Glycolipids from a colloid chemical point of view

Journal of Biotechnology 124 (2006) 284–301

Glycolipids from a colloid chemical point of view P.H. Thiesen a , H. Rosenfeld b,∗ , P. Konidala a , V.M. Garamus b , L. He c , A. Prange b , B. Niemeyer a,b a

Helmut-Schmidt-University/University of the Federal Armed Forces Hamburg, Hamburg, Germany Holstenhofweg 85, D-22043 Hamburg, Germany b GKSS Research Centre, Max-Planck-Str., D-21502 Geesthacht, Germany c University of Queensland, St. Lucia, Brisbane Qld 4072, Australia

Received 18 October 2005; received in revised form 24 February 2006; accepted 29 March 2006

Abstract Glycolipids are a group of compounds with a broad range of applications. Two types of glycolipids (alkylpolyglycosides and gangliosides) were examined with regard to their physicochemical properties. Despite their structural differences, they have in common that they are amphiphilic molecules and able to aggregate to form monolayers, bilayers, micelles, lyothropic mesophases or vesicles. The structures of glycolipid micelles were investigated by different experimental techniques in addition to molecular dynamic simulations. The knowledge of the physicochemical properties of gangliosides enables a better understanding of their biological functions. Structural features were obtained for the monosialogangliosides GM1, GM2 and GT1b from bovine brain by means of mass spectrometry. Further the aggregation behaviour was determined by small-angle neutron and dynamic light scattering experiments. Interaction studies of these compounds were carried out by means of surface plasmon resonance using gangliosides incorporated liposomes. They were used as model membranes that interact with the lectins WGA, RCA and HPA. The interaction of lectins immobilized to a modified silicon surface was investigated by in-situ ellipsometry. © 2006 Elsevier B.V. All rights reserved. Keywords: Glycolipids; Alkylpolyglycosides; Gangliosides; Micelles; Molecular dynamic simulations; Biospecific interaction

1. Introduction Thomas Graham coined the term “colloid” following the greek “kollan” for glue, a protein rich colloidal system (Graham, 1861). In 1914 Wolfgang Ostwald described colloid chemistry as a “world of ∗

Corresponding author. Tel.: +49 4152 87 2831. E-mail address: [email protected] (H. Rosenfeld).

0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.03.032

neglected dimensions” (Ostwald, 1914) and pointed out the importance of particle size and surface chemistry on the properties of colloidal systems. Nowadays colloid chemistry is an inherent part of science interfaced with different fields of chemistry, physics and life sciences. Colloid chemistry describes the properties of systems “implying that the molecules or polymolecular particles dispersed in a medium have at least in one direction a dimension roughly between 1 nm and

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1 ␮m or that in a system discontinuities are found at distances of that order” (Everett, 1972). Such systems are dominated by the surface chemistry and not by the properties of the bulk phase. Background information is given elsewhere (Lagaly et al., 1997; Hunter, 1989). Direct technical applications of surface and colloidal phenomena are in lubrication, foams, emulsions or the use for purification like sugar refining, products manufactured as colloids, surface active materials (soaps and detergents), and in physiological applications such as blood transport, emulsification of nutrient, enzymes and cell membranes (Myers, 1999). Referring to their influence in surface chemistry and their ability to aggregate as association colloids by self-assembling, surfactants are of special interest in colloid chemistry. They are amphiphilic molecules with a chemical structure comprising a hydrophobic portion, usually a long alkyl chain, attached to hydrophilic or water solubility enhancing functional groups. An approach to quantify the correlation of the chemical structure of surfactant molecules with their surface activity is the hydrophil-lipophil balance (HLB) (Griffin, 1949, 1954), the packing parameters (p) and the critical micellization concentration (CMC). Experimentally available parameters to characterize surfactants are the micelle size, shape and aggregation number. Many other structural and kinetic properties (e.g. accessible surface area of the micelle, probability distribution functions of monomer/hydrocarbon tail, radial distribution functions, dynamics of monomers in the micelle as well as internal motion of atoms in the monomers and time correlation functions) which are difficult to measure are accessible readily from the computer simulations. The intra- and inter-molecular motions of monomers and lipids in the colloidal solution are rather complex and have a direct influence on the macroscopic properties. These underlying dynamics between the surfactant molecules at the nanoscale cause the experimental investigations difficult to interpret quantitatively and also qualitatively for the properties of a colloidal system. Numerous theoretical research works have been carried out in the last decade to address these specific properties with the molecular dynamic (MD) simulations. Application of these tools proved very efficient in studying the static and dynamic properties of the surfactant molecules (Tieleman et al., 2000; Marrink et al., 2000; Bogusz et al., 2001).

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Kuhn et al. reported the structural properties of sodium octanoate micelles from the MD simulations while Bruce et al. studied the distribution of counterions and also the behaviour of water surrounding their sodium dodecyl sulfate micelle (Kuhn and Rehage, 1997; Bruce et al., 2002). “Glycolipids are glycosyl derivatives of lipids such as acylglycerols, ceramides and phenols. They are collectively part of a larger family of substances known as glycoconjugates” (Chester, 1998). From the colloid chemical point of view, they are surfactants with a hydrophilic head group, consisting of a carbohydrate structure connected to a lipophilic tail. Two representing groups are selected for this work: The one are synthetic alkyl glucosides, which find applications because of their amphiphilic properties. The others are glycosphingolipids, which have a biological origin. For the physiological important glycolipids, the carbohydrate structure is of special interest, because they may serve as the third code system which can transfer biological information beside nucleic acids and proteins (Gabius, 2000). Alkyl glucosides are glycolipids with a more simple structure (Fig. 1). They were synthesized due to the reaction of a polyfunctional sugar with a hydrophobic alcohol exhibiting an alkyl chain ranging from octyl (C8) up to octadecyl (C18) carbon atoms. They get industrially manufactured in varying compositions, because they provide remarkable properties, which differ in some cases from other non-ionic surfactants (Hill et al., 1997). These properties were exploited e.g. in hard surface cleaners and laundry detergents. They are used for agricultural applications, because they are biodegradable and exhibit low ecotoxicity. Other applications were found in cosmetics and personal care products, because of their dermatological compartibility. The phase and aggregation behaviour of alkyl glucopyranosides and alkyl maltopyranosides have been studied by different authors (Aoudia and Zana, 1998; Dupuy et al., 1997; Zhang et al., 1996). The phase diagram of n-octyl-␤-glucopyranoside in water exhibit micellar, hexagonal, cubic and lamellar phases (Nilsson et al., 1996). Although some thermodynamic properties such as CMC and aggregation number have been determined (Aoudia and Zana, 1998; Antoneli et al., 1994; Frindi and Michels, 1992; Kameyama and Takagi, 1990; Shinoda et al., 1959).

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Fig. 1. Structure of n-octyl-␤-glucopyranoside (OG), n-octyl-␤-maltopyranoside (OM), n-dodecyl-␤-maltopyranoside (DDM).

Utilization of the MD to the n-octyl-␤-d-glucopyranoside systems showed that the micelle shape had been changed over the nanoseconds range, leading the micelle to a less spherical shape (Tieleman et al., 2000). The accessible surface area of an n-octyl␤-glucopyranoside micelle head is high and remains constant in the solution at 0.62 M concentration region studied. The structural properties calculated from the simulations are in the isotropic solution region which is far from the CMC. Moreover, to study the surfactant systems at the CMC will be computationally expensive due to the very large number of water molecules needed to solvate the solute molecules. Nevertheless the accuracy of the specially optimized parameters used in the MD simulations reflects the specific properties calculated which gives satisfactory correlation with the experimental data. In the coming years with the steep increase in the computational hardware power and also faster calculation methods it will be possible to explore the systems in the near CMC-region to understand the real behaviour in the colloidal liquid phase. The increase of system size, length of the simulation and the accurate parameterization of the molecules used in the simulations obtained from other sources like quantum chemical calculations or from the experiments answering many questions of the colloidal systems in the future with greater accuracy and reliability.

Gangliosides are the structurally most versatile glycosphingolipids (structures are listed in Table 1). They feature oligosaccharide chains containing at least one sialic acid residue (Fig. 2). The lipid tail consists of the aminoalcohol sphingosine and a fatty acid attached to it. These fatty acid derivates of shingosine are called ceramides. They are mainly located in brain and nerve tissues. The glyco moiety of these molecules participates in many biological mechanisms such as cell–cell recognition and play a major role in cell growth and differentiation (Hakomori, 2000). Gangliosides serve as surface receptors for viruses and bacteria and are transducers of biological signals (Zhang and Kiechle, 2004). The ganglioside expression of cancer cells changes dramatically (Lloyd and Furukawa, 1998). Based on these biological functions, gangliosides can be used as diagnostic tools and therapeutics for various diseases in humans. The knowledge of the physicochemical properties of natural surfactants enables a better understanding of their applications in biological and environmental processes (von Rybinski, 2001) and the need to study the specific physicochemical and thermodynamic properties of unknown surfactants remains challenging which keeps colloidal chemistry still active to date. The studied glycolipids have in common that they are amphiphilic molecules and able to aggregate to mono-

P.H. Thiesen et al. / Journal of Biotechnology 124 (2006) 284–301 Table 1 Abbreviations and symbols of gangliosides (refer also to Fig. 2) Name/structure

Abbreviation

Ceramide Glucose N-acetylgalactosamin Galactose Sialic acid Trisialoganglioside Neu5Ac␣3Gal␤3GalNAc␤4(Neu5Ac␣8Neu5Ac␣3)Gal␤4GlcCer Monosialoganglioside GalNAc␤4(Neu5Ac␣3)Gal␤4GlcCer Monosialoganglioside Gal␤3GalNAc␤4(Neu5Ac␣8Neu5Ac␣3)Gal␤4GlcCer

Cer (IUPAC, Chester, 1998) Glc (IUPAC, Chester, 1998) GalNAc (IUPAC, Chester, 1998) Gal (IUPAC, Chester, 1998) NeuAc (IUPAC, Chester, 1998) GT1b (Svennerholm, 1963) (IUPAC, Chester, 1998) GM2 (Svennerholm, 1963) (IUPAC, Chester, 1998) GM1 (Svennerholm, 1963) (IUPAC, Chester, 1998)

Fig. 2. Structure of the gangliosides: GM1 (a), GM2 (b), and GT1b (c).

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layers, bilayers, micelles, lyothropic mesophases or vesicles. For a bright range of applications, the surface active properties of glycolipids are promising. From the view point of biotechnology, these amphiphilic properties cause problems in downstream processing. A detailed knowledge of the colloid chemical properties of glycolipids is essential to overcome this obstacle. 2. Materials and methods 2.1. Materials N-octyl-␤-glucopyranoside (purity > 98%) and noctyl-␤-maltopyranoside (purity > 98%) were purchased from Sigma Chemical (St. Louis, MO, USA) and the sialogangliosides GT1b, GM1a and GM2 as well as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were obtained from Sigma (Steinheim, Germany) and used without further purification. The gangliosides were supplied with a purity of 95% as lyophilized powder. D2 O (Deuteration grade > 99.8%) was obtained from Merck (Darmstadt, Germany). Water was cleaned by a high purity water system with a conductivity smaller than 0.05 ␮S. Other materials were of analytical grade. 2.2. Methods 2.2.1. Colloid chemical parameters 2.2.1.1. Electrospray ionization tandem mass spectrometry (ESI-MS/MS). Mass spectra were obtained using an API QStar Pulsar from Applied Biosystems/MDS SCIEX (Darmstadt, Germany) with nano-electrospray ionization technique (ESI) with subsequent tandem quadrupole mass spectrometry (MS/MS) and time of flight (TOF) analyzer. Specific fragmentation pattern were obtained in the negative ion

mode with an ion spray voltage of −1100 V. Gangliosides were detected as intact molecules and dissolved in a methanol:water mixture 8:2 (v:v) at a concentration of 1 mg/ml. The collision energy was set to −90 eV. 2.2.1.2. Light scattering. Dynamic light scattering experiments (Fig. 3) were carried out on a HPPS particle sizer from the company Malvern Instruments (Worcestershire, UK) equipped with a 633 nm heliumneon laser. Detection occurred at a 173◦ angle in glass cuvettes. A latex standard of uniform particle size of 60 nm was measured to evaluate the accuracy and precision of the measurements before the micelle solutions were analyzed. Different concentrations of GM1, GM2 and GT1b were measured with the light scattering technique. The gangliosides were dissolved in methanol. Aliquots of 20 ␮g were filled in glass cuvettes and dried under a nitrogen stream, filtered over a particle filter to prevent from oil droplets of the gas compressor. Samples were picked up with 2 ml water (Millipore) slightly shaken and measured. The concentrations were comparable to the ones that were applied to the SANS experiments. 2.2.1.3. Small-angle neutron scattering (SANS). The SANS experiments were performed on the instrument SANS-1 at the Geesthacht Neutron Facility, GKSS Research Centre, Geesthacht, Germany, as previously described (Stuhrmann et al., 1995). Briefly, four sample-to-detector distances (from 0.7 to 7 m) were employed to cover the range of scattering vectors q (q = 4π sin θ/λ, where 2θ gives the scattering angle ˚ −1 . In and λ is the wavelength) from 0.01 to 0.25 A ˚ all experiments, the neutron wavelength λ was 8.1 A, and a wavelength resolution of 10% (full width at half maximum value) was used. The samples were kept in quartz cells (Hellma, M¨uhlheim, Germany) with a

Fig. 3. Scattering experiments – principle experimental set-up.

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path-length of 1 mm, which were put in a thermostatic sample holder for isothermal conditions. Backgrounds from the solvent and sample cell were subtracted from the raw spectra by conventional procedures which taking into account the transmission of the samples. The two-dimensional isotropic scattering spectra were azimuthally averaged, converted to an absolute scale, and corrected for detector efficiency by using the incoherent scattering spectra of pure water (Wignall and Bates, 1987) which was measured with a 1 mm path-length quartz cell. The smearing induced by the different instrumental set-ups was included in the data analysis. For each instrumental setting the appropriate resolution function was applied to smear the ideal model scattering curves when the model scattering intensity was compared to the measured one using the least-squares method (Pedersen et al., 1990). 2.2.1.4. Computational investigations. The simulations were performed with the CHARMM (Chemistry at HARvard Macromolecular Mechanics) molecular dynamic tool (Brooks et al., 1983). The partially ordered spherical micelle used in the simulation was initially created with the Insight II graphical tool from Accelry’s Inc. (San Diego, USA). The minimized micelle structure was later soaked into the center of the equilibrated Rhombic Dodecahedron (RHDO) water cell, developed independently from the CHARMM scripts (Dixon et al., 2002). Carbohydrate solution forcefield parameters have been used for the n-octyl␤-glucopyranoside (Bogusz et al., 2001; Kuttel et al., 2002) and TIP3P potentials for the water (Durell et al., 1994). Periodic boundary conditions (PBC) were applied to the central cell to mimic the influence of the bulk solvent. The van der Waals nonbonded interac˚ with a smooth switchtions were terminated at 14 A ˚ and a distance depening function starting at 10 A dent dielectric constant. Particle-Mesh Ewald (PME) method was used for the calculation of electrostatic interactions (Essmann et al., 1995). The bond lengths containing hydrogen atoms were fixed with the Shake algorithm, thus a higher time step of 2 fs was used for all of our simulations. Several minimizations have been performed to ensure that the system is at lower potential energy configurations prior to the heating and equilibration stage. Finally, 40 ps of heating stage was carried out by increasing the temperature in small steps from 0 to

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298.15 K with the NPT (constant number, pressure, temperature) ensemble maintained at the reference pressure of 1 atm, pressure piston of 3000 amu and a collision frequency of 25 ps−1 in the system. The heating stage was followed by the equilibration of the system with the reference temperature maintained at 298.15 K using the “Hoover thermostat” (Hoover, 1985). The simulations were performed for 11 and 6 ns time scales with the HP Itanium2 and SGI Octane2 processors. The dynamic trajectories stored during the simulations were analyzed for the structural properties of the micelle in an aqueous environment. 2.2.2. Biospecific interactions 2.2.2.1. Surface plasmon resonance. Interaction experiments were performed with a Biacore 3000 instrument, the data analyses occurred with the BiaEvaluation software. The sensor chip L1 was used for the immobilization of gangliosides incorporated into liposomes. This equipment was obtained from Biacore International (Uppsala, Sweden). The preparation of liposomes was conducted by the extrusion technique (MacDonald et al., 1991). For liposomes preparation 1 mg total lipid was inserted, which was dissolved in chloroform:methanol 2:1 (v:v). 0.2 mg phosphatidylethanolamine (PE) and 0.8 mg phosphatidylcholine (PC) were combined for the reference cell. Gangliosides incorporated liposomens contain 0.2 mg PE 0.4 mg PC and 0.4 mg ganglioside standard. GM2, GM1 and GT1b were used for interaction studies. The lipid mixtures were dried under a N2 stream and picked up with 150 ␮l of 0.3 M sucrose solution. After 1 h incubation 850 ␮l H2 O were added and centrifuged for another hour at 17,500 g. The pellet was picked up with 0.01 M phosphate buffer (PBS), pH 7.2 and 0.15 M NaCl. Extrusion through a 100 nm membrane was done with an extruder of Avestin Europe (Mannheim, Germany). The flow cell 1 was applied as the reference cell to detect unspecific binding. The lectins HPA, RCA and WGA served as interactions partners. Interaction studies were performed in PBS pH 7.2 containing 0.15 M NaCl. The buffer was filtered and degassed before use. A short impulse of Bovine Serum Albumine (BSA) was employed to block free hydrophobic binding sites. The regeneration of the immobilized liposomes was accomplished with a competing sugar solution for the lectins. HPA was detached

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with 0.3 M N-acetyl-galactosamine (GalNAc) solution, WGA with 0.4 M N-acetyl-glucosamine (GluNAc) solution and RCA with 0.2 M lactose solution. The flowrate was set to 30 ␮l/min to minimize mass transport limitations. 2.2.2.2. In situ ellipsometry. The in situ ellipsometric studies were performed with an EL X-02 C – High Precision Ellipsometer (DRE – Dr. Riss Ellipsometerbau GmbH, Ratzeburg, Germany), using the Minsearch Algorithm, based on a very fast error corrected stepper motor (360,000 steps/rotation) and equipped with a HeNe-Laser emitting a wavelength of 632.8 nm. The interaction studies were performed in a cell with anisotropy stabilized windows, a volume of 2 ml and a magnetic stirrer. After a slide was inserted into the cell 1.5 ml PBS (pH 7.2) was added and the baseline was taken. The changes of delta and psi was traced, when 500 ␮l lectin solution was added. The surface film was washed with buffer inside the cell. While still measuring the delta and psi values, 500 ␮l glycoconjugate solution was added, followed by the elution with a sugar inhibiting the activity of the lectin.

3. Results and discussion To get more insight into the colloid chemical behaviour of glycolipids, especially alkyl glycosides and gangliosides, it is necessary to investigate their aggregation properties. For example, the CMC or the shape of micelles depend on the chemical structure. 3.1. Hydrophilic-lipophilic balance (HLB-concept) An approach to appraise the principle properties of surfactants is to calculate the packing parameter and

the hydrophilic-lipophilic balance. The HLB-concept is the bridge between the molecular structure and the amphiphilic properties of the molecules in an aqueous solution. The HLB-concept assigns a dimensionless number between 0 and 20. Surfactants with an HLBvalue of 10 are said to be hydrophilically-lipophilically balanced (Mollet and Grubenmann, 2000). HLBvalue =

MH × 20 M

(1)

where MH is the molar mass of the hydrophilic part and M is the molar mass of the detergent. For the synthetic alkyl glycosides, the structure is well known and the HLB-values are readily to be calculated (Table 2). A problem in calculating the HLB-value of gangliosides extracted from biological material like brain is the alteration in structure. Because of this reason, the molar mass of the concerned molecule parts was determined experimentally. Mass spectrometry techniques were successfully employed for structural elucidation of glycosphingolipids (Adams and Ann, 1993; Costello and Vath, 1990; Levery et al., 2000; Meisen et al., 2004; Metelmann et al., 2000). A single mass spectrometry (MS) scan of the GM1 standard exhibits two major molecule peaks (M-H)− at m/z 1544.7 amu and m/z 1572.7 amu (data not shown). With the product ion scan it is possible to obtain structural information about these molecules (Sullards, 2000). The fragments of these molecule ions exhibit a GM1 fragmentation pattern shown in Fig. 4. The loss of sialic acid ions at m/z 290.0 result in a fragment bearing the mass-to-charge ratio (m/z) at 1253.6 amu. The ion of the ceramide moiety (Y0 ) is designated at m/z 564.4, is corresponding to a composition of a C18:0 fatty acid and a d18:1 sphingosine (Metelmann et al., 2000). m/z 888.5 is the Y2 -NeuAc ion and m/z 726.5 represents the Y1 fragment. The fragmentation pattern of the GM1 ion with the m/z ratio of 1572.7 amu reveals the same car-

Table 2 HLB-values and associated application (D¨orfler, 2002) of glycolipids Sample

HLB-value

O/W-emulsifier

Mediator for aqueous solvents

n-Octyl-␤-glucopyranoside n-Octyl-␤-maltopyranoside n-Dodecyl-␤-maltopyranoside GM1 GM2 GT1b

12.4 15.0 14.1 12.7 11.9 14.8

X X X X X X

X X X X (x) X

Active cleansing agent X X

X

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Fig. 4. Nano electrospray ionization quadrupole time of flight tandem mass spectrometry of monosialoganglioside GM1. Two different molecules can be detected exhibiting the same glycosylation pattern differing in the acyl chain length. Fragmentation pattern of the m/z 1544 molecule-ion in the small picture (a) mentions the names of the fragments according to Costello and Vath (1990) and shows the collision induced fragmentation of the m/z 1572 parent ion (b).

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bohydrate moiety but it contains a larger ceramide ion m/z of 592.5 amu (Y0 ). Analog results were found for GM2 and GT1b standards (data not shown). Indicating that the ganglioside standards contain at least two different molecules which differ in the acyl chain length of the ceramide moiety (C18:0 and C20:0) (Metelmann et al., 2000). It contains two homologous ganglioside species. From the ESI MS/MS data the HLB-value can be calculated and the results are listed in Table 2. The HLB-values for the synthetic alkyl glycosides between 12.4 and 15.0 indicate an application as an emulsifier for oil-in-water emulsions, as a detergent or as a solubilizer. The HLB-values of the gangliosides suggest similar properties. However, they are more difficult to handle in aqueous solutions because of the lipophilic double chain. It is not expected, that gangliosides will be applied as a detergent in the future, but nevertheless, they behave like a detergent or emulsifier in other applications. 3.2. Packing parameter (p) The packing parameter concept makes predictions about the final shape of the aggregate. It implies the structural extensions of the amphiphilic molecules (area of the headgroup and volume resp. length of the lipophilic tail) and assigns the molecule a dimensionless number between 0 and 1. It is calculated with the equation p=

v al

where v is the volume and l is the maximum length of the hydrocarbon chain; a describes the area of the hydrophilic headgroup. Molecules with small packing parameters (<1/3) form spherical micelles, non-spherical micelles will be generated with a packing parameter between 1/3 and 1/2. For larger packing parameters (1/2–1) bilayers are detected. Gangliosides were reported to have a packing parameter in the region of non-spherical micelles, however they are close to 1/2 and therefore highly sensitive to even slight changes in the ganglioside molecule conformation (Cantu et al., 1996; Corti et al., 1988). The heterogeneity of the biological material is proofed with the mass-spectrometry analysis. The used ganglioside standard contains at least two different homo-

logues differing in the acyl chain length of the ceramide portion. 3.3. Critical micelle concentration (CMC) The phase behaviour of n-octyl-␤-glucopyranoside (OG) in water has been investigated by several authors. Some thermodynamic properties such as CMC and aggregation number have been determined (Aoudia and Zana, 1998; Antoneli et al., 1994; Frindi and Michels, 1992; Kameyama and Takagi, 1990; Shinoda et al., 1959). Double chain surfactants like phospholipids normally do not form micelles in aqueous solutions. However, gangliosides are an exception, because they consist also of a bulky head group, which protects the long alkyl chains from the polar solvent. If the ceramide portion is exposed to water, the surrounding water molecules are strictly ordered. The driving force for the micellization is the gain in entropy arising from the loss of these highly ordered water molecules. Consequently the CMC for gangliosides is low and ranges between 10−4 and 10−9 M in literature, depending on the determination method and the purity of the gangliosides (Sonnino et al., 1994). 3.4. Micelle size, shape and aggegation number The combination of SANS and SAXS investigations allows a detailed understanding of the structure of the colloid particles. The investigations of alkylglycosides by small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) are described elsewhere (He et al., 2002). The results are summarized in Table 3. They performed SANS and SAXS experiments to study the micelle structure of n-octyl-␤maltopyranoside at changing temperature and concentration. The micelle size does not increase with increasing concentration up to 188 mM, and the structure of the micelle is not sensitive to temperature from 10 to 50 ◦ C. In H2 O and D2 O, n-octyl-␤-maltopyranoside micelles have similar structures. Comparison of the nonionic glycolipids: n-octyl-␤-maltopyranoside, n-octyl␤-glucopyranoside, and n-dodecyl-␤-maltopyranoside shows, that the hydrophilic force plays an important role in the determination of the micelle structure. Gangliosides have both a large hydrophobic volume and a bulky head group. Thus they form micelles in aqueous solutions. The aggregation behaviour is stud-

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Table 3 Micelle structures of alkylpyranosides (He et al., 2002) Sample

n-Octyl-␤-maltopyranoside n-Dodecyl-␤-maltopyranoside n-Octyl-␤-glucopyranoside

Aggregation number

26 132 90

Shape

Sphere Oblate ellipsoid Cylinder

Micelle size Total micelle

Hydrocarbon core

˚ RT = 23.7 A ˚ RT = 34.4 A ε = 0.59 ˚ RT = 12.7 A

˚ RC = 11.5 A ˚ RC = 28.2 A εC = 0.59 ˚ RT = 8.5 A

ied with dynamic light scattering (DLS) and SANS. With light scattering, diameters of 10.3 ± 1.8 nm for GM1 micelles, and 13.9 ± 2.1 nm for GT1b micelles, and 13.2 ± 1.6 nm for GM2 micelles, respectively are found (Fig. 5). The light scattering experiments reveal that the GT1b monomers form slight larger micelles than GM1 and GM2, even though they have the smallest packing parameter. In general a larger packing parameter result in a larger aggregation number and vice

versa (Corti et al., 1988). Since the chain length is similar in the used gangliosides and GT1b has got the largest headgroup, it has therefore the smallest packing parameter. The concept of the packing parameter is based on geometrical considerations but does not account for the diverse repelling and attractive forces between the multifarious headgroups and tails that may exist between the amphiphilic monomers. The higher sialic acid content in GT1b which will be three-fold charged in solution, causes that the headgroups cannot be packed too tightly because of the repelling negative charges. The radius of gyration (Rg ) is an overall parameter characterizing the size and the shape of aggregates. It was obtained from the analysis of GM1 according to the SANS data by Indirect Fourier Transformation method to Rg = 3.64 ± 0.01 nm with an approximated Dmax of 10 nm for the upper limit of the particle size. The radius of the sphere is calculated to 4.7 nm. Fig. 6 depicts the scattering vectors q plotted against the scattering cross section d(q)/d. The data are in good

Fig. 5. Dynamic light scattering measurements of different solutions of GM1 (a), of GM2 (b) and of GT1b (c).

Fig. 6. Fitted curves of experimental SANS data using the spherical model (continuous line). Radius of the sphere 4.7 nm.

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agreement with the spherical model. Thus the form of the micelles is to be expected spheroidal. The investigated radius of GM1 micelles with SANS experiments is 4.7 nm and is in good agreement to the DLS data which provide a diameter of 10.3 nm of the aggregates. This is also in accordance with the results of Corti et al. who found hydrodynamic radii of 5.9 nm for GM1, 6.6 nm for GM2, and 5.3 nm for GT1b micelles, respectively (Corti et al., 1988). 3.5. Molecular dynamics simulations Recent advances in the MD methods allowed to characterize the properties of the colloidal surfactant systems accurately from the trajectories calculated during the simulation runs. The structural properties like micelle shape, radius of gyration, accessible surface area and radial distribution functions (pair correlation functions) were calculated for the n-octyl-␤glucopyranoside molecule and the results were analyzed. The micelle size and shape had been frequently reported in the literature. The radius of gyration calculated from the simulations ensures that the micelle size is not growing significantly in the solution with respect to the simulation time. An average value of ˚ was calculated for the n-octyl-␤19.4 and 19.8 A glucopyranoside micelle from the 11 and 6 ns dynamic trajectories. Because of its small hydrocarbon tails and fixed geometric areas of the head group, the molecule exerts strong hydration forces from the aqueous solution in order to stabilize the size. The radius of gyration from the simulations is consistent with the small-angle neutron scattering (SANS) experiments (D’Aprano et

al., 1996) and other theoretical work (Bogusz et al., 2001). The SANS experiments measured cylindrical ˚ at a micelles with the gyration radius Rg of about 19 A concentration region very close to the CMC. Previous ˚ for the aggregate MD results report an Rg of 17.6 A size of 75 monomers (Bogusz et al., 2001), which is in good agreement with the present simulations of 92 monomers. However, the micelle grows very quickly near to the CMC, when it covers the surface with enough glucose head groups in consistence with the molecular geometry and free energy requirements of the system. A further increase in the number of monomers won’t increase the micelle size; instead it starts releasing monomers back into the solution in the isotropic concentration region (Fig. 7). Summarizing these results state that the micelles are more favoured to be small and short ranged and are not extending to bilayer or extended rod like shape (e = 1) in the isotropic solution phase (≤2.0 M) (Nilsson et al., 1996). The principal moment of inertia ratios (I1 /I3 , I2 /I3 and I1 /I2 ) confirm the anisotropic shape of an n-octyl␤-glucopyranoside micelle in the aqueous solution (Tieleman et al., 2000; Gao and Wong, 2001). Moments of inertia tensors were calculated from the coordinates of the system and the corresponding eigenvalues are diagonalized to get the values for the three principle moments of inertias. The ratios of these three principal moment of inertias will provide the accurate shape transformations of the aggregate during the dynamic simulations. The average principle moments of inertia ratios obtained from both simulations are 1.4, 1.3, 1.1 and 1.3, 1.2, 1.1, respectively. The lowest ratio of the principal moment of inertias (I1 /I2 ) compared

Fig. 7. Snapshot of the n-octyl-␤-glucopyranoside micelle during the MD simulation at (a) 0 ns (b) 5 ns and (c) 10 ns time scales.

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to the other two ratios indicates that the micelle is a prolate ellipsoid (semi-axes a = b). Similar moment of inertia ratios and the shape change at the nanoseconds range were also observed by Tieleman et al. (2000) for the dodecylphosphocholine (DPC) micelle of 40 monomers. Furthermore, a couple of monomer release from the micellar aggregate has been observed from the dynamic trajectories at frequent intervals of time (Fig. 7). At some instants the released monomers also diffused out of the simulation cell while others reentered into the central cell in the opposite direction by the application of periodic boundary conditions. We anticipate from these observations that the monomer release from the micelle and the interaction between the micelle and the free monomers in the solution are in the time scale of nanoseconds. The monomers staying next to the surface of the micelle might interact with it or rejoin into the aggregate at higher time scales depending on the surface properties and the available free energy of the system. Moreover, these underlying processes/interactions can be further hypothesized to the micelle collisions, occur most probably at a time much longer than nanoseconds (in the range of microseconds) of simulation, which is rather a difficult task to accumulate the trajectories for such large systems with the current state of MD development. The hydrophilic-hydrophobic character and the surface roughness of the aggregated colloidal lipids in the aqueous solution can be quantified by the accessible surface area (ASA) calculations. Lee and Richards introduced this method for studying hydrophobic side chains in the proteins that were preferential buried away from the external solvent (Lee and Richards, ˚ similar to the water 1971). A probe radius of 1.4 A radius is being rolled over the surface of the n-octyl␤-glucopyranoside micelle to calculate the ASA’s in the present work. With an initial increase for few hundred ps time steps at the beginning of the simulation, the ASA of the n-octyl-␤-glucopyranoside heads, tails and the micelle as a whole were constant for both simulations with a mean average value of 12.8, 3.2 and ˚ 2 , respectively (Konidala et al., 2005). This ini16.0 A tial increase is due to the shape transformations of a partially constructed spherical micelle at the start of the MD simulations. Despite some changes in the shape at several infrequent intervals at above 2 ns time scale, the ASA of the n-octyl-␤-glucopyranoside micelle was not affected. The constant ASA over the time states that the

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contribution of the glucose head to the total monomer ASA is high due to the presence of surface roughness and the anisotropic shape of the micelle (Bruce et al., 2002). The roughness of the micelle enables water molecules to enter easily into the micelle surface and interact with the glucose head group atoms. Whereas the contribution of the hydrocarbon tail to the total monomer ASA is comparatively low, because a large portion of these chains were buried away from the bulk solvent thus facing to the direction of the hydrocarbon core (similar to the oil droplet), overall avoiding to contact with water. Nevertheless, this result ensures that the micelle shape fluctuations as a whole occurred at a longer time scale of nanoseconds will not significantly affect the local structure of the aggregated monomers (Tieleman et al., 2000). To understand the specific interactions of n-octyl-␤glucopyranoside with water several radial distribution functions (RDF) were constructed between oxygen, carbon atoms of the n-octyl-␤-glucopyranoside and oxygen atoms of the water molecule (Gao and Wong, 2001). The interaction of oxygen atoms of the glucose head with the water oxygen atoms concludes that the hydroxyl atoms in the n-octyl-␤-glucopyranoside head are primarily involved in the hydrogen bond formation with the water and therefore for hydration of the micelle surface. The appearance of sharp pronounced ˚ (Fig. 8) for these atom types in the peaks at 2.8 A RDF plots are due to a ␤-conformation of the n-octyl␤-glucopyranoside monomer (Fig. 1) (Nilsson et al., 1996) and the overall shape and surface of the micelle. ˚ the number of As seen in Fig. 8 (g(r) ≈ 2 at r = 2.8 A) water molecules near to the n-octyl-␤-glucopyranoside hydroxyl oxygen atoms is about two times higher than the oxygen number in the bulk water. The water molecules are organized onto the micelle surface with a distortion in the water hydrogen bonding network unlike bulk solvent (Bruce et al., 2002). The increasing number of hydrogen bonds with the micelle atoms makes glycolipids more water soluble, which indeed, indirectly facilitates the solubilization and isolation of the membrane proteins. RDF peaks for the acetal and ring oxygen atoms (O1 and O5 in Fig. 1) in the first hydration shell is reduced to almost one third of the magnitude compared to the hydroxyl oxygen atoms (Fig. 8). The steric hindrances caused by the micelle outermost surface atoms lowers the contact of water in the first hydra-

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Fig. 8. Radial distribution functions of hydroxyl (O2, O3, O4, O6), ring (O5), acetal (O1) oxygen atoms, anomeric carbon (C1) atom and the whole hydrocarbon chain with the water oxygen (Ow ) atoms. For the nomenclature of the different atoms refer to Fig. 1.

tion shell for these atom types, where most of these oxygen atoms are facing inwards (away from the bulk water) to the direction of the micellar hydrocarbon core. ˚ under these peaks Integration of the areas within 5.0 A ensures that O1 and O5 atoms are 2.2 times lower in average hydration number than the hydroxyl oxygen atoms (Konidala et al., 2005). The similar behaviour has been also observed for the anomeric carbon atom C1 as shown in the Fig. 8. In addition, the RDF for the whole hydrocarbon chain and water is also shown in the same plot, where all the peaks are diffused meaning that there was no considerable number of water molecules involved in the interaction with the octyl hydrocarbon chains.

4. Biospecific interactions The biospecific interaction studies were focused on selected gangliosides interacting with the lectins wheat germ agglutinin (WGA), Helix pomatia agglutinin (HPA) or Ricinus communis agglutinin (RCA). 4.1. Surface plasmon resonance The immobilized amount of the liposomes that remains stable on the surface of the sensor chip after treatment with 10 ␮l of 50 mM NaOH was 7722 resonance units (RU) for the PE/PC liposomes, 5740 RU for GM2-liposomes, 6083 RU in the case of GM1liposomes, and 4935 RU for GT1b-containing lipo-

somes, respectively. The binding event of gangliosides with lectins, in which parts of the branched carbohydrate moiety of the ganglioside headgroup interacts with the carbohydrate recognition domain (CRD) of the lectin, is a fast reaction. Kinetic data of the interaction cannot be evaluated with the applied technique. This becomes obvious examining the steep slope at the beginning of the binding curve for all investigated interacting molecules. Fig. 9a shows the fast interaction between RCA and GM1 incorporated liposomes, while Fig. 9b depicts the HPA-GM2 interaction and Fig. 9c illustrates the binding characteristics of the lectin WGA with GT1b liposomes. The adsorption isotherms of these interacting compounds are shown in Fig. 9d and constitutes the basis for the calculations of the Kd values. Another feature of the lectingangliosides interaction is that the recognition is highly specific. HPA only binds to GM2 and displays no interaction with GM1 and GT1b. HPA has an affinity to GalNAc and this monosaccharide is not accessible in the GM1 and GT1b molecules. The amount of HPA that remains attached on the GM2 liposomes after the injection-stop can be desorbed with an impulse of 0.3 M GalNAc solution. The binding constant amounts Kd = 1.9 × 10−6 M. RCA binds only to GM1 because it recognizes the terminal galactose residue. A NeuAc residue is attached to the terminal galactose of GT1b molecules and prevents the binding of the galactose residue to RCA. The Kd value of the GM1 – RCA binding is Kd = 2.7 × 10−5 M. Remarkable is the binding of WGA to different gangliosides. WGA provides an

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Fig. 9. Different binding studies with surface plasmon resonance: RCA binding of varying concentrations to GM1 containing liposomes (a). Immobilized GM2 liposomes bind to HPA lectin (b). WGA adsorbs to GT1b incorporated into liposomes (c). Isotherms of the different binding events (d).

affinity to sialic acids, thus it should have an affinity to nearly all gangliosides, which are defined as sialic acid containing glycosphingolipids. But effectively WGA discriminates between different gangliosides. It looks like that the sialic acid residue should protrude to a certain extend, that it is accessible to the WGA carbohydrate recognition domain. That could be the reason for the high affinity to GT1b and low affinity to GM1 and GM2 (Fig. 10). Kd = 9.5 × 10−5 is the dissociation constant for the WGA-GT1b binding.

the buffer. To calculate the thickness of the ganglioside layer, the protein coated surface was defined as the substrate for the calculation. The relative thickness of the protein layer adsorbed and desorbed, followed by the ganglisoside adsorption and specific desorption were displayed in Fig. 11. The data were extrapolated

4.2. In situ ellipsometry Surface plasmon resonance is a very sensitive technique to investigate interactions in general and in our case that of lectins and gangliosides but it is always limited to a metallic or in practice a gold surface which is a prerequisite for the appearance of the surface plasmons. In situ ellipsometry is adaptable to each reflecting surface even silicon wafers or glass plates. The relative thicknesses are calculated with the program ELX-02 (DRE, Ratzeburg, Germany) using a complex refractive index of 1.40 − j0.02 for the protein layer, of 1.49 − j0.00 for the ganglioside layer and 1.33 for

Fig. 10. Binding characteristics of WGA to the gangliosidesliposomes GT1b, GM2 and GM1. The differences in the binding behaviour can be explained due to protrusion of the NeuAc residues and therefore the accessibility to the WGA binding domain.

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Fig. 11. In situ ellipsometric study of the interaction of the lectine WGA with the ganglioside GM1 and GT1b: Scheme of the experimental set-up (a). Adsorption of WGA at a tresyl activated silicon slide (b), washing with buffer (c) adsorption of the gangliosides GM1, and GT1b, respectively (d) and specific desorption with GluNAc-solution, and after the exchange of the sugar solution against buffer (e).

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Table 4 In situ ellipsometric study: relative thickness of the adsorbed and desorbed protein and ganglioside layers Lectin

WGA WGA a b c

Ganglioside

GM1 GT1b

Lectin rel. thickness (nm)

Ganglioside rel. thickness (nm)

Ads.

Des.b

Ads.

Des.

c

3.60 2.45

3.10 1.21

0.93 1.02

0.45 0.77

0.48 0.25

Interactiona rel. thickness (nm) 0.15 0.21

Interaction = specific desorbed Ganglioside/immobilized lectin. The thickness after desorption = immobilized protein. Specific desorbed Ganglioside = (ads. − des.).

to time zero for adsorption and to the end point of desorption, respectively. The relative thickness based on interaction is the amount of adsorbed ganglioside, which can be desorbed specifically with a monosaccharide fitting into the CRD of the lectin – in our case GluNAc. To compare the interactions, it is necessary to take the ratio of the relative thicknesses of the protein adsorption to the interaction (Table 4). The relative interaction between WGA immobilized to a tresylated silica surface and GM1 is 0.30, the value for GT1b is 0.84. The relative interaction can be used to compare qualitatively the interaction between immobilized lectins and gangliosides, but not to calculate the binding constant, because some experimental uncertainty have to be taken into account. First, the use of a refractive index for the different gangliosides is a simplification, which was necessary. Second, because of optical reasons, the laser beam has to pass through the solution and consequently the measurement is only possible for diluted solutions when the impact caused by the adsorption of one component on the refractive index of the solution can be neglected. This is, for example not the case during the washing procedure with GalNAc. Because of this reason, the measurement of the relative thickness of unspecific adsorbed gangliosides has to be performed after exchanging the monosaccharide solution against buffer solution free of sugar. Having in mind the experimental uncertainties, in situ ellipsometry offers the possibility to investigate the influence of very different surfaces on the activity of immobilized lectins.

5. Conclusions Glycolipids are a family of surfactants, offering promising properties for technical and pharmacolog-

ical applications. Glycopyranosides are non-ionic surfactants. They are alternative detergents to commercial applications like O/W-emulsifier, mediator for aqueous solvents or as an active cleaning agent. Gangliosides are anionic surfactants, playing a role in cell differentiation and development and minimal changes in the structure can cause big effects in biology. A mixture of at least two structural homologs was used for the depicted experiments. The colloid chemical behaviour of glycolipids was characterized with different methods, starting with the HLB-concept in combination with the analytical structure determination by tandem mass spectrometry, a method required to obtain the molecular mass of the hydrophilic and hydrophobic part of the ganglioside molecules. But the structure analysis with mass spectrometry reveal the problems that encounter in case of working with biological material. The purity of the compounds has to be considered. The alteration of structure and distribution in living organism increase the variation of the determined data significantly, what is typical for natural substance investigations. An alternative technique to semi-empirically investigate such complex ganglioside-lectin properties can be performed with the molecular dynamics simulations. The expressiveness of such methods was exemplified by the n-octyl-␤-glucopyranoside micelle simulations reported in the present work. In the future also more complex systems like gangliosides and the interaction between surfaces and glycolipids will be accessible by such theoretical methods. Beside the properties of a surfactant, glycolipids like gangliosides can interact specifically with the CRD of lectins. The interaction studies are multifaceted, depending on the aggregation state of the amphiphiles. Interaction studies were performed by surface plasmon resonance. If the influence of different surfaces

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on the activity of immobilized lectins has to be investigated, in situ ellipsometry is the method of choice. To resume, when applying glycolipids in biotechnology the amphiphilic character and the colloid chemical properties have to receive attention.

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