Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. II. Formation of membrane leaks in ghost membranes after limited proteolysis of skeletal proteins by trypsin.

Limited proteolysis of human erythrocyte ghost membranes by low levels of trypsin (10-240 ng/ml) added bilaterally at 0 degrees C together with the proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF) before resealing at 37 degrees C leads to a graded digestion of spectrin and ankyrin and the disappearance of band 4.1 protein, while band 3 is cleaved only to a very low extent. These alterations are accompanied by an increase of membrane permeability of the resealed ghosts to hydrophilic nonelectrolytes (erythritol to sucrose), taken to reflect impaired resealing. Moreover, the membrane begins to vesiculate. Shedding of vesicles during the efflux measurements can not be responsible for the increased release of test solutes, since the ghosts do not loose hemoglobin and discriminate the nonelectrolytes according to their size. Moreover, the vesiculation site itself does not seem to act as the leak site, since ghosts prepared from erythrocytes pretreated with a carbodiimide which induces membrane rigidification still exhibit a pronounced protein degradation and vesiculation while the permeability enhancement induced by trypsination is markedly suppressed. The trypsin-induced leak has the properties of an aqueous pore as indicated, besides size selectivity, by its inhibition by phloretin and the very low activation energy. In analogy with concepts developed in the preceding paper (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)) the impaired resealing after limited proteolysis is assumed to be related to a perturbation of interactions of membrane skeletal elements with themselves and/or with the bilayer domain constituting the permeability barrier.     

Formation and properties of aqueous leaks induced in human erythrocytes by electrical breakdown

Leaks were induced in human erythrocytes by brief (tau = 1-40 microseconds) discharges of high electric fields (3-20 kV/cm). Leak permeabilities were characterized by measuring (a) net and tracer fluxes of K+ and nonelectrolytes under protection of the cells against colloid-osmotic lysis, or (b) rates of colloid osmotic lysis in various salt solutions. The induced permeabilities are essentially stable for hours at 0-2 degrees C. Leak permeability P increases exponentially with the breakdown voltage ED according to a function of the general type P = bED. The basis b varies with the pulse length. A log-linear presentation reveals a biphasic linear relationship with a break at which the slope (= log b) decreases markedly. Elevated ionic strengths of the suspension medium during the electric discharge enhance leak formation. Leak permeability exhibits an apparent activation energy of 29 +/- 5 kJ/mol, indicative of diffusion through aqueous pathways. Somewhat differing equivalent pore radii emerge from measurements with different probes: 0.6-0.8 nm from tracer fluxes of polyols (Mr = 3600, ED = 4-7 kV/cm) and 0.8-1.9 nm from osmotic protection studies with polyethylene glycols (Mr = 200-3300, ED = 6-10 kV/cm). These numbers and the non-monoexponential increase of leak permeability with the field strength suggest a dual mechanism for the increase of leak permeability: an increase of the number of pores at low breakdown voltage and an additional increase of pore size at higher voltage. Estimated numbers of pores range from 1 to 10 per cell, which suggests dynamic fluctuating structural defects to be involved. The leaks discriminate small monovalent inorganic ions in the sequence of free solution mobility. Organic anions are discriminated according to size and charge. Common properties of these electrically induced defects and of chemically induced leaks (diamide, periodate, t-butylhydroperoxide) in the erythrocyte membrane suggest close similarities in the molecular organization.     

Xanthine oxidase-catalyzed crosslinking of cell membrane proteins

Isolated erythrocyte membranes exposed to protease-free xanthine oxidase plus xanthine and ferric iron undergo lipid peroxidation and protein crosslinking (appearance of high molecular weight aggregates on sodium dodecyl sulfate (SDS) gel electrophoresis). Spectrin is more susceptible to crosslinking than the other polypeptides. Thiol-reducible bonds (disulfides) as well as nonreducible bonds are generated, the former type relatively rapidly (detected within 10-20 min) and the latter type more slowly (usually detected after 1 h). Reducible crosslinking is inhibited by catalase, but not by superoxide dismutase, desferrioxamine, butylated hydroxyltoluene, and mannitol; whereas nonreducible crosslinking, like free radical lipid peroxidation, is inhibited by all of these agents except mannitol. Zinc(II) also inhibits lipid peroxidation, but stimulates disulfide bond formation to the virtual exclusion of all other crosslinking. Our results indicate that disulfide formation is dependent on H2O2, but not O2- or iron. However, O2-, H2O2, and iron are all required for lipid peroxidation and nondisulfide crosslinking, suggesting the intermediacy of OH generated via the iron-catalyzed Haber-Weiss reaction. The possible role of malonaldehyde (MDA, a by-product of lipid peroxidation) in the latter type of crosslinking was examined. Solubilized samples of xanthine/xanthine oxidase-treated membranes showed a strong visible fluorescence (emission maximum 450 nm; excitation 390 nm). This resembled the fluorescence of membranes treated with authentic MDA, which forms conjugated imine linkages between amino groups. Fluorescence scanning of SDS gels from MDA-treated membranes showed a strong signal coincident with crosslinked proteins and also one in the low molecular weight, nonprotein region, suggestive of aminolipid conjugates. Similar scanning on xanthine/xanthine oxidase-reacted membranes indicated that all fluorescence is associated with the lipid fraction. Thus, nonreducible protein crosslinks in this system do not appear to be of the MDA-derived, Schiff base type.     

Selective phosphorylation of erythrocyte membrane proteins by the solubilized membrane protein kinases.

This report describes the substrate and phosphoryl donor specificities of solubilized erythrocyte membrane cyclic adenosine 3',5'-monophosphate (cAMP)-independent protein kinases toward human and rabbit erythrocyte membrane proteins. Three types of substrate preparations have been utilized: heat-inactivated ghosts, isolated spectrin, and 2,3-dimethylmaleic anhydride (DMMA)-extracted membranes. A 30 000-dalton protein kinase, extracted from either human or rabbit erythrocyte membranes, catalyzes the phosphorylation of heat-inactivated membranes in the presence of ATP. The resulting phosphorylation profile is analogous to that of the autophosphorylation of membranes with ATP (in the absence of cAMP). These kinases also phosphorylate band 2 of isolated spectrin and band 3, but not glycophorin, in the DMMA-extracted ghosts. The ability of the 30 000-dalton kinases to use GTP as a phosphoryl donor appears to be related to the substrate or some other membrane factor. A second kinase, which is 100 000 daltons and derived from rabbit erythrocyte membranes, uses ATP or GTP to phosphorylate membrane proteins 2, 2.1, 2.9-3 in heat-inactivated ghosts, band 2 in isolated spectrin, glycophorin, and to a lesser extent, band 3 in the DMMA-extracted ghosts.     

Resolution of erythrocyte membrane proteins by two-dimensional electrophoresis.

A two-dimensional electrophoresis method has been developed which solubilizes erythrocyte membrane proteins, and which resolves the components of the band that migrates in detergent gels as if its molecular mass were 95,000 daltons. This method uses gel electrophoresis with sodium dodecyl sulfate in the first dimension and phenol, aqueous urea, and acetic acid in the second dimension. The 95,000 dalton band is known to contain several different membrane proteins, including those associated with anion transport, glucose transport, and (Na+,K+) transport. Two-dimensional electrophoresis resolved this band into one major spot and several minor ones. Pronase digestion of whole erythrocytes, followed by preparation of ghosts and two-dimensional electrophoresis, showed that only the major component of this band was digested by pronase.     

Storage-induced changes in erythrocyte membrane proteins promote recognition by autoantibodies

Physiological erythrocyte removal is associated with a selective increase in expression of neoantigens on erythrocytes and their vesicles, and subsequent autologous antibody binding and phagocytosis. Chronic erythrocyte transfusion often leads to immunization and the formation of alloantibodies and autoantibodies. We investigated whether erythrocyte storage leads to the increased expression of non-physiological antigens. Immunoprecipitations were performed with erythrocytes and vesicles from blood bank erythrocyte concentrates of increasing storage periods, using patient plasma containing erythrocyte autoantibodies. Immunoprecipitate composition was identified using proteomics. Patient plasma antibody binding increased with erythrocyte storage time, while the opposite was observed for healthy volunteer plasma, showing that pathology-associated antigenicity changes during erythrocyte storage. Several membrane proteins were identified as candidate antigens. The protein complexes that were precipitated by the patient antibodies in erythrocytes were different from the ones in the vesicles formed during erythrocyte storage, indicating that the storage-associated vesicles have a different immunization potential. Soluble immune mediators including complement factors were present in the patient plasma immunoprecipitates, but not in the allogeneic control immunoprecipitates. The results support the theory that disturbed erythrocyte aging during storage of erythrocyte concentrates contributes to transfusion-induced alloantibody and autoantibody formation.     

Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. III. Permeability of spectrin-depleted inside-out membrane vesicles to hydrophilic nonelectrolytes. Formation of leaks by chemical or enzymatic modification of membrane proteins.

Spectrin-depleted inside-out vesicles (IOV's) prepared from human erythrocyte membranes were characterized in terms of size, ground permeability to hydrophilic nonelectrolytes and their sensitivity to modification by SH reagents, DIDS and trypsin. IOV's proved to have the same permeability of their lipid domain to erythritol as native erythrocytes, in contrast to resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)), which have a residual leak. On the other hand, IOV's have a slightly elevated permeability for mannitol and sucrose, nonelectrolytes which are almost (mannitol) or fully (sucrose) impermeant in the native membrane. These increased fluxes, which have a high activation energy and can be stimulated by phloretin, are, however, also much smaller than the corresponding leak fluxes observed in resealed ghosts. In view of these differences, formation of IOV's can be concluded to go along with partial annealing of barrier defects persisting in the erythrocyte membrane after preparation of resealed ghosts. Oxidation of SH groups of the IOV membrane by diamide produces an enhancement of permeability for hydrophilic nonelectrolytes which is much less pronounced than that induced by a similar treatment of erythrocytes or ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)). Moreover, proteolytic treatment of the vesicle membrane, although leading to a marked digestion of integral membrane proteins, only induces a minor, saturating increase of permeability, much lower than that in trypsinized resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 137-142 (Part II of this series)). Since absence of the cytoskeletal proteins, spectrin and actin, is the major difference between IOV's and resealed ghosts, these results may be taken as further evidence for a dependence of the barrier properties of the erythrocyte membrane bilayer domain on its interaction with cytoskeletal elements. In contrast, these barrier properties seem to be rather insensitive to perturbations of integral proteins.     

Cross-linking of phospholipids to proteins in the erythrocyte membrane

Erythrocytes treated with the cross-linking agents difluorodinitrobenzene and suberimidate are rendered refractory to lysis. When ghosts are treated with these reagents 8.4% and 2.3% of the total lipid phosphate is cross-linked to protein by difluorodinitrobenzene and suberimidate respectively. This represents 20 and 5.8% of the amino-phospholipids. The lipids extracted from treated ghosts do not react with ninhydrin as do lipids extracted from control ghosts. Thus essentially all the amino-phospholipids of the ghosts react with these cross-linking agents and up to 20% becomes cross-linked to proteins.     

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.     

Radiation-induced changes in the structure of erythrocyte membrane proteins

Structural changes in proteins of erythrocyte membranes induced by gamma-radiation at doses of 10-10(3) Gy were studied using the method of tryptophan fluorescence quenching by acrylamide. It was found that the exposure to ionizing radiation leads to a decrease in intramolecular dynamics of membrane proteins.     

Passive shedding of erythrocyte antigens induced by membrane rigidification

Rigidification of the cell membrane lipid bilayer can lead to an increase in the degree of exposure of membrane proteins to either side of the membrane. It is shown in this study that excess increase of the membrane lipid microviscosity (‘hyper-rigidification’) in intact human erythrocytes can cause the release of Rh₀(D) and A blood group antigens from the cell surface which can then be collected from the supernatant by affinity chromatography. The most efficient antigen shedding was achieved upon incorporation of cholesteryl hemisuccinate (CHS) (incubation for 2 h at 37 °C in a mixture of 200 μg/ml CHS, 3.5% polyvinylpyrrolidone 1% bovine serum albumin, 0.5% glucose in phosphate-buffered saline) followed by application of hydrostatic pressure (1 500 atm, 5 min) which increases the lipid microviscosity by about 2-fold. This technique can be of general application for isolation of membrane proteins without disruption of the cells or the use of detergents.