Accepted Manuscript Title: A Langmuir monolayer study of the action of phospholipase A2 on model phospholipid and mixed phospholipid-GM1 ganglioside membranes Author: Wiebke Schulte Monika Orlof Izabella Brand Beata Korchowiec Ewa RogalskaTel.: +48 12 663 22 51; fax: +48 12 634 05 15. PII: DOI: Reference:

S0927-7765(13)00785-6 http://dx.doi.org/doi:10.1016/j.colsurfb.2013.12.032 COLSUB 6184

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

13-8-2013 4-12-2013 19-12-2013

Please cite this article as: W. Schulte, M. Orlof, I. Brand, B. Korchowiec, E. Rogalska, A Langmuir monolayer study of the action of phospholipase A2 on model phospholipid and mixed phospholipid-GM1 ganglioside membranes, Colloids and Surfaces B: Biointerfaces (2013), http://dx.doi.org/10.1016/j.colsurfb.2013.12.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract (for review)

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Graphical abstract

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Page 1 of 32

Highlights (for review)

Highlights

Phospholipases A2 (PLA2) are dependent on the properties of the lipid system



The rate of the PLA2 catalyzed hydrolysis of DLPC decreases in the presence of GM1



The decrease of the reaction rate may be due to H-bonding of the lipid polar heads

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Page 2 of 32

*Revised Manuscript

A Langmuir monolayer study of the action of phospholipase A2 on model phospholipid and mixed phospholipid-GM1 ganglioside membranes

Carl von Ossietzky University of Oldenburg, Center of Interface Science (CIS), Department

of Pure and Applied Chemistry, D-26111 Oldenburg, Germany

Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian

University, ul. R. Ingardena 3, 30-060 Krakow, Poland

Structure et Réactivité des Systèmes Moléculaires Complexes, UMR CNRS 7565, Faculté

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Wiebke Schultea, Monika Orlofb, Izabella Branda, Beata Korchowiecb,* and Ewa Rogalskac,**

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des Sciences et Techniques, Université de Lorraine, 54506 Vandœuvre-lès-Nancy, France

Key-words: lipid monolayer; phospholipid; ganglioside; Langmuir isotherms; polarization-

*

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modulation infrared reflection-absorption spectroscopy

Corresponding author. Tel.: +48 12 663 22 51; fax: +48 12 634 05 15. Corresponding author. Tel.: +33 3 83 68 43 45; fax: +33 3 83 68 43 22. E-mail addresses: [email protected] (E. Rogalska), [email protected] (B. Korchowiec) **

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ABSTRACT Polarization-modulation infrared reflection-absorption spectroscopy, surface pressure measurements and thermodynamic analysis were used to study enzymatic hydrolysis of lipid monolayers at the air/water interface. The Ca2+-requiring pork pancreatic phospholipase A2

mixed

1,2-dilauroyl-sn-glycero-3-phosphocholine

-

ip t

was used as a catalyst. The substrates were pure 1,2-dilauroyl-sn-glycero-3-phosphocholine or monosialotetrahexosylganglioside

cr

Langmuir films. The physicochemical properties of the monolayers were established with the

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aim of a correlation with enzyme activity.

The infrared spectra were acquired upon the advancement of the catalysis; the latter was

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studied at a controlled surface pressure and area of the film. Changes of the intensity and frequency of different infrared signals characteristic for the two lipids were correlated with

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modification of the properties of the monolayer due to hydrolysis. The amide I signal characteristic for peptides permitted detecting the enzyme adsorbed at the interface. The

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thermodynamic and infrared results indicate that monosialotetrahexosylganglioside increases H-bonding of the lipid polar heads in the films. This effect, which may be responsible for the

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low activity of phospholipase A2 in the mixed films, could be used for developing enzyme-

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resistant lipid systems.

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1. Introduction Gangliosides [1] have drawn special attention owing to their predominance as the major sialoglycoconjugate category within the nervous system. Relatively little is known about gangliosides

structure,

physical

properties

and

function.

In

the

case

of

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monosialotetrahexosylganglioside (GM1; Fig. 1), an array of functions has already been

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revealed; many of them are related to cell membranes [1]. Because phospholipases represent the largest group of lipid-modifying enzymes, which influence membrane structure and

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function [2], it is interesting to get more understanding of the phospholipase-ganglioside interaction [3].

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Among different phospholipases, the best understood are phospholipases A2 (PLA2; EC

M

3.1.1.4) [4-8]. Numerous crystal structures are available for this class of enzymes, which utilize in their action a catalytic histidine in a so-called dyad or a catalytic serine in either a

ed

dyad or a triad [9, 10]. The mechanism of catalysis [4, 11], as well as the emerging biological functions of these enzymes [12, 13] were reviewed recently. PLA2 catalyze acyl ester yielding fatty acids and

ce pt

hydrolysis at the sn-2 position of phosphoglycerides

lysophospholipids [7], both of which may alter cell function [6, 14]. PLA2 are best known for their role in initiating the arachidonic acid (AA) cascade [15, 16]. AA is the precursor of a

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large family of compounds known as the eicosanoids including prostaglandins and leukotrienes involved in inflammatory reactions [17]. The other compound released by PLA2 from membrane phospholipids, the 2-lysophospholipid, is at the origin of

formation of

another inflammatory mediator, namely platelet-activating factor (PAF, 1-O-alkyl-2-acetylsn-glycero-3-phosphocholine) [18]. Thus, PLA2 is an attractive target for drug development because if one could inhibit PLA2, the synthesis of all three inflammatory mediators (the prostaglandins, leukotrienes and PAF) could potentially be blocked [14]. From this point of view, the recent findings indicating that PLA2 activity depends on the minute organization of

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Page 5 of 32

lipid molecules is of particular interest. Indeed, it was shown that PLA2 is sensitive to the physics of its substrate and is regulated by the presence of lipid domains [19-24]; this observation may offer leads for controlling PLA2 activity by adjusting the properties of the lipid aggregates.

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Recently, the activity of the enzyme was correlated with the effect of meloxicam, piroxicam, and tenoxicam on the properties of phospholipid monolayer, namely on hydration

cr

of the lipid polar heads, orientation of the molecules, and morphology of the domains [25].

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The latter indicates that the anti-inflammatory action of oxicams may be related to interference with phospholipase activity in addition to cyclooxygenase inhibition. A decreased

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rate of PLA2 catalyzed lipolysis observed in phospholipid monolayers in the presence of trivalent aluminum (Al) cations compared to the Al-free systems was interpreted in terms of a

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lower microheterogeneity of the former [26]. It cannot be excluded that in physiological conditions modulation of the enzyme action by the Al-bound membranes is among the

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reasons for Al toxicity. A decreased rate of PLA2 catalyzed lipolysis was observed as well in mixed phospholipid-glycosphingolipid (ganglioside) monolayers [27]. This effect was

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interpreted in terms of a higher polarity, higher hydration and higher homogeneity of the mixed films compared to pure phospholipids [28]. The sialic acid-containing glycosphingolipid gangliosides are components of most cells,

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but they are especially abundant in the plasma membrane of neuronal cells [29-32]. GM1 is a member of the ganglio series of gangliosides [1], which contain one sialic acid residue. There is evidence that GM1 has antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems [32]. It was observed that GM1 at 5-7 mol% is miscible with both dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and distearoyl-snglycero-3-phosphocholine bilayers and forms the most stable vesicles in the presence of cholesterol [33]. This effect could be due to shielding of the negative charge in GM1 by a

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bulky, neutral, hydrophilic sugar moiety, and thus to a low protein adsorption [34]. However, differential scanning calorimetry and X-ray diffraction studies of the thermotropic and structural properties of ganglioside GM1 alone and in a binary system with DPPC indicate that the presence of the negatively charged sialic acid moiety of the GM1 oligosaccharide at

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the bilayer surface (even at a low molar concentration, 5.7 mol %) is sufficient to produce the charge repulsion effect responsible for continuous interbilayer hydration [29]. The surface

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Fig. 1

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behavior of glycosphingolipids in biomembranes was reviewed by Maggio [35].

Here, the activity of pork pancreatic PLA2 [36], which is a Ca2+-requiring extracellular

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enzyme, was studied in pure 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC; Fig. 1), or mixed DLPC-GM1 Langmuir films. Surface pressure and polarization-modulation infrared

ed

reflection-absorption spectroscopy (PM-IRRAS) were used to monitor the advancement of the reaction. It was shown that different PM-IRRAS signals characteristic for DLPC, GM1 and

ce pt

PLA2 can be used to observe the changes of the physicochemical properties of the film due to enzymatic hydrolysis.

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2. Materials and methods

2.1. Materials and reagents (β-Gal-(1-3)-β-GalNac-(1,4)-[α-Neu5Ac-(2,3)]-β-Gal-(1,4)-βGlc-(1,1)-Cer) (≥95%pure),

1,2-dilauroyl-sn-glycero-3-phosphocholine

(DLPC)

(~99%

(GM1) pure),

spectrophotometric grade chloroform and methanol (both 99.9% pure) used for preparing phospholipid solutions were from Sigma-Aldrich. The GM1 and DLPC were dissolved in chloroform/methanol mixture (3:1 v/v) to achieve a final concentration of 0.5 mg ml-1. The

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stock solution of the GM1 and DLPC were used for preparing 0.05, 0.1, 0.15 and 0.2 GM1 mole fraction (xGM1) mixtures. The compound solutions were stored at 4 ºC. The subphases were prepared with pure Milli-Q water (resistivity 18.2 MΩ·cm at 25 °C; surface tension of 72.8 mN m-1 at 20 ºC; pH 5.6). Dihydrous calcium chloride (CaCl2·2H2O, purity 99%) was

ip t

from Sigma-Aldrich.

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2.2. Compression isotherms

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The surface pressure measurements were carried out with a KSV 2000 instrument (KSV Instruments, Ltd., Helsinki, Finland). A Teflon trough [7.5 cm (l) × 36 cm (w) × 0.5 cm (d)]

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with two hydrophilic Delrin barriers (symmetric compression) was used in compression isotherm experiments. The system was equipped with an electrobalance and a platinum

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Wilhelmy plate (perimeter 3.94 cm) as a surface pressure sensor. The apparatus was closed in a Plexiglas box, and temperature was kept constant at 20 ± 0.1 °C. Before each run, the trough

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and the barriers were washed using cotton soaked in chloroform and ethanol and then rinsed with Milli-Q water. The platinum Wilhelmy plate was cleaned between each experiment by

ce pt

rinsing with purified water and ethanol, and heating to a red-hot glow in a propane flame to eliminate any organic contaminants. All solvents used for cleaning the trough and the barriers were of analytical grade. All impurities were removed from the subphase surface by sweeping

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and suction. When the surface pressure fluctuation was found to be lower than 0.2 mN m-1 during a compression stage, monolayers were spread from solutions of accurate concentrations using a microsyringe (Hamilton Co., USA). After the equilibration time of 15 min, the films were compressed at the rate of 10 mm min−1 by two symmetrically moving barriers (5 mm min−1 per barrier; in the case of pure DLPC it corresponds to ~2 and ~2.7 Å2 molecule−1 min−1 at the beginning and at the end of the compression, respectively). A PC computer and KSV software were used to control the experiments. Each compression

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Page 8 of 32

isotherm was performed at least three times. The standard deviation was 0.5 Å2 for mean molecular area and 0.2 mN m-1 for surface pressure measurements. The compression isotherms allowed determining the compressibility modulus (Cs-1 = -A(∂Π/∂A)T) [37]. The collapse parameters, Πcoll and Acoll, were determined directly from the compression isotherms.

ip t

Free Gibbs energy of mixing, Gmix [38, 39], was calculated from Π-A isotherms using the 

following formula: G

mix

  A12  x1 A1  x2 A2  d ; A12 is a mean molecular area in the

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0

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mixed monolayer at a given surface pressure, A1 and A2 are mean molecular areas of, respectively, pure components 1 and 2 at the same surface pressure, and x1 and x2 are mole

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fractions of the two components in the mixed film. It should be noted that Gmix is defined in our work as an excess energy relative to the additive energy of pure components present in the

2.3. Enzymatic lipolysis

ed

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film.

Porcine pancreatic phospholipase PLA2 (P6534, Sigma; 1020 U mg-1 protein, conc. 3.4

ce pt

mg mL-1) was used as received in DLPC lipolysis experiments. In the case of kinetics studies, the experiments were performed at a constant surface pressure of 15 mN m-1; the subphase used contained CaCl2 (concentration 5 mM, pH 6.0). The experiments were realized with a

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KSV 2000 Langmuir balance (KSV, Helsinki, Finland) and a zero-order trough with a symmetric compression. A zero-order trough was composed of a reaction compartment [5.5 cm (w) × 3.0 cm (l) × 0.5 cm (d)] and two reservoir compartments [16 cm (l) × 7.5 cm (w) × 0.5 cm (d)] communicating by means of two narrow surface channels [40, 41]. The reaction rate was monitored using a KSV software, which allows maintaining a constant surface pressure via barrier movement.

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Page 9 of 32

The enzyme was injected with a Hamilton syringe (2 μL of the commercial enzyme solution) under the film in the reaction compartment only, whereas the film covered all three compartments. The reservoir compartments contained mobile barriers, which were used to compensate for substrate molecules removed from the film in the reaction compartment by

ip t

enzyme hydrolysis, thereby keeping surface pressure constant. Surface pressure was measured in the reservoir compartment with a Wilhelmy plate (perimeter 3.94 cm) attached to an

cr

electromicrobalance, connected in turn to a computer controlling the movement of the mobile

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barriers. The subphase was thermostatically maintained at 20 °C and was continuously

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agitated in the reaction compartment with a 1 cm magnetic stirrer moving at 250 rpm.

2.4. Polarization-modulation infrared reflection-absorption spectroscopy.

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The monolayers were spread as described in Section 2.2. The PM-IRRAS measurements were performed during PLA2 catalyzed hydrolysis maintaining a constant surface pressure of

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15 mN m-1 at a decreasing area of the film contained between the barriers, or at a constant area of the film and a decreasing surface pressure. In the first case, the surface concentration

ce pt

of the DLPC molecules is constant in the pure DLPC film; in the mixed DLPC-GM1 films, the surface concentration of DLPC decreases, while that of GM1 increases compared to their initial concentrations. In the second case, the surface concentration of DLPC decreases during

Ac

hydrolysis in the pure and mixed films, while GM1 surface concentration is constant. The PM-IRRAS [42-46] spectra were recorded at 20 °C. The Teflon trough dimensions were 36.5 cm (l)  7.5 cm (w)  0.5 cm (d). A KSV PMI 550 instrument (KSV Instruments Ltd, Helsinki, Finland) was used in the experiments. The PMI 550 contains a compact Fourier Transform IR-spectrometer equipped with a polarization-modulation (PM) unit on one arm of a goniometer, and a MCT detector on the other arm. The incident angle of the light beam was 75°. The spectrometer and the PM

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Page 10 of 32

unit operate at different frequencies, allowing separation of the two signals at the detector. The PM unit consists of a photoelastic modulator (PEM), which is an IR-transparent, ZnSe piezoelectric lens. The incoming light is continuously modulated between s- and ppolarization at a frequency of 74 kHz. This allows simultaneous measurement of spectra for

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the two polarizations, the difference providing surface specific information, and the sum providing the reference spectrum. As the spectra are measured simultaneously, the effect of

cr

water vapor is largely removed. The PM-IRRAS spectra of the film-covered surface, R(f), as

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well as that of the pure water, R(w), were measured and the normalized difference ΔR/R = [R(f) – R(w)] / R(w) is reported. 6000 interferogram scans (10 scans per second) were

an

acquired for each spectrum.

It is important to mention that the spectra were acquired upon the advancement of the

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enzymatic hydrolysis, at low surface concentrations of the lipids. Consequently, the intensity of the signals was low and the signal to noise ratio high. The ΔR/R spectra were corrected

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with a polynomial fitted spectrum from the air-water interface. In the mid-IR region, the wavenumber at which the half-wave retardation takes place can

ce pt

be freely selected. Here, the maximum of PEM efficiency was set either to 1500 or to 2900 cm-1 for analyzing the carbonyl stretching or methylene stretching regions of the spectra,

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respectively. The spectral range of the device is 800-4000 cm-1 and the resolution is 8 cm-1.

3. Results and discussion 3.1. Compression isotherms Pure DLPC, GM1, or their mixtures were used to spread monolayers on a pure water subphase, or on a 5 mM CaCl2 solution. The presence of Ca2+ ions in the subphase is necessary, as PLA2 used for catalyzing lipid hydrolysis is Ca2+-dependent [4, 8, 17]. Because

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Page 11 of 32

no meaningful impact of Ca2+ on the Π-A isotherms was observed, only those obtained with monolayers formed on the pure water subphase are presented here. The isotherms of pure DLPC or GM1 and of their mixtures are plotted as surface pressure () versus area per molecule (A) (Fig. 2). It can be observed that the -A isotherm of GM1 is

ip t

shifted to higher molecular areas compared to the isotherms of pure DLPC and of the DLPCGM1 mixtures. Interestingly, the xGM1 = 0.05 monolayer is slightly expanded compared to

cr

pure DLPC, while an increasing content of GM1 results in a shift of the isotherms to lower

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molecular areas (Fig. 2), indicating a condensing effect of the ganglioside. On the other hand, the mixed films are less rigid (lower Cs-1) compared to DLPC, except the xGM1 = 0.05 mixture.

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The characteristic parameters of the isotherms at the collapse and at 15 mN m-1 are collected

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in Table 1.

Fig. 2

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Table 1

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3.2. Interaction between monolayer components To better understand the interaction between the monolayer components, which non-ideal mixtures are here examined, the analysis of mean molecular area (MMA) and Gibbs energy of

Ac

mixing (ΔGmix) were plotted as a function of xGM1 (Fig. 3). In ideal mixtures, intermolecular forces are the same between each pair of molecular kinds. In classical, bulk thermodynamics, this is the reference case against which are examined corresponding mixings of non-ideal materials. The thermodynamic models applied to monolayers are extrapolated from bulk systems; such extrapolation is delicate and needs clarifying the terms used. In our work, the additive values of pure compounds are used as reference in MMA and Gmix versus xGM1 plots (Fig. 3). As a consequence, Gmix in monolayers formed with miscible components can be positive [47] or negative [48], which is

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Page 12 of 32

contrary to the situation in the bulk, where Gmix is always negative for miscible and equals zero for immiscible systems [49].

Fig. 3

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A comparison between the MMA of additive and experimental mixing as a function of the GM1 mole fraction at three arbitrary chosen surface pressures: 10, 15 and 20 mN m-1 is

cr

shown in Fig. 3A. For most mixtures, deviation from linearity of MMA occurs for all surface

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pressures. Adding small amounts of the ganglioside (xGM1 = 0.05) to a pure DLPC matrix leads to a slight increase of MMA values. Upon a further increase of the GM1 content, the

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MMA values decrease significantly indicating monolayer condensation. Overall, the MMA results obtained show that GM1 significantly affects packing of the phospholipid molecules in

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the monomolecular film.

The П-A isotherms allowed calculating the Gibbs energy of mixing, ΔGmix. The ΔGmix

ed

plot as a function of the composition of the mixed DLPC-GM1 monolayers at 10, 15 and 20 mN m-1 is presented in Fig. 3B. It can be seen that ΔGmix decreases compared to pure films;

ce pt

the ΔGmix ≠ 0 values indicate that for xGM1 = 0.05 ÷ 0.2 DLPC and GM1 are miscible. These differences in the ΔGmix values can be attributed to negative enthalpy effects, which are sufficiently important to overcome the negative entropic effects [49] expected in the mixed

Ac

films undergoing condensation and ordering, as observed with MMA (Fig. 3A). The negative values of ΔGmix (Fig. 3B) suggest that H-bonding and charge-charge interaction between the polar head groups in the mixed films are favored compared to the pure films.

3.3. Enzymatic reaction The impact of GM1 on PLA2 activity was followed using pure DLPC and mixed DLPCGM1 monolayers. DLPC was used as a model substrate because it yields water-soluble

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Page 13 of 32

products [40, 50, 51]; the latter is necessary for monitoring enzyme activity [25, 52, 53]. Since PLA2 used in this study is calcium dependent, the hydrolysis reactions were performed in the presence of Ca2+ in the subphase. The kinetics was performed at 15 mN m-1 (Fig. 4), because PLA2 shows the highest activity at this surface pressure [25]. In Fig. 4, the variation of the

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reaction rate is presented as a function of xGM1. It is clearly seen that the rate of DLPC hydrolysis decreases dramatically in the presence of small amounts of GM1. Interestingly, the

cr

decrease of the reaction rate is twofold for the film with xGM1 0.05 compared to pure DLPC

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Fig. 4

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and is less important for the higher contents of GM1.

M

3.4. PM-IRRAS spectra

PLA2 catalyses the hydrolysis of the ester bond in the sn-2 position of phospholipids. The

ed

progress of the enzymatic reaction was monitored with PM-IRRAS using pure DLPC or mixed DLPC-GM1 (xGM1 = 0.1) films as substrates; the pure GM1 film was used as a blank.

ce pt

PLA2 was injected beneath monolayers compressed to = 15 mN m-1. As expected, in the case of pure GM1, the surface pressure and the film area between the barriers was stable

Ac

in the presence of PLA2.

3.4.1. Hydrolysis of a pure DLPC monolayer at the air-water interface at a constant area of the film The impact of PLA2 on the properties of the pure DLPC monolayer compressed to the surface pressure  = 15 mN m-1 was monitored by means of the PM-IRRAS at a constant area of the monolayer contained between the barriers. In these conditions, the surface pressure

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Page 14 of 32

decreases, because the products of the enzymatic hydrolysis are water-soluble and the surface concentration of DLPC decreases. Fig. 5

ip t

Fig. 5 shows the PM-IRRAS spectra of the pure DLPC monolayer at the air-water interface during the enzymatic reaction. The stretching modes of the methylene groups in acyl

cr

chains are observed in the 3000–2800 cm-1 region (Fig. 5A) [25]. In the spectrum

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corresponding to = 15 mN m-1 (Fig. 5A, curve 1), the as(CH2) and s(CH2) modes are located at 2920 and 2851 cm-1, respectively. These signals are accompanied by low intensity

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signals centered at 2938 cm-1 and 2863 cm-1. The two sets of signals point to the co-existence of hydrocarbon chains in two different states, the former corresponding to higher and the

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latter to lower chain ordering [18, 25, 54-56]. These results indicate that the DLPC monolayer is in a liquid state and are in accordance with the film properties determined using

ed

compression isotherms. The intensity of these signals decreases with the advancement of the reaction that is a decrease of the number of DLPC molecules at the air-water interface (Fig.

ce pt

5A, curves 2 and 3).

Fig. 5B shows the PM-IRRAS spectra in the 1800–1500 cm-1 region of the DLPC monolayer undergoing hydrolysis catalyzed by PLA2. In this spectral region DLPC molecules

Ac

have two overlapping absorption bands (C=O) 1729 and 1743 cm-1 modes (Fig. 5B, curve 1), which originate from the hydrated (H-bonded) and non-hydrated (non-H-bonded) ester groups, respectively [54, 57, 58]. In the PM-IRRAS spectra obtained at Π = 8 mN m-1 (Fig. 5B, curve 2) a red-shift of the (C=O) to 1723 and 1738 cm-1 is observed compared to Π = 15 mN m-1 (Fig. 5B, curve 1). The red-shift was observed for the consecutive decreasing surface pressures as well. This result indicates an increased hydration of the ester groups. At low surface pressure (Fig. 5B, curves 4 and 5), the intensity of (C=O) is significantly lower

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Page 15 of 32

compared to 15 mN m-1, which is in accordance with the decreasing surface concentration of DLPC. It can be noticed that new absorption bands located at 1575 and 1530 cm-1 appear transitorily in the PM-IRRAS spectra upon the enzymatic reaction (Fig. 5B, curves 2 and 3).

ip t

These two weak absorption modes may arise from the as(COO-) stretching mode of the carboxylic group interacting with Ca2+ ions. The latter is in accordance with the results

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published recently in the literature [59]. This observation indicates that the presence of lauric

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acid cleaved from DLPC can be detected with PM-IRRAS. These bands disappear upon reaction progress (Fig. 5B, curves 4 and 5), because lauric acid is soluble in water [40, 50];

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the decreasing intermolecular interaction in the film with decreasing surface concentration of DLPC favors desorption of the acid from the interface.

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The overlapping absorption bands appearing at Π = 8 mN m-1 between 1690 and 1600 cm-1 (Fig. 5B, curve 2) may be due to the amide I mode of PLA2 interacting with the

ed

phospholipid monolayer. The main absorption band centered at 1657 cm-1 has two shoulders at 1670 and 1629 cm-1. The well-defined band at 1657 cm-1 indicates that the secondary

ce pt

structure of PLA2 is predominantly composed of -helices with some disordered fragments [60, 61]. The two weaker bands may correspond to the -sheet structure of the protein [61]. This observation is in accordance with X-ray crystallography as well as Fourier transform

Ac

infrared spectroscopy studies showing that calcium dependent phospholipases A2 contain ca. 50% -helices, 35% -sheets and 15% random coil [61].

3.4.2. Hydrolysis of a mixed DLPC-GM1 monolayer at the air-water interface at a constant area of the film The PM-IRRAS spectra of a mixed DLPC-GM1 (xGM1 = 0.1) monolayer undergoing catalytic hydrolysis at a constant area of the film contained between the barriers are shown in

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Page 16 of 32

Fig. 6. The number of the DLPC molecules in the film decreases upon hydrolysis leading to a decrease of the surface pressure and to an increase of the surface concentration of GM1 relative to DLPC.

ip t

Fig. 6 The ester groups present in DLPC show a (C=O) mode centered at 1730 cm-1. This

cr

signal is blue-shifted with the progress of the enzymatic reaction (Fig. 6, curves 1 – 5).

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Indeed, at  = 0.5 mN m-1 the (C=O) shifts to 1735 cm-1 indicating dehydration of the DLPC ester groups (Fig. 6, curve 5); the latter may be due to the interaction between the polar

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heads of DLPC and GM1. A strong absorption in the amide I and II mode regions appear in the PM-IRRAS spectra at low surface pressure values (Fig. 6, curve 5). This signal may be

M

due to GM1, which does not undergo catalytic hydrolysis, and thus its surface concentration increases relative to DLPC. The amide I and amide II signals are clearly visible in the pure

ed

GM1 spectrum presented in Fig. 7. On the other hand, at low surface pressures, a contribution

ce pt

to the amide signals from the PLA2 adsorbed to the film cannot be excluded [61].

3.4.3. Hydrolysis of a mixed DLPC-GM1 monolayer at the air-water interface at a constant surface pressure

Ac

The hydrolysis of DLPC in the mixed DLPC-GM1 (xGM1 = 0.1) film performed at a constant surface pressure results in an increase of the concentration of GM1 relative to DLPC. The surface pressure was kept constant at 15 mN m-1 by decreasing the area of the monolayer. The PM-IRRAS spectra obtained are presented in Fig. 7.

Fig. 7

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Page 17 of 32

The PM-IRRAS spectra in the 1800 - 1500 cm-1 region of the pure GM1 film, as well as the mixed DLPC-GM1 monolayer during PLA2 catalyzed hydrolysis are presented in Fig. 7A and 7B, respectively. In the pure GM1 monolayer, the three amide groups present in GM1 give rise to the amide I mode centered at 1681, 1657 and 1636 cm-1 (Fig. 7A). In the mixed

ip t

film (Fig. 7B), the amide I bands are centered at 1685 and 1654 cm-1 and the amide II band at 1557 cm-1. During hydrolysis of DLPC in the mixed film, the amide I modes arising from

cr

GM1 increases in intensity (Fig. 7B, curves 1 – 5), and corresponds to the modes observed in

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the pure GM1 monolayer (Fig. 7A). Indeed, as described in the literature, the GM1 amide I mode in monolayers and vesicle suspensions is complex and composed of three overlapping

an

absorptions; positions of these bands are strongly dependent on the hydration of three amide groups present at different sites in the large polar head group [62].

M

The GM1 as (COO-) mode is split into two bands appearing at 1580 and 1537 cm-1 in the pure monolayer (Fig. 7A). These bands are not observed in the monolayer containing 10 % of

ed

GM1 (Fig. 7B, curve 1). However, for higher GM1 concentrations, the carboxylic group has

ce pt

two as(COO-) modes at 1573 and 1535 cm-1 (Fig. 7B, curve 5), which is similar to the pure GM1 monolayer.

The ester carbonyl group at the DLPC gives an asymmetric absorption band centered at 1730 cm-1 (Fig. 7B). During the enzymatic reaction, the DLPC (C=O) mode shifts to higher

Ac

frequencies indicating dehydration of the ester groups (Fig. 7B, curves 1 – 5). The latter may be due to an increasing interaction with the GM1 polar head groups, as the surface concentration of GM1 increases [54].

16

Page 18 of 32

4. Conclusions PLA2 was found to be less active on mixed DLPC-GM1 films compared to pure DLPC. The thermodynamic analysis indicates an increased H-bonding between the polar head groups

ip t

in the mixed films compared to pure DLPC and pure GM1; the PM-IRRAS results support this conjecture. The increased H-bonding in the mixed films may hinder desorption of DLPC

cr

from the interface and transfer into the enzyme active site, leading to a decreased hydrolysis

us

rate. This observation may be helpful for providing protection against enzymatic degradation in different lipid based developments. On the other hand, the red-shift of the DLPC (C=O)

an

signals upon PLA2 action together with the increasing intensity of GM1 amide modes and the carboxylic group signals, which appear in the mixed films with the advancement of the

ce pt

Acknowledgments

ed

M

hydrolysis may be used for monitoring PLA2 action.

Monika Orlof acknowledges the Polish Minister of Science and Higher Education for financial support for the best PhD students. We also thank Małgorzata Kuniewicz and

Ac

Stephane Parant for their technical assistance. The work was supported by German Science Foundation DFG project BR 3961/2, and a Procope French-German project operated by the Ministère des Affaires étrangères (MAE) and the Ministère de l'Enseignement supérieur et de la Recherche (MESR), as well as the Deutscher Akademischer Austauschdienst (DAAD).

17

Page 19 of 32

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Ac

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cr

ip t

[1] R.W. Ledeen, G. Wu, Neurochem. Res. 35 (2010) 1867-1874. [2] W.J. Brown, K. Chambers, A. Doody, Traffic (Oxford, U. K.) 4 (2003) 214-221. [3] M.A. Perillo, A. Guidotti, E. Costa, R.K. Yu, B. Maggio, Mol. Membr. Biol. 11 (1994) 119-126. [4] E.A. Dennis, J. Cao, Y.-H. Hsu, V. Magrioti, G. Kokotos, Chem. Rev. (Washington, DC, U. S.) 111 (2011) 6130-6185. [5] M. Murakami, Y. Taketomi, Y. Miki, H. Sato, T. Hirabayashi, K. Yamamoto, Prog. Lipid Res. 50 (2011) 152-192. [6] M. Murakami, Y. Taketomi, H. Sato, K. Yamamoto, J. Biochem. 150 (2011) 233-255. [7] D.A. Six, E.A. Dennis, Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 1488 (2000) 119. [8] H.M. Verheij, A.J. Slotboom, G.H. De Haas, Rev. Physiol., Biochem. Pharmacol. 91 (1981) 91-203. [9] L.W. Tjoelker, C. Eberhardt, J. Unger, H.L. Trong, G.A. zimmerman, T.M. McIntrye, D.M. Stafforini, S.M. Prescott, P.W. Gray, J. Biol. Chem. 270 (1995) 25481-25487. [10] D.L. Scott, S.P. White, Z. Otwinowski, W. Yuan, M. Gelb, P.B. Sigler, Science 250 (1990) 1541-1546. [11] V.D. Mouchlis, E. Barbayianni, T.M. Mavromoustakos, G. Kokotos, Curr. Med. Chem. 18 (2011) 2566-2582. [12] S.M. Moghimi, A.C. Hunter, J.C. Murray, Pharmacol. Rev. 53 (2001) 283-318. [13] M. Murakami, Y. Taketomi, C. Girard, K. Yamamoto, G. Lambeau, Biochimie 92 (2010) 561-582. [14] J. Balsinde, M.A. Balboa, P.A. Insel, E.A. Dennis, Annu. Rev. Pharmacol. Toxicol. 39 (1999) 175-189. [15] D.W. Gilroy, J. Newson, P. Sawmynaden, D.A. Willoughby, J.D. Croxtall, FASEB J. 18 (2004) 489-498. [16] N.V. Bogatcheva, M.G. Sergeeva, S.M. Dudek, A.D. Verin, Microvasc. Res. 69 (2005) 107-127. [17] E.A. Dennis, J. Biol. Chem. 269 (1994) 13057-13060. [18] F. Snyder, Biochem. J. 305 (1995) 689-705. [19] K. Wagner, G. Brezesinski, Curr. Opin. Colloid Interface Sci. 13 (2008) 47-53. [20] M. Grandbois, H. Clausen-Schaumann, H. Gaub, Biophys. J. 74 (1998) 2398-2404. [21] P. Hoyrup, K. Jorgensen, O.G. Mouritsen, Europhys. Lett. 57 (2002) 464-470. [22] O.G. Mouritsen, T.L. Andresen, A. Halperin, P.L. Hansen, A.F. Jakobsen, U.B. Jensen, M.O. Jensen, K. Joergensen, T. Kaasgaard, C. Leidy, A.C. Simonsen, G.H. Peters, M. Weiss, J. Phys.: Condens. Matter 18 (2006) S1293-S1304. [23] C. Leidy, L. Linderoth, T.L. Andresen, O.G. Mouritsen, K. Jorgensen, G.H. Peters, Biophys. J. 90 (2006) 3165-3175. [24] C. Leidy, J. Ocampo, L. Duelund, O.G. Mouritsen, K. Jorgensen, G.H. Peters, Biophys. J. 101 (2011) 90-99. [25] K. Czapla, B. Korchowiec, M. Orlof, J.R. Magnieto, E. Rogalska, J. Phys. Chem. B 115 (2011) 9290-9298. [26] Y. Corvis, B. Korchowiec, G. Brezesinski, S. Follot, E. Rogalska, Langmuir 23 (2007) 3338-3348. [27] B. Maggio, Chem. Phys. Lipids 132 (2004) 209-224. [28] I.D. Bianco, G.D. Fidelio, R.K. Yu, B. Maggio, Biochemistry 30 (1991) 1709-1714. [29] R.A. Reed, G.G. Shipley, Biophys. J. 70 (1996) 1363-1372.

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[30] A. Olivera, S. Spiegel, Glycoconjugate J. 9 (1992) 110-117. [31] A.A. Rampersaud, J.L. Oblinger, R.K. Ponnappan, R.W. Burry, A.J. Yates, Biochem. Soc. Trans. 27 (1999) 415-422. [32] M. Hadjiconstantinou, N.H. Neff, J. Neurochem. 70 (1998) 1335-1345. [33] F.K. Bedu-Addo, L. Huang, J. Pharm. Sci. 85 (1996) 714-719. [34] A. Gabizon, D. Papahadjopoulos, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 69496953. [35] B. Maggio, Prog. Biophys. Mol. Biol. 62 (1994) 55-117. [36] F.F. Davidson, E.A. Dennis, J. Mol. Evol. 31 (1990) 228-238. [37] J.T. Davies, E.K. Rideal, Interfacial Phenomena, 2nd ed., Academic Press, New York, 1963. [38] K.J. Bacon, G.T. Barnes, J. Colloid Interface Sci. 67 (1978) 70-77. [39] F.C. Goodrich, Proc. Int. Congr. Surf. Act., 2nd 1 (1957) 85-91. [40] G. Zografi, R. Verger, G.H. De Haas, Chem. Phys. Lipids 7 (1971) 185-206. [41] R. Verger, G.H. De Haas, Chem. Phys. Lipids 10 (1973) 127-136. [42] D. Blaudez, T. Buffeteau, J.C. Cornut, B. Desbat, N. Escafre, M. Pezolet, J.M. Turlet, Thin Solid Films 242 (1994) 146-150. [43] D. Blaudez, T. Buffeteau, B. Desbat, J. Marie Turlet, Curr. Opin. Colloid Interface Sci. 4 (1999) 265-272. [44] D. Blaudez, J.-M. Turlet, J. Dufourcq, D. Bard, T. Buffeteau, B. Desbat, J. Chem. Soc., Faraday Trans. 92 (1996) 525-530. [45] I. Cornut, B. Desbat, J.M. Turlet, J. Dufourcq, Biophys. J. 70 (1996) 305-312. [46] J. Saccani, T. Buffeteau, B. Desbat, D. Blaudez, Appl. Spectrosc. 57 (2003) 12601265. [47] B. Korchowiec, A. Ben Salem, Y. Corvis, J.-B. Regnouf de Vains, J. Korchowiec, E. Rogalska, J. Phys. Chem. B 111 (2007) 13231-13242. [48] B. Korchowiec, J. Korchowiec, M. Hato, E. Rogalska, Biochim. Biophys. Acta, Biomembr. 1808 (2011) 2466-2476. [49] I. Prigogine, R. Defay, Chemical Thermodynamics, Longmans, Green, and Co., London, 1954. [50] Z. Bilkadi, R.D. Neuman, J. Colloid Interface Sci. 82 (1981) 480-489. [51] V. Point, A. Benarouche, I. Jemel, G. Parsiegla, G. Lambeau, F. Carriere, J.-F. Cavalier, Biochimie 95 (2013) 51-58. [52] Y. Corvis, W. Barzyk, G. Brezesinski, N. Mrabet, M. Badis, S. Hecht, E. Rogalska, Langmuir 22 (2006) 7701-7711. [53] K. Wieclaw, B. Korchowiec, Y. Corvis, J. Korchowiec, H. Guermouche, E. Rogalska, Langmuir 25 (2009) 1417-1426. [54] G. Sautrey, M. Orlof, B. Korchowiec, J.-B. Regnouf de Vains, E. Rogalska, J. Phys. Chem. B 115 (2011) 15002-15012. [55] X. Bi, C.R. Flach, J. Perez-Gil, I. Plasencia, D. Andreu, E. Oliveira, R. Mendelsohn, Biochemistry 41 (2002) 8385-8395. [56] R.A. MacPhail, H.L. Strauss, R.G. Snyder, C.A. Elliger, J. Phys. Chem. 88 (1984) 334-341. [57] R.N. Lewis, R.N. McElhaney, W. Pohle, H.H. Mantsch, Biophys. J. 67 (1994) 23672375. [58] A. Blume, W. Hubner, G. Messner, Biochemistry 27 (1988) 8239-8249. [59] M. Grandbois, B. Desbat, C. Salesse, Biophys. Chem. 88 (2000) 127-135. [60] J.L.R. Arrondo, F.M. Goni, Prog. Biophys. Mol. Biol. 72 (1999) 367-405. [61] D.F. Kennedy, M. Crisma, C. Toniolo, D. Chapman, Biochemistry 30 (1991) 65416548.

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cr

ip t

[62] M. Roeefzaad, T. Kluener, I. Brand, Phys. Chem. Chem. Phys. 11 (2009) 1014010151.

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Page 22 of 32

Figure captions

Fig. 1. Structure of a) ganglioside GM1 and b) DLPC.

Fig. 2. -A isotherms of monolayers formed with pure DLPC, pure GM1 and their binary

ip t

mixtures. Black, red, green, blue, cyan and magenta curves correspond to xGM1 = 0, 0.05, 0.1,

cr

0.15, 0.2 and 1.0, respectively. Subphase: pure water; temperature: 20 °C. Inset: zoom of the -A isotherms. (For interpretation of the references to color in this figure legend, the reader is

an

us

referred to the web version of the article.)

Fig. 3. Miscibility analysis of DLPC-GM1 monolayers based on the isotherms presented in

M

Fig. 2. (A) MMA-xGM1; (B) Gex-xGM1. Π = 10 (■), 15 (●) and 20 mN m-1 (▲). Dotted lines:

ed

additive mixing.

Fig. 4. The rate of PLA2 catalyzed hydrolysis as a function of the composition of DLPC-GM1

ce pt

monolayers. Π = 15 mN m-1.

Fig. 5. PM-IRRAS spectra of pure DLPC monolayer in the 3000–2800 (A) and 1800–1500

1

Ac

cm-1 region (B). PLA2 was injected beneath the monolayer compressed initially to 15 mN m . (A) spectra 1 - 3 were recorded at 15.0, 8.0, and 1.0 mN m-1, respectively; (B) spectra 1 - 5

were recorded at 15.0, 8.0, 5.0, 4.5 and 4.3 mN m-1, respectively.

Fig. 6. PM-IRRAS spectra of a mixed DLPC-GM1 (xGM1 = 0.1) monolayer in the 1800–1500 cm-1 region. PLA2 was injected beneath the monolayer compressed initially to 15 mN m -1. Spectra 1 - 5 were recorded at 14.0, 7.0, 2.5, 1.2 and 0.5 mN m-1, respectively.

21

Page 23 of 32

Fig. 7. The PM-IRRAS spectra in 1800–1500 cm-1 region (ester, amide and carboxyl groups). (A) pure GM1 monolayer spread on pure water. (B) mixed DLPC-GM1 (xGM1 = 0.1) monolayer upon enzymatic hydrolysis of DLPC; spectra 1 - 5 were recorded at 0 (enzyme

Ac

ce pt

ed

M

an

us

cr

ip t

injection), 10, 20, 40 and 90 min, respectively.  = 15 mN m-1.

22

Page 24 of 32

O

O

HO

ip t

Figure 1 revised

N H

O

O O

O

O

D-glucose

O

HO

P

O

O

us

HO O

N+

b)

OH

O

D-galactose

O

O

O

HO

_ O COO

HO

O

HO O

N-acetylneuraminidate (sialic acid)

HN

O

HO

OH

OH

HO

ed

D-galactose

OH

M

N-acetyl-D-galactosamine

NH

an

HO

O

cr

H OH

O

HO

OH

Ac

Fig. 1

ce pt

a)

Page 25 of 32

Figure 2 revised

80 70 60

ip t

-1

 (mN m )

50 40 30

cr

20

0 40

60

80

100

120

140

A (Ų)

160

Ac

ce pt

ed

M

an

Fig. 2

us

10

Page 26 of 32

Figure 3 revised

A

90

)

75

2

80

MMA (Å

85

70 65

0.00

0.05

0.10

0.15

0.20

xGM1

cr

B -20

us

-40 -60 -80

-100 0.00

0.05

an

mix

(J

mol

-1

)

0

G

ip t

60

0.10

0.15

0.20

ed

M

xGM1

Ac

ce pt

Fig. 3

Page 27 of 32

0.03

2

-1

reaction rate (umoles cm min )

Figure 4 revised

0.02

ip t

0.01

0.00 0.0

0.2

0.4

0.6

0.8

1.0

cr

xGM1

Ac

ce pt

ed

M

an

us

Fig. 4

Page 28 of 32

Figure 5 revised

R/R 2x10

-4

as(CH2)

A

R/R 5x10

Amide I

3

2

2

1

1

3000

2950

2900

2850

2800

1800

1750

1700

an

-1

Wavenumber (cm )

as(COO ) Ca

+2

B

cr

4

us

R / R

R/R

5

3

- ...

ip t

(C=O)ester

s(CH2)

B

-4

1650

1600

1550

1500

-1

M

Wavenumber (cm )

Ac

ce pt

ed

Fig. 5

Page 29 of 32

Figure 6 revised

R/R

5x10

-4

Amide I

Amide II

5

ip t

4 3 2

1

1800

1750

1700

1650

1600

1550

1500

-1

an

us

Wavenumber (cm )

cr

R/R

(C=O)ester

Ac

ce pt

ed

M

Fig. 6

Page 30 of 32

Figure 7 revised

R/R 2x10

Amide I

-4

A -

R / R

as(COO ) (C=O)acid

2+

(C=O)ester

B

Amide II

5 4 3 2 1

1800

1750

1700

1650

1600

1550

-1

an

Wavenumber (cm )

1500

cr

- ..

as(COO ) Ca

Amide I

-4

us

R / R

R/R 5x10

ip t

Amide II

Ac

ce pt

ed

M

Fig. 7

Page 31 of 32

Table 1 revised

Table 1 Isotherm parameters at 15 mN m-1 and collapse obtained with films formed on pure water  = 15 mN m-1

Collapse

A (Å2)

Cs-1 (mN m–1)

Acoll (Å2)

coll (mN m–1)

Cs-1 (mN m–1)

0.00 0.05 0.10 0.15 0.20 1.00

73 74 73 72 71 91

61.7 62.1 61.4 60.2 59.8 36.8

51 51 50 49 48 53

46.5 47.6 46.6 46.3 46.2 63.7

114.5 114.7 107.5 103.9 97.6 173.7

Ac

ce pt

ed

M

an

us

cr

ip t

xGM1

Page 32 of 32