6.3. Globosides

6.2. Cerebrosides

228 Cell Culture

potential.

and cerebrosides forming a homogeneous mixture.

between head-groups of neighboring molecules.

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

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

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

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 carbohydrate exposure in the subphase.

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 SSEA-1 and DPPC.

#### 6.4. Lipopolysaccharides

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 compression. The variation of the collapse pressure with composition provided evidence that Re-LPS and DPPC were miscible. Plots of mean molecular area vs. composition at three surface pressures all showed negative deviations from ideality and hence evidence for attractive interactions between Re-LPS and DPPC. Pure monolayers of DPPC show a coexistence region between a LE and a LC phase in which distinct microdomain formation occurs. Addition of increasing amounts of Re-LPS increased the transition pressure and caused a decrease in domain size along with a change to less distinct shapes. Pure monolayers of Re-LPS did not show a phase transition upon compression. Addition of lung surfactant protein A beneath mixed monolayers of Re-LPS and DPPC induced segregation and domain formation. In a later study, Re-LPS was extracted from Salmonella Minnesota strain R595 and studied by surface pressure isotherms and by XRR and GIXD [89]. On pure phosphate buffer solution of pH 7.2, the surface pressure for LPS began to rise at 370 Å2 molecule<sup>1</sup> ; addition of 50 mM CaCl2 to the subphase reduced this area to 315 Å2 molecule<sup>1</sup> as the divalent counterions cross-linked the sugar units of LPS molecules. As the surface pressure increased, the monolayer became thicker and there was a change in the conformation of the sugar head-groups. At higher surface pressures, the hydrocarbon chain packing became more oblique and the size of ordered domains became smaller.

Glycopeptidolipids (GPLs) are present in their bacterial cell wall of mycobacteria. In one study, the GPLs were extracted from mycobacteria and studied in mixed monolayers with phospholipids [93]. Three GPLs were studied as monolayers and found by surface potential measurements to undergo a conformational change upon compression but not a phase transition. Addition of GPL dispersions beneath egg phosphatidylcholine monolayers resulted in insertion of GPL into the monolayer as registered by significant surface pressure increases. A subsequent study used PM-IRRAS to study mixed monolayers of GPLs with 1,2-di(perdeuteropalmitoyl)phosphatidylcholine [94]. It was concluded that phase segregation occurred between the GPLs and the phospholipid, driven by the ability of GPLs to form β-sheet structures, and that the phase segregation was more pronounced for the GSLs that were more heavily glycosylated.

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In addition to studies of the monolayer behavior of natural occurring carbohydrate containing lipids, significant effort has been devoted to the study of synthetic derivatives, some being structural analogs of naturally occurring lipids and others being new structures. For example, changes in the structure of ganglioside and sphingosine derivatives have been found to alter the activity of phospholipase enzymes against mixed monolayers containing dilauroylphosphatidylcholine [95]. In these experiments, enzyme is injected into the subphase with the monolayer maintained at a surface pressure of 12 mN m<sup>1</sup> and the decrease in molecular area due to hydrolysis of the ester bond of the phospholipid is followed as a function of time. These studies illustrate the role of gangliosides and glycophingolipids in regulating enzyme-based signaling at membrane surfaces. The surface pressure and surface potential behavior of these derivatives was studied in detail separately [96]. One of the structural changes investigated was removal of the sialic acid from ganglioside GM1 resulting in asialo-GM1, which was found to form a LC phase not seen for monolayers of GM1. Surface potential data revealed that monolayers of asialo-GM1 had a significantly larger dipole moment perpendicular to the

Mixed monolayers of DMPC and derivatives of N-acetylglucosamine were formed and their interaction with the lectin wheat germ agglutinin were examined. It was observed that the synthetic glycolipids were miscible with DMPC at higher surface pressures, and that the lectin could only bind to derivatives with a spacer group between the hydrocarbon chains and the sugar [97]. The binding of the lectin to the monolayer was seen to result in a significant increase in surface pressure for the derivatives with a long enough spacer. Subsequently, the interaction of monolayers of synthetic glycolipids bearing either N-acetyl-D-glucosamine or L-fucose with three lectins (wheat germ, Ulex europaeus I, and Lotus tetragonolobus agglutinins) was examined. Mixed monolayers of the synthetic glycolipids with DMPC were able to bind lectins injected into the subphase resulting in an increase in surface pressure with time and a shift of the

Synthetic glycolipids derived from glycerol bearing two hydrocarbon chains, a triethyleneglycol spacer, and an N-acetyl-D-glucosamine head-group were used to form monolayers, with the

6.5. Synthetic glycolipids

water surface than did monolayers of GM1.

monolayer isotherms to higher molecular areas [98].

Monolayers of LPS extracted from Pseudomonas aeruginosa were studied using surface pressure measurements and found to be stable [90]. When monovalent or divalent salts were added to the subphase, LPS molecules adopted a compact conformation. Addition of increasing amounts of CaCl2 to the subphase destabilized the monolayer and caused the collapse pressure to decrease. Upon compression to 45 mN m<sup>1</sup> , surface pressure relaxation to about 43 mN m<sup>1</sup> was seen over a period of about 90 min. The RcLPS form of LPS, whose structure contains seven sugars of the inner and outer core polysaccharide, was extracted from Escherichia coli, and used to form monolayers that were studied using surface pressure, BAM, XRR and neutron reflectivity, and GIXD [91]. The surface pressure isotherm showed a steady increase and no obvious phase transition, and the BAM observation showed a homogeneous surface. At 20 mN m<sup>1</sup> , a monolayer thickness of 41 Å was calculated. A hexagonal oblique arrangement of hydrocarbon chains was observed at all surface pressures. The calculated arrangement of rcLPS at the air-water interface shows that the molecules are overall tilted by 15–29 and that the tails occupy a thickness of 12 Å, and that the inner carbohydrate and amide and ester linkages occupy 14 Å of thickness and the outer entirely carbohydrate part of the head-group occupies 15 Å of thickness.

Interaction of plasticins with LPS monolayers was studied [92]. Plasticins are linear antimicrobial peptides with a repeated GXXXG motif where G is glycine and X is any amino acid. The interaction of plasticins with mixed monolayers of LPS (both smooth LPS and Re-LPS) and phospholipids was studied to gain insight into how the antimicrobial peptide interacts with bacterial membranes. A combination of surface pressure measurements, BAM, GIXD and AFM performed on films transferred to mica was applied. Both plasticins studied were highly surface active. Smooth LPS formed unstable monolayers but Re-LPS formed stable monolayers. The monolayers appeared homogeneous to BAM. The cationic plasticin was able to significantly penetrate the LPS monolayers. Plasticin insertion was able to introduce disorder into the monolayers, as seen by changes in the X-ray correlation lengths.

Glycopeptidolipids (GPLs) are present in their bacterial cell wall of mycobacteria. In one study, the GPLs were extracted from mycobacteria and studied in mixed monolayers with phospholipids [93]. Three GPLs were studied as monolayers and found by surface potential measurements to undergo a conformational change upon compression but not a phase transition. Addition of GPL dispersions beneath egg phosphatidylcholine monolayers resulted in insertion of GPL into the monolayer as registered by significant surface pressure increases. A subsequent study used PM-IRRAS to study mixed monolayers of GPLs with 1,2-di(perdeuteropalmitoyl)phosphatidylcholine [94]. It was concluded that phase segregation occurred between the GPLs and the phospholipid, driven by the ability of GPLs to form β-sheet structures, and that the phase segregation was more pronounced for the GSLs that were more heavily glycosylated.

### 6.5. Synthetic glycolipids

compression. The variation of the collapse pressure with composition provided evidence that Re-LPS and DPPC were miscible. Plots of mean molecular area vs. composition at three surface pressures all showed negative deviations from ideality and hence evidence for attractive interactions between Re-LPS and DPPC. Pure monolayers of DPPC show a coexistence region between a LE and a LC phase in which distinct microdomain formation occurs. Addition of increasing amounts of Re-LPS increased the transition pressure and caused a decrease in domain size along with a change to less distinct shapes. Pure monolayers of Re-LPS did not show a phase transition upon compression. Addition of lung surfactant protein A beneath mixed monolayers of Re-LPS and DPPC induced segregation and domain formation. In a later study, Re-LPS was extracted from Salmonella Minnesota strain R595 and studied by surface pressure isotherms and by XRR and GIXD [89]. On pure phosphate buffer solution of pH 7.2, the surface pressure for LPS began

315 Å2 molecule<sup>1</sup> as the divalent counterions cross-linked the sugar units of LPS molecules. As the surface pressure increased, the monolayer became thicker and there was a change in the conformation of the sugar head-groups. At higher surface pressures, the hydrocarbon chain

Monolayers of LPS extracted from Pseudomonas aeruginosa were studied using surface pressure measurements and found to be stable [90]. When monovalent or divalent salts were added to the subphase, LPS molecules adopted a compact conformation. Addition of increasing amounts of CaCl2 to the subphase destabilized the monolayer and caused the collapse pressure to decrease.

a period of about 90 min. The RcLPS form of LPS, whose structure contains seven sugars of the inner and outer core polysaccharide, was extracted from Escherichia coli, and used to form monolayers that were studied using surface pressure, BAM, XRR and neutron reflectivity, and GIXD [91]. The surface pressure isotherm showed a steady increase and no obvious phase

layer thickness of 41 Å was calculated. A hexagonal oblique arrangement of hydrocarbon chains was observed at all surface pressures. The calculated arrangement of rcLPS at the air-water interface shows that the molecules are overall tilted by 15–29 and that the tails occupy a thickness of 12 Å, and that the inner carbohydrate and amide and ester linkages occupy 14 Å of thickness and the outer entirely carbohydrate part of the head-group occupies 15 Å of thickness. Interaction of plasticins with LPS monolayers was studied [92]. Plasticins are linear antimicrobial peptides with a repeated GXXXG motif where G is glycine and X is any amino acid. The interaction of plasticins with mixed monolayers of LPS (both smooth LPS and Re-LPS) and phospholipids was studied to gain insight into how the antimicrobial peptide interacts with bacterial membranes. A combination of surface pressure measurements, BAM, GIXD and AFM performed on films transferred to mica was applied. Both plasticins studied were highly surface active. Smooth LPS formed unstable monolayers but Re-LPS formed stable monolayers. The monolayers appeared homogeneous to BAM. The cationic plasticin was able to significantly penetrate the LPS monolayers. Plasticin insertion was able to introduce disorder

transition, and the BAM observation showed a homogeneous surface. At 20 mN m<sup>1</sup>

into the monolayers, as seen by changes in the X-ray correlation lengths.

packing became more oblique and the size of ordered domains became smaller.

; addition of 50 mM CaCl2 to the subphase reduced this area to

, surface pressure relaxation to about 43 mN m<sup>1</sup> was seen over

, a mono-

to rise at 370 Å2 molecule<sup>1</sup>

230 Cell Culture

Upon compression to 45 mN m<sup>1</sup>

In addition to studies of the monolayer behavior of natural occurring carbohydrate containing lipids, significant effort has been devoted to the study of synthetic derivatives, some being structural analogs of naturally occurring lipids and others being new structures. For example, changes in the structure of ganglioside and sphingosine derivatives have been found to alter the activity of phospholipase enzymes against mixed monolayers containing dilauroylphosphatidylcholine [95]. In these experiments, enzyme is injected into the subphase with the monolayer maintained at a surface pressure of 12 mN m<sup>1</sup> and the decrease in molecular area due to hydrolysis of the ester bond of the phospholipid is followed as a function of time. These studies illustrate the role of gangliosides and glycophingolipids in regulating enzyme-based signaling at membrane surfaces. The surface pressure and surface potential behavior of these derivatives was studied in detail separately [96]. One of the structural changes investigated was removal of the sialic acid from ganglioside GM1 resulting in asialo-GM1, which was found to form a LC phase not seen for monolayers of GM1. Surface potential data revealed that monolayers of asialo-GM1 had a significantly larger dipole moment perpendicular to the water surface than did monolayers of GM1.

Mixed monolayers of DMPC and derivatives of N-acetylglucosamine were formed and their interaction with the lectin wheat germ agglutinin were examined. It was observed that the synthetic glycolipids were miscible with DMPC at higher surface pressures, and that the lectin could only bind to derivatives with a spacer group between the hydrocarbon chains and the sugar [97]. The binding of the lectin to the monolayer was seen to result in a significant increase in surface pressure for the derivatives with a long enough spacer. Subsequently, the interaction of monolayers of synthetic glycolipids bearing either N-acetyl-D-glucosamine or L-fucose with three lectins (wheat germ, Ulex europaeus I, and Lotus tetragonolobus agglutinins) was examined. Mixed monolayers of the synthetic glycolipids with DMPC were able to bind lectins injected into the subphase resulting in an increase in surface pressure with time and a shift of the monolayer isotherms to higher molecular areas [98].

Synthetic glycolipids derived from glycerol bearing two hydrocarbon chains, a triethyleneglycol spacer, and an N-acetyl-D-glucosamine head-group were used to form monolayers, with the increase in the alkyl chain length to C16 resulting in a highly organized surface arrangement [99]. Monolayers containing one of these glycolipids together with immunoglobulin G were successfully formed by spreading from vesicle dispersions [100]. The presence of the immunoglobulin G in transferred monolayers was subsequently confirmed by infrared spectroscopy, and it was proposed that on compression the immunoglobulin G re-orients from a flat to standing orientation [101]. Monolayers of these derivatives were studied in mixed monolayers with phospholipids by GIXD, and it was found that addition of the glycolipid reduced the correlation length and hence the extent of ordering for the phospholipid component [102].

7. Conclusions

Author details

USA

References

2009. pp. 1-22

2017;18:361-374

Bishal Nepal and Keith J. Stine\*

\*Address all correspondence to: kstine@umsl.edu

Journal of Experimental Medicine. 1925;41(4):439-443

Biophysics and Biomolecular Structure. 2003;32(1):257-283

hydrate Nanotechnology. New York: Wiley; 2016. pp. 233-66

ion in Chemical Biology. 2007;11(6):581-587

Lipids. 2014;177(Supplement C):8-18

The study of monolayers containing glycolipids provides many insights into the molecular arrangement of glycolipids in membranes. The physical state of the monolayers influences the interaction of glycolipids with binding partners, which can be studied directly by introducing these partners into the subphase. The modulation of these binding interactions is significant to the membrane biochemistry of glycolipids, and monolayers at the water surface provide a

Monolayers of Carbohydrate-Containing Lipids at the Water-Air Interface

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233

Department of Chemistry and Biochemistry, University of Missouri—St. Louis, St. Louis, MO,

[1] Gorter E, Grendel F. On bimolecular layers of lipoids on the chromocytes of blood. The

[2] Bar RS, Deamer DW, Cornwell DG. Surface area of human erythrocyte lipids: Reinvestigation of experiments on plasma membrane. Science. 1966;153(3739):1010-1012

[3] Edidin M. The state of lipid rafts: From model membranes to cells. Annual Review of

[4] Varki A, Sharon N. Historical background and overview. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME, editors. Essentials of Glycobiology. 2nd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press;

[5] Stine KJ. Glycans in mesoporous and nanoporous materials. In: Stine KJ, editor. Carbo-

[6] Sezgin E, Levental I, Mayor S, Eggeling C. The mystery of membrane organization: Composition, regulation and roles of lipid rafts. Nature Reviews Molecular Cell Biology.

[7] Chan Y-HM, Boxer SG. Model membrane systems and their applications. Current Opin-

[8] Patil YP, Jadhav S. Novel methods for liposome preparation. Chemistry and Physics of

uniquely convenient and controllable environment in which to conduct such studies.

A homologous series of dialkylglycerylethers and their β-D-glucoside and β-D-cellobioside derivatives were studied as monolayers. It was found that introduction of the sugars expanded the monolayers, and acted in opposition to the effect of increasing chain length [103]. Using pentaerythritol as a building block, a gemini glycolipid was synthesized with two C16 alkyl chains and two N-acetyl-β-D-glucosamine units [104]. Surface pressure-area isotherms showed that monolayers of the compound underwent an expanded to condensed phase transition. In a related study, glucoside lipids were created with a single hydrocarbon chain and either one or two glucose units in the head-group, presented in a branched geometry [105]. The bivalent glucoside achieved maximal binding to lectin at a lower surface fraction than did the monovalent glucoside lipid, when studied in mixed monolayers with an analog lacking sugar units.

Some studies in which glycolipid systems were transferred onto solid supports have been reported. Synthetic glycolipids bearing lactose, Lewis X, and sialyl Lewis X were synthesized containing partially fluorinated chains [106]. Mixed monolayers with phospholipids were observed by fluorescence microscopy and found to display phase separated microdomains. The monolayers were transferred onto silanized glass slides and it was found that adherence of Chinese hamster ovary cells to the supported monolayers varied with the composition and extent of microdomain formation. Langmuir-Blodgett films containing polydiacetylene derivatives that undergo a color change upon a binding induced conformational change are of interest for development of biosensors. Dioctadecyl glyceryl ether-β-glucosides (DGG) were used to form mixed monolayers with 10,12-pentacosadiynoic acid (PCDA) or tricosa-2,4-diynoic acid (TCDA) [107]. BAM showed that mixed monolayers with TCDA could be uniform, but those with PCDA showed phase separated domains. The excess Gibbs free energy of mixing was determined under varied subphase conditions. Another study reported mannosyl derivatives of PCDA in which BAM revealed highly structured dendritic like domains indicating the presence of a highly ordered phase [108]. Absorbance spectroscopy, carried out directly at the water-air interface, showed a shift from blue to red upon irradiation of the monolayers.

Synthetic derivatives of galactosyl ceramides with varied chain length between 11 and 33 carbons were synthesized and used to prepare monolayers, the phase behavior of the monolayers varied with overall chain length becoming more condensed with increasing chain length [109]. Synthetic glycolipids based on glycerol with two hydrocarbon chains and one, two, or three lactose units as the head-group were used to make monolayers and the interfacial viscosity was measured. With one lactose unit, a highly viscoelastic monolayer was obtained, with two a more fluid monolayer was observed, and with three a transition from viscous to elastic was observed [110]. In this study, the glycolipid monolayers were considered as a model for the cellular glycocalyx.
