Membrane Mechanical Stability Research Articles

Molecular Characterization of Erythrocyte Glycophorin C Variants

Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. Immunochemical and serologic studies have identified a number of glycophorin C variants that include the Yus, Gerbich, and Webb phenotypes. We report here the molecular characterization of these variants. Amplification of glycophorin C mRNA from the Yus phenotype, using two oligonucleotide primers that span the coding domain, generated a 338-bp fragment compared with a 395-bp fragment generated by amplification of normal glycophorin C mRNA. Sequencing of the mutant 338-bp fragment identified a 57-bp deletion that corresponds to exon 2 of the glycophorin C gene. Similar analysis showed deletion of 84-bp exon 3 in the Gerbich phenotype. In contrast to the generation of shorter than normal DNA fragments from mRNA amplification in the Yus and Gerbich phenotypes, amplification of mRNA from the Webb phenotype generated a normal-sized fragment. Sequencing of this DNA fragment showed an A----G substitution at nucleotide 23 of the coding sequence, resulting in the substitution of asparagine by serine. This modification accounts for the altered glycosylation of glycophorin C seen in this phenotype. These results have enabled us to characterize glycophorin C variants in three different phenotypes that involve deletions of exons 2 and 3 of the glycophorin C gene, as well as a point mutation in exon 1 that results in altered glycosylation of this protein.

Abnormal membrane physical properties of red cells in McLeod syndrome

McLeod red cells (RBCs) lack Kx antigens and have weak expression of the Kell antigens. Individuals who carry the McLeod phenotype have acanthocytic RBCs and a compensated hemolytic state. To elucidate the role of the protein on which the Kx antigens reside in maintaining membrane deformability, the rheologic properties of McLeod RBCs were determined by ektacytometry. RBCs were obtained from normal individuals and from four patients with McLeod syndrome. Osmotic gradient deformability profiles of McLeod RBCs showed decreased whole cell deformability. Resealed ghosts from McLeod RBCs also showed decreased deformability, partly because of the decreased cell surface area and partly because of an intrinsic membrane stiffness in this syndrome. For the measurement of membrane mechanical stability, resealed ghosts were subjected to constant high shear stress in the ektacytomer, and deformability was recorded continuously as the deformable ghosts fragmented into rigid spherical vesicles. Membranes from McLeod RBCs showed a noticeable increase in mechanical stability. Acquired causes of acanthocytosis, such as liver disease, did not cause the rheologic abnormalities observed in McLeod cells. Other abnormalities noted in McLeod RBCs were decreased RBC potassium content and an increased number of dense RBCs, as determined by centrifugation on a discontinuous density gradient. The data indicate that McLeod RBCs are rigid and have decreased surface area and that their membranes are intrinsically rigid with increased mechanical stability. These abnormalities may account for the reduced RBC survival observed in McLeod syndrome. The protein that carries the Kx surface antigen seems to be required for the maintenance of the normal physical function of RBC skeletal proteins.

Molecular analysis of insertion/deletion mutations in protein 4.1 in elliptocytosis. I. Biochemical identification of rearrangements in the spectrin/actin binding domain and functional characterizations.

Protein 4.1 (80 kD) interacts with spectrin and short actin filaments to form the erythrocyte membrane skeleton. Mutations of spectrin and protein 4.1 are associated with elliptocytosis or spherocytosis and anemia of varying severity. We analyzed two mutant protein 4.1 molecules associated with elliptocytosis: a high molecular weight 4.1 (95 kD) associated with mild elliptocytosis without anemia, and a low molecular weight 4.1 (two species at 68 and 65 kD) associated with moderate elliptocytosis and anemia. 4.1(95) was found to contain a approximately 15-kD insertion adjacent to the spectrin/actin binding domain comprised, at least in part, of repeated sequence. 4.1(68/65) was found to lack the entire spectrin-actin binding domain. The mechanical stability of erythrocyte membranes containing 4.1(95) was identical to that of normal membranes, consistent with the presence of an intact spectrin-actin binding domain in protein 4.1. In contrast, membranes containing 4.1(68/65) have markedly reduced mechanical stability as a result of deleting the spectrin-actin binding domain. The mechanical stability of these membranes was improved following reconstitution with normal 4.1. These studies have thus enabled us to establish the importance of the spectrin-actin binding domain in regulating the mechanical stability of the erythrocyte membrane.

Decreased membrane mechanical stability and in vivo loss of surface area reflect spectrin deficiencies in hereditary spherocytosis.

Whereas marked variations in the clinical manifestations of hereditary spherocytosis have long been recognized, we have only recently begun to define the molecular basis for this heterogeneity. An important unanswered question is whether decreased spectrin results in reduced membrane mechanical stability, and if this reduction in membrane mechanical stability can be related to in vivo surface area loss. Using the ektacytometer, we quantitated membrane surface area and stability in erythrocytes from 18 individuals with hereditary spherocytosis and deficiencies of spectrin (30-80% of normal spectrin level). Membrane mechanical stability was reduced and the magnitude of the reductions correlated with the spectrin content. Moreover, the reductions in mechanical stability correlated with in vivo loss of membrane surface area. These data indicate that decreased spectrin content results in reduced membrane mechanical stability and surface area loss in vivo. We conclude that partial deficiencies of spectrin, reductions in membrane mechanical stability, and loss of membrane surface area are directly related and are major features determining the heterogeneous clinical manifestations of hereditary spherocytosis.

Normal membrane function of abnormal beta-related erythrocyte sialoglycoproteins.

Red cells totally deficient in beta and gamma sialoglycoproteins (the Leach type of Gerbich-negative) are elliptocytic and have altered membrane physical properties as evidenced by marked decreases in both membrane mechanical stability and membrane deformability. Red cells from individuals who are of the Gerbich and Yus phenotypes of Gerbich-negative are also deficient in beta and gamma sialoglycoproteins, but possess abnormal beta-related sialoglycoproteins. In order to determine if these beta-related sialoglycoproteins can functionally substitute for normal sialoglycoproteins, we measured membrane deformability and stability of red cells of the Gerbich and Yus phenotypes. In contrast to the red cells of the Leach phenotype, cells of Gerbich and Yus phenotypes were found to have normal membrane deformability and stability. Moreover, flow cytometric analysis using a monoclonal anti-beta antibody revealed that the Gerbich and Yus phenotype red cells expressed the beta-related sialoglycoprotein to the same extent as its normal counterpart on normal cells. Based on these data, we suggest that the abnormal beta-related sialoglycoproteins can functionally substitute for normal beta and gamma sialoglycoproteins.

Identification of a functional role for human erythrocyte sialoglycoproteins beta and gamma

Four distinct erythrocyte membrane sialoglycoproteins (SGPs) denoted alpha, beta, gamma, and delta have been described, but their functions have not yet been defined. Recent evidence suggests that several of these SGPs associate with membrane skeletal proteins. Because the membrane skeletal protein network plays an important role in regulating the membrane material properties of deformability and mechanical stability, we wanted to determine whether the SGPs, through their interaction with the membrane skeleton, can modulate these membrane properties. We measured membrane mechanical stability and membrane deformability of erythrocytes that were deficient in either alpha, or delta or beta and gamma SGPs. Only erythrocytes deficient in beta and gamma SGP had altered membrane properties, as evidenced by marked decreases in both membrane mechanical stability (50% of normal) and membrane deformability (40% of normal). Erythrocytes deficient in either alpha or delta SGP had normal deformability and stability. Based on these data, we suggest that an interaction of beta and/or gamma SGP with the membrane skeleton plays a functionally important role in regulating normal erythrocyte membrane properties.

Identification of a Functional Role for Human Erythrocyte Sialoglycoproteins β and γ

Four distinct erythrocyte membrane sialoglycoproteins (SGPs) denoted alpha, beta, gamma, and delta have been described, but their functions have not yet been defined. Recent evidence suggests that several of these SGPs associate with membrane skeletal proteins. Because the membrane skeletal protein network plays an important role in regulating the membrane material properties of deformability and mechanical stability, we wanted to determine whether the SGPs, through their interaction with the membrane skeleton, can modulate these membrane properties. We measured membrane mechanical stability and membrane deformability of erythrocytes that were deficient in either alpha, or delta or beta and gamma SGPs. Only erythrocytes deficient in beta and gamma SGP had altered membrane properties, as evidenced by marked decreases in both membrane mechanical stability (50% of normal) and membrane deformability (40% of normal). Erythrocytes deficient in either alpha or delta SGP had normal deformability and stability. Based on these data, we suggest that an interaction of beta and/or gamma SGP with the membrane skeleton plays a functionally important role in regulating normal erythrocyte membrane properties.

Stabilization of erythrocyte membranes by polyamines.

Using a laser diffraction technique, we have studied the effects of putrescine, spermidine, and spermine, the three physiologic polyamines, on the deformability and mechanical stability of human erythrocyte membranes. Ghosts resealed with polyamines were subjected to high fluid shear stress in an ektacytometer. All polyamines increased the membrane shear modulus (decreased deformability) in a concentration- and time-dependent manner. The order of effectiveness was spermine greater than spermidine greater than putrescine. At 10 microM, spermine appreciably decreased membrane deformability. For the measurement of membrane mechanical stability, resealed ghosts were subjected to constant high shear stress in the ektacytometer and deformability was continuously recorded as the deformable ghosts fragmented into rigid spherical vesicles. Polyamines, especially spermine, caused a noticeable increase in the t1/2 for fragmentation. These effects could not be ascribed to proteolysis or Ca2+-induced transglutamination. That the effects of polyamines were specific and not simply due to their positive charge was demonstrated by the finding that Ca2+ and Mg2+ destabilized the erythrocyte membrane as evidenced by decreasing the t1/2 for fragmentation. Extracellular polyamines were not effective except under conditions that caused significant accumulation inside the cell. The data indicate that intracellular physiologic polyamines, especially spermine, decrease erythrocyte membrane deformability and stabilize the membrane skeleton, making it more resistant to fragmentation.

A Technique to Detect Reduced Mechanical Stability of Red Cell Membranes: Relevance to Elliptocytic Disorders

A method has been developed for determining the relative tendency of red cell membranes to fragment under shear stress. For these measurements, resealed ghosts were subjected to constant, high fluid shear stress in an ektacy-tometer. The deformability signal, which this instrument provides, progressively declined as the deformable cells were transformed into less deformable, more spherical cell fragments. The deformability signal for normal ghost preparations decayed over a narrowly defined time period; this decay was accompanied by the production of cell fragments, as shown by microscopic examination and analysis of particle volume distribution. The method was used to study cells from patients with various hemolytic disorders. Membrane preparations from patients with various types of nonhemolytic congenital elliptocytoses showed a reduction in the time required for fragmentation to about half the normal value. Membrane preparations from patients with hemolytic elliptocytosis associated with a severe deficiency of the membrane skeletal protein band 4.1 showed an even greater reduction in fragmentation time, distinct from the values for nonhemolytic samples. In elliptocytoses secondary to myelofibrosis, the fragmentation times were within the normal range. In hereditary spherocytosis (HS), heterogeneity in fragmentation times was observed; marginally decreased or normal fragmentation times were seen for cells from 8 patients, but a pronounced shortening in fragmentation time was found for one other HS patient. A defect was previously found in spectrin-band 4.1 binding for this same patient. These observations suggest that this membrane fragmentation technique may be useful for the identification of certain specific disorders or their clinical variants in which the integrity of membrane skeletal interactions has been perturbed in such a way that the mechanical stability of the membrane is reduced.