Langmuir 1999, 15, 2493-2499
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Cluster Organization of Glycosphingolipid GD1a in Lipid Bilayer Membranes: A Dielectric and Conductometric Study F. De Luca, C. Cametti,* and A. Naglieri Dipartimento di Fisica, Universita` di Roma “La Sapienza”, Piazzale A. Moro 5, I-00185 Rome, Italy, and Istituto Nazionale per la Fisica della Materia (INFM), Unita’ di Roma1, Rome, Italy
F. Bordi Sezione di Fisica Medica, Dipartimento di Medicina Interna, Universita` di Roma “Tor Vergata”, Rome, Italy, and Istituto Nazionale per la Fisica della Materia (INFM), Unita’ di Roma1, Rome, Italy
R. Misasi and M. Sorice Dipartimento di Medicina Sperimentale, Universita` di Roma “La Sapienza”, Rome, Italy Received July 28, 1998. In Final Form: December 15, 1998 The dielectric and conductometric properties of mixed ganglioside/phospholipid vesicle aqueous suspensions were measured in the frequency range from 10 kHz to 10 MHz, where a pronounced dielectric dispersion due to surface bilayer polarization occurs. The concentration of the ganglioside employed, a disialoglycosphingolipid GD1a, was varied from 50 to 500 µg/mL (corresponding to a molar ratio [GD1a]/ [DPPC] from 0.2 × 10-3 to 2 × 10-3) and the excess surface polarizability, attributed to ganglioside organization within the lipid bilayer, was evaluated. The dependence of the ganglioside concentration shows that a clusterization process occurs, giving rise to the formation of large in-phase aggregates whose coordination number, of the order of a few hundred dipoles, depends on temperature. The relevance, from a biological point of view, of a nonuniform distribution of GD1a in a lipid bilayer membrane is briefly discussed.
1. Introduction Gangliosides are sialic acid containing glycosphingolipids located primarily on the outer leaflet of the lipid bilayer of biological membranes.1 They are composed of hydrophilic carbohydrate chains linked to ceramide, which are hydrophobic moieties composed of a sphingoid base and a long-chain fatty acid. Gangliosides are ubiquitous constituents of cell membranes, where they show cell typespecific and differentiation expression patterns.2,3 Functionally, gangliosides act as specific binding sites for bacterial toxins4 and viruses5 and are involved in membrane receptor modulation6,7 and in the control of the cell cycle.8 Moreover, these molecules appear to be relevant in cell-cell interactions, antigen recognition, cell activation, and signal tranduction.9-11 * To whom correspondence should be addressed. (1) Hakomori, S. Biochem. Soc. Trans. 1993, 21, 583-595. (2) Hakomori, S. Annu. Rev. Biochem. 1981, 50, 733-764. (3) Hannun, Y. A.; Bell, R. M. Science 1989, 234, 500-507. (4) Fishman, P. H. J. Membr. Biol. 1982, 69, 85-97. (5) Markwell, M. A.; Moss, J.; Hom, B. E.; Fishman, P. H.; Svennerholm, L. Virology 1986, 155, 356-364. (6) Bremer, E. G.; Hakomori, S.; Bowen-Pope, D. F.; Raines, E.; Ros, R. J. Biol. Chem. 1984, 259, 6818-6825. (7) Bremer, E. G.; Schlessinger, J.; Hakomori, S. J. Biol. Chem. 1986, 261, 2434-2440. (8) Usuki, S.; Hoops, S.; Sweely, C. C. J. Biol. Chem. 1988, 263, 1059510599. (9) Whisler, R. L.; Yates, A. J. J. Immunol. 1980, 125, 2106-2111. (10) Norihisa, Y.; McVicar, D. W.; Ghosh, P.; Houghton, A. N.; Longo, D. L.; Creekmore, S. P.; Blake, T.; Ortaldo, J. R.; Young, H. A. J. Immunol. 1994, 152, 485-495.
Although there is much information available on the chemistry of gangliosides, relatively little is known about their interactions with phospholipids and particularly about their organization and distribution either in synthetic or in biological membranes. For example, it has recently been reported that in A431 cells, a monosialoganglioside, GM1, appeared to be nonuniformly distributed over the plasma membrane, showing a peculiar localization in noncoated invaginations, identified as caveolae by the presence of the VIP-21 protein caveolin.12,13 In our previous work,14 we have investigated the electrical conductivity of lipid bilayers containing, at various concentrations, different gangliosides with the same hydrophobic part but with different lengths of the carbohydrate chains. It is well-known that headgroups of the ganglioside molecules adopt a perpendicular orientation toward the axis of the ceramide chain. This particular orientation of the polar head groups allows lateral interactions with other lipid molecules. Since the conformation and the electrical interactions among the headgroups of ganglioside molecules in the lipid bilayer are related to the presence and to the position of hydrogen donor and acceptor groups, it seems likely that the number and the peculiar position of negatively charged neuraminic (11) Gouy, H.; Debr, P.; Bismuth, G. J. Immunol. 1984, 155, 51605166. (12) Parton, R. G. J. Histochem. Cytochem. 1994, 42, 155-166. (13) Parton, R. G. Curr. Opin. Cell Biol. 1996, 8, 542-548. (14) Cametti, C.; De Luca, F.; Macri’, M. A.; Maraviglia, B.; Misasi, R.; Sorice, M.; Pavan, A.; Garofolo, T.; Pontieri, G. M.; Bordi, F.; Zimatore, G. Colloids Surf. B 1996, 7, 39-46.
10.1021/la9809473 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/11/1999
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acids in these molecules play a crucial role for the overall electrical behavior of the model membrane. In this paper, we have investigated the dielectric and conductometric properties of aqueous vesicle suspensions, built up with ganglioside-phospholipid mixed bilayers, over a frequency range from 10 kHz to 10 MHz, where marked surface polarization effects occur, resulting in welldefined dielectric dispersions. We employed a disialoganglioside, GD1a, a glycosphyngolipid with two sialic acid residues in the polysaccharide polar head, mixed with dipalmitoylphosphatidylcholine (DPPC), at different concentrations, up to 500 µg/mL. The surface polarization excess, over that due to surface organization of phospholipid head groups, has been estimated from the usual dielectric theory of heterogeneous systems based on the Maxwell-Wagner interfacial effect and compared with that derived from the measured dielectric dispersion. These results suggest that ganglioside GD1a does not distribute uniformly in the lipid bilayer, but rather it prefers to clusterize, forming domains of different extensions, depending on temperature and concentration, with a high electrical dipole moment correlation. These findings confirm our previous, but preliminary, studies based on electrical conductivity measurements14 in aqueous multilamellar bilayers, where aggregation and clusterization effects were found. The relevance from a biological point of view of a nonuniform distribution of gangliosides, in particular GD1a, in lipid bilayer membranes is briefly discussed. 2. Experimental Section 2.1. Material and Vesicle Preparation. Dipalmitoylphosphatidylcholine (DPPC) and disialoganglioside GD1a were purchased from Sigma Chem. Co. (St. Louis, MO). Mixtures of appropriate aliquots of DPPC and GD1a were dissolved in chloroform-methanol (2:1, v/v) and then dried under high vacuum by rotatory evaporation in order to remove residuals of organic solvents. The dried GD1a/DPPC mixtures at different ganglioside concentrations (from 0 to 500 µg/mL, corresponding to a molar ratio varied from 0 to 2 × 10-3) were suspended in lowconductivity deionized water and heated at a temperature above the main transition temperature (Tc ) 42 °C) for 2 h. The lipid concentration in the different suspensions investigated was kept constant to the value of 10% wt/wt throughout the experiments. Unilamellar vesicles were produced by extrusion technique (Lipex Biomembranes Extruder, Canada) employing two stacked polycarbonate filters with 0.1 µm pore size (Nucleopore Corp. Pleasanton, CA). The extrusion process was performed at a temperature above the main transition temperature in order to get solution of low viscosity and was repeated for a total of 10 passes. 2.2. Dynamic Light Scattering Characterization. Vesicle size and size distribution of unilamellar vesicles was determined by dynamic light scattering measurements15 performed utilizing a 128-channel digital correlator with a 10 mW helium-neon laser at a wavelength of 632.8 nm. The process is characterized by the correlation function of the scattered intensity G(τ) defined as
G(τ) ) 〈I(τ)I(t + τ)〉
(1)
For particles of the same size, the time-dependent part of G(τ) is a single exponential with a time constant τc ) 1/(q2D), where D is the translational diffusion coefficient and q is the magnitude of the momentum transfer. In the limit of very diluted samples, the hydrodynamic radius RH of the particles is given by the Stokes-Einstein relation (15) Pecora, R. Dynamic light scattering. Applications to photon correlation spectroscopy; Plenum Press: New York, 1985.
RH ) KBT/6πηD
(2)
where KBT is the thermal energy and η the viscosity of the solvent. For all the samples investigated, at the different ganglioside concentrations employed, the average hydrodynamic radius of the vesicles is RH ) 850 nm. The polydispersity index, defined as (σRH/RH) ) K21/2/K1, where K1 and K2 are the first and second cumulant of the expansion of G(τ), assumes a value of about 0.15. Figure 1 shows a typical correlation function of GD1a/DPPC mixed vesicles in aqueous suspension at the temperature of 20 °C and the hydrodynamic radius RH resulting from the first cumulant of the expansion. As can be seen, its value remains constant over long period of time, without the presence of aggregation processes. 2.3. Dielectric and Conductivity Measurements. The dielectric measurements were carried out in the frequency range from 10 kHz to 10 MHz by means of a Hewlett-Packard lowfrequency impedance analyzer model HP 4192A, in the temperature interval from 5 to 50 °C. The conductivity cell consists of a coaxial line terminated by a sample-filled circular waveguide excited below its cutoff frequency. The cell constants were determined by means of appropriate calibration measurements of standard liquids of known dielectric constant and conductivity. The overall accuracy of the whole experimental setup is better than 5% in the permittivity ′ and 0.1% in the electrical conductivity σ.
3. Theoretical Background 3.1. Dielectric Relaxation of a Heterogeneous System (a Triphasic Mixture Suspension). The dielectric properties of the vesicle suspension investigated can be described in the light of the Maxwell-Wagner theory of heterogeneous systems16 developed for spherical particles uniformly dispersed in a continuous phase in a complete form by Pauly and Schawn.17 For a triphasic system composed of water core particles covered by a thin phospholipid bilayer and dispersed in an aqueous continuous medium, the interfacial polarization theory predicts a complex spectrum, resulting from two different relaxation regions. In the general case, the complex dielectric constant of the vesicle suspension *(ω) can be written as
*(ω) ) /m(ω)
A + iωE + (iω)2B C + iωF + (iω)2D
(3)
where A, B, C, D, E, and F are appropriate quantities given in ref 17 depending on the dielectric properties of the core particle (*p(ω) ) p + σp/(i0ω)), the surface phospholipid bilayer (*s(ω) ) s + σs/(i0ω)), and the aqueous continuous medium (*m(ω) ) m + σm/(i0ω)), besides geometrical and composition factors (the radius R of the particle, the thickness δ of the lipid bilayer, and the fractional volume φ of the dispersed phase). Here, is the permittivity and σ the electrical conductivity. Finally, 0 is the dielectric constant of free space and ω the angular frequency of the applied field. The frequency dependence of the dielectric constant *(ω) given in eq 3 can be equivalently written as the sopraposition of two Debye-type relaxation functions
*(ω) ) ∞ +
(0) - i i - ∞ σ0 + + 1 + iωτ1 1 + iωτ2 iω0
(4)
where ∆1 ) (0) - i and ∆2 ) i - ∞ are the dielectric increments of the dispersions characterized by relaxation (16) Takashima, S. Electrical properties of biopolymers and membranes; Adam Hilger: Bristol, UK, 1989. (17) Pauly, H.; Schwan, H. P. Z. Naturforsch. 1959, 14b, 125-131.
Glycosphingolipid GD1a in Lipid Bilayer Membranes
Langmuir, Vol. 15, No. 7, 1999 2495
observable in the whole frequency range investigated. The particular structure of the heterogeneous system studied, where the lipid bilayer disjoins the same aqueous phase, makes the dielectric spectrum simpler and allows the surface polarization effects in the lipid bilayer directly observable. Finally, the dc electrical conductivity σ0, with the same above assumptions, is given by
σ0 ) σm
Figure 1. (A) Correlation function C(τ) as a function of the correlation time τ of GD1a/DPPC mixed vesicles in aqueous suspension at the temperature of 20 °C. The ganglioside concentration is 50 µg/mL. The inset shows the deviation from a single-exponential decay correlation function. The polydispersity can be evaluated to be (σRH/RH) ) K21/2/K1 ) 0.15. (B) Hydrodynamic radius RH as a function of time of GD1a/DPPC mixed vesicles, calculated from the first term of the expansion of the correlation function C(τ).
times τ1 and τ2 (with τ2 e τ1 and hence with relaxation frequencies ω1 e ω2), respectively, σ0 is the low-frequency electrical conductivity, and ∞ the high-frequency limit of the permittivity ′(ω). Equating eq 4 to eq 3 yields, after somewhat cumbersome calculation, the full description of the dielectric behavior of a triphasic system in terms of the dielectric parameters ∆1, ∆2, τ1, τ2, and σ0. For a unilamellar vesicle suspension, such as that investigated in this work, some simplification are possible and in particular we have assumed, (i) the electrical conductivity of the water core equals that of the external medium, σp ) σm; (ii) the electrical conductivity σs of the lipid bilayer is negligible compared to that of the aqueous phase, σs , σm; (iii) the thickness δ of the lipid bilayer is very small in comparison with the particle radius R, δ/R , 1. Under the above assumptions, the dielectric dispersion at the higher frequency is characterized by a relaxation time
m τ2 ) 0 σm
(5)
depending only on the bulk properties of the aqueous phase and by a dielectric increment ∆2 ) i - ∞ approaching zero. The first dielectric dispersion (occurring at the lower frequency) is characterized by the following parameters
τ1 ) ∆1 ) s - i )
0s
(6)
(2 + φ)δ/Rσm 9φs (2 + φ)2δ/R
-
3φ (2 + φ) m
(7)
and represents the unique relaxation mechanism directly
3σs + 2(1 - φ)δ/Rσm 3σs + (2 + φ)δ/Rσm
(8)
3.2. Contribution to the Surface Polarizability Due to the Lipid Bilayer Structure. Equations 6-8 hold for a suspension composed of homogeneous media, each of them characterized by an appropriate dielectric constant. In the present case, however, the structure of the interface between the aqueous components is rather complex, owing to the presence of a polar head group layer due to the phospholipid component and to the ganglioside molecules with large hydrophilic regions inserted within it. In order to take into account the contribution to the interfacial polarizability due to the presence of such a structured hydrophilic layer, we will follow the model proposed by Kaatze et al., Henze and Pottel et al.18-21 According to the basic assumptions of this model, the lipid-ganglioside interface contributes with two different effects, the first due to the presence of lipid domains with high orientational correlation and originated by diffuse thermal rotational motion of the zwitterionic phosphorylcholine groups21 and the second one due to the alignment of ganglioside polar head groups, enhanced by cooperative motion, giving rise to the formation of large inphase aggregates. These effects should, in principle, occur also at the inner phospholipid bilayer interface since, in model membrane systems, i.e., unilamellar vesicles, it has been shown that gangliosides distribute between the two monolayer surfaces.22,23 However, owing to the depolarizing electric field, the resulting dipole moment is small and thus its contribution to the observed dielectric dispersion will be neglected in the following analysis. Each of the above contributions produces, under the influence of an external electrical field, a surface polarization characterized by different relaxation times. Assuming these mechanisms independent each other, the whole electrical frequency-dependent surface polarizability R*(ω) can be written as
R*(ω) ) R∞ +
∆RL ∆RG + 1 + iωτL 1 + iωτG
(9)
where ∆RL and ∆RG are the relaxation strength associated to the phospholipid rotational motion and ganglioside correlation, with τL and τG their relaxation times, respectively, and R∞ the high-frequency limit contribution. Since we are interested in the effects induced by the ganglioside organization in the lipid bilayers, we will address our attention to the relaxation strength ∆RG and the relaxation time τG of the ganglioside polarization. (18) Kaatze, U.; Muller, S. C.; Eibl, H. Chem. Phys. Lipids 1980, 27, 263-280. (19) Kaatze, U.; Henze, R.; Pottel, R. Chem. Phys. Lipids 1980, 25, 149-177. (20) Henze, R. Chem. Phys. Lipids 1979, 27, 165-177. (21) Pottel, R.; Gopel, K. D.; Henze, R.; Kaatze, U.; Uhlendorf, V. Biophys. Chem. 1984, 19, 233-244. (22) Maggio, B.; Montich, G. G.; Cumar, F. A. Chem. Phys. Lipids 1988, 46, 137-146. (23) Cestaro, B.; Barenholz, Y.; Gatt, S. Biochemistry 1980, 19, 615619.
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According to the model proposed by Kaatze et al., and Henze and Pottel et al.,18-21 the anionic groups of the ganglioside hydrophilic regions are replaced by moveable charges undergoing diffusive correlated motions relative to the bilayer surface, resulting in a surface polarization described by the parameters
∆RG ) gn(eξ)2/2KBT
(10)
τG ) ξ2/uKBT
(11)
where g represents the orientational correlation factor in the ganglioside cluster (the number of neighboring head groups with the same orientation), n the mean surface number density of ganglioside molecules in the lipid bilayer, ξ the distance over which charge displacement occurs, u the mobility of the anionic groups, e the electronic charge, and KBT the thermal energy. Transforming the vesicle surface polarization due to the above-stated mechanisms into a volume polarizability of the bilayer homogeneous medium allows to completely describe the dielectric behavior of the system; i.e., the vesicle bilayer with polarizable surface layer can be replaced by a spherically shaped homogeneous layer of complex dielectric constant
*s(ω) ) s +
4πR*(ω) 3R
(12)
where R is the mean radius of the bilayer aggregates. Substituting eq 12 into eq 3 gives the complete frequency dependence of the complex dielectric constant *(ω). Owing to the frequency dependence of the above polarization contributions occurring in the lipid bilayer, the whole behavior of the vesicle suspension results in very complex expressions. However, if we consider only the excess dielectric increment due to the presence of gangliosides, over that of the ganglioside-free vesicle suspension, these expressions are markedly simplified and the total dielectric increment ∆ attributable to ganglioside correlation can be written, to a first approximation, as
(
9φ s + ∆ )
)
4πng(eξ)2 6RKBT 2
(2 + φ) δ/R
- m
3φ (2 + φ)
(13)
This expression contains the characteristic parameters of the model, i.e., the correlation factor g, the mobility u, and the distance ξ, that can be evaluated from the experimental spectra. 4. Results and Discussion In the frequency range investigated, the vesicle suspensions display complex dielectric spectra, resulting from the sovraposition of a very marked dispersion (with dielectric increments of the order of 102-103) due to the electrode polarization effect and a generally lower dispersion (with dielectric increment of the order of 10-50) due to molecular polarization of the lipid-water interface and/ or to particular orientational processes involving the polar head group components. Other relaxation processes generally observed in heterogeneous systems such those investigated here fall in a higher frequency range and will be left out in the present study. A typical dielectric spectrum in the frequency range from 10 kHz to 10 MHz of GD1a/DPPC vesicle aqueous suspensions is shown in Figure 2.
Figure 2. A typical dielectric spectrum of GD1a/DPPC mixed vesicles in aqueous suspension as a function of frequency in the interval from 104 to 107 Hz: the permittivity ′(ω) (A), the dielectric loss ′′(ω) (B), and the Cole-Cole plot (C), resulting from the overlapping of the electrode polarization dispersion (at lower frequencies) and vesicle surface polarization (at higher frequencies). The full lines represent the calculated values on the basis of eq 14, taking into account the contribution due to the electrode polarization.
Curve fitting based on a nonolinear least-squares fitting procedure was done using a Cole-Cole relaxation function to which an electrode polarization dispersion obeying a power law is added, according to the expression
*(ω) ) ′(ω) + i′′(ω) ) Aω-m + ∞ +
∆ + 1 + (iωτ)R σ0 (14) iω0
where ∆ and τ are the dielectric increment and the relaxation time of the main relaxation process, respectively, σ0 is the dc electrical conductivity, ∞ is the highfrequency limit of the permittivity, and ω is the angular frequency. As already stated, the steep rise of ′(ω) at frequencies below 105 Hz was attributed to an electrode polarization
Glycosphingolipid GD1a in Lipid Bilayer Membranes
Figure 3. Dielectric increment ∆ ) s - ∞ of vesicle aqueous suspensions as a function of ganglioside concentration, at some selected temperatures: (3) T ) 10 °C; (4) T ) 20 °C; (]) T ) 30 °C; (0) T ) 40 °C; (O) T ) 50 °C. The full line represents the value of the dielectric increment due to the polarization of the lipid bilayer, in absence of ganglioside. The dotted lines serve to guide the eye, only.
Figure 4. Relaxation frequency ν ) 1/(2πτ) of vesicle aqueous suspensions as a function of ganglioside concentration, at some selected temperatures: (3) T ) 10 °C; (4) T ) 20 °C; (]) T ) 30 °C; (0) T ) 40 °C; (O) T ) 50 °C. The dotted lines serve to guide the eye only.
type dispersion and the parameters A and m characterize its power-law frequency dependence.24,25 Since the dielectric relaxation of water occurs at frequencies well above 10 MHz, the term ∞ in eq 14 reflects contributions upon placing vesicles in aqueous phase and finally, the last term in eq 14 takes into account ohmic contributions to the total imaginary part of the complex dielectric constant. The strong reduction of the highfrequency permittivity ∞ compared with the value of the pure water (∞/H2O of the order of 0.8) cannot be explained by a simple dilution of the solvent (fractional volume of the dispersed phase) and must be attributed to an internal depolarizing field effect. These effects are particularly relevant when a zwitterionic head group interface disjoins nonpolar material (the bilayer core) and highly polar material (the external aqueous solution), giving rise to heterogeneous lipid particle suspension. Figures 3 and 4 show the dielectric parameters (the dielectric increment ∆ and the relaxation time τ) of the GD1a/DPPC mixed vesicles in aqueous suspension as a function of ganglioside concentration derived from the fitting of eq 14 by means of a nonlinear least-squares minimization to the experimental data. Figure 5 shows the relaxation time spread parameter R as a function of (24) Schwan, H. P. Determination of biological impedance. In Physical Techniques in Biological Research; Nastuk, W. L., Ed.; Academic Press: New York, 1963; pp 323-407. (25) Asami, K.; Irimajiri, A. Biochim. Biophys. Acta 1984, 778, 570578.
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Figure 5. Relaxation time spread parameter R of vesicle aqueous suspensions as a function of temperature, at different ganglioside concentrations: (O) 50 µg/mL; (0) 100 µg/mL; (]) 150 µg/mL; (4) 200 µg/mL; (3) 300 µg/mL; (2) 400 µg/mL; ([) 500 µg/mL. These concentrations correspond to a [GD1a]/ [DPPC] molar ratio fromn 0.2 × 10-3 to 2 × 10-3.
temperature for the various ganglioside concentrations investigated. 4.1. Dielectric Behavior. As can be seen in Figure 3, above a dielectric increment of about 20 dielectric units, due to surface lipid bilayer polarization, a ganglioside concentration-dependent dielectric dispersion appears, of the order of few ten dielectric units, whose maximum occurs at about a ganglioside concentration of 200 µg/mL, corresponding to a molar ratio concentration of about [GD1a]/[DPPC] ) 8 × 10-4. This effect is attributed to an excess surface polarizability caused by a surface ganglioside organization, and a comparison of the observed excess dielectric increment with that predicted by eq 13 allows the correlation factor g to be estimated and hence the size of the ganglioside domains. In order to carry out this analysis, the knowledge of some parameters concerning the structure of the lipid interface is required. The fractional volume φ of the vesicles can be calculated according to the expression
φ)C
VLNR MLδ
(15)
where VL and ML are the molecular volume and the molecular weight of DPPC, N is the Avogadro number, and C is the DPPC concentration expressed in mass per unit volume of suspension. By assuming a bilayer thickness δ of about 62 Å and a volume VL ) 404 Å3,26 at a lipid concentration of C ) 0.1 g/mL as that employed in this work, the fractional volume is φ ) 0.143, sufficiently small to neglect vesicle-vesicle interaction and to allow the applicability of the Maxwell-Wagner interfacial polarization theory.16 The mean surface number density of ganglioside molecules inserted in the lipid bilayer, assuming that no monodisperse molecules or micellar aggregates are present in the aqueous phase, is given by
n)
δML mG VLMG mL
(16)
where MG is the molecular weight of GD1a and the ratio mG/mL gives the ganglioside concentration expressed in mass per unit mass of DPPC. This quantity has been varied from 3 × 1011 to 3 × 1012 ganglioside per unit surface of a vesicle (corresponding to changes in the ganglioside concentration from 50 to 500 µg/mL). (26) Cunningham, B. A.; Shimotake, J. E.; Tamura-Lis, W.; Mastran, T.; Kwok, W. M.; Kaufman, J. W.; Lis, L. J. Chem. Phys. Lipids 1986, 39, 135-143.
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Figure 7. Electrical conductivity σ of vesicle aqueous suspension as a function of ganglioside concentration, at some selected temperatures: (O) T ) 5 °C; (0) T ) 10 °C; (3) T ) 15 °C; (]) T ) 20 °C; (4) T ) 25 °C; (b) T ) 30 °C; (9) T ) 35 °C; ([) T ) 40 °C; (2) T ) 45 °C; (1) T ) 50 °C. The dotted lines serve to guide the eye only.
Figure 6. (A) Correlation factor g as a function of ganglioside concentration for GD1a/DPPC mixed vesicle aqueous suspensions, at two different temperatures. The values are calculated on the basis of the excess surface polarizability due to ganglioside organization, assuming a displacement ξ ) 10 Å. (B) The mobility u as a function of ganglioside concentration, for GD1a/ DPPC mixed vesicle aqueous suspensions. The values are calculated on the basis of the excess surface polarizability due to ganglioside organization, assuming a displacement ξ ) 10 Å. The data concerning the relaxation times (Figure 4) do not allow a temperature dependence to be properly evaluated.
Fitting eq 13 to the measured excess dielectric increment yields the values of the model parameter g(ξ)2 related to the cooperativity factor g through the distance ξ. Similarly, eq 11 furnishes the value of the ratio u/ξ2 between the mobility u and the displacement ξ. The diffusion path length ξ of the anionic groups is not known so that the correlation factor g and the mobility u cannot be explicitly calculated. However, if ξ is assumed to be at most 10 Å, the minimum values of the correlation factor g and the maximum values of the mobility u can be properly evaluated. Their dependence on the ganglioside concentration is shown in Figure 6. As can be seen, large ganglioside clusters form, at concentrations between 100 and 200 µg/mL (molar ratio concentration between 0.5 × 10-4 and 1 × 10-4), depending on temperature.The correlation factor g varies from about g ) 100 at the lowest temperature investigated to about g ) 230 at T ) 50 °C and correspondingly the ganglioside concentration decreases from about 200 µg/mL to about 100 µg/mL, indicating that temperature favors more extended clusters. Our findings strongly suggest that, depending on temperature and concentration, gangliosides may exist in ganglioside-rich and/or ganglioside-poor domains of variable number and size. The mechanisms for domain formation are largely unknown, but domains of relatively small size (of the order of 10-20 nm) as those evidenced in these systems can form readily because of very small differences in energies of interaction between gangliosides and phospholipids, the process being favored at higher temperature, owing to the increased ganglioside mobility in the lipid bilayer. The presence of microdomains in cell surface membranes is biologically interesting since this effect could be one of the most important issues in order
to understand membrane organization and their structure-function relationships. Microdomains have been detected in cell membranes and model systems using various techniques and particularly glycosphingolipids are likely to form specialized microdomains.27,28 More recently, the existence of monoganglioside-enriched domains in biological membranes has been strongly supported by Vie’ et al.29 on the basis of atomic force microscopy measurements on 1:1 dioleoylphosphatidylcholine (DPOC)/ dipalmitoylphosphatidylcholine (DPPC) films. A more detailed analysis concerning the size cluster distribution and their mean distance will be carried out next. At concentrations higher than 300 µg/mL, the excess surface polarizability is progressively decreased and this clusterization process tends to disappear and gangliosides appear to be more uniformly distributed in the lipid matrix. These findings may have important implications from a biological point of view, taking into account that gangliosides could form balanced hydrophobic-hydrophilic pores and thus modulate the membrane permeability. 4.2. Low-Frequency Conductivity Behavior. The low-frequency conductivity behavior of the whole vesicle suspension as a function of ganglioside concentration is shown in Figure 7, at some selected temperatures from 5 to 50 °C. Also in this case, a maximum in the conductivity σ appears, in correspondence to a ganglioside concentration of about 200-300 µg/mL. Similar results have been reported in an our previous work.14 Within the interfacial polarization theory, eq 8 should describe the low-frequency conductivity behavior of GD1a/DPPC mixed vesicle suspension. However, since an anionic charged ganglioside has been incorporated into the uncharged DPPC bilayer, the vesicle surface becomes negatively charged and in the absence of excess counterions a contribution due to surface conductivity must be properly considered. The alteration of the outer surface structure of vesicles induced by insertion of GD1a molecules could be taken into account by introducing a surface conductance term λs, similar to what is generally done to describe the electrical double layer properties of charged colloidal particles in aqueous suspensions. Within this framework, eq 8 should be replaced by (27) Simon, K.; van Meer, G. Biochemistry 1980, 81, 234-248. (28) Brown, D.; Rose, J. K. Cell 1992, 27, 6197-6202. (29) Vie’, V.; van Mau, N.; Lesniewska, E.; Goudonnet, J. P.; Heitz, F.; Le Grimellec, C. Langmuir 1998, 14, 4574-4583.
Glycosphingolipid GD1a in Lipid Bilayer Membranes
( (
) )
σs + Ks + 2(1 - φ)δ/R σm σ0 ) σm σs 3 + Ks + 2(1 + φ)δ/R σm 3
(17)
where a dimensionless surface conductivity parameter Ks ) λs/σmR has been introduced. For the system investigated, at a volume fraction φ ) 0.141, eq 17 implies that the suspension conductivity would vary within the interval 0.82 e σ/σm e 1, whereas the observed changes in the conductivity, as can be seen in Figure 7, are well above these boundaries. This means that a single surface conductance term added to the bilayer conductance is, in this case, inappropriate to give a full description of the conductivity behavior. Then the question arises how to relate the surface conductance contribution to the characteristic of the GD1a/DPPC mixed bilayer. This can be done according to the theory developed by O’Brien30 for the electrical conductivity of dilute dispersions of spherical particles with relatively thin ionic double layer, where the low-frequency limit of the electrical conductivity, in a first approximation and in the limit of small fractional volume φ, can be written as30
(
σ0 ) σm + 3φσm -
3Ks 1 + 2 2(1 + Ks)
)
Langmuir, Vol. 15, No. 7, 1999 2499
(18)
The first term between parentheses reflects the decrease of the overall conductivity upon placing low-conducting shelled vesicles in aqueous solution and the second term its increase associated with surface conductance. In this case, eq 18 is able to account for the observed conductivity behavior over the whole ganglioside concentration interval investigated. The surface conductivity λs, as a function of temperature, derived from the comparison with the experimental data, is shown in Figure 8, for different ganglioside concentrations. This parameter, that at a given concentration increases monotonously with temperature, shows, as a function of ganglioside concentration, the presence of a maximum at about 200-300 µg/mL, indicating that, above this concentration, the ganglioside organization results in a reduced cooperative clusterization, favoring a more molecular dispersion of ganglioside in the lipid bilayer. The ganglioside concentration at which the maximum occurs is in agreement with that deduced from the excess dielectric increment and that corresponding to the maximum in the relaxation frequency. 5. Conclusions Since glycosphingolipids are generally present in biological cell membranes at low concentration, it would seem reasonable that these molecules would be uniformly distributed in the lipid matrix. On the contrary, there is some experimental evidence that these molecules might cluster in the lipid bilayer owing to their capability to form a large number of interlipid hydrogen bonds, especially if the anionic sialic residues are sufficiently shielded. The significance of these features has been the subject of considerably speculation.31 (30) O’Brien, R. W. J. Colloid Interface Sci. 1980, 68, 533-541.
Figure 8. Surface conductance λs deduced from the comparison of eq 18 with the observed electrical conductivity, for the different ganglioside concentrations studied: (O) 50 µg/mL; (0) 100 µg/mL; (]) 150 µg/mL; (3) 200 µg/mL; (4) 300 µg/mL. The inset shows the surface conductance λs for the two higher concentrations investigated: (O) 400 µg/mL; (0) 500 µg/mL. The above-quoted concentrations correspond to a [GD1a]/ [DPPC] molar ratio from 0.2 × 10-3 to 2 × 10-3.
The present work, on the basis of dielectric spectroscopy measurements, provides further evidence to this picture and suggests that GD1a molecules, despite their ionic character, self-associate in cluster, depending on the concentration and temperature. At temperatures close to the main transition temperature Tc, cluster size increases. The biological significance of this ganglioside clustering might be relevant, since these structures might have a role as recognition sites that mediate the interaction of cells with their surroundings. The intermolecular arrangements of GD1a, characterized by closer than expected molecular packing and by the formation of clusterization, can induce membrane fusion,32 give rise to modification in the ion permeability, even in view of the considerable effect that gangliosides have on the water structure,33 or finally lead to changes in the functional membrane properties involved in different processes such as neurotransmitter release, cellular transformation, modulation of cell growth, and various cell-associated functions mediated by gangliosides in the plasma membrane.34 Dielectric spectroscopy measurements offer a powerful tool to study ganglioside clusterization in model membrane and to investigate a variety of membrane-mediated processes where gangliosides, in view of their organization, could acquire properties capable of conferring to the whole system particular physicochemical behavior. These investigations will be extended to a series of different glycosphingolipids (closely related chemically) with the some hydrophobic portion but with different polar head groups to study the interfacial conformation and their molecular packing in lipid bilayers. LA9809473 (31) Curatolo, W. Biochim. Biophys. Acta 1987, 906, 111-136. (32) Lucy, J. A. In Membrane fusion; Poste, G., Nicolson, G. L., Eds.; Elsevier: The Netherlands, 1978, pp 267-304. (33) Curatolo, W.; Small, D. M.; Shipley, G. G. Biochim. Biophys. Acta 1977, 468, 11-20. (34) Tettamanti, G.; Riboni, L. Adv. Lipid Res. 1993, 25, 235-267.
