Augusta Brovelli

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.

In vivo behaviour of neuraminidase-treated rabbit erythrocytes and reticulocytes.

51Cr rabbit erythrocytes were treated with different amount of neuraminidase and reinjected into the animal. The survival curves after the removal of more than 50% membrane sialic acid show a characteristic behaviour: after a rapid decrease, blood radioactivity increases again reaching a maximum level 50-80 h after reinjection, then tends to decrease with a slope similar to that of control curves. Liver radioactivity determined before the rise of blood radioactivity is evidently higher than the value determined after radioactivity elevation. Similar results were obtained with phenylhydrazine-induced young erythrocytes.

Modification of membrane protein organization during in vitro aging of human erythrocytes

In in vitro aged human erythrocytes, the presence of protein clusters can be found on the membrane; these clusters are made up of peptides held together by disulfide bridges, since they can be nearly completely dissociated by dithiothreitol treatment. SDS-polyacrylamide gel electrophoresis after dithiothreitol dissociation indicates that the aggregates are made of peptide fragments with a molecular weight ranging from 20 to approximately 110 kdalton; none of these fragments correspond to an intact protein component of the membrane. Their formation results from oxidation and proteolysis of membrane, and perhaps cytoplasmic proteins.

Membrane Properties of Senescent and Carrier Human Erythrocytes

Studies about human red cell senescence have shown that the viability and post-transfusion survival of red cells is related to the structure of their plasma membrane.1 In an attempt to analyze the survival potential in the circulation of red cells manipulated for loading with drugs or biosubstances, we addressed our investigation to the identification of new parameters useful to describe membrane characteristics of red cells with a decreased life expectancy. In this study we have analyzed membrane properties of young, middle-aged, and senescent red cells, and compared them with those of red cells manipulated for loading, in order to discern the membrane structural lesions leading to a decreased survival potential. Removal from the circulation of senescent red cells seems to be triggered by the binding of autologous antibodies2 recognizing band 3 (B3) protein3–5, α-galactosyl groups, probably belonging to glycolipids6, and other epitopes not yet defined.5 Although the role played by autoantibodies in the removal of senescent cells has not been completely elucidated, their presence on the surface of senescent erythrocytes focused on plasma membrane studies about cell aging and raised questions about the mechanisms leading to the expression on the cell surface of the senescence antigens. Among the processes modifying the structure of membrane components and described to occur during red cell senescence, oxidation seems to play an important role.7–11 Therefore we have analyzed the oxidative state of membrane proteins in young, middle-aged and senescent normal red cells and tried to relate it with the functional activity of B3 protein12, considering that the involvement of B3 in the expression of the senescence antigen has been recognized by different authors.3–5,11 The same investigation was carried out on red cells submitted to hypotonic dialysis and resealed. The aim of this investigation was to identify steps of cell loading processes producing cell suffering and decrease of the survival potential, in order to prevent or minimize the cellular damage with appropriate protocols.

Erythrocyte Membrane Damage in Hemolytic Anemias

Molecular defects directly affecting a membrane component can modify the shape of the red cell and reduce its survival capacity; the same effect can result from molecular lesions at the level of a cytoplasmic protein or enzyme. In some diseases where the primary defect does not involve a membrane component, a secondary membrane lesion develops, contributing to or causing red cell death. For instance, in some hemoglobinopathies, a less stable anomalous hemoglobin interacts with the membrane and modifies lipid and proteins; in erythroenzymopenias, such as glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase (PK) deficiency, the altered structure of a key enzyme involved in glycolysis or the pentose pathway, impairs the ability of red cells to withstand stress conditions, and there are clues that lead us to suspect a membrane lesion secondary to the enzyme deficiency. In this review, membrane processes found in the three above-mentioned groups of diseases will be described: in particular, we will deal with two hemoglobinopathies, thalassemia and sickle-cell anemia; with the enzymopathies glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency; and with the hereditary membrane skeleton diseases spherocytosis, elliptocytosis, and pyropoikilocytosis. A summary of membrane lesions found in each disease will be presented, more detailed reviews concerning each of these topics being available [see Wagner et al. (1985), Hebbel et al. (1985), and Hebbel (1986) for sickle cell anemia; Rachmilewitz et al. (1985) for thalassemia; Palek and Lux (1983), Becker and Lux (1985), Palek (1985) for hereditary defects of membrane skeleton; Beutler (1983), Valentine et al. (1983) for G6PD and PK deficiencies]. Membrane damage associated with an impaired survival capacity of pathological red cells will be compared with membrane properties of senescent normal red cells (see Bartosz, this volume). The general aim of the review is to analyze which membrane lesions in erythrocyte pathology shorten red cell life span, and to find insights into membrane processes that would be worth investigating in those diseases, where indirect evidence suggests the presence of membrane damage. Structural integrity of the membrane allows the erythrocyte to perform its biological role of oxygen transport from the lungs throughout the body. The ability to deform reversibly during flow is crucial to the red cell for performing its function. This property, known as cellular deformability, is determined by membrane material characteristics, cell surface-area-to-volume ratio, and cytoplasmic viscosity, and is strictly related to the red cell survival capacity (Mohandas et al., 1983). Since structure and function of the membrane are highly involved in determining cell deformability (through the maintenance of ion gradients and water distribution, and through the transport of nutrients and catabolites), the life span of a red cell ultimately depends on the integrity of its membrane.

Red Cell Ageing

The mammalian erythrocyte is unique among other cell types of the organism, being devoid of nucleus and other intracellular organelles. This cell lacks the transcriptional and translational apparatus for protein synthesis, and it therefore constitutes a good model for studying cell ageing. The mechanisms for the replacement or repair of damaged macromolecules are in fact absent, or minimal, thus permitting the analysis the age-related modifications of the cell and its constituents without major interference.

Supramolecular Assembly of Membrane Constituents During in Vitro Aging of Human Erythrocytes

Abstract Aging ‘in vitro’ of human erythrocytes produces structural modifications concerning glycoprotein structure and the supramolecular organization of proteins in the membrane. These processes can be evidentiated using both intact cells and right-out vesicles and can be partially inhibited by physiological concentrations of ATP and GSH. The possibility that the loss of sialopeptides and protein clustering are related phenomena was studied. To mimic the structural modifications which take place during ‘in vitro’ aging the external side of the membrane was modified by enzyme treatment and it was investigated if a clustering of proteins can be detected in these conditions by gel filtration of SDS-dissociated membranes on a Sepharose 2B column. Neuraminidase and trypsin treatment of both intact cells and right-out vesicles does not induce any detectable protein aggregation on the membrane. At present no relationship can be stated between sialopeptides release and protein clustering.

Structural modifications in membrane glycoproteins during the erythrocyte life-span

SummaryErythrocyte membrane glycoproteins undergo various types of modification during the life of the cell in the circulation; when only sialic acid is removed, the younger red cells can be repaired in the liver and return to the circulation. Otherwise, when an autolytic mechanism removing a sialopeptide becomes active as a consequence of the metubolic impairment of the cell, the erythrocyte is probably trapped by the hemocatheretic organs and destroyed.