Protein J (2013) 32:593–600 DOI 10.1007/s10930-013-9521-2
Effect of Human Serum Albumin Upon the Permeabilizing Activity of Sticholysin II, a Pore Forming Toxin from Stichodactyla heliantus Gloria Celedo´n • Gustavo Gonza´lez • Felipe Gulppi • Fabiola Pazos • Marı´a E. Lanio • Carlos Alvarez • Cristian Caldero´n • Rodrigo Montecinos Eduardo Lissi
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Published online: 6 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Sticholysin II (St II) is a haemolytic toxin isolated from the sea anemone Stichodactyla helianthus. The high haemolytic activity of this toxin is strongly dependent on the red cell status and the macromolecule conformation. In the present communication we evaluate the effect of human serum albumin on St II haemolytic activity and its capacity to form pores in the bilayer of synthetic liposomes. St II retains its pore forming capacity in the presence of large concentrations (up to 500 lM) of human serum albumin. This effect is observed both in its capacity to produce red blood cells haemolysis and to generate functional pores in liposomes. In particular, the capacity of the toxin to lyse red blood cells increases in the presence of human serum albumin (HSA). Regarding the rate of the pore forming process, it
Electronic supplementary material The online version of this article (doi:10.1007/s10930-013-9521-2) contains supplementary material, which is available to authorized users. G. Celedo´n Departamento de Fisiologı´a, Facultad de Ciencias, Universidad de Valparaı´so, Valparaiso, Chile G. Gonza´lez F. Gulppi Facultad de Ciencias, Instituto de Quı´mica, Pontificia Universidad Cato´lica de Valparaı´so, Valparaiso, Chile F. Pazos M. E. Lanio C. Alvarez Facultad de Biologı´a, Centro de Estudios de Proteı´nas, Universidad de la Habana, Habana, Cuba C. Caldero´n E. Lissi (&) Facultad de Quı´mica y Biologı´a, Universidad de Santiago de Chile (USACH), Santiago, Chile e-mail:
[email protected] R. Montecinos Facultad de Quı´mica, Pontificia Universidad Cato´lica de Chile, Santiago, Chile
is moderately decreased in liposomes and in red blood cells, in spite of an almost total coverage of the interface by albumin. All the data obtained in red cells and model membranes show that St II remains lytically active even in the presence of high HSA concentrations. This stubbornness can explain why the toxin is able to exert its haemolytic activity on membranes immersed in complex plasma matrixes such as those present in living organisms. Keywords Sticholysin Human serum albumin Haemolytic activity Liposome permeation Abbreviations HSA Human serum albumin St II Stycholysin II RBC Red blood cell HA Haemolytic activity DPPC Dipalmitoylphosphatidylcholine SM Sphingomyelin LUVs Large unilamellar vesicles
1 Introduction Stycholysin II is a member of the protein family named actinoporins [1, 2] due to their ability to form channels in natural and model membranes [3–7]. This activity has been tested under several experimental conditions being toxin function extremely sensitive to its modification and the characteristics of the target membranes. In fact, the poreforming capacity of the toxin in red blood cells can be tuned by pH, ionic strength, membrane composition, association to surfactants, and partial oxidation [2], Ca2? [8], and site directed mutation [9]. Similarly, its action on RBC is extremely dependent upon the cell age [10], pre-oxidation
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status [11], ion and water transport capacity [12], and lipid translocation [10]. In spite of the large number of works aimed to establish the relationship between experimental conditions and St II activity, no studies have been performed regarding the effect of serum proteins upon the toxin activity in RBC membranes. This aspect is particularly relevant since the presence of serum proteins, such as albumin, can influence both the toxin capacity to bind to the membrane bilayer and to achieve the competent conformation required to form the pores needed to trigger the osmotic shock. Furthermore, the toxin haemolytic action in living organisms takes place in the presence of large amounts of albumins. Human serum albumin is the most abundant protein in plasma, reaching concentrations up to 600 lM [13]. In the present work we report data bearing on the effect of HSA upon the capacity of St II to generate pores in RBC and in model membranes comprising dipalmitoyl phosphatidylcholine and sphingomyelin. Albumin is known to affect the activities of hemolytic toxins. Given the variety of factors that can affect the efficiency of the lytic process, it is not surprising that both enhancements and protection is afforded by albumin in different systems. For example, it has been reported that HSA interacts with the amino terminal peptide (23 residues) of gp41 of human HIV-1 virus leading to a dramatically reduction in the haemolytic activity of the virus [14]. Conversely, interaction with HSA enhances the haemolytic activity of Vivrio vulnificus hemolysin [15] and phospholipase C from Pseudomonas aeruginosa [16]. To know the effect of HSA addition upon St haemolytic activity could be useful to assess the possibility of employing a toxin-HSA adduct in the construction of therapeutic aptamer conjugates [17].
2 Materials and Methods 2.1 Materials Human serum albumin (Sigma, fatty acid free, pI = 4.7) was used as received. St II toxin (Swiss Protein Data Bank P07845) was purified according to Lanio et al. [18].
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absence and presence of HSA. HA was evaluated turbidimetrically at 620 nm at room temperature (25 ± 1 °C) in a microplate reader (Labsystems, Helsinki, Finland). The process was started by adding 10 lL of the toxin solution to a RBC suspension (280 lL final volume), and the decrease in absorbance was recorded at different times. Loss of turbidity was quantitatively related to the toxin HA. Assays were carried out after pre-incubation of RBC in the presence of HSA (5–200 lM, final concentration) during 15 min at 25 °C. Controls were performed in the absence of HSA. 2.4 RBC Ensemble Osmotic Fragility Assay Osmotic fragility of the RBC ensemble was evaluated by the standard procedure in the absence and in the presence of 200 lM HSA. RBC suspensions (20 % hematocrit) were added to solutions containing increasing NaCl concentrations (from 27 to 154 mM) to achieve a final 0.2 % hematocrit. After 60 min incubation at room temperature, suspensions were centrifuged (30 s, 12,000g) and haemolysis was determined spectrophotometrically from supernatant readings at 405 nm in a Labsystems microplate reader [19]. 2.5 Evaluation of HSA Association to RBC Efficiency of HSA association to RBC was assessed by centrifugation of the cell ensemble (10 % hematocrit, 2 min at 12,000g) after pre-incubation (30 min at 25 ± 1 °C) in the presence of the protein at different concentrations. Free HSA was determined by measuring the intrinsic fluorescence intensity of the supernatants in a Shimadzu spectrofluorometer (Japan) employing a 1 cm path length cell and 10 nm excitation and emission slits. In order to record predominantly tryptophan fluorescence emission, minimizing tyrosine fluorescence [20], supernatants were excited at 295 nm and the emission was recorded at 335 nm. The fluorescence of the supernatant, measured in blank assays without HSA addition, was negligible. 2.6 Evaluation of the Association of St II with HSA
2.2 RBC Isolation Fresh human blood was obtained from healthy donors following informed consent. RBC were isolated by centrifugation (30 s, 12,000g) and washed three times with PBS (145 mM NaCl, 5 mM phosphate buffer, pH 7.4). 2.3 Haemolytic Activity Assay RBC suspensions were diluted to an absorbance of 1.0 at 600 nm with addition of PBS (&2 9 106 cell/mL) in the
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Microcentrifugation experiments were carried out at 37 °C, pH 7.4 (50 mM Tris/HCl buffer), using Millipore Ultra free-MC centrifugal filter units (nominal cut-off 30 kDa). The conditions of centrifugation (2 min, 2,300g) were selected in order to permeate ca. 100 lL of solution. Control measurements were carried out to ensure that the toxin was not retained by the membrane and that HSA quantitatively remains in the upper solution. Toxin concentration in the filtrate was determined by Lowry‘s assay [21].
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2.8 Binding of HSA to Liposomes
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Large unilamellar liposomes were prepared by extrusion of a solution containing multilamellar liposomes in 0.05 mM Tris/HCl buffer prepared using a 1:1 molar mixture of DPPC and SM. Briefly, phospholipids were dissolved using a 2:1 dichloromethane/methanol mixture and the organic solvents evaporated in a rotary evaporator (40–50 °C, vacuum). The phospholipid layer was resuspended in 0.05 mM Tris/HCl buffer containing 80 mM calcein. The resulting suspension was subjected to 10 freezing/thawing cycles (heating at 50° C and cooling in liquid nitrogen) and extruded through a 0.4 microns pore size Isopore filter (Millipore Inc.) by applying nitrogen pressure. Extruded liposomal suspensions were eluted through a column of Sephadex G-25 equilibrated with 0.05 mM Tris/HCl buffer pH = 7.4, to remove unincorporated calcein. Average diameter of liposomes in the absence and presence of HSA was evaluated by dynamic light scattering employing a Malvern zetasizer nano S-90 instrument. Pore forming activity of St II was estimated in absence and presence of HSA by following the time course of the increase in calcein fluorescence intensity at 538 nm (kex = 485 nm) after St II addition to a suspension of calcein entrapped DPPC/SM liposomes suspended in 0.05 mM Tris/HCl buffer pH = 7.4. Total calcein release was obtained by adding a St II excess (approximately 600 nM).
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Fig. 1 a Representative plot of the effect of HSA on the time profile of the lytic process elicited by toxin addition. b Times required to lyse 50 % of the cells in the assay relative to control,(t/tc) as a function of albumin concentration. Controls were performed in the absence of HSA. St II: 1 nM. * p \ 0.008 compared to control values. N = 6
HSA association with the bilayer was evaluated by measuring the amount of protein remaining in the supernatant after centrifugation of liposomes/protein mixtures. Liposomes/protein mixtures were incubated during 10 min at room temperature and centrifuged (10 min at 9,300g). Protein concentration was determined by Lowry0 s method [21]. A light dispersion of the supernatant equal to that of the buffer was taken as indicative of total liposome sedimentation.
complexes were ranked using energy criteria based on steric and electrostatic surface interactions.
2.9 Molecular Docking Calculations
3 Results
The HSA-St II interactions were evaluated using HEX v6.3 docking molecular program [22]. The structures 1E78 and 1GWY, both obtained from Protein Data Bank, were used for HSA and St II, respectively [23, 24]. Surface charges of each protein were described using 3-D spherical harmonic basis functions. The fast Fourier transformation methodology was used to scan the global rotational and translational space for both proteins. Structures of 100 HSA-St II
3.1 Effect of HSA on St II Haemolytic Activity
2.10 Statistical Analysis Data were subjected to a Wilcoxon test and are expressed as mean ± SEM of independent samples. Duplicate or triplicate measurements were performed on each sample.
Haemolytic activity of St II was assessed by measuring the percentage of cells lysed by the toxin after relatively long reaction times (10–15 min). These evaluations were carried out in controls (without HSA) and RBC ensembles preincubated with different HSA concentrations. Results obtained are shown in Figs. 1 and 2.
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Fig. 2 Relative haemolysis values measured at long incubation times (15 min) after preincubation of RBC with increased concentrations of HSA during 5 min. St II: 0.7 nM. * p \ 0.003 compared to control values. N = 6
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Figure 1a shows data obtained at a toxin concentration high enough to lyse the whole RBC ensemble. These data allow an evaluation of t, (the time required to lyse 50 % of the cells). This parameter is related to the rate of the toxin organization in the membrane and the capacity of the cells to delay the lytic process. The data, included in Fig. 1b, show that HSA (at concentrations higher than 30 lM) reduces the rate of the lytic process, suggesting a reduced rate of toxin organization and/or an increase of the cells capacity to prevent the colloid osmotic shock. Figure 2 comprises HA data obtained at smaller toxin concentrations. Under these conditions in the absence of HSA, only a fraction of the components of the red cell ensemble is lysed (ca. 70 %) even at relatively long incubation times (15 min). The limiting values, measured at the plateau, are related to the efficiency of the toxin organization and/or the capacity of the red cell to avoid the lytic process [19]. The data shown in Fig. 2 indicate that the presence of albumin increases the lytic efficiency of the toxin. This suggests that, under physiological conditions, the haemolytic activity of St II could be even larger than that measured in albumin-free solutions. In summary, the data given in Figs. 1 and 2, indicate a slower rate and a higher efficiency of toxin-promoted cell disruption in the presence of HSA. The modification of the rate and/or efficiency of the lytic process, associated with the presence of HSA can result from a variety of factors. Among them, it can be mentioned: 1. 2. 3.
Changes of the toxin efficiency to form competent aggregates resulting from its association with HSA; Competitive binding of HSA and St II to the RBC membrane; and Changes in the resistance of the RBC membrane to osmotic driven haemolytic shocks.
In order to assess these possibilities, we measured St II/ HSA association, HSA association with RBC and liposomes, and the effect of albumin on RBC osmotic fragility.
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Fig. 3 Adsorption isotherm of St II (24 lM) to HSA obtained by microcentrifugation. Slope = 0.2; R2 [ 0.98
Also, we evaluated how the presence of HSA modifies St II pore forming capacity in DPPC/SM large unilamellar liposomes. 3.2 St II Association with HSA HSA can bind a large number of molecules and macromolecules, among them proteins [25]. In spite of the biological relevance of this process, very few evaluations of protein/HSA associations have been reported. Most of these evaluations have been performed to explain the effect of albumin in enzymatic catalysis [26] and, to our understanding, there are not reported studies of actinoporin/HSA association and how this might modify toxin function. Microcentrifugation of HSA/St II mixtures allows a direct measurement of their association. The adsorption isotherm obtained is shown in Fig. 3. The binding constant, defined as K ¼ Stbound =ðStfree Þ HSA -3
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has a value of 2 9 10 . This implies that ca. 19 % of the toxin (total St concentration 24 lM) is bound to albumin in presence of 100 lM HSA. Molecular docking calculations were performed in order to obtain further insight regarding the St II/HSA association. This treatment allows us to rank 100 complexes according to their energy values. The ten most stable structures where analysed in detail. In particular, we evaluated the exposure of the surface cluster of aromatic residues and the N-terminal segment of St II that play a pivotal role in the pore formation mechanism [24, 29–34]. Figure 4 shows the most stable HSA-St II complex structure. In the figure, is noticeable that both St II regions are oriented towards the external solvent. It is also remarkable
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[HSA]free ( M) Fig. 5 HSA adsorption to RBC (10 % hematocrit) at 25 °C. Each point is the average of 10 independent assays. Slope = 0.21; R2 = 0.98
membrane to the osmotic pressure is almost independent of albumin presence. 3.5 Effect of HSA on St II Pore Formation in Liposomes Fig. 4 Structure of the lowest energy St II/HSA complex determined by molecular docking calculations. HSA and St II structures are shown in light and dark gray respectively. The aromatic cluster and N-terminal segment of St II are shown in black (color scheme available as an electronic supplementary material)
that, although the other nine complex structures exhibit different binding regions, in all of them, both the aromatic cluster and the N-terminal segment of St II remains exposed to the external solvent and hence the toxin could be active even after binding to the albumin. 3.3 HSA Association to RBC Under physiological conditions, albumin reversibly binds to RBC membranes and most of the binding sites on the membrane are occupied by the protein [31]. This adsorption inhibits cell aggregation [32]. Binding of HSA to RBC was estimated by measuring the protein concentration in the supernatant after centrifugation of a RBC suspension incubated with different albumin concentrations. The adsorption isotherm, at 10 % hematocrit, is shown in Fig. 5.
In order to assess the effect of HSA on the pore forming capacity of the toxin in a model system, we evaluated its effect upon calcein release from DPPC/SM LUVs. This allows to evaluate how the presence of albumin modifies the fraction of disrupted LUVs at a given toxin concentration and the time required to form a pore in 50 % of the disruptable vesicle population (t). Figure 6 shows the effect of albumin upon t values. The efficiency of pore formation can be estimated from the fraction of the vesicles that losses their calcein at long incubation times. The effect of albumin upon this efficiency is shown in Fig. 7. Data given in this figure show that HSA presence decreases the pore forming capacity of the toxin. The data obtained in the present work by centrifugation experiments are given in Fig. 8, plotted as an adsorption isotherm. Data of this figure show that the HSA adsorption is highly cooperative, and that there is saturation at high protein concentrations. This massive adsorption of the protein increases the average size of the particles (Fig. 9).
3.4 Effect of HSA on RBC Osmotic Fragility 4 Discussion The osmotic fragility of RBC is not modified by the presence of albumin (data not shown). This implies that the resistance of the red cells to an osmotic misbalance is not substantially modified by HSA addition up to concentrations of 200 lM, indicating that the resistance of the
The main results obtained in the present work regarding the effect of HSA addition upon St II capacity to form pores in membranes, leading to an osmotic driven lysis of RBC, can be summarized as follows:
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Fig. 8 Adsorption of HSA on DPPC/SM liposomes (1 mg/mL) determined by centrifugation
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St II moderately binds to HSA (Fig. 3) without a great compromising of binding relevant fragments of the toxin (Fig. 4) HSA is associated to liposomes and RBC in amounts such that suggest an almost total coverage of their external leaflets (Figs. 5, 8) The presence of HSA reduces the rate of pore formation in liposomes (Fig. 6) and the rate of lysis of the RBC ensemble (Fig. 1) The presence of HSA reduces the pore formation extent in liposomes: and HSA addition increases the fraction of RBC lysed at a given StII/RBC ratio
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Fig. 9 Hydrodynamic diameter distribution (in number) of unmodified liposomes and liposomes in the presence of HSA. Left peak Control without albumin (filled square). Right peak In presence of 500 lM albumin (empty square). Integration of each peak normalized to 100 %. Lipids = 1 mg/mL
The data given in Fig. 3 imply a moderate interaction between both proteins, in spite of their different isoelectric points: ca. 9.5 for St II [18] and 4.7 for HSA. This moderate effect of albumin addition contrast with the large effect reported for the haemolytic amiloid peptide Aß25–35. In fact, it has been found that HSA, at concentrations as low as 0.37 lM, completely prevents RBC lysis induced by up to 20 lM Aß25–35 and that ca 89 % of circulating peptide in blood is bound to albumin [25, 27, 28]. Results (3) and (4) can be explained in terms of a diminution in the free (active) toxin concentration. This implies that the free toxin presents a pore forming capacity
Effect of Human Serum Albumin
almost independent of the albumin presence, in spite of the large amounts of HSA bound to the external leaflets of the bilayers. Asociation of HSA to liposomes is extremely sensitive to the liposome composition, size and preparation techniques [36–41]. Law et al. [42–44] working with phosphatidylcholine vesicles and bovine serum albumin (albumin/lipid concentration ratio &10-4) concluded that a fraction of albumin molecules penetrate the bilayer up to the hydrocarbon region, a process governed by hydrophobic forces, while the non-penetrating moieties remain coating the liposomes. In some systems this coating constitutes a barrier to the incorporation of solutes [45]. The data given in Fig. 8 show that, when the analytical albumin concentration is 500 lM, ca. 325 lM of protein is associated with the liposomes. If it is assumed that each lipid molecule contributes with 0.6 nm2 to the lipid/water interface, adsorption of 300 lM of HSA on 1 mg of lipids renders ca. 10 layers of albumin coating the liposomes (assuming an area of ca. 29 nm2 per adsorbed HSA molecule). The data given in Fig. 5 show that, under the employed experimental conditions (10 % hematocrit), ca. 15 % of the albumin is associated to the RBC. This corresponds to ca. 35 9 106 HSA molecules per red cell when albumin concentration is 200 lM. This quantity is sufficient to form an albumin monolayer on the red cell surface [35]. It is then surprising that this massive presence of HSA in RBC and liposomes does not precludes St II capacity to generate pores in the bilayer. This stubbornness allows the toxin to exert its HA even on membranes immersed in complex plasma matrixes, such as those present in living organisms. This increase of the damage inflicted to target cells in presence of albumins has been reported for the hemolytic activity of Vivrio vulnificus hemolysin [15] and phospholipase C from Pseudomonas aeruginosa [16]. A remarkable aspect of the present data is the opposite effect of albumin upon the HA in RBC and pore formation in liposomes. In order to explain this apparent controversy, it has to be considered that calcein exit is only a measured of the rate of pore formation, while the HA is determined by both the rate of pore formation and the rate of lysis of the pore bearing red cells. This last step is a very complex process [2] and its promotion by albumin could be the rationale of the increased HA elicited by protein addition. Conversely, this effect should not be operative in the much simpler process of salt promoted osmotic shock.
5 Conclusions St II retains its pore forming capacity in the presence of large amounts of HSA. This effect is observed both in its
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capacity to produce RBC haemolysis and to generate functional pores in model membranes. Even more, the haemolytic activity of the toxin increases in presence of albumin. Regarding the rate of the pore forming process, it is moderately decreased in liposomes and in RBC, in spite of an extensive binding of albumin to the interfaces. This small reduction in the rate of RBC lysis and pore forming in liposomes can be account in terms of a diminution of in the toxin free concentration due to albumin-toxin association. This association was supported by docking simulations. Acknowledgments This work is part of a Chile-Cuba collaboration program. CC thanks CONICYT for a doctoral fellowship. This work has been partially supported by the Iberoamerican CYTED Network BIOTOX 212RT0467.
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