Interaction between glycophorin and ganglioside GM1 on liposomal membranes. Effect of the interaction on the susceptibility of membranes to HVJ

Liposomes could bind and fuse efficiently to human erythrocytes in the presence of HVJ when they contained glycophorin isolated from human erythrocytes (Umeda, M., et al. (1983) J. Biochem. 94, 1955). In the present work we demonstrated that HVJ-induced fusion between liposomes containing glycophorin and erythrocytes was suppressed when GM1 coexisted with glycophorin in the same liposomal membranes. Asialo-GM1 and other gangliosides such as GM3 and sialosylparagloboside did not affect the fusion between the liposomes and erythrocytes. An intermolecular interaction between glycophorin and GM1 was suggested by the ESR spectrum obtained from liposomes containing glycophorin and a ganglioside GM1 analog carrying a nitroxyl spin label in the fatty acyl chains (5SL-gangliosidoide). The overall splitting value (2A parallel) observed in the ESR spectrum of liposomes containing 5SL-gangliosidoide increased with increase of the amount of glycophorin, whereas 2A parallel of spin-labeled phosphatidylcholine was not changed. The increase of 2A parallel of 5SL-gangliosidoide suggests that the mobility of the fatty acyl chain of the gangliosidoide was restricted by the interaction with glycophorin. It can be concluded that GM1 located near glycophorin, a receptor of the virus, interferes with the activity of viral F protein, inhibiting the fusion of liposome to erythrocyte.     

Effect of lipid composition on HVJ-mediated fusion of glycophorin liposomes to erythrocytes

We previously reported that liposomes containing glycophorin or gangliosides, both of which were isolated from human erythrocytes, are efficiently fused to erythrocyte membranes in the presence of HVJ (Umeda, M. et al., J. Biochem. 94, 1955-1966 (1983), and Virology 133, 172-182 (1984]. In the present work, the effect of lipid composition in glycophorin liposomes on their sensitivity to fusion with erythrocytes was studied. Very little fusion occurred when glycophorin liposomes composed of dipalmitoylphosphatidylcholine-dicetylphosphate (9:1), dimyristoylphosphatidylcholine-dicetylphosphate (9:1), or egg yolk phosphatidylcholine-dicetylphosphate (9:1) were incubated with human erythrocytes in the presence of HVJ at 37 degrees C. Addition of cholesterol into these liposomal membranes greatly enhanced the sensitivity of the liposomes to fusion. The presence of phosphatidic acid and phosphatidylethanolamine in liposomes also enhanced the sensitivity, whereas the presence of lysophosphatidylcholine had no significant effect on the ability of the liposomes to fuse. The fusion efficiency of liposomes was also enhanced by the presence of glucosylceramide. Change of lipid composition in liposomes had, however, no appreciable influence on the HVJ-mediated binding of liposomes to erythrocytes, suggesting that the interaction between HANA protein of HVJ and glycophorin in liposomes was not affected by the lipid composition of the liposomes.     

Studies on the interaction of Sendai virus with liposomal membranes. Sendai virus-induced agglutination of liposomes containing glycophorin

Liposomes constituted with the major sialoglycoprotein of human erythrocytes, glycophorin, were used as models for studies on cell-virus interactions. Liposomes composed of egg yolk phosphatidylcholine, cholesterol and glycophorin were found to interact with the paramyxovirus HVJ to form aggregates. The aggregation process was temperature dependent: it was maximal at 0 degrees C and decreased with increase of the incubation temperature. The activity of viral neuraminidase is also temperature dependent, and it increases with increase of the incubation temperature; release of N-acetylneuraminic acid was negligible at 0 degrees C. Shift-up of the incubation temperature immediately cancelled HVJ-induced agglutination of liposomes. Viruses attached to liposomes seemed to be released into the supernatant when the 'virus-liposome' complex formed at 0 degrees C was incubated at 37 degrees C, possibly as a result of breakdown of the 'binding' site by neuraminidase. The characteristics of the interaction of HVJ with liposomes containing glycophorin appeared to be phenomenologically similar to those of HVJ-cell interaction.     

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.     

Kinetic analysis of fusion of hemagglutinating virus of Japan with erythrocyte membrane using spin-labeled phosphatidylcholine

HVJ* (hemagglutinating virus of Japan containing spin-labeled phosphatidylcholine in its envelope around 10 mol %) was adsorbed onto erythrocytes or erythrocyte ghosts at various doses, and the ESR spectrum of the virus-cell system was measured at 37 degrees C. The peak-height increase for the HVJ*-ghost system was satisfactorily analyzed on the basis of envelope fusion by a first-order kinetic equation with two different rate constants. The rate constant was obtained as k1 = 0.84 min-1 and k2 = 0.011 min-1, independent of the virus dose. The fraction of virus fused at the rate constant k1 decreased with the dose. However, the average number of fast-fusing viruses per cell was nearly independent of the dose, and the value was one to two. The peak-height increase in the HVJ*-erythrocyte system was caused by both envelope fusion and phospholipid exchange catalyzed by the virus-induced hemolyzate. At lower doses, where the virus-induced hemolysis was small and, therefore, the rate of phospholipid exchange was small, the peak-height increase could be analyzed by the same kinetic equation with nearly the same rate constant value for k1 as that for HVJ*-ghosts. However, the k2 was larger than that for HVJ*-ghost, owing to the additional transfer by phospholipid exchange.     

HVJ (Sendai virus)-induced envelope fusion and cell fusion are blocked by monoclonal anti-HN protein antibody that does not inhibit hemagglutination activity of HVJ

Two kinds of monoclonal antibodies against HN protein of HVJ were isolated. In competitive binding assay, binding of one of these antibodies to HN protein did not inhibit binding of the other antibody to the same molecule. One of the antibodies, named HN-1 antibody, inhibited hemagglutination activity of HVJ and also blocked neuraminidase activity of the virus when fetuin and Ehrlich ascites tumor cells were used as substrates, but it did not inhibit the activity when neuramine-lactose was used as substrate. The other antibody, HN-2, did not inhibit hemagglutination activity or neuraminidase activity, but blocked HVJ-induced viral envelope-cell fusion, cell-cell fusion and hemolysis. The mechanism by which HN-2 antibody blocked the fusion process is discussed.     

Characterization of the fusogenic properties of Sendai virus: kinetics of fusion with erythrocyte membranes

A novel fluorescence assay [Hoekstra, D., De Boer, T., Klappe, K., & Wilschut, J. (1984) Biochemistry 23, 5675-5681] has been used to characterize the fusogenic properties of Sendai virus, using erythrocyte ghosts and liposomes as target membranes. This assay involves the incorporation of the "fusion-reporting" probe in the viral membrane, allowing continuous monitoring of the fusion process in a very sensitive manner. Fusion was inhibited upon pretreatment of Sendai virus with trypsin. Low concentrations of the reducing agent dithiothreitol (1 mM) almost completely abolished viral fusion activity, whereas virus binding was reduced by ca. 50%, indicating that the fusogenic properties of Sendai virus are strongly dependent on the integrity of intramolecular disulfide bonds in the fusion (F) protein. Pretreatment of erythrocyte ghosts with nonlabeled Sendai virus inhibited subsequent fusion of fluorophore-labeled virus irrespective of the removal of nonbound virus, thus suggesting that the initial binding of the virus to the target membrane is largely irreversible. As a function of pH, Sendai virus displayed optimal fusion activity around pH 7.5-8.0. Preincubation of the virus at suboptimal pH values resulted in an irreversible diminishment of its fusion capacity. Since virus binding was not affected by the pH, the results are consistent with a pH-induced irreversible conformational change in the molecular structure of the F protein, occurring under mild acidic and alkaline conditions. In contrast to virus binding, fusion appeared to be strongly dependent on temperature, increasing ca. 25-fold when the temperature was raised from 23 to 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the target membrane structure in fusion with Sendai virus

Fusion between membranes of Sendai virus and liposomes or human erythrocytes ghosts was studied using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We considered only viral fusion that reflects the biological activity of the viral spike glycoproteins. The liposomes were made of phosphatidylcholine, and the effects of including cholesterol, the sialoglycolipid GD1a, and/or the sialoglycoprotein glycophorin as receptors were tested. Binding of Sendai virus to those liposomes at 37 degrees C was very weak. Fusion with the erythrocyte membranes occurred at a 30-fold faster rate than with the liposomes. Experiments with biological and liposomal targets of different size indicated that size did not account for differences in fusion efficiency.     

Action of polyethylene glycol on the fusion of human erythrocyte membranes

Summary Factors affecting the polyethylene glycol (PEG)-induced membrane fusion were examined. Human erythrocyte membrane ghosts, cytoskeleton-free vesicles budded from erythrocytes, mechanically disrupted erythrocyte vesicles, and recombinant vesicles from glycophorin and egg phosphatidylcholine were used as models. Fusion was monitored by darkfield light microscopy and by freeze-fracture electron microscopy. Osmotic swelling was found necessary for fusion between membrane ghosts following PEG treatment. The sample with the highest fusion percentage was sealed ghosts incubated in hypotonic media after at least 5 min of treatment in <25% PEG. At similar osmolarity, glycerol, dextran and PEG produced progressively more pronounced intramembranous particle (IMP) patching, correlating with their increasing fusion percentages. The patching of IMP preceded cell-cell contact, and occurred without direct PEG-protein interaction. The presence of cytoskeletal elements in small vesicles had no significant effect on fusion, nor on the aggregation of intramembranous particle (IMP) upon PEG treatment. Disrupting the membrane by lysolecithin, dimethylsulfoxide, retinol or mild sonication resulted in the fragmentation of ghosts without an increase in fusion percentage. The purity of the commercial PEG used had no apparent effect on fusion. We concluded that the key steps in PEG-induced fusion of cell membrane are the creation of IMP-free zones, and the osmotic swelling of cells after the formation of bilayer contacts during the PEG treatment. Cell cytoskeleton affects PEG-induced fusion only to the extent of affecting IMP patching.     

Interaction of Sendai virions with resealed human erythrocyte ghosts. Lateral mobility of the viral glycoproteins in the cell membrane following fusion

Two independent methods demonstrated that resealed human erythrocyte ghosts undergo Sendai virus-mediated cell-cell fusion to a much lower degree (about 4%) than intact erythrocytes, in spite of similar levels of viral envelope-cell fusion in the two preparations. Fluorescence photobleaching recovery (FPR) showed similar lateral mobilities of the viral glycoproteins following fusion with either ghosts or whole erythrocytes. It is suggested that although viral glycoprotein mobilization in the cell membrane is essential for cell-cell fusion, the target cell properties are also important; in the absence of the required cellular parameters, the mobilization may not be a sufficient condition.     

Stimulation of virus-induced fusion of Ehrlich ascites tumor cells by 3',5'-cyclic AMP.

HVJ(Sendai virus)-induced fusion of Ehrlich ascites tumor cells was found to be stimulated by treatments which increase the intracellular level of cyclic AMP. This stimulation was optimal at an external concentration of Ca⁺⁺ of about 0.5 mM. During the process of cell fusion, the intracellular concentration of cyclic AMP was increased with a maximum at 2 min after the initiation of the fusion reaction.Evidence is also presented which suggests that the increase of the cyclic nucleotide is a part of control mechanism of HVJ-induced fusion of eukariotic cells. Thus, this cyclic AMP-stimulated process could be one of the step(s) requiring ATP and Ca⁺⁺, both of which are necessary for the overall fusion process of the tumor cells.     

Studies on the role of goat heart galectin-1 as an erythrocyte membrane perturbing agent

Galectins are β-galactoside binding lectins with a potential hemolytic role on erythrocyte membrane integrity and permeability. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its hemolytic actions on erythrocyte membrane. When exposed to various saccharides, lactose and sucrose provided maximum protection against hemolysis, while glucose and galactose provided lesser protection against hemolysis. GHG-1 agglutinated erythrocytes were found to be significantly hemolyzed in comparison with unagglutinated erythrocytes. A concentration dependent rise in the hemolysis of trypsinized rabbit erythrocytes was observed in the presence of GHG-1. Similarly, a temperature dependent gradual increase in percent hemolysis was observed in GHG-1 agglutinated erythrocytes as compared to negligible hemolysis in unagglutinated cells. The hemolysis of GHG-1 treated erythrocytes showed a sharp rise with the increasing pH up to 7.5 which became constant till pH 9.5. The extent of erythrocyte hemolysis increased with the increase in the incubation period, with maximum hemolysis after 5 h of incubation. The results of this study establish the ability of galectins as a potential hemolytic agent of erythrocyte membrane, which in turn opens an interesting avenue in the field of proteomics and glycobiology.     

Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.     

Calpain activation is essential for membrane fusion of erythrocytes in the presence of exogenous Ca2+.

The membrane mobility agent, 2-(methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) promotes the fusion of rat, rabbit, and human erythrocytes in the presence of exogenous Ca2+. Under these conditions, the high sensitivity form of calcium-activated neutral protease (mu-calpain) in erythrocytes is activated autolytically. mu-Calpain is activated in accordance with fusion; that is, both erythrocyte fusion and autolytic activation of mu-calpain are induced in rat erythrocytes at 30 min, in rabbit erythrocytes at 150 min, and in human erythrocytes at 240 min after the addition of A2C and Ca2+. When erythrocytes are preincubated with the Ca2+ ionophore A23187, both fusion and autolytic activation start earlier. A leupeptin analogue, Cbz-Leu-Leu-Leu-aldehyde (ZLLLal), inhibits both the autolytic activation of mu-calpain and fusion induced by A2C and Ca2+. These results indicate that treatment of erythrocytes with A2C and Ca2+, results in first an influx of Ca2+ into the cells, followed by autolytic activation of mu-calpain, proteolysis of membrane proteins, exposure of fusion-sites, and, finally, fusion of erythrocytes.     

Mechanism of human erythrocyte hemolysis induced by short-chain phosphatidylcholines and lysophosphatidylcholine

The incorporation and accumulation of a certain amount of short-chain phosphatidylcholine or lysophosphatidylcholine into lipid bilayers of erythrocyte membranes is the first step causing membrane perturbation in the process of hemolysis. Accumulation of dilauroylglycerophosphocholine into membranes makes human erythrocytes "permeable cells"; Ions such as Na+ or K+ can permeate through the membrane, though large molecules such as hemoglobin can not. The "pore" formation was partially reproduced in liposomes prepared from lipids extracted from human erythrocyte membranes; C12:0PC induced the release of glucose from liposomes but did not significantly induce the release of dextran. It was suggested that the phase boundary between dilauroylglycerophosphocholine and the host membrane bilayer or dilauroylglycerophosphocholine rich domain itself behaves as "pores." Erythrocytes could expand to 1.5 times the original cell volume without any appreciable hemolysis when incubated with C12:0PC at 37 degrees C. The capacity of the erythrocytes to expand was temperature dependent. The capacity may play an important role in the resistance of the cells against lysis. The "permeable cell" stage could be hardly observed when erythrocytes were treated with didecanoylglycerophosphocholine and lysophosphatidylcholine. Perturbation induced by accumulation of didecanoylglycerophosphocholine or lysophosphatidylcholine may cause non specific destruction of membranes rather than formation of a kind of "pore."     

Insertion of lipid domains into plasma membranes by fusion with erythrocytes

After prelabeling the plasma membrane with several lipid-specific fluorescent probes, erythrocytes with symmetric lipid bilayers were fused with culture cells using either poly(ethylene glycol) or Sendai virus as fusogen. Several nonspecific probes were transferred to, and became uniformly distributed within, the culture cell membrane upon fusion. In contrast, when merocyanine 540, which displays preferential binding to bilayers in which the lipids are loosely packed, was used to prelabel erythrocytes, fluorescence remained localized within a small confined area of the membrane, even 24 h after fusion. These results suggest that insertion of the lipids of the erythrocyte membrane into the plasma membrane of the culture cell can produce discrete domains which persist as such for long periods following fusion. Because the inserted proteins of the erythrocyte membrane similarly do not freely diffuse throughout the culture cell membrane, interactions between membrane proteins and lipids may be involved in this singular compartmentalization.     

Transmembrane phospholipid motions induced by F glycoprotein in hemagglutinating virus of Japan.

Transfer of phospholipid from the envelope of hemagglutinating virus of Japan (HVJ) to erythrocyte (RBC) membrane and the virus-induced transfer of phospholipid between RBC membranes were studied using spin-labeled phosphatidylcholine (PC). The transfer of PC from membranes labeled densely with PC to unlabeled membranes was followed by the peak height increase in the electron spin resonance spectrum. The two kinds of transfer reactions took place very rapidly as reported previously. To obtain further details, the transfer reactions were studied with HVJ, HVJ inactivated by trypsin, HVJ harvested early, HVJ grown in fibroblast cells, the fibroblast HVJ activated by trypsin, influenza virus, and glutaraldehyde-treated RBCs. The results demonstrated that the viral F glycoprotein played a crucial role in the transmembrane phospholipid movements as well as in the fusion and hemolysis of RBCs. The transfer from HVJ to RBC's occurred partially through an exchange mechanism not accompanying the envelope fusion. This was shown by a decrease in the exchange broadening of the electron spin resonance spectrum of released spin-labeled HVJ (HVJ) and also by an increase in the ratio of PC to viral proteins incorporated into RBC membranes. HVJ modified RBC membrane so as to be able to exchange its phospholipids with those of inactive membranes such as fibroblast HVJ, influenza virus, glutaraldehyde-treated RBC'S, and phosphatidylcholine vesicles. HVJ affected the fluidity of RBC membranes markedly, the environments around PC being much fluidized. The virus-induced fusion was discussed based on close apposition of the membranes by HANA proteins and on the destabilization and fluidization of RBC membranes by F glycoproteins.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus).

A large number of viral materials are associated with the surface of cells after cell fusion with HVJ at 37 °C for 30 min. This is due to fusion of viral envelopes with the cell membrane. Studies were made on the process from viral adsorption to cell-cell, or cell-viral envelope fusion. On incubation at low temperatures, such as 0–15 °C, no envelope fusion or cell fusion was observed, although there was some interaction between the virus and cells. This interaction resulted in loss of hemadsorption (HA) activity of the cells and partial damage of the ion barrier of the cell membrane. The viral particles seem to come close to the lipid layer of the cell membrane at the low temperatures and to distort the non-flexible membrane structure. On incubation of the cell-virus complex at 37 °C, the cells rapidly became HA-positive and the HA activity was maximal within 5 min. At this stage there was much leakage of ions through the cell membrane. On further incubation the damage to the ion barrier of the cell membrane was repaired completely with completion of cell fusion. This process may be correlated with fusion of viral envelopes with cell membranes and restoration of the cell membrane fused with them.     

Characterisation of myogenic cell membrane: II. Dynamic changes in membrane lipids during the differentiation of mouse C2 myoblast cells

Proliferating mouse C2 myoblast cells resist haemagglutinating virus of Japan, Sendai virus (HVJ) mediated cell fusion. However, differentiating C2 cells can be induced to fuse by HVJ, suggesting that the rigid membrane of C2 cells changes during the differentiation. To investigate this phenomenon, changes in membrane lipids which affect fluidity were examined. Membrane cholesterol gradually decreased with the differentiation of C2 cells. However, spontaneous fusion to form myotubes and artificial fusion induced by HVJ were both inhibited when the level of cholesterol was prevented from falling in the cell membrane. The membranes of differentiating C2 cells contained more unsaturated fatty acids than those of proliferating cells. Thus, when differentiating C2 cells were treated with stearate (a saturated fatty acid), they failed to form myotubes and were insensitive to HVJ-mediated fusion. Whereas, if proliferating C2 cells were given linolenate (an unsaturated fatty acid), they became capable of HVJ-induced fusion. These results indicate that differentiating C2 cells change their fusion sensitivity by decreasing cholesterol, probably at the same time as they increase the unsaturated fatty acid content of the cell membrane.     

Heat-induced alterations in monkey erythrocyte membrane phospholipid organization and skeletal protein structure and interactions

Rhesus monkey erythrocytes were subjected to heating at 50 degrees C for 5-15 min, and the heat-induced effects on the membrane structure were ascertained by analysing the membrane phospholipid organization and membrane skeleton dynamics and interactions in the heated cells. Membrane skeleton dynamics and interactions were determined by measuring the Tris-induced dissociation of the Triton-insoluble membrane skeleton (Triton shells), the spectrin-actin extractability at low ionic strength, spectrin self-association and spectrin binding to normal monkey erythrocyte membrane inside-out vesicles (IOVs). The Tris-induced Triton shell dissociation and spectrin-actin extractability were markedly decreased by the erythrocyte heating. Also, the binding of the heated erythrocyte membrane spectrin-actin with the IOVs was much smaller than that observed with the normal erythrocyte spectrin-actin. Further, the spectrin structure was extensively modified in the heated cells, as compared to the normal erythrocytes. Transbilayer phospholipid organization was ascertained by employing bee venom and pancreatic phospholipases A2, fluorescamine, and Merocyanine 540 as the external membrane probes. The amounts of aminophospholipids hydrolysed by phospholipases A2 or labeled by fluorescamine in intact erythrocytes considerably increased after subjecting them to heating at 50 degrees C for 15 min. Also, the fluorescent dye Merocyanine 540 readily stained the 15-min-heated cells but not the fresh erythrocytes. Unlike these findings, the extent of aminophospholipid hydrolysis in 5-min-heated cells by phospholipases A2 depended on the incubation time. While no change in the membrane phospholipid organization could be detected in 10 min, prolonged incubations led to the increased aminophospholipid hydrolysis. Similarly, fluorescamine failed to detect any change in the transbilayer phospholipid distribution soon after the 5 min heating, but it labeled greater amounts of aminophospholipids in the 5-min-heated cells, as compared to normal cells, after incubating them for 4 h at 37 degrees C. These results have been discussed to analyse the role of membrane skeleton in maintaining the erythrocyte membrane phospholipid asymmetry. It has been concluded that both the ATP-dependent aminophospholipid pump and membrane bilayer-skeleton interactions are required to maintain the transbilayer phospholipid asymmetry in native erythrocyte membrane.