MODIFICATION OF ERYTHROCYTE MEMBRANE PROTEINS BY POLYETHYLENE GLYCOL 1500

PEG-1500

Polyethylene glycols (PEG) of various molecular weights are low toxic biocompatible polymers widely used in biology, medicine and pharmaceuticals [1].Typically, PEG is used as a precipitating agent for the isolation and purification of proteins, nucleic acids and viruses.Fusogenic properties of the polymer provide a preparation of hybridoma cell cultures by the fusion of the two cell types.Surface modification by covalently attached PEG is the basis of the camouflage biotechnology, designed to reduce the immunogenicity of molecules and cells in order to extend their survival in vivo.The process of pegylation enhances the solubility of macromolecules in water and improves biocompatibility, that is important for drug development.In combination with other components the PEG is used to form hydrogels, which allow a controlled release of therapeutic agents, moreover they are also applied in regenerative medicine and some medical devices.
PEG has also demonstrated cryoprotective properties in respect of certain cell types, providing strong protection against effects of extreme environmental factors associated with freezing and thawing processes [2,3].Such factors include [4]: the formation of crystals, the growth of salt concentration in the supercooled liquid, increasing the osmotic pressure, phase transitions and lateral separation of lipids in membrane systems, dehydration of macromolecules.PEG of m.w.1500 (PEG-1500) does not cross the plasma membrane, that is referred to the exocellular type of cryoprotectants, which cause considerable interest among researchers [2][3][4][5][6], because on their base washing-free methods of cell cryopreservation can be developed.PEG-1500 provides effective protection of the erythrocytes during freezing down to ultralow temperatures (-196 C) [2].However, the transfer of cryopreserved erythrocytes to physiological conditions reveals latent membrane injuries that may be caused by a modification of the structural and functional state of the individual components of the membrane under the influence of PEG-1500.Taking into account the prospects of PEG-1500 using for erythrocyte cryopreservation and establishment of blood banks, the research of membrane modification in the cryoprotectant presence may be the key condition to understanding and leveling of the adverse effects of the polymer interaction with cells by adjusting the processing conditions of cells with cryoprotective medium or introducing additional components into it.
It is known that the stability of erythrocytes largely depends on the state of membrane proteins associated by point contacts to cytoskeletal components into an integrated protein network.One of these integral proteins involved in forming connections with the cytoskeleton is a surface marker CD44 [7].It can be assumed that the effect of PEG-1500, communicating only with the outer surface of the membrane, on integral proteins is stipulated by disturbance of their interactions with cytoskeleton proteins.Application protein-linking reagents, in particular diamide, would assist in clarifying the involvement of cytoskeletal proteins to a change in the integral protein CD44 expression under the influence of PEG-1500.It is known that the diamide causes oxidation of sulfhydryl groups and the formation of disulfide bridges between proteins [8].In erythrocytes the diamide induces crosslinking of mainly spectrin polypeptides, which are the main component of the erythrocyte cytoskeleton, and, to a lesser extent, the main component of integral membrane proteins -band 3 [9].Restraining the dynamics of protein-protein interactions in membrane-cytoskeleton complex in such a manner, it is possible to evaluate the role of this structure in changing of the СD44 characteristics in erythrocytes under the influence of the cryoprotectant.It should also consider the fact that protein-protein interactions in membrane-cytoskeleton complex of erythrocytes are substantially dependent on intracellular Ca 2+ level, which is regulated by the only one element of active transport of this cation in erythrocytes: plasma membrane Ca 2+ -ATPase.
The aim of the work was to study the effect of PEG-1500 on Ca 2+ -ATPase activity and an integral membrane protein CD44, involved in forming of contacts with erythrocyte cytoskeleton.Application of diamide, restraining the dynamics of protein-protein interactions, will help to clarify the role of cytoskeletal proteins in the alteration of CD44 expression in PEG-1500 presence.
The objects of the study were donor's blood erythrocytes, obtained from the Blood Center (Kharkiv), with a shelf life not more than 5 days at 3-5 С.Erythrocytes were washed from plasma and leukocyte components with salt medium A (150 mM NaCl, 10 mM Tris-HCl, pH 7,4), as described previously [10].
The erythrocyte membranes (ghosts) were isolated by the method [11].The ghosts were sealed in the medium, used for determining of an enzymatic activity of Ca 2+ -ATPase [12], which included 135 mM KCl, 10 mM Tris, 10 mM HEPES (pH 7.4), 0.037 mM MgCl 2 , 4 mM ATP, 1 mM EGTA, 1.5 mM PMSF, and necessary amounts of CaCl 2 , added at the rate of free [Ca i 2+ ] at the level of 2-410 -6 M (the medium B).For the calculation of free Ca 2+ concentration the software MAXChelator (http://www.standford.edu/~cpatton/maxc.html) was used.The produced ghosts divided into two equal parts and incubated in media with or without CaCl 2 (only in the presence of EGTA) at 37 С.After sealing stage the restored cells were pelleted and washed with a saline medium A. Before the beginning of the experiment the aliquots of ghosts from each sample were taken to determine the base level of inorganic phosphate (P i ) and protein concentration.Restored cells (100 μl) were incubated in solutions of PEG-1500 [5-30% (0,033-0,2 M)], prepared on the basis of the medium A, for 20 min at 22 С.The medium A was used as a control when assessing the Ca 2+ -ATPase activity.The changes in the Ca 2+ -ATPase activity were assessed by the difference in the accumulation of P i in Са 2+ -containing and Са 2+ -free media after subtracting baseline values P i .Enzymatic reaction was stopped by adding cold TCA solution (5% final concentration).Protein was precipitated by centrifugation.The P i in the supernatant was examined by the method [13].Protein concentration was determined by the method of Bradford [14].
Effect of PEG-1500 on the erythrocytes membrane surface marker CD44 was evaluated similarly to the previously described procedures [10], by incubating the cells for 20 hrs in solution of 30% PEG-1500, prepared on the base of Ringer medium with adding 5 mM glucose, 125 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 32 mM HEPES (pH 7.4).As a control in this series of experiments the Ringer-glucose medium was used.A part of the samples before exposure to PEG-1500 was incubated in the presence of 2.5 mM diamide at 37 С for 1 hr in Ringer medium.The erythrocyte CD44 parameters were determined by flow cytometry (FACS Calibur, Becton Dickenson, USA).In each measurement 30,000 events were counted.Data were analyzed using WinMDI 2.8 software.
Our findings were statistically analyzed using the software package Statgraphics plus 2.1.Data are presented as M ± SE (mean value ± standard error).Statistical significance of differences between experimental groups was evaluated using Fisher's rank test for multiple comparison of sample with the least significant difference.Each series comprised at least 8 experiments.

Results and Discussion
Assessment of Ca 2+ -ATPase activity in erythrocytes in the presence of PEG-1500 showed that the adding even a small amount of the cryoprotectant into a medium led to inhibition of Ca 2+ -ATPase activity (Fig. 1).
In the presence of significant concentrations of polymer (20-30%), which are capable of exerting a pronounced cryoprotective effect, there was observed a decrease in the rate of P i accumulation approximately 3 times.Evidently, the inhibition of Ca 2+ -ATPase activity in erythrocytes, exposed to hypertonic medium of PEG-1500, will entail an increase in intracellular Ca 2+ and can affect the proteinprotein interactions in membrane-cytoskeleton complex.
In the study of changes in surface marker CD44 in erythrocytes under the influence of the cryoprotectant by flow cytometry we assessed two parameters: composition of cell suspensions characterized by the ratio of CD44 + -and CD44 --erythrocytes (for convenience the percentage of only CD44 +erythrocytes was assessed), and level of surface marker expression for CD44 + -erythrocytes, i.e. the amount or density of CD44 molecules in membranes of individual cells.
The characteristic of the marker CD44 expression in cells is the median of the distribution histograms showing a value, relative to which, cells in the suspension are divided into two equal size parts.Since only a part of erythrocytes in suspension binds the label CD44-FITC, it was necessary to assess the distribution of native erythrocytes, which were not incubated with CD44-FITC, within FL1 fluorescence channel to clearly identify the area of СD44 --erythrocytes (negative control).Cutting off СD44 --cells (Fig. 2, curve 1), we identified the marker margins, corresponding to CD44 + -erythrocyte area (Fig. 2, curve 2).
In the samples of native erythrocytes the restraining of dynamics of protein-protein interactions in membrane-cytoskeleton complex, caused by diamide-induced crosslinks, entails a decrease in the expression level of СD44 (9.6 ± 0.5 a. u.IF) without significant changes (75,9 ± 4,1) in the amount of CDD44 +cells (Fig. 2, curve 3).
Exposure of erythrocytes in the medium containing 30% PEG-1500 (Fig. 3, curve 2) was accompanied by a decrease in CD44 expression (9.1 ± 1.6 a. u.IF) in membranes and reducing of CD44 + -erythrocyte amount (41.9 ± 4.8%) in the range of the marker standard margins M1 with simultaneous appearance of a small amount of cells (2.3 ± 1.0%) with CD44 expression above the control values (median value 157.3 ± 15.1 a.u.IF) corresponding to the margins M2 in histograms.Pretreatment of cells with diamide exerted more significant impact on CD44 in erythrocytes, exposed to 30% cryoprotectant solution (Fig. 3, curve 3) as compared to the control.Amount of CD44 + -cells decreased to 22.5 ± 5.3, and the surface marker expression level was only 5.7 ± 0.4 a. u.IF.However, pretreatment of cells with diamide prevented the development of processes in membrane, resulting in the formation of the erythrocyte subpopulation with higher expression level of CD44, corresponding to the margins M2.
Thus, an exposure of erythrocytes in 30% PEG-1500 causes a change in the Ca 2+ -АТРase activity and affects the characteristics of the integral protein associated with cytoskeleton and simultaneously presenting the surface marker CD44.Using diamide, which forms disulfide cross-links between the major proteins of membrane-cytoskeleton complex, confirms, as exemplified by СD44, the ability of PEG-1500 to change the structural and functional properties of membranes via modifying of regulatory effects of the cytoskeletal proteins.
A model of reconstructed erythrocytes provides the possibility to assess the Са 2+ -ATPase activity affected by exocellular compound under the conditions allowing it to prevent a direct contact with cytoplasmic domains of an enzyme.Effects of organic compounds, many of those as know to exhibit cryoprotective properties, on the Са 2+ -ATPase activity were investigated in various biological models.In particular, the stimulation of enzymatic activity of isolated Са 2+ -ATPase derived from erythrocyte membranes was demonstrated in presence of PEG, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol [15].However, the observed effect is rather associated with the characteristics of model system than reflects the influence of these substances on the Са 2+ -ATPase activity in a cell, because the stimulation of its activity was due to the recovery of hydrophobic interactions within membrane domains of isolated enzyme under the influence of amphiphilic substances [15].It was shown that in various experimental models trends of changing in the Са 2+ -ATPase activity under the influence of organic solvents could be disagreed.In particular, despite the fact that DMSO stimulates the Са 2+ -ATPase activity of isolated purified enzyme [15], it does not change the basic Са 2+ -ATPase activity in membranes of erythrocyte ghosts and inhibits calmodulin-stimulated enzyme activity [16].The effect of glycerol on the Са 2+ -ATPase in the sealed ghosts and saponin-permeabilized erythrocytes were also different [17].Since the ability of Са 2+pump to transport ions Са 2+ is primarily determined by its catalytic activity, one can expect an increase in the level of intracellular Са 2+ in the erythrocytes incubated in PEGcontaining solutions.Growing of Са 2+ level in the PEG presence is promoted via water loss by cells under the influence of osmotic gradient due to a high concentration of the non-penetrating across membrane polymer.Ions Са 2+ , functioning as a second messenger, undoubtedly play a role in cell survival under stress conditions.
Increasing in the Са 2+ level, which has been observed even in the presence of an isotonic solution of PEG-1500 [18], should affect the structural state of membrane components, and changes in a surface marker expression can be resulted from occurring structural rearrangements caused by stress factors.Whereas the cryoprotective agent is referred as "compatible cosolvents" and it is capable to maintain the structure of protein macromolecules, membranes and cells in a state close to native without causing denaturation or impaired structural order [19][20][21], but it has been still able to induce structural and functional changes in erythrocytes, which during a prolonged exposure resulted in lowering of CD44 + -cells amount and marker expression level.The most likely cause of the mentioned changes in erythrocytes in cryoprotectant presence is the loss of membrane material in the form of vesicles with the capture of molecules of the surface marker CD44 in their composition.For erythrocyte membranes the vesiculation is a recognized  mechanism of cell response to either stress or aging [22][23][24].The changes, affecting CD44, may depend on structural modification of lipid bilayer and proteins of the membranecytoskeleton complex.In particular, the lipid packing mismatch in neighboring areas, caused for example by phase transitions in specific membrane areas, affected by dehydration processes, are able to induce the formation of vesicles [25].Furthermore, the CD44 molecules are known to exist in the membrane as individual molecules or a part of the intricate protein complexes.In particular, CD44 may be associated with the cytoskeleton network via ankyrin and protein 4.1 [26].These links are dynamic and may vary in different parts of membrane.Non-associated with the cytoskeleton the CD44 molecules can be captured easier into the vesicles forming on the erythrocyte membranes under stress.It should be noted that changes in the intracellular Ca 2+ level play a key role in initiation of these processes, since Ca 2+ and calmodulin decrease the affinity of interaction between the protein 4.1 and CD44 [26], whereby the latter becomes a free membrane protein or gets able to form a link with ankyrin.Expected redistribution of interaction within network of membranecytoskeleton protein complex involving CD44, ankyrin, protein 4.1 and band 3 in the presence of cryoprotectants can affect the physical properties of membrane.
Important information to understand the cryoprotectant mechanism actions on erythrocyte membrane can be provided by an evidence on the specificity of changes in the marker CD44 expression under the effect of a 30% PEG-1500 solution, which is characterized by the appearance of cells with higher expression of the marker CD44 (M2).This subpopulation emergence upon the absence of capability for protein synthesis in erythrocytes can be associated with structural changes in membranes wherein there is partial or complete fusion of membrane systems, that leads to the enrichment of individual cells with the marker.It is known that PEG contributes to an aggregation and fusion of cells [27,28].However, PEG-1500 has been previously shown [29] does not cause a fusion of erythrocytes.For this purpose, PEG-6000 is used at a concentration of about 40%, furthermore cell fusion requires additional conditions, one of those is cellular rehydration [30], not presented in our experiments.Nevertheless, in the erythrocyte membranes on the contact with high PEG-1500 concentrations the structural changes can be initiated that eventually lead to the formation of sufficient area with a modified structure that allows fusion of individual membranes.In the described experimental conditions, fusion of vesicles, carrying the marker, with the cells seems the most probable.
The crosslinking of proteins with diamide can, to a certain extent, serve as confirmation of the PEG-1500 effect on the surface marker CD44 by modifying the bonds between proteins of membrane-cytoskeleton complex in the cryoprotectant presence.Assuming that the cryoprotectant affects not only the lipid bilayer, but also a system of protein-protein interactions and thereby control changes in the CD44 parameters, it can be expected that the modification of proteins with diamide will entail either slowing or intensifying loss of the CD44 marker by cells.As shown by experiments, previous oxidation of proteins by diamide really leads to a significant intensification of the CD44 loss in erythrocytes exposed to PEG-1500, compared to the similar variants of the control samples.It was also noted that the treatment with diamide prevented the formation of erythrocyte subpopulation with higher level of CD44 expression in the presence of 30% PEG-1500 solution.This is obviously related to the fact that the proteins "cross-linked" with diamide impede the formation of protein-free areas in the membrane, indispensable for membrane fusion.In other words, the diamide stabilizes the lipid bilayer through control of its structure by "crosslinked proteins" and complicates the PEGinduced fusion of membrane.Meanwhile the loss of CD44 in erythrocytes, treated by diamide, only amplified under these conditions, confirming the cryoprotectant effect on the surface marker via its impact on proteins of membrane-cytoskeleton complex.The inhibitory effect of PEG-1500 on Ca 2+ -ATPase activity and a decrease in CD44 expression in erythrocytes may be stipulated by physicochemical properties of the polymer solution.It is known that PEG causes a change in surface potential [31] and dielectric constant of the solution [32], exhibits amphiphilic (hydrophilic and hydrophobic) properties as it is dissolved not only in water but also in some nonpolar organic solvents [33,34].In aqueous solutions the PEG binds 2-3 water molecules per monomer unit [33] that determines its dehydrating effect against various macromolecules and membranes.Further, the PEG molecules can be distributed at the air-water interface forming stable monolayers [35,36].Surface-active properties of the polymer, its dehydrating action and changing in the polarity in the presence of PEG may have some influence on membrane surface [37] that may cause a decrease in membrane fluidity [38].It has been shown that the adding of PEG modifies the surface potential of lipid monolayers [33] and causes a shift of temperature of the phase lipid transition [39].It has also been found that PEG molecules induce dehydration of multilamellar lipid vesicles that is accompanied by a reduction in effective size of the polar headgroups of the membrane phospholipids and enhanced van der Waals interactions between the acyl chains of the matrix lipid [40,41].These structural changes are typically compensated by an increased thickness of the membrane and decrease in the mobility of lipid molecules that leads to segregation of different types of membrane lipids.Probably similar changes may affect some areas of erythrocyte membranes and exert a certain negative impact on the stability of membranes in the presence of PEG-1500 under stressful conditions of cryopreservation.
The observed changes in Ca 2+ -ATPase activity in erythrocytes under the influence of PEG-1500 does not give a comprehensive view on its mechanism of inhibition and requires further assessment of its functioning characteristics, in particular the analysis of Ca 2+ -transporting capacity in presence of PEG-1500 in the medium.In future such a study could potentially give a clearer insight in the state and role of Ca 2+ -regulating constituents in changes of structural and functional state of the cells at using of exocellular cryoprotectants and low temperature effects.Especially important issue is the assessment of changes in the level of intracellular Ca 2+ , which can be both a regulator of the structural state of membrane and trigger of apoptosis under stressful conditions.Effect of PEG-1500 on the surface marker CD44 apparently is implemented in various ways, and the change in the level of intracellular Ca 2+ , which determines the modification of protein-protein interactions in the membrane-cytoskeleton complex, plays definitely an important role.The consequence of the modification of the structural state of the membrane components in the presence of PEG-1500 may be resulted in changing the erythrocyte stability under stress conditions.
Thus, the experiments revealed a decrease in the Ca 2+ -ATPase activity, as well as a reduction in the expression of surface markers CD44 and CD44 + -cell amount on the exposure of erythrocytes in the presence of PEG-1500.A decrease in the Ca 2+ -ATPase activity may be caused by a modification of the membrane structure under the influence of physical and chemical properties of 30% PEG-1500 solution.The consequence of reducing of the Ca 2+ -ATPase activity is rising intracellular Ca 2+ level that can lead to a modification of the interactions between integral proteins and cytoskeleton components and, eventually, contribute to membrane vesiculation with an inclusion into them integral proteins not associated with the cytoskeleton, thereby reducing the expression of surface marker CD44.The appearance of a small amount of erythrocytes with the higher expression of CD44 indicates structural changes in the membrane, whereat a partial or a complete fusion of the membranes may take place (probably, fusion of cells with vesicles carrying the marker).The treatment with diamide impedes the formation of the erythrocyte subpopulation with higher level of the CD44 expression in the presence of PEG-1500, apparently, due to the fact that proteins "cross-linked" with diamide inhibit the formation of protein-free areas in the membranes indispensible for membrane fusion.Despite the fact that the diamide stabilized the lipid bilayer affecting its structure by "cross-linked proteins" and complicated the fusion of membrane constituents the loss of CD44 in erythrocytes, treated with diamide, only amplified.And this loss can serve as a strong argument in the evidence of the cryoprotectant effect on the surface marker via its action on proteins of membranecytoskeleton complex.

Fig. 1 .Fig 2 .
Fig. 1.Change in Ca 2+ -ATPase activity in the sealed erythrocytes depending on the PEG-1500 concentration All the experimental values significantly different from control with a significance level Р < 0.001