The comparison of zymosterol vs cholesterol membrane properties –The effect of zymosterol on lipid monolayers

Colloids and Surfaces B: Biointerfaces 123 (2014) 524–532

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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

The comparison of zymosterol vs cholesterol membrane properties –The effect of zymosterol on lipid monolayers a,∗ a ˛ ´ Katarzyna Hac-Wydro , Paweł Wydro b , Michał Flasinski a b

Department of Environmental Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 3, 30-387, Kraków, Poland Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060, Kraków, Poland

a r t i c l e

i n f o

Article history: Received 9 July 2014 Received in revised form 14 September 2014 Accepted 25 September 2014 Available online 5 October 2014 Keywords: Zymosterol Langmuir monolayers BAM images Membrane activity

a b s t r a c t In this work systematic investigations on the influence of zymosterol (zymo), which is one of cholesterol precursors, on lipid monolayers were done. The aim of these studies was to perform thorough comparison of zymosterol vs cholesterol membrane activity and fill the gap in the studies on the effect of sterols on membranes. The Langmuir monolayers experiments combined with Brewster angle microscopy studies were performed on binary (SM:zymo, POPC:zymo, GM3:zymo) and ternary (SM:POPC:zymo, SM:GM3:zymo) films differing in the sterol content. The obtained results evidenced differences in the influence of both sterols on lipid monolayers, which was manifested in the parameters calculated based on the isotherms as well is in monolayers morphology. It was found that zymosterol is of condensing, ordering and domain promoting abilities thus this molecule can be included to the group of membrane active sterols. However, zymosterol is much less effective than cholesterol as condensing and ordering agent. These findings were attributed to the differences in the structure of both sterols and their ability to pack tightly with other lipids in the mixed systems. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Sterols form a large group of compounds having a tetracyclic ring system as a basic structural element of the molecule (Scheme 1). Modifications in the ring systems and/or at C3 position and/or in the structure of the side chain differ each other particular sterols molecules. These compounds are vitally important components of cellular membranes, which act as regulators of membrane organization and properties and are crucial for the formation of highly ordered domains in membranes [1]. However, the respective molecules vary significantly as regards their interactions and tight packing ability with membrane lipids and resulting capacity of altering membrane properties as well as in domainspromoting activity. These differences are directly connected with the structure of sterol molecule and its stereochemistry. It seems that cholesterol – the major animal sterol, has optimal structure to be highly potent as condensing, ordering and domains promoting agent, therefore the membrane properties of other sterols are usually classified in reference to cholesterol [2]. A number of experiments performed on artificial membrane systems evidenced that some sterols similarly to cholesterol are able to pack closely with

∗ Corresponding author. Tel.: +48 0 12 664 67 97; fax: +48 0 12 634 05 15. ˛ E-mail address: [email protected] (K. Hac-Wydro). http://dx.doi.org/10.1016/j.colsurfb.2014.09.054 0927-7765/© 2014 Elsevier B.V. All rights reserved.

membrane lipids and lead to the formation of domains (dihydrocholesterol, epicholesterol, sitosterol), while some of sterols behave as anti-cholesterol and inhibit domains formation (e.g. coprostanol or androstenol) [2–5]. To systematize the relationship between the membrane activity of sterol and its molecular structure, some structural criteria probably decisive for sterols to be membraneactive were formulated [6]. However, these criteria evolve, being continuously verified by the experiments [3,5]. Indeed, despite intensive investigations on sterols membrane activity, there are still some gaps or inconsistencies as regards membrane properties of particular compounds. The problems concern both discrepancies concerning the properties reported for the respective compounds (a good example is desmosterol, which in some experiments has been recognized as able to replace cholesterol in membranes [7], while the other studies excluded such a possibility [8]) and serious deficiency of information concerning membrane activity of particular sterols. Considering the latter issue, a good example of sterol, which has not been thoroughly investigated in membrane systems is zymosterol. Although zymosterol similarly to desmosterol and lanosterol is a precursor of cholesterol in the synthesis pathway [9], the information on its membrane properties are very scarce as compared to the knowledge on the properties of the remaining forerunners. The experiments done on lipid vesicles evidenced that zymosterol stabilizes SM or DPPC domains, however, in a lesser degree than cholesterol [10] and its domains promoting abilities

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H3C CH3 CH3 H

HO

CH3 CH3

H H

H3C CH3

cholesterol CH3 CH3

525

made of filter paper (ashless Whatman Chr1) connected to an electrobalance. The subphase temperature (20 ◦ C) was controlled thermostatically (±0.1 ◦ C) by a circulating water system. For the experiments Ultrapure Milli-Q water was used. Brewster angle microscopy experiments were performed with UltraBAM instrument (Accurion GmbH, Goettingen, Germany) equipped with a 50 mW laser emitting p-polarized light at a wavelength of 658 nm, a 10x magnification objective, polarizer, analyzer and a CCD camera. The spatial resolution of BAM was 2 ␮m. In order to notice anisotropy in the reflected images, the analyzer was rotated to specific angles in the directions opposite from p-polarization.

CH3 H

2.2. The studied monolayers

zymosterol

HO Scheme 1. The structure of cholesterol and zymosterol.

were defined as intermediate [11]. In fact, the effect of zymosterol on membranes and its interactions with particular membrane components have never been studied in a systematic way and thus it is very difficult to compare the properties of this sterol with those reported for the other sterols. This issue is also highly important from the point of view of possible disorders in synthesis of cholesterol. Namely, any disturbances in cholesterol biosynthesis result in the accumulation of potentially harmful cholesterol intermediates (e.g. zymosterol) in cell and membranes. This kind of disorders in embryonic development leads to various human malformation syndromes [12] for example Conradi-Hunermann syndrome [12] or holoprosencephaly [13]. The studies performed herein were aimed at investigating the influence of zymosterol on sphingomyelin, phosphatidylcholine and ganglioside monolayers as well as on sphingomyelin/ phosphatidylcholine or sphingomyelin/ganglioside systems. In our experiments the Langmuir monolayer technique was applied. Since the influence of sterols on membranes is directly connected with the packing and interactions of sterols molecules with other membrane lipids, this method, allowing for the analysis of miscibility and lipid–lipid interactions as well as condensation and ordering of a monolayer, is highly appropriate for this kind of studies. Although Langmuir films technique was widely used to study the effect of various sterols on membrane, this method has never been applied in the investigations of zymosterol-containing films.

The experiments were done for zymosterol and its mixtures with sphingomyelin (SM), phosphatidylcholine (POPC) and ganglioside (GM3) containing 33, 50 and 66% of sterol (lipid:zymo = 2:1; 1:1 and 1:2 mixtures). Then, the influence of zymosterol on binary SM:POPC = 1:1 films was studied at 33; 50 and 67% of sterol in the systems (SM:POPC:zymo = 1:1:1; SM:POPC:zymo = 1:1:2 and SM:POPC:zymo = 1:1:4). Finally, the effect of zymosterol on sphingolipids mixture (SM + GM3) was verified. In the studied SM:GM3:zymo monolayers the SM + GM3 to zymosterol proportion was estimated to 2:1, and the system contained only 5% of ganglioside. To be able to compare the effect of zymosterol vs cholesterol on the investigated monolayers similar experiments were performed for cholesterol-containing films.

2.3. Data analysis From the surface pressure-area (-A) isotherms recorded upon compression of the monolayers the compression modulus values, were calculated from Eq. (1) [14] CS−1 = −A(d/dA)

(1)

wherein A is the mean area per molecule value at a given surface pressure . To analyze the condensation, miscibility and interactions between molecules in the studied monolayers the excess areas of mixing (AExc ) values were calculated from Eq. (2) [15] AExc = A − Aid

(2)

2. Experimental 2.1. Materials and methods Synthetic sphingomyelin (N-palmitoyl-d-erythro-sphingosylphosphorylcholine, SM), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), ganglioside GM3 (Milk, Bovine-Ammonium Salt) and zymosterol were purchased from Avanti Polar Lipids Inc., USA. Cholesterol was supplied by Sigma. All the compounds were the products of high purity (≥99%). Spreading solutions of SM, POPC and GM3 were prepared in chloroform:methanol 9:1 v/v mixture, while the sterols were dissolved in chloroform. The solvents were purchased from Aldrich (HPLC grade, ≥99.9%). Mixtures were prepared from the respective stock solutions and deposited onto water subphase with the Hamilton micro syringe (±2.0 ␮L). After spreading the films were left for 10 min before the compression was initiated (barrier speed of 20 cm2 /min). The experiments were performed with NIMA (UK) Langmuir trough (total area = 300 cm2 ) placed on an anti-vibration table. Surface pressure was measured (±0.1 mN/m) using Wilhelmy plate

where A are the values of the mean area per molecule derived from the isotherms at a given surface pressure, while Aid are the areas corresponding to ideal mixing defined as (Eq. (3)): Aid =



Ai Xi

(3)

where Ai is the mean area per molecule for the respective one component films, Xi is the mole fraction of the respective component in the mixed monolayer. Ai calculated from the above equation means a linear combination of the areas of all the respective single components and their molar fractions in the mixtures. The errors for these parameters were calculated with the exact differential method and their maximal values were shown in the Figs. 3 and 4. The properties of the monolayers were discussed based on the analysis of AExc , compressional modulus and BAM images at various surface pressures (5; 15 and 30 mN/m), with special interest to the ␲ region, at which the lipids monolayers properties correlate with those of lipids bilayers [16].

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π, mN/m

75

SM SM:zymo=2:1 SM:zymo=1:1 SM:zymo=1:2 zymo

50

25

0

35

2

70

105

A, Å /molecule POPC POPC:zymo=2:1 POPC:zymo=1:1 POPC:zymo=1:2 zymo

π, mN/m

50

25

0

0

35

70

A, Å2/molecule

π, mN/m

75

105

GM3 GM3:zymo=2:1 GM3:zymo=1:1 GM3:zymo=1:2 zymo

50

25

0

0

35

70

A, Å2/molecule

105

Fig. 1. The surface pressure–area isotherms for lipid:zymosterol monolayers.

3. Results 3.1. The influence of zymosterol on SM, POPC, ganglioside GM3 monolayers In Fig. 1 the surface pressure area isotherms recorded during compression of the monolayers formed by zymosterol and its mixtures with sphingomyelin (SM), phosphatidylcholine (POPC) and ganglioside (GM3) are shown. At first glance the curve for one component zymosterol film is very similar to that for cholesterol monolayer (the isotherms collected for cholesterol-containing monolayers are presented in supplementary materials Fig. S1), however, the collapse surface pressure is visibly lower and both isotherms differ in their slope. Considering the latter difference, it reflects well in the values of the compression modulus (CS −1 ),

which were calculated directly from –A curves. The comparison of the values of this parameter for both sterols indicates that at the same surface pressure CS −1 values are lower for zymosterol than for cholesterol (e.g. at 30 mN/m, CS −1 ≈ 800 mN/m for cholesterol vs CS −1 ≈ 650 mN/m for zymosterol). These differences in CS −1 values prove that cholesterol molecules form monolayers of higher condensation and ordering than zymosterol. The comparison of BAM pictures taken for zymosterol and cholesterol evidenced additionally strong dissimilarities in the morphology of both films (In Fig. 2 we present the images taken for zymosterol, while those for cholesterol are widely published in literature, see e.g. [17]). In the pictures for zymosterol monolayer the mosaic textures can be observed in the whole range of the surface pressure. This is in contrast to cholesterol film being homogenous in the wide range of the film compression. It was also found that BAM pictures for zymosterol monolayer evidenced strong optical anisotropy resulting from the surface domains formed by molecules tilted in different directions. The addition of zymosterol into SM, POPC and GM3 (Fig. 1) monolayer shifts the curves for one component lipid films to smaller areas and visibly changes the shape of the isotherms to be more similar to that for sterol film. Qualitatively similar effect on the studied lipid films induces the incorporation of cholesterol (please see Supplementary Materials Figs. S1 and S2). However, to perform a deeper verification of the properties of lipid:zymosterol monolayers and compare them with lipid:cholesterol films the values of AExc were analyzed and BAM pictures taken at various surface pressures for the respective monolayers were studied. The results of AExc calculations for lipid:zymosterol films and, to facilitate the comparison, for lipid:cholesterol monolayers, are shown in Fig. 3. The compression modulus values for the investigated films were also analyzed (please see Supplementary Materials Fig. S3). The excess area per lipid values are negative for all the mixed systems, which means nonideal behaviour of the films and more favorable interactions between the molecules in the mixtures than in particular one-component monolayers. As regards SM:sterol films at 1:1 lipids proportion the values of AExc are nearly identical for both sterols-containing systems. However, AExc are the lowest (the most negative) at 2:1 for SM:zymosterol, while 1:2 for SM:cholesterol mixtures. The mentioned above minima of AExc values indicate the lipids proportion ensuring the most favorable mixing and the strongest interactions between molecules. This parameter is also a measure of the condensing potency of sterol. Since AExc at SM:sterol = 2:1 mixtures are more negative for SM:cholesterol film, it can be concluded that cholesterol at this lipids proportion is of stronger condensing ability than zymosterol. On the other hand, at the prevailing content in the monolayer (SM:zymosterol = 1:2) the condensation induced by zymosterol on SM is stronger as compared to that caused by cholesterol, which reflects in more negative AExc values for zymosterol-containing system. Incorporation of zymosterol into SM monolayer leads also to the increase of the compression modulus values (Supplementary Materials Fig. S3). This means that sterol molecules cause ordering of lipids in the mixed system. This “ordering” effect of zymosterol on SM is weaker as compared to the influence of cholesterol since the increase of CS −1 values for SM:zymosterol monolayers is lower than that for SM:cholesterol films (Supplementary Materials Fig. S3). The analysis of BAM pictures taken for SM:zymosterol monolayers evidenced that independently on sterol content in the system, there is a similar trend as regards the variations of the monolayers morphology during films compression. In Fig. 2 a selected pictures for one of the studied SM:zymosterol films are shown. As it can be observed in a wide range of the surface pressure the mixed monolayers are in homogenous condensed state and only at large molecular areas the coexistence of gaseous and

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Fig. 2. BAM images for zymosterol monolayer and selected lipid:zymosterol mixed films.

condensed phase can be observed. It is also important that the images for SM:zymosterol monolayers are similar to the pictures for SM:cholesterol (please see Supplementary Materials Fig. S2). As regards POPC:zymosterol monolayers the collected results lead us to very similar conclusions, from a qualitative point of view, on the effect of zymosterol on phosphatidylcholine (POPC) as it was mentioned above for SM-containing monolayers. Namely, the interactions of this sterol with POPC and its condensing efficiency become stronger with the increase of zymosterol content in the mixture. Analyzing the AExc values for POPC:zymosterol film it can be seen (Fig. 3) that the higher fraction of zymosterol in the mixed monolayer with POPC the more negative values of this parameter. The minimum of AExc , which indicates the monolayer of the most favorable mixing and the strongest intermolecular forces, was found for POPC:zymosterol = 1:2 system. As it can be noticed in Fig. 3 in the case of cholesterol-containing systems the most favorable mixing occurs at 1:1 POPC:cholesterol proportion. BAM pictures taken for POPC:zymosterol films evidenced that these monolayers are in homogenous liquid-like state in a wide range of the surface pressure (from ca. 1 mN/m to the collapse). Only at large molecular areas (corresponding to  = 0 mN/m) the coexistence of gaseous and liquid phase occurs. The observed morphology is identical to those found for POPC:cholesterol mixtures (in Fig. 2 selected

images for POPC:zymosterol, while in Fig. S2 for POPC:cholesterol are presented). Finally, considering the properties of GM3:zymosterol systems, it can be concluded that the interactions in the mixed system and condensing effect are the weakest at 2:1 proportion of GM3:zymosterol and generally they become stronger with the increase of the content of zymosterol in the mixed film. Interestingly, at the lowest (33%) content of zymosterol in the mixed monolayer, the condensation causes by zymosterol molecule is weaker as compared to the influence of cholesterol, however, at 1:1 as well as 2:1 GM3:zymosterol proportion the effect of zymosterol on ganglioside film is stronger than the effect of cholesterol. In Fig. 2 BAM pictures for GM3:zymosterol = 1:1 films are shown as representative images for all the studied mixtures, which are generally of very similar morphology. Namely, at large molecular areas (low surface pressures) a gaseous and liquid phase coexist at the interface. With the increase of the surface pressure the monolayer becomes homogenous (that is at ca. 1 mN/m at 2:1 GM3:zymosterol proportion and at ca. 0.1 mN/m at 1:1 GM3:zymosterol ratio) and finally the domains of condensed phase are formed. These condensed domains appear at ca. 20 mN/m at 2:1 GM3:zymosterol proportion and at ca. 6 mN/m at 1:1 GM3:zymosterol ratio. In the case of 1:2 GM3:zymosterol monolayer the condensed phase is

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Fig. 4. The isotherms for POPC:SM:zymosterol monolayers (a), the excess area per molecule values (b) and the variations in the compressional modulus values (c) for POPC:SM:zymosterol vs POPC:SM:cholesterol films.

Fig. 3. The excess area per mixing values for lipid:sterol monolayers at various surface pressures.

formed at very low surface pressures (ca. 0.2 mN/m) and in fact the region of the film homogeneity was not detected in the pictures. Similarly to the discussed above SM:sterol and POPC:sterol monolayers also in the case of GM3:sterol films the morphology of zymosterol vs cholesterol-containing mixtures is nearly identical (Supplementary Materials, Fig. S2). The only difference is the surface pressure values corresponding to the formation of condensed domains in the respective mixtures. Namely, in GM3:cholesterol monolayers the condensed phase appears at lower surface pressures as compared to GM3:zymosterol films. 3.2. The effect of zymosterol on POPC/SM systems In Fig. 4a the surface pressure-area isotherms for POPC:SM = 1:1 monolayers and POPC:SM:zymosterol mixtures differing in the level of sterol (1:1:1; 1:1:2 and 1:1:4 lipids proportion) are shown (the surface pressure - area isotherms for cholesterol-containing

mixtures investigated for the comparison at the same experimental conditions are shown in supplementary materials, Fig. S4). The properties of these monolayers were analyzed at  = 30 mN/m based on AExc and compressional modulus values as well as BAM pictures taken for these systems. The calculated parameters for POPC:SM:zymosterol and for the comparison for cholesterolcontaining systems are shown in Fig. 4 b and c. In Fig. 5 BAM images taken for POPC:SM:zymosterol differing in the content of sterol are shown (the pictures for cholesterol-containing films are presented in Supplementary Materials, Fig. S5). Let us start the analysis of the collected data from the monolayer devoid of sterol (POPC:SM = 1:1). The AExc values obtained for this film are highly positive indicating that the area per molecule values are larger than those resulting from additivity rule. The pictures taken for this film evidenced completely homogenous monolayer in a wide range of the surface pressure (thus they are not presented herein). Similar results were obtained previously for compositionally similar binary egg SM/POPC = 0.9 monolayers [17]. As it can be observed in Fig. 4a the addition of zymosterol shifts the isotherms to smaller area

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Fig. 5. BAM images taken for POPC:SM:zymosterol mixed films.

and decreases the collapse surface pressure (similar effect induces cholesterol, see Fig. S4). The influence of zymosterol on POPC:SM films manifests in a decrease of AExc values (they are negative for all ternary mixtures investigated) and in the increase of CS −1 (Fig. 4b and c). This proves that zymosterol causes condensation and ordering of POPC:SM film. However, this favourable effect of zymosterol is much weaker as compared to the influence of cholesterol. In the case of POPC:SM:cholesterol mixtures both AExc values are always visibly more negative as well as the changes of CS −1 are stronger than those for zymosterol-containing monolayers. However, both for zymosterol and cholesterol the condensing effect induced on POPC:SM film is the strongest at POPC:SM: cholesterol = 1:1:1. The analysis of the morphology of this monolayer (POPC:SM: zymosterol = 1:1:1) evidences that at large molecular areas gaseous, fluid and condensed phase coexist. With monolayer compression gaseous regions vanish and condensed phase dispersed within fluid matrix is seen in the pictures (Fig. 5). Then, the condensed domains enlarge joining together and finally at ca. 10 mN/m they cover whole interface and the film is homogenous. Then, small domains are formed and they exist up to the collapse. Similar morphological features reflect the pictures for POPC:SM:cholesterol = 1:1:1 films (Supplementary Materials, Fig. S5). The strongest differences between the images for zymosterol vs cholesterol-containing films appear at the highest content of particular sterol in the mixture. In this case within POPC:SM: zymosterol = 1:1:4 monolayers even at low surface pressures 3D crystallites manifested as bright points in the pictures, are formed. This kind of phenomena was not found for POPC:SM:cholesterol = 1:1:4 films.

3.3. The comparison of the effect of zymosterol and cholesterol on SM:GM3 films The surface pressure-area isotherms for (SM + GM3):sterol = 2:1 (the system contain 62% of SM, 5% of GM3 and 33% of sterol) monolayers as well as for corresponding SM:GM3 film and SM monolayers in Fig. S6 (Supplementary Materials) are shown. The properties of these systems were analyzed based on AExc values and BAM (Fig. 6) pictures taken at different stages of their compression. As proved the collected data the incorporation of even small amount of GM3 into SM film results in the condensation of the monolayer (for the studied herein SM:GM3 mixture containing 7.5% of ganglioside the AExc = −1.1 A˚ 2 /molecule) and evidences favorable mixing of these two lipids (SM:GM3). The images taken for binary SM:GM3 film show that at the initial step of the compression in a wide range of the surface pressure the monolayer is in homogenous fluid phase. Then, at ca. 14 mN/m the domains of condensed phase are formed. Initially they are oval and small, however, with film compression they enlarge and change their shape. These domains gather together and at ca. 30 mN/m the condensed phase covers practically whole interface. However, the monolayers remain inhomogeneous up to 60 mN/m. The addition of 33% of sterol into SM:GM3 monolayer results in the shift of the isotherms to lower areas, however, this effect is significantly more pronounced for cholesterol-containing monolayer. The AExc values calculated for these systems at 30 mN/m are more negative for SM:GM3:chol (AExc = −3.0 A˚ 2 /molecule) than for SM:GM3:zymo (AExc = −0.9 A˚ 2 /molecule) monolayers. This evidences stronger condensing effect of cholesterol vs zymosterol on SM:GM3 films. The pictures taken for both these systems prove that

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Fig. 6. BAM images taken for SM:GM3 and SM:GM3:sterol monolayers at different stages of compression.

the presence of sterol in the system causes strong modification in the morphology of SM:GM3 monolayer, however, the images for SM:GM3:chol vs SM:GM3:zymo films are very similar. The only difference is that the domains of condensed phase form at ca. 5 mN/m for zymosterol-containing film, while at 7 mN/m for cholesterolcontaining monolayer. 4. Discussion Sterols are vitally important components of membranes and in general the influence of these compounds on membranes and lipids is intensively studied. However, it seems that these investigations are focused on major sterols like cholesterol, ␤-sitosterol or ergosterol, while the properties of those steroids, which are minor membrane components are less explored. To the latter group undoubtedly belongs one of cholesterol precursors namely zymosterol [18]. The aim of our experiments was to discuss the condensing and ordering effect of zymosterol and its interactions with lipids in comparison with cholesterol. The studies were done in lipid Langmuir films being a model of membranes [19,20]. Already at the initial step of our experiments strong differences in monolayer properties of zymosterol vs cholesterol were

identified. The monolayers formed by zymosterol were found to be less ordered than cholesterol films and their morphology was strongly different. Both compounds form highly condensed monolayers, however, BAM pictures for zymosterol evidenced strong heterogeneity of the film at a whole stages of compression, while cholesterol monolayer was uniform. The differences between these two sterols manifest strongly in their influence on lipids in the mixed systems. The analysis of the excess area per molecules values and the compressional modulus values derived from the isotherms as well as BAM images taken for the studied films shown that zymosterol molecules condense and order SM, POPC and GM3 monolayers and favorably interact with the foregoing lipids. Thus, zymosterol similarly to cholesterol can be classified as membrane active sterol. However, when the effect of zymosterol vs cholesterol on the studied monolayers was analyzed quantitatively important differences were found. Considering the results collected at higher surface pressures, which is of special importance from the point of view of the correspondence between lipids monolayers and bilayers properties, it was found that the AExc values for lipid:zymosterol films decrease (becomes more negative) with the increase of sterol in the mixed film. This evidences that the condensing properties of zymosterol are the stronger the

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more sterol is in the system. For all the lipid:zymosterol monolayers the minimum of AExc appears at 1:2 = lipid:zymosterol ratio, which proves that zymosterol is the most potent as a condensing agent at the mentioned above monolayer composition. On the other hand, the results for lipid:cholesterol monolayers shown that cholesterol induces the strongest condensation at its 33% (SM:cholesterol and GM3:cholesterol) or 50% (POPC:cholesterol) level and with further increase of sterol content in the system the condensation becomes weaker. This enables one to conclude that zymosterol is less effective as a condensing agent as compared to cholesterol. This sterol is also of lower ordering abilities since it provokes lower increase of the compressional modulus values than cholesterol. Moreover, comparing the AExc values for SM:zymosterol vs POPC:zymosterol vs GM3:zymosterol monolayers at the minimum (AExc = −1.9; −5.7 and −5.0 A˚ 2 /molecule for SM:zymosterol vs POPC:zymosterol vs GM3:zymosterol = 1:2) with the values obtained for lipid:cholesterol mixtures (AExc = −2.7 and −7.0 A˚ 2 /molecule for SM:cholesterol and GM3:cholesterol = 2:1, while AExc = −5.7 A˚ 2 /molecule for POPC:cholesterol = 1:1,) it can be deduced ones again that zymosterol increases the condensation of the monolayers less effectively than cholesterol. Only in the case of POPC films both sterol similarly condense the lipid film, however, also in this case to achieve this effect higher level of zymosterol vs cholesterol is required. The condensing and ordering properties of zymosterol were also verified in ternary SM:POPC:sterol monolayers, which are frequently considered as a raft like systems [21]. The obtained results evidenced that the addition of zymosterol into POPC:SM = 1:1 monolayer causes condensation of the system, which is reflected in the negative values of AExc as well as in morphological changes observed in the pictures for POPC:SM:zymosterol mixtures vs SM:POPC system. The latter is based on the formation of condensed phase at the interface after addition of sterol. Similar condensing and ordering effect on SM:POPC film cholesterol induces, however, also in this case the influence of cholesterol is much stronger as compared to zymosterol. Less favorable effect of zymosterol vs cholesterol on SM:POPC monolayer is especially manifested for POPC:SM: sterol = 1:1:4 monolayers. Namely, for zymosterol-containing film the 3D structures are formed within the monolayer. This kind of phenomena was not found for POPC:SM:cholesterol = 1:1:4 films. Interestingly, in contrast to discussed above binary lipid:sterol monolayer, in the case of ternary films the effect of both sterols is the strongest at 1:1:1 lipids proportion in the systems. Finally, to investigate the effect of ganglioside on SM:sterol interactions the properties of (SM + GM3):zymosterol = 2:1 mixture was analyzed. In the studied monolayers ganglioside comprised only 5% of lipids in the system. Our data evidence stronger condensation and more favourable interactions in SM:GM3:chol vs SM:GM3:zymo monolayers, which again proves that condensing properties of cholesterol prevail on the properties of zymosterol. Based on these experiments it can be also noticed that the presence of ganglioside in the system strengthens the interactions in the mixed film. This can be deduced from the fact that the AExc values for SM:sterol = 2:1 mixtures are less negative than those for (SM + GM3): sterol = 2:1 films. Namely, AExc = −0.9 A˚ 2 /molecule vs −0.5 A˚ 2 /molecule for (SM + GM3): zymosterol = 2:1 vs SM:zymosterol= 2:1 and for cholesterolcontaining mixtures: AExc = −3.0 A˚ 2 /molecule vs −2.65 A˚ 2 /molecule for (SM + GM3): cholesterol = 2:1 vs SM:cholesterol= 2:1. Systematic studies on the properties of mixed systems composed of sterols and membrane lipids of various classes allow one to conclude that zymosterol is able to favorably interact with SM, POPC and ganglioside in the monolayers and to induce condensation and ordering of molecules in the studied systems. However, the condensing and ordering properties of this sterol are weaker

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as compared to those of cholesterol. Since the foregoing influence of sterols on membranes is connected with optimal packing abilities of the molecules in the system ensuring favorable interactions between sterols and other lipids (that is the interactions between sterol hydroxyl group and lipids polar heads as well as the van der Waals forces between the hydrophobic part of sterol molecule and lipids chains), it can be claimed that the observed differences in the effect of zymosterol vs cholesterol on monolayers result from the structural differences between both molecules. As it can be noticed in Scheme 1, cholesterol and zymosterol differ both in the structure of the ring system (i.e. the position of a double bond) and in the structure of the side chain (the presence of a double bond in zymosterol molecule). We postulate that the membrane activity of zymosterol and cholesterol is differentiated mainly by the structure of the side chain, which determines a geometry of both molecules. As it was evidenced for other sterols [22], the presence of a double bond in the side chain of sterol increases diameter of molecule and decreases its length, making the molecule bulkier. The impact of this kind of modification in steroid molecule on the interactions of sterol with membrane lipids is easy to notice for cholesterol vs ˇ-sitosterol and stigmasterol (the latter being plant sterols), since their molecules differ only in the structure of the side chain. Namely, both these phytosterols have an additional, as compared to cholesterol, ethyl group, while stigmasterol also a double bond, in the side chain. In the consequence the side chain in plant sterols is branched and less flexible and the molecules are of larger diameter as compared to cholesterol. The results of theoretical calculations performed for cholesterol, ˇ-sitosterol and stigmasterol evidenced that cholesterol molecule has smaller cross-sectional area and is longer as compared to phytosterols. This in turn impedes the tight packing of phytosterols with other molecules and makes van der Waals interactions between sterol and the chains of other lipids difficult. The effect of these structural differences on the sterol:lipid interactions was confirmed in various experiments on model membrane systems [23–28]. The investigated herein zymosterol similarly to the mentioned above plant sterols has structural modification in the side chain, which weakens the condensing and ordering potency of this compound on lipids acyl chains. The packing properties of zymosterol are also in some degree affected by the position of a double bond in the ring system. This conclusion can be drawn in relation to the results of differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopic studies performed for lathosterol/DPPC bilayers [29]. Lathosterol is also a cholesterol precursor differing from cholesterol only in the position of double bond in the ring system. As it was found the molecules of this compound less favorably mix with DPPC and the bilayer is less tightly packed as compared to the system containing cholesterol [29].

5. Conclusions The results of our systematic monolayer experiments on the interactions of zymosterol with lipids evidenced that this cholesterol precursor possesses condensing and ordering abilities. This allows one to include this molecule to the group of membrane active sterols. However, a quantitative examination of the effect of zymosterol on model membranes evidenced a lower effectiveness of this compound as compared to cholesterol in condensation and ordering of lipids. The differences in the effect of both sterols manifest also in the morphology of the studied monolayers. The observed effects were attributed to the differences in the structure of zymosterol vs cholesterol, which determines the ability of both molecules to tight packing with other lipids in the mixed systems.

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Acknowledgment This work was financed by grant Iuventus Plus (decision number: 0514/IP3/2013/72) from the Ministry of Science and Higher Education. Appendix A. Supplementary data

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Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2014. 09.054.

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