins increases: this was better shown by the assay of methionine sulphoxide residues rather than by the thiol titration, when studies were carried out on ghost membranes. After separation of individual membrane proteins by SDS-PAGE, decreased accessibility of DACM to thiol groups of band 3 and of the main proteins of the membrane skeleton was evident in senescent erythrocytes. These results show that during aging, band 3 and membrane skeleton proteins undergo conformational changes and/or oxidation. Similar results were obtained when thiol distribution was studied in membrane proteins separated by SDS-PAGE in both reducing and non-reducing conditions.

Conformational changes and oxidation of membrane proteins in senescent human erythrocytes.

Human red cells spend 120 days in the circulation and are then removed in an age-dependent manner (1). Since cell destruction is age-dependent, studies about red cell senescence focused on the mechanisms by which the aging of the cell leads to its destruction. The presence of autoantibodies on the surface of senescent cells produced the development of the autoimmune hypothesis for senescent cell removal from the circulation (2–4), and raised questions about the presence of senescence markers on the cell surface that permit such recognition and the mechanisms of their development during red cell life span. Studies on surface changes taking place during red cell senescence have been carried out mainly on density-separated red cells (5). A reduction in membrane surface area in the dense cell population is evident as a decrease in membrane cholesterol and phospholipid content (6,7) and in acetylcholinesterase activity and sialic acid content (8). Cell deformability decreases (9–12) and at the level of the membrane slight modifications of the covalent structure of some components have been described, produced by processes like oxidation (13–15), proteolysis (16, 17), glycation (18), methylation and transamidation (19), phosphorylation (20), and modifications of phospholipid asimmetry (21) and of topology and topography of proteins have been reported or hypothesized (22–25). Most of these modifications are effective in promoting autoantibody binding and/or phagocytosis in vitro, thus supporting a possible role of these mechanisms in determining recognition and removal of senescent cells. Investigations carried out with in vivo (26,27) and in vitro models (28,29) for red cell senescence and studies with mutant erythrocytes showed that oxidation plays a relevant role in determining surface properties of senescent cells and of many pathological cells with a decreased life span (30–32). Since the oxidative state of membrane proteins in human red cells of different age has not been investigated in detail in the past, we tried to quantitate the oxidative lesion the membrane proteins undergo during red cell life-span, in an attempt to understand what kind of membrane processes expressed in senescent red cells can be related to oxidation.

On the association of lipid rafts to the spectrin skeleton in human erythrocytes.

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.

Resistance of human erythrocyte membranes to Triton X-100 and C12E8.

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°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.

Neutrophil granulocytes uniquely express, among human blood cells, high levels of Methionine-sulfoxide-reductase enzymes.

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.