MODIFICATION OF MODEL MEMBRANES UNDER THE INFLUENCE OF OLIGOMERIC LYSOZYME

Kastornaya A.P., Yudintsev A.V., Trusova V.M., Gorbenko G.P.

V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv, 61077, Ukraine

MODIFICATION OF MODEL MEMBRANES UNDER THE INFLUENCE OF OLIGOMERIC LYSOZYME

The correlation between neurodegenerative diseases (Parkinson’s, Alzheimer’s and Huntigton’s diseases), type II diabetes, systemic amyloidosis, etc. and amyloid aggregation in brain tissue has long been established. A growing body of evidence has demonstrated that amyloid protein-membrane interactions may underlie the cytotoxic effects elicited by amyloid proteins. A number of recent studies suggest that amyloid toxicity arises primarily from a soluble oligomeric form of the peptide rather than amyloid monomers or mature fibrils. Membrane-associated mechanisms of amyloid cytotoxicity include membrane depolarization, bilayer destabilization, pore or ion channel formation, and membrane-associated free radical generation [1,2]. However, the membrane effects of mature fibrils so far are poorly understood. In view of this, the present study has been undertaken to ascertain the influence of fibrillar lysozyme on the structure of model membranes (liposomes) composed of phosphatidylcholine (PC) and its mixture with cardiolipin (CL) (5 and 10 mol%) and cholesterol (30 mol%). To this end, fluorescent probe 6-Lauroyl-2-(N,N-dimethylamino)naphthalene (Laurdan), highly sensitive to the environmental polarity, has been employed. The structure of Laurdan molecule and its fluorescence spectra in PC liposomes are shown in Fig. 1. Unilamellar lipid vesicles composed of PC and its mixtures with CL or cholesterol were prepared by the extrusion method. Amyloid fibers of lysozyme were obtained by protein incubation in 80% ethanol under continuous agitation during 30 days.

Quantifying of Laurdan partition coefficient

For quantitative description of Laurdan binding to liposomes of varying composition the results of fluorimetric titration were treated in terms of partition model. The partition coefficient, K_P, is defined as

(

where n_Land n_Ware the molar concentrations of the probe in lipid and water phases respectively, V_L and V_W are the volumes of respective phases.


Fig. 1. Laurdan structure and fluorescence spectra in PC liposomes

Based on fluorescence data K_p can be calculated from equation

(2)

where ΔI – fluorescence intensity change , I_L, I_W – fluorescence intensities in lipid and in water phases, respectively, I_max – maximum fluorescence intensity of the probe in a lipid environment [3]. The recovered in such a manner partition coefficients are presented in Table 1. The results obtained are indicative of rather high Laurdan affinity for lipid bilayers.

Table 1. Partition coefficients of Laurdan in different lipid systems

Liposome composition	K_P	ΔI_max
PC	1.4·10⁴±5.4·10³	1.3·10³±3.1·10²
PC:CL (5%)	2.1·10⁴±9.7·10³	5.8·10²±1.5·10²
PC:CL (10%)	9.2·10³±3.2·10³	7.6·10²±1.9·10²
PC:Chol (30%)	5.6·10³±3.2·10³	1.4·10²±7.0·10²

Analysis of Laurdan fluorescence spectra

Laurdan is an amphiphilic fluorescence probe, whose fluorescence spectra are sensitive to the environmental polarity (hydration level). In the solvents of high polarity, Laurdan shows a considerable shift of its emission spectrum to longer wavelengths due to dipolar relaxation processes. When the local environment of Laurdan is a phospholipid phase, the emission depends on the packing of the lipid chains. At temperatures below the phase transition (gel state) the emission maximum is near 440 nm. At temperatures above the phase transition (liquid crystalline state) the emission maximum is red-shifted to 490 nm. In the lipid bilayer the Laurdan molecule is strongly anchored in the hydrophobic core of the bilayer by hydrophobic interactions between its lauric acid tail and the lipid alkyl tails while its fluorescing moiety is located at the glycerol level of the phospholipid headgroups. The changes in the emission spectrum of Laurdan can be characterized by the generalized polarization value (GP). It has been shown that the GP value decreases when water penetration into the bilayer increases; this is due to the foregoing red shift of the Laurdan fluorescence spectrum caused by dipole–dipole interactions and reorientation of available water molecules in the vicinity of the Laurdan probe in the bilayer [4]. The generalized polarization was calculated according to the equation

(3)

where I_B and I_R are the maximum fluorescence intensities of the blue and red spectral components, respectively. To obtain the value of this parameter, fluorescence intensities at 440 (I_B) and 490 (I_R) nm were used.


Fig. 2. Generalized fluorescence polarization (GP) of Laurdan lipid probe emission in vesicles of different composition as a function of amyloid lyzozyme concentration

Excitation wavelength was 364 nm. The GP of Laurdan in different lipid vesicles as a function of amyloid protein concentration is shown in Fig. 2. As evident from represented data, the GP was negative (about -0.08 – -0.1) in liposomes composed of phosphatidylcholine and its mixture with cardiolipin, while it turned out to attain positive values in the vesicles from PC mixture with cholesterol. This effect could be explained by condensing influence of cholesterol on the lipid bilayer. In all types of liposomes increase of fibrillar lysozyme concentration resulted in the increment of the generalized polarization value. These findings reveal that amyloid fiblills cause decrease of polarity and increase of lipid packing density in the model membranes.

To summarize, the present study provides evidence for modifying effect of mature lysozyme fibrils on the structure of model membranes. Regardless of the membrane composition, fibrillar aggregates of lysozyme brought about reduction of bilayer polarity originated presumably from the increment of lipid packing density. The most pronounced polarity decrease (GP increase by ~ 40 %) were observed for PC/CL liposomes, while in PC/Chol bilayer lysozyme fibrils produced weaker polarity changes (GP increase by ~ 18 %).

This work was supported in part by the grant #4534 from the Science and Technology Center in Ukraine and Fundamental Research State Fund (project number F.28.4/007).

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