6. Survey of studies of monolayers containing glycolipids

#### 6.1. Gangliosides

4.7. Infrared spectroscopy

224 Cell Culture

ATR-FTIR spectroscopy.

thermodynamics.

Infrared Spectroscopy is another important technique widely used to characterize the conformation and orientation of monolayers transferred onto the solid support or in situ at the airwater interface. It is desirable to carry out the experiments in situ to avoid problems due to artifacts during transfer. Infrared reflection absorption spectroscopy (IRRAS) and polarization modulated (PM)-IRRAS are two versions of Fourier transform infrared spectroscopy (FTIR)

In IRRAS, the sample is irradiated with an IR beam and the intensity of the reflected beam is measured as a function of wavelength. The measurements can be carried out with p- and spolarized light at various angles of incidence above or below the Brewster angle. If the samples are on metal substrates, a "surface selection" rule is followed which states that only vibrational dipole moments oriented perpendicular to the substrate are observed. IRRAS has a disadvantage while studying Langmuir films, the strong absorption of water vapor conceals the spectral regions with desired molecular information. PM-IRRAS, which is insensitive to IR absorption of water vapor, was introduced to avoid such problems. In PM-IRRAS, the incident beam is alternated between s- and p-polarization at a frequency of tens of kHz [63] and differential reflectivity is calculated. Besides these two IR spectroscopic techniques, there are others such as surface-enhanced infrared-absorption spectroscopy (SEIRA) and attenuated total reflection

5. Thermodynamics of mixed monolayers at the water-air interface

The monolayer deposited can be made of two or more surface active molecules at varying composition. In this chapter, we are concerned with mixed monolayers of glycolipids and other membrane components including membrane phospholipids such as dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidycholine (DMPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylethanolamine (DPPE) and others. Three main types of interactions exist between such molecules in the mixed monolayer, van der Waal attractions between the hydrocarbon chains, and hydrogen-bonding and ionic interaction between the head-groups. These interactions, especially the ionic interactions, determine the stability of the mixed monolayer and are also responsible for the deviations from ideality in mixing. A quantitative study of such forces can be done by applying the concepts of

The excess Gibbs free energy of mixing, ΔGexc is one such measure which helps determine the stability of the mixed monolayer. ΔGexc can be determined by using the following relation.

A1,2 is mean molecular area of the mixed monolayer, A<sup>1</sup> and A<sup>2</sup> are the molecular areas of individual monolayer components, and X<sup>1</sup> and X<sup>2</sup> are their mole fractions, respectively. The

ð Þ A1, <sup>2</sup> � x1A<sup>1</sup> � x1A<sup>2</sup> dπ: (3)

<sup>Δ</sup>Gexc <sup>¼</sup>

ðπ 0

frequently employed for the study of Langmuir monolayers [62].

Gangliosides have been the subject of many studies as monolayers and we survey some of the reported results. In an early study using surface pressure isotherms of gangliosides GM1, GM2, GM3, GD1a, GD3 and GT1, it was found that increasing the number of sialic acid residues caused an increase in the prevalence of the LE phase and that the surface pressure isotherms shifted to higher molecular areas and the monolayers became more compressible [64]. This trend was attributed to increasing electrostatic repulsions with introduction of additional negatively charged sialic acids in the structures. In a related study using surface pressure and surface potential measurements, it was found that the strength of interaction of Ca2+ ions in the subphase with gangliosides depended on the number and arrangement of the sialic acid units [65]. A study using surface pressure isotherms of mixed monolayers of GM1, GD1a, and GT1 with phospholipids showed positive deviations from ideality at 30 mN m�<sup>1</sup> for DPPE, a phosphatidylinositol and a phosphatidylserine, but negative deviations from ideality for mixtures with DPPC [66]. In contrast, the interaction of gangliosides with neutral glycosphingolipids was found to be favorable. Mixed monolayers of ceramide or of glucosyl ceramide with gangliosides were found to show favorable interactions, while those of lactosyl ceramide and gangliosides showed immiscibility [67]. The activity of the enzyme phospholipase A2 injected into the subphase against mixed monolayers of dilauroylphosphatidylcholine and gangliosides GM1, GD1a, and GT1b was found to be strongly inhibited [68]. Gangliosides were also found to inhibit the activity of phospholipase C against model membrane systems including monolayers [69]. The sensitivity of monolayer parameters to trace impurity of peptide materials in isolated gangliosides has been emphasized along with provision of methods for rigorous purification [70].

Gangliosides are known for their ability to form microdomains in biological membranes. The partitioning of ganglioside, GM1 in phase separated DOPC/DPPC LB films transferred to freshly cleaved mica at the approximated physiological pressure of 37 mN m<sup>1</sup> and varying concentration of GM1 ranging from 0.2 to 4 mol% was studied using AFM [71]. It was found that below 1 mol% GM1 preferred the DPPC LC phase as evident from nanometer scale patches of round and elongated shapes at the center and periphery of DPPC macrodomains. At 3 mol% concentration of GM1 the size of DPPC macrodomains decreased and patches in their center and on their periphery were more pronounced, and further increasing the concentration to 4 mol% resulted in separation of macrodomains into smaller domains and the elongated patches were arranged around their boundary forming a fencelike structure. According to the authors, this was the first direct determination of distribution of GM1 in phase separated lipid mixtures. A similar study was conducted in DPPC and 2:1 DPPC/ cholesterol monolayer. For DPPC monolayer, the experiments were conducted in a pure gel phase (at 45 mN m<sup>1</sup> ) and a mixture of LE and LC phase (at 7 mN m<sup>1</sup> ) while DPPC/ cholesterol monolayers were in a homogenous liquid-ordered phase at both lower and higher pressure [72]. Ganglioside rich microdomains that were small and circular were observed at the center and the edges of LC and gel phase that coalesced at higher ganglioside concentration to form filamentous structures in the center and larger patches around the edges. In the case of liquid-ordered 2:1 DPPC/cholesterol monolayer, the addition of GM1 gave rise to small round domains of varying diameter (50–150 nm) which coalesced to give long filaments that covered 30–40% of the monolayer surface when the GM1 concentration was increased to 10 mol%. The results indicated that biologically relevant concentrations of GM1 led to formation of microdomains in the model membranes, which is suggestive of their raft forming nature in cholesterol-rich regions of biological membranes.

Another important ganglioside found predominantly in the early immature nervous system of mammals and birds is GD3. GD3 constitutes 3–8% of total ganglioside in adult human brain and is vital for cell growth and proliferation. A combined AFM-thermodynamic analysis was performed to study the aggregation process of GD3 in DPPC [75]. The results obtained revealed a very different aggregation behavior of GD3 from those observed for GM1. In contrast to GM1, GD3 molecules were found to be miscible with DPPC. The excess Gibbs free energy values, calculated for four mole fractions (X = 0.2, 0.4, 0.6 and 0.8) of GD3, were all negative with a minimum at X = 0.4. The mean molecular area showed negative deviations indicative of attractive interactions. AFM images did not show any significant changes in domain diameter or height in the LC region of DPPC up to X = 0.4. A significant morphological change was observed at X = 0.6, a compact LC domain of ≈2 μm in diameter from which ≈0.14 μm wide stripes were extended. This trend became more pronounced at X = 0.8, with the disappearance of stripes and formation of a filamentous network covering the whole surface. The authors suggested that the appearance of stripes indicated a critical point in the phase

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Gangliosides are able to act as receptors for binding bacterial toxins and mediate their entry into cells. For example, cholera toxin binds specifically to GM1, whereas tetanus toxin and botulinum neurotoxin A have strong binding affinity for trisialogangliosides. The in-plane and out-of-plane structure of pure and mixed monolayers of GT1b, a trisialoganglioside, in DPPC/ DPPE were investigated using GIXD and XRR measurements [77]. These studies showed that the phospholipids were able to incorporate up to 20 mol% of GT1b without any phase separation. The finding suggested the binary monolayers can be employed as a model for the study

Much remains to be learned about the lateral organization of gangliosides and how it influences the neighboring lipids. As evident from the above examples of binary DPPC/GM1 mixtures, GM1 has a condensing effect on DPPC, but not enough is known about which interactions are responsible for this effect. This effect has been credited to the complimentary geometrical structures of GM1 and DPPC, intermolecular hydrogen-bonding between the sugar groups and to alignment of the dipole moment of DPPC with the negative charge on the sialic acid residue. Recently, a study was conducted to understand the impact of gangliosides on the surrounding lipids and see if previous knowledge of the GM1/DPPC system can be used for generalization over a range of other gangliosides [78]. Three gangliosides with the same ceramide backbone but different numbers of sialic acid were investigated. A trend seen in GM1/DPPC, of condensation at lower concentration (<20 mol%) followed by fluidization at higher concentration (20 mol%), was observed, but less DPPC was required to condense gangliosides of larger cross-sectional area. A model was proposed to explain the observed result in which the authors took into account two competing factors: electrostatic repulsion between sialic acid groups and their perpendicular dipole moments, μ. It was suggested that with the increase in number of sialic acids the effect of a positive perpendicular dipole moment, μ is more pronounced and hence requires a smaller proportion of DPPC with

diagram [76].

of toxin membrane binding and penetration.

negative μ to stabilize a proportionally small electrostatic repulsion.

The influence of subphase ionic strength on mixed monolayers of GM1 and either stearoyloleoyl-phosphatidylcholine (SOPC) or DPPC was analyzed by surface pressure measurements [73]. Both phospholipids chosen had similar zwitterionic head-groups, but SOPC exists in a fluid LE phase and DPPC exist in a condensed phase at higher surface pressure. It was observed that mixed monolayers were more expanded on buffer than on pure water. The author suggested that a change in GM1 orientation at the interface was responsible. The binding of wheat germ agglutinin (WGA), a dimeric lectin recognizing GM1, with the monolayer was studied. The binding of WGA to GM1 was reduced in the mixed monolayer with DPPC as compared to that with SOPC, attributed to a higher packing density of the SOPC monolayer.

The structure of the two-dimensional mixed monolayer of glycolipid, GM1 + DPPE at airwater interface was studied using GXID and XRR [74]. Pure DPPE, GM1 and mixtures of 5, 10, and 20 mol% of GM1 with DPPE were studied. It was observed that GM1 was accommodated within the DPPE matrix without distorting the in-plane and out-of-plane packing structure. The observed results were in contrast with the previous finding in which the lipids with hydrophilic head groups altered the packing of the phospholipid monolayer. Based on their observation, the author suggested that X-ray scattering technique in combination with monolayer bearing GM1 as a probe can be utilized for studying interaction of proteins such as amyloid β, myelin-based protein and cholera toxin.

Another important ganglioside found predominantly in the early immature nervous system of mammals and birds is GD3. GD3 constitutes 3–8% of total ganglioside in adult human brain and is vital for cell growth and proliferation. A combined AFM-thermodynamic analysis was performed to study the aggregation process of GD3 in DPPC [75]. The results obtained revealed a very different aggregation behavior of GD3 from those observed for GM1. In contrast to GM1, GD3 molecules were found to be miscible with DPPC. The excess Gibbs free energy values, calculated for four mole fractions (X = 0.2, 0.4, 0.6 and 0.8) of GD3, were all negative with a minimum at X = 0.4. The mean molecular area showed negative deviations indicative of attractive interactions. AFM images did not show any significant changes in domain diameter or height in the LC region of DPPC up to X = 0.4. A significant morphological change was observed at X = 0.6, a compact LC domain of ≈2 μm in diameter from which ≈0.14 μm wide stripes were extended. This trend became more pronounced at X = 0.8, with the disappearance of stripes and formation of a filamentous network covering the whole surface. The authors suggested that the appearance of stripes indicated a critical point in the phase diagram [76].

freshly cleaved mica at the approximated physiological pressure of 37 mN m<sup>1</sup> and varying concentration of GM1 ranging from 0.2 to 4 mol% was studied using AFM [71]. It was found that below 1 mol% GM1 preferred the DPPC LC phase as evident from nanometer scale patches of round and elongated shapes at the center and periphery of DPPC macrodomains. At 3 mol% concentration of GM1 the size of DPPC macrodomains decreased and patches in their center and on their periphery were more pronounced, and further increasing the concentration to 4 mol% resulted in separation of macrodomains into smaller domains and the elongated patches were arranged around their boundary forming a fencelike structure. According to the authors, this was the first direct determination of distribution of GM1 in phase separated lipid mixtures. A similar study was conducted in DPPC and 2:1 DPPC/ cholesterol monolayer. For DPPC monolayer, the experiments were conducted in a pure gel

) and a mixture of LE and LC phase (at 7 mN m<sup>1</sup>

cholesterol monolayers were in a homogenous liquid-ordered phase at both lower and higher pressure [72]. Ganglioside rich microdomains that were small and circular were observed at the center and the edges of LC and gel phase that coalesced at higher ganglioside concentration to form filamentous structures in the center and larger patches around the edges. In the case of liquid-ordered 2:1 DPPC/cholesterol monolayer, the addition of GM1 gave rise to small round domains of varying diameter (50–150 nm) which coalesced to give long filaments that covered 30–40% of the monolayer surface when the GM1 concentration was increased to 10 mol%. The results indicated that biologically relevant concentrations of GM1 led to formation of microdomains in the model membranes, which is suggestive of their raft forming

The influence of subphase ionic strength on mixed monolayers of GM1 and either stearoyloleoyl-phosphatidylcholine (SOPC) or DPPC was analyzed by surface pressure measurements [73]. Both phospholipids chosen had similar zwitterionic head-groups, but SOPC exists in a fluid LE phase and DPPC exist in a condensed phase at higher surface pressure. It was observed that mixed monolayers were more expanded on buffer than on pure water. The author suggested that a change in GM1 orientation at the interface was responsible. The binding of wheat germ agglutinin (WGA), a dimeric lectin recognizing GM1, with the monolayer was studied. The binding of WGA to GM1 was reduced in the mixed monolayer with DPPC as compared to that with SOPC, attributed to a higher packing density of the SOPC

The structure of the two-dimensional mixed monolayer of glycolipid, GM1 + DPPE at airwater interface was studied using GXID and XRR [74]. Pure DPPE, GM1 and mixtures of 5, 10, and 20 mol% of GM1 with DPPE were studied. It was observed that GM1 was accommodated within the DPPE matrix without distorting the in-plane and out-of-plane packing structure. The observed results were in contrast with the previous finding in which the lipids with hydrophilic head groups altered the packing of the phospholipid monolayer. Based on their observation, the author suggested that X-ray scattering technique in combination with monolayer bearing GM1 as a probe can be utilized for studying interaction of proteins such

nature in cholesterol-rich regions of biological membranes.

as amyloid β, myelin-based protein and cholera toxin.

) while DPPC/

phase (at 45 mN m<sup>1</sup>

226 Cell Culture

monolayer.

Gangliosides are able to act as receptors for binding bacterial toxins and mediate their entry into cells. For example, cholera toxin binds specifically to GM1, whereas tetanus toxin and botulinum neurotoxin A have strong binding affinity for trisialogangliosides. The in-plane and out-of-plane structure of pure and mixed monolayers of GT1b, a trisialoganglioside, in DPPC/ DPPE were investigated using GIXD and XRR measurements [77]. These studies showed that the phospholipids were able to incorporate up to 20 mol% of GT1b without any phase separation. The finding suggested the binary monolayers can be employed as a model for the study of toxin membrane binding and penetration.

Much remains to be learned about the lateral organization of gangliosides and how it influences the neighboring lipids. As evident from the above examples of binary DPPC/GM1 mixtures, GM1 has a condensing effect on DPPC, but not enough is known about which interactions are responsible for this effect. This effect has been credited to the complimentary geometrical structures of GM1 and DPPC, intermolecular hydrogen-bonding between the sugar groups and to alignment of the dipole moment of DPPC with the negative charge on the sialic acid residue. Recently, a study was conducted to understand the impact of gangliosides on the surrounding lipids and see if previous knowledge of the GM1/DPPC system can be used for generalization over a range of other gangliosides [78]. Three gangliosides with the same ceramide backbone but different numbers of sialic acid were investigated. A trend seen in GM1/DPPC, of condensation at lower concentration (<20 mol%) followed by fluidization at higher concentration (20 mol%), was observed, but less DPPC was required to condense gangliosides of larger cross-sectional area. A model was proposed to explain the observed result in which the authors took into account two competing factors: electrostatic repulsion between sialic acid groups and their perpendicular dipole moments, μ. It was suggested that with the increase in number of sialic acids the effect of a positive perpendicular dipole moment, μ is more pronounced and hence requires a smaller proportion of DPPC with negative μ to stabilize a proportionally small electrostatic repulsion.

#### 6.2. Cerebrosides

The interaction of cerebrosides with cholesterol in mixed monolayers has been investigated [79]. A lactosylceramide with a C7 chain, and maltosylceramides with C8 and C18 chains were studied in mixed monolayers with cholesterol; also studied were GalCer and GlcCer from bovine brain. Cholesterol was found to condense the dihexosyl cerebroside monolayers, shifting the isotherms to lower molecular areas. Cholesterol oxidase served as a probe of the cerebrosidecholesterol interactions by injecting the enzyme into the subphase. The molecular area increased as more cholesterol in the monolayer was oxidized. Stronger interaction with the lipid served to protect the cholesterol from oxidation. It was found that cholesterol interacted more strongly with the monolayers containing dihexosyl ceramides than those contain monohexosyl ceramides. In another study, mixed monolayers of porcine galactocerebrosides with plamitic acid were examined [80]. The excess Gibbs free energy of mixing was negative for most compositions indicating favorable interactions attributed to head-group hydrogen-bonding, and palmitic acid condensed the cerebroside monolayers. BAM showed a gas + LC coexistence with palmitic acid and cerebrosides forming a homogeneous mixture.

AFM has been applied in a few studies of cerebroside monolayers. Bovine brain cerebrosides were spread as monolayers and first examined on compression by fluorescence microscopy [84] revealing formation of branched, fractal-like domains of a LC phase. Transfer of the monolayers onto mica and imaging by AFM revealed the presence of rod-like structures that were concluded to be single bilayer nanotubes based on their size. Cerebrosides extracted from Bohadschia argus (sea cucumber) were investigated by surface pressure and surface potential isotherms, BAM, fluorescence microscopy and AFM [85]. Miscibility of the cerebrosides with DPPC was confirmed and surface pressure isotherms as a function of composition suggested a negative azeotrope in the phase diagram. AFM on the pure cerebrosides showed circular domains of LC and LE phase coexisting. BAM and fluorescence microscopy confirmed misci-

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Monolayers of globotriaosylceramides (Gb3), which contain two galactose units and a glucose unit, were examined by surface pressure isotherms, BAM, XRR, and GIXD [86]. Analogs of Gb3 with variable acyl chains (22:0, 22:1, 14:0) and the lysolipid form were studied in mixtures with DSPC and DPPE. The isotherms and BAM observations indicated that the molecules were miscible in the monolayer. Electron density profiles were calculated from the XRR data and inplane structure determined by GIXD. The carbohydrate region was found to extend into the water, by 10 Å for the 4:1 DSPC/Gb3 mixture, and also was extended for the 4:1 DPPE/Gb3 mixture. The thickness of the monolayer alkyl chains was correlated with the length of the Gb3 acyl chains. For mixed monolayers with DSPC, a segment of the carbohydrate of Gb3 was located within the phospholipid head-group region, an observation that has implications for Gb3 binding of Shiga toxin. Longer acyl chains on the Gb3 analog resulted in greater carbohy-

Surface pressure isotherms were used to study mixed monolayers of stage-specific embryonic antigen-1 (SSEA-1) and DPPC [87]. The SSEA-1 was isolated from Japanese quail intestine and contained a mixture of chain lengths on the ceramide. A LE to LC phase transition was observed in the mixed monolayers. The isotherms as a function of composition showed behavior resembling that of a negative azeotrope which indicated favorable interactions between

The outermost membranes of gram negative bacteria contain lipopolysaccharides. Lipid A, a glucosamine based phospholipid serves as a hydrophobic anchor for LPS. Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) domains are present in the structure as well possibly additional core and O-antigen sugars. Monolayers of LPS have been investigated. The smallest LPS that are active are known as Re-LPS. The miscibility of Re-LPS with monolayers of DPPC was studied using fluorescence microscopy [88]. The fluorescent lipid probe 1-palmitoyl-1-[12-[(7-nitro-2-1,3 benzoxadizole-1-yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) was used at 1 mol%. The surface pressure for monolayers of pure Re-LPS began rising around 400 Å2 molecule<sup>1</sup> upon

bility of the cerebrosides and DPPC.

drate exposure in the subphase.

SSEA-1 and DPPC.

6.4. Lipopolysaccharides

6.3. Globosides

Mixed monolayers of cerebrosides bearing glucosyl head-groups mixed with cholesterol or with cholesteryl sulfate were examined by surface pressure isotherms, surface potential isotherms, and fluorescence microscopy [81]. The surface pressure and surface potential isotherms were analyzed as a function of composition resulting in the conclusion that these cerebrosides were not miscible with cholesterol but were miscible with cholesteryl sulfate. Fluorescence microscopy images supported these conclusions. The same group reported a study of six cerebrosides extracted from the blue sea star Linckia laevigata [82]. Mixed monolayers with DPPC were examined by surface pressure and surface potential isotherms and fluorescence microscopy and miscibility of the cerebrosides with DPPC was established. It was found that the nature of the hydrocarbon chain had a significant influence on the surface potential.

The thermodynamic behavior and structure of monolayers of three galactocerebrosides was examined using surface pressure isotherms, BAM, GIXD, and IRRAS [83]. The high prevalence of galactocerebrosides in myelin membranes motivated the study. The galactocerebrosides contained a galactose head-group, a sphingosine backbone, and a fatty acid chain that was varied between C24 (GalCer C24:0), C24 hydroxylated on the 2-position (GalCer C24:0 (2-OH), and a C24 with a double bond at position 15 (GalCer C24:1). The isotherms of GalCer 24:0 and GalCer 24:1 showed plateaus indicating LE + LC phase coexistence above 38 and 20C, respectively. In contrast, the GalCer 24:0 2-OH derivative at all temperatures showed direct transition from a gas-like phase to a LC phase, with the condensation effect assigned as due to additional interactions of the 2-OH groups. BAM showed appearance of flower-like domains on compression of GalCer 24:0, of round domains on compression of GalCer 24:1 which also showed a kinetic overshoot in the compression isotherms at the LE to LC transition. Using a twodimensional analogy of the Clapeyron equation and the phase transition pressures found in the isotherms as a function of temperature, ΔH for the phase change was estimated. Together, for GalCer 24:0, the GIXD and IRRAS data confirmed a rigid phase with hydrogen-bonding between head-groups of neighboring molecules.

AFM has been applied in a few studies of cerebroside monolayers. Bovine brain cerebrosides were spread as monolayers and first examined on compression by fluorescence microscopy [84] revealing formation of branched, fractal-like domains of a LC phase. Transfer of the monolayers onto mica and imaging by AFM revealed the presence of rod-like structures that were concluded to be single bilayer nanotubes based on their size. Cerebrosides extracted from Bohadschia argus (sea cucumber) were investigated by surface pressure and surface potential isotherms, BAM, fluorescence microscopy and AFM [85]. Miscibility of the cerebrosides with DPPC was confirmed and surface pressure isotherms as a function of composition suggested a negative azeotrope in the phase diagram. AFM on the pure cerebrosides showed circular domains of LC and LE phase coexisting. BAM and fluorescence microscopy confirmed miscibility of the cerebrosides and DPPC.
