Carbohydrate Head Group Research Articles

Surface-Templated Glycopolymer Nanopatterns Transferred to Hydrogels for Designed Multivalent Carbohydrate-Lectin Interactions across Length Scales.

Multivalent interactions between carbohydrates and proteins enable a broad range of selective chemical processes of critical biological importance. Such interactions can extend from the macromolecular scale (1-10 nm) up to much larger scales across a cell or tissue, placing substantial demands on chemically patterned materials aiming to leverage similar interactions in vitro. Here, we show that diyne amphiphiles with carbohydrate headgroups can be assembled on highly oriented pyrolytic graphite (HOPG) to generate nanometer-resolution carbohydrate patterns, with individual linear carbohydrate assemblies up to nearly 1 μm, and microscale geometric patterns. These are then photopolymerized and covalently transferred to the surfaces of hydrogels. This strategy suspends carbohydrate patterns on a relatively rigid polydiacetylene (persistence length ∼ 16 nm), exposed at the top surface of the hydrogel above the bulk pore structure. Transferred patterns of appropriate carbohydrates (e.g., N-acetyl-d-glucosamine, GlcNAc) enable selective, multivalent interactions (KD ∼ 40 nM) with wheat germ agglutinin (WGA), a model lectin that exhibits multivalent binding with appropriately spaced GlcNAc moieties. WGA binding affinity can be further improved (KD ∼ 10 nM) using diacetylenes that shift the polymer backbone closer to the displayed carbohydrate, suggesting that this strategy can be used to modulate carbohydrate presentation at interfaces. Conversely, GlcNAc-patterned surfaces do not induce specific binding of concanavalin A, and surfaces patterned with glucuronic acid, or with simple carboxylic acid or hydroxyl groups, do not induce WGA binding. More broadly, this approach may have utility in designing synthetic glycan-mimetic interfaces with features from molecular to mesoscopic scales, including soft scaffolds for cells.

Comprehensive Glycan Analysis of Sphingolipids in Human Serum/Plasma.

Glycosphingolipids (GSLs) are glycolipids with ceramide and carbohydrate head groups that play an important role in numerous biological processes. Previously, we performed GSL-glycan analysis of various cell lines and virus-infected cells using a glycoblotting approach. Recently, we developed several methods for sialic acid linkage-specific chemical modification to distinguish sialylated glycan isomers by mass spectrometry. In this chapter, we describe a method for analyzing GSL-glycans in human serum/plasma using glycoblotting combined with aminolysis-SALSA (sialic acid linkage-specific alkylamidation) and lactone-driven ester-to-amide derivatization (LEAD)-SALSA for comprehensive and detailed structural glycomics.

Linking glycosphingolipids to Alzheimer's amyloid-ß: extracellular vesicles and functional plant materials.

Glycosphingolipids (GSLs) are a specialized class of membrane lipids composed of a ceramide and a carbohydrate head group. GSLs are localized in cell membranes and were recently found to be enriched in the membrane of neuron-derived exosomes, which are a type of extracellular vesicle. Our studies demonstrated that exosomal GSLs may be associated with the amyloid-ß (Aß) peptide, a principal agent of Alzheimer's disease (AD), and act to clear Aß by transporting Aß into brain phagocytic microglia. In this review, we summarize and discuss the function of exosomal GSLs in Aß homeostasis in AD pathology. Improvement in Aß clearance is a potent strategy for AD prevention and therapy. Dietary glucosylceramides (GlcCer) isolated from plants are absorbed into the body as various metabolites, including ceramides. Our recent work demonstrated that dietary GlcCer accelerates neuronal exosome production, which facilitates Aß clearance in mice. Furthermore, studies of AD model mice and human clinical trials have found that oral administration of plant-type GlcCer attenuates the Aß burden in the brain. We also introduce the development of plant-type GlcCer as functional food materials to prevent AD.

Ganglioside isomer analysis using ion polarity switching liquid chromatography-tandem mass spectrometry.

Gangliosides are ubiquitously present on cell surface. They are more abundantly expressed in nerve cells and tissues and involved in pathology of various diseases. Diversity of molecular structures in the carbohydrate head group, fatty acyl, and long chain base increases the complexity of analyzing gangliosides. In this study, an ultrahigh-performance liquid chromatography-tandem mass spectrometry method is developed for analysis of the co-eluting ganglioside isomers, which uses ion polarity switching to integrate glycan head isomer identification, ceramide isomer differentiation, and quantification of ganglioside into one analysis. The method is facilitated with an extensive ganglioside target list by combining the various glycan head groups, long chain bases, and the experimentally determined fatty acyls. Correlation between the retention time of ganglioside and its ceramide total carbon number is experimentally validated and used to predict retention time of ganglioside target list for scheduling the final multiple reaction monitoring method. This method was validated according to the FDA guidelines: 96.5% of gangliosides with good accuracy (80-120%), precision (< 15%), and linearity R2 > 0.99. The authenticated gangliosides were quantified from mouse brain by isotope dilution. Overall, 165 gangliosides were quantified using 10mg mouse brain tissue, including 100 isomers of GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, and GT1b.

Differential recognition of lipid domains by two Gb3-binding lectins

The two lectins LecA from Pseudomonas aeruginosa and the B-subunit of Shiga toxin from Shigella dysenteriae (StxB) share the glycosphingolipid globotriaosylceramide (Gb3) as receptor. Counterintuitively, we found that LecA and StxB segregated into different domains after recognizing Gb3 at the plasma membrane of cells. We hypothesized that the orientation of the carbohydrate head group of Gb3 embedded in the lipid bilayer differentially influences LecA and StxB binding. To test this hypothesis, we reconstituted lectin-Gb3 interaction using giant unilamellar vesicles and were indeed able to rebuild LecA and StxB segregation. Both, the Gb3 fatty acyl chain structure and the local membrane environment, modulated Gb3 recognition by LecA and StxB. Specifically, StxB preferred more ordered membranes compared to LecA. Based on our findings, we propose comparing staining patterns of LecA and StxB as an alternative method to assess membrane order in cells. To verify this approach, we re-established that the apical plasma membrane of epithelial cells is more ordered than the basolateral plasma membrane. Additionally, we found that StxB recognized Gb3 at the primary cilium and the periciliary membrane, whereas LecA only bound periciliary Gb3. This suggests that the ciliary membrane is of higher order than the surrounding periciliary membrane.

Molecular characterization of the outer membrane of Pseudomonas aeruginosa

Discovery of new antibiotics effective against Gram-negative bacteria is hampered by the low permeability of the bacterial two-membrane cell envelopes. The outer membrane (OM) is an asymmetric bilayer of lipopolysaccharides (LPS) and phospholipids and is a significant permeability barrier to noxious compounds. Molecular characterization of the OM is essential for understanding how antibiotics penetrate this barrier, and for the development of new therapeutic strategies and rational drug design. We carry out all-atom (AA) and coarse-grained (CG) molecular dynamics (MD) simulations to characterize the fully assembled OM bilayer of Pseudomonas aeruginosa, a human pathogen resistant to most clinical antibiotics. These simulations were run using AA and CG parameters that we developed to model the most abundant LPS of P. aeruginosa, specifically the A band O-antigen. Our simulations reproduce several experimentally determined physical properties such as area per lipid, membrane stiffness and increased stability due to the interaction with divalent cations. These tested parameters allowed us to study various OM properties including the effect of divalent cations and OM electroporation. In addition, the massive and extended carbohydrate head groups increase water permeation, which propagates towards the aliphatic tails of the inner leaflet. This effect contributes to the low ordering of the lipids in the inner leaflet. Further, evaluation is needed to understand whether the increased solvation of outer membrane could modulate permeation of highly polar drugs.

Worm-like micelles and vesicles formed by alkyl-oligo(ethylene glycol)-glycoside carbohydrate surfactants: The effect of precisely tuned amphiphilicity on aggregate packing

Carbohydrates are appealing non-ionic surfactant head-groups as they are naturally abundant, generally biocompatible and biodegradable, and readily functionalized. Recent work has produced a promising molecular candidate for the formation of viscoelastic worm-like micellar solutions: a tri(ethylene glycol)-linked oleyl-β-D-glucoside surfactant (GlcC18:1) exhibited near ideal Maxwell behavior at low concentrations (2.9 wt%) without additives at room temperature. Here, fourteen surfactants have been synthesized with structural variations based around GlcC18:1. Each contain an oligo(ethylene glycol) linker of varying length (2, 3, 4, 6 EO units) between a carbohydrate head-group (glucose, galactose, mannose, maltose, lactose, cellobiose) and a cis-unsaturated alkyl tail-group (oleyl, linoleyl, erucyl). The aqueous adsorption kinetics and self-assembly of these surfactants was explored using tensiometry and small-angle neutron scattering (SANS), respectively. With SANS we observed the formation of worm-like micelles for four surfactants, and vesicles for two surfactants which exhibited behavior similar to insoluble lipids. We also observed temperature-induced micellar elongation due to dehydration of the oligo(ethylene glycol) linker, resulting in a further three surfactants forming worm-like micelles at 50 °C. Worm-like micellar fluids were further characterized using rheology to reveal two surfactants with vastly superior viscoelastic properties compared to GlcC18:1, with >2 orders of magnitude increase in viscosity and >3 orders of magnitude increase in stress relaxation time. These results provide insight into structure-function relationships for non-ionic surfactants and demonstrate a class of designed amphiphiles with a special propensity for forming viscoelastic worm-like micellar solutions at low concentrations.

Photoswitchable Glycolipid Mimetics: Synthesis and Photochromic Properties of Glycoazobenzene Amphiphiles.

Glycolipids as constituents of cell membranes play an important role in cell membrane functioning. To enable the structural modification of membranes on demand, embedding of photosensitive glycolipid mimetics was envisioned and novel amphiphilic glycolipid mimetics comprising a photoswitchable azobenzene unit were synthesized. In this study, the photochromic properties of these glycolipid mimetics were analyzed by means of UV/Vis spectroscopy and reversible photoswitching. The glycolipids were based on a racemic glycerolipid derivative to be comparable in DPPC (dipalmitoylphosphatidylcholine) phospholipid membrane monolayers. Carbohydrate head groups were altered between a β-glucoside and a β-lactosyl unit, as well as acyl chain lengths between C12 and C16, resulting in altered photoswitching. Langmuir isotherms showed that photoswitching of Langmuir films comprising the synthetic photosensitive glycoamphiphiles was successful.

Physicochemical and Biological Characterisation of Azobenzene-Containing Photoswitchable Surfactants

Abstract Surfactants are a versatile and widely used class of molecules, due to their valuable adsorption and self-assembly properties. In particular, surfactants that can respond to stimuli are of interest in modulating wetting, controlling delivery, and exploring mechanistic aspects of biological processes. Incorporating azobenzene into surfactants is a classic approach to rendering molecules that respond to light as an external stimulus; these molecules find wide utility in the precise spatiotemporal control of dispersed systems, from DNA to graphene. More recently, the creation of diverse libraries of such molecules has been achieved by coupling azobenzene-containing hydrophobic tail-groups to hydrophilic carbohydrate head-groups. Such a synthetic strategy offers fine control over adsorption and aggregation, as evidenced by physicochemical characterization of these molecules, uncovering rich phase behavior and diverse biological response. This article covers recent advances in the field of both ‘traditional’ and new azobenzene-containing photosurfactants, and offers directions for future study and use of this unique class of molecule.

Cholesterol Enriched Archaeosomes as a Molecular System for Studying Interactions of Cholesterol-Dependent Cytolysins with Membranes.

Archaeosomes are vesicles made of lipids from archaea. They possess many unique features in comparison to other lipid systems, with their high stability being the most prominent one, making them a promising system for biotechnological applications. Here, we report a preparation protocol of large unilamellar vesicles, giant unilamellar vesicles (GUVs), and nanodiscs from archaeal lipids with incorporated cholesterol. Incorporation of cholesterol led to additional increase in thermal stability of vesicles. Surface plasmon resonance, sedimentation assays, intrinsic tryptophan fluorescence measurements, calcein release experiments, and GUVs experiments showed that members of cholesterol-dependent cytolysins, listeriolysin O (LLO), and perfringolysin O (PFO), bind to cholesterol-rich archaeosomes and thereby retain their pore-forming activity. Interestingly, we observed specific binding of LLO, but not PFO, to archaeosomes even in the absence of cholesterol. This suggests a new capacity of LLO to bind to carbohydrate headgroups of archaeal lipids. Furthermore, we were able to express LLO inside GUVs by cell-free expression. GUVs made from archaeal lipids were highly stable, which could be beneficial for synthetic biology applications. In summary, our results describe novel model membrane systems for studying membrane interactions of proteins and their potential use in biotechnology.

Tuning the position of head groups by surfactant design in mixed micelles of cationic and carbohydrate surfactants

HypothesisEmerging applications of carbohydrate/cationic surfactant mixtures require not only synergistic mixing, but also accessible sugar headgroups at the exterior of micelles. A previous study showed that the glucoside headgroups of octyl-β-d-glucopyranoside aggregate at the interior of mixed micelles with equimolar cetyltrimethylammonium bromide rather than mixing with trimethylammonium groups at the corona. The current study tests the hypothesis that structural characteristics of the surfactants (the relative lengths of the alkyl tails and the type of linker) can be tuned to shift the carbohydrate groups to micelle surfaces. ExperimentsThe structural arrangement of 30 mM equimolar mixed micelle solutions in D2O is investigated using NMR. The dynamics in different regions are probed using 1H spin-lattice (T1) and spin-spin (T2) relaxation measurements, and relative positioning by nuclear Overhauser effect spectroscopy (NOESY). Additional micellar properties are determined using solvatochromic fluorescent probes. FindingsMatching surfactant alkyl tail lengths is found ineffective at “pushing out” the carbohydrate headgroups due to a large mismatch in interactions between the headgroups and D2O. However, inserting a novel polar triazole group between the carbohydrate head group and the hydrophobic tail (e.g. in n-octyl-β-d-xylopyranoside) using click chemistry is able to “pull out” the carbohydrate, thus giving accessible sugar moieties at the surface of mixed micelles.

Photoswitchable Janus glycodendrimer micelles as multivalent inhibitors of LecA and LecB from Pseudomonas aeruginosa

The first example of the self-assembly and lectin binding properties of photoswitchable glycodendrimer micelles is reported. Light-addressable micelles were assembled from a library of 12 amphiphilic Janus glycodendrimers composed of variable carbohydrate head groups and hydrophobic tail groups linked to an azobenzene core. Spontaneous association in water gave cylindrical micelles with uniform size distribution as determined by dynamic light scattering (DLS) and small angle neutron scattering (SANS). Trans-cis photoisomerization of the azobenzene dendrimer core was used to probe the self-assembly behaviour and lectin binding properties of cylindrical micelles, revealing moderate-to-potent inhibition of lectins LecA and LecB from Pseudomonas aeruginosa.

Resorcinarene-Based Facial Glycosides: Implication of Detergent Flexibility on Membrane-Protein Stability.

As a membrane-mimetic system, detergent micelles are popularly used to extract membrane proteins from lipid environments and to maintain their solubility and stability in an aqueous medium. However, many membrane proteins encapsulated in conventional detergents tend to undergo structural degradation during extraction and purification, thus necessitating the development of new agents with enhanced properties. In the current study, two classes of new amphiphiles are introduced, resorcinarene-based glucoside and maltoside amphiphiles (designated RGAs and RMAs, respectively), for which the alkyl chains are facially segregated from the carbohydrate head groups. Of these facial amphiphiles, two RGAs (RGA-C11 and RGA-C13) conferred markedly enhanced stability to four tested membrane proteins compared to a gold-standard conventional detergent. The relatively high water solubility and micellar stability of the RGAs compared to the RMAs, along with their generally favourable behaviours for membrane protein stabilisation described here, are likely to be, at least in part, a result of the high conformational flexibility of these glucosides. This study suggests that flexibility could be an important factor in determining the suitability of new detergents for membrane protein studies.

Photoswitchable carbohydrate-based fluorosurfactants as tuneable ice recrystallization inhibitors

Cryopreservation is an important technique employed for the storage and preservation of biological tissues and cells. The limited effectiveness and significant toxicity of conventionally-used cryoprotectants, such as DMSO, have prompted efforts toward the rational design of less toxic alternatives, including carbohydrate-based surfactants. In this paper, we report the modular synthesis and ice recrystallization inhibition (IRI) activity of a library of variably substituted, carbohydrate-based fluorosurfactants. Carbohydrate-based fluorosurfactants possessed a variable mono- or disaccharide head group appended to a hydrophobic fluoroalkyl-substituted azobenzene tail group. Light-addressable fluorosurfactants displayed weak-to-moderate IRI activity that could be tuned through selection of carbohydrate head group, position of the trifluoroalkyl group on the azobenzene ring, and isomeric state of the azobenzene tail fragment.

Inverted Micelle-in-Micelle Configuration in Cationic/Carbohydrate Surfactant Mixtures.

Nuclear magnetic resonance is applied to investigate the relative positions and interactions between cationic and non-ionic carbohydrate-based surfactants in mixed micelles with D2 O as the solvent. This is accomplished by using relaxation measurements [spin-lattice (T1 ) and spin-spin (T2 ) analysis] and nuclear Overhauser effect spectroscopy (NOESY). This study focuses on the interactions of n-octyl β-d-glucopyranoside (C8G1) and β-d-xylopyranoside (C8X1) with the cationic surfactant hexadecyltrimethylammonium bromide (C16 TAB). Whereas the interactions between carbohydrate and cationic surfactants are thermodynamically favorable, the NOESY results suggest that both of the sugar head groups are located preferentially at the interior core of the mixed micelles, so that they are not directly exposed to the bulk solution. The more hydrophilic sugar headgroups of C8G1 have more mobility than sugar heads of C8X1 owing to increased hydration. Herein, an inverted carbohydrate configuration in mixed micelles is proposed for the first time and supported by fluorescence spectroscopy experiments. This inverted carbohydrate headgroup configuration would limit the use of these mixed surfactants when access to the carbohydrate headgroup is important, but may present new opportunities where the carbohydrate-rich core of the micelles can be exploited.

Design and synthesis of fluorescent glycolipid photoaffinity probes and their photoreactivity

Glycolipid–protein interactions at the cell surface are implicated in various biological processes. Toward the investigation of glycolipid binding proteins, we designed and synthesized trifunctional photoaffinity probes, which present a sugar head group with a triazole linkage to the lipid tail unit containing a photoreactive group and a fluorescent tag. The glycolipid photoaffinity probes bearing benzophenone group or diazirine group were evaluated for their photocrosslinking reactivity toward a carbohydrate head group specific protein. The diazirine based glycolipid photoaffinity probe was found to be more effective than the benzophenone-based probe in a comparative analysis involving a competitive ligand to distinguish a specific binding protein.

Separation and analysis of mono-glucosylated lipids in brain and skin by hydrophilic interaction chromatography based on carbohydrate and lipid moiety.

Mono-glycosylated sphingolipids and glycerophospholipids play important roles in diverse biological processes and are linked to a variety of pathologies, such as Parkinson disease. The precise identification of the carbohydrate head group of these lipids is complicated by their isobaric nature and by substantial differences in concentration in different biological samples. To overcome these obstacles, we developed a zwitterionic (ZIC)-hydrophilic interaction chromatography (HILIC) electrospray ionization tandem mass spectrometry method. ZIC-HILIC preferentially retains inositol, followed by glucose- and galactose-featuring lipids. Comparison with unmodified silica gel HILIC stationary phase revealed different retention specificity. To evaluate the precision of ZIC-HILIC, we quantified glucosyl- (GlcCer) and galactosylceramides (GalCer) in seven different regions of the mouse brain and discovered that GlcCer and GalCer concentrations are inversely related. The highest GalCer (lowest GlcCer) content was found in the medulla oblongata and hippocampus, whereas the highest GlcCer (lowest GalCer) content was found in other regions. With a neutral loss scan, ZIC-HILIC resolved glucosylceramide species featuring non-hydroxylated fatty acid, hydroxylated fatty acid, and trihydroxy sphingoid bases in mouse epidermis samples. This demonstrates that our ZIC-HILIC-based approach is a valuable tool for characterizing the structural diversity of mono-glucosylated lipids in biological material and for quantifying these important lipids.

Molecular Insights into the Low Permeability Barrier of Gram-Negative Pathogens

Discovery of new antibiotics effective against Gram-negative bacteria is hampered by the low permeability barrier provided by two-membrane cell envelopes. The outer membrane (OM) is an asymmetric bilayer of lipopolysaccharides (LPS) and phospholipids and is highly impermeable to noxious compounds. Molecular characterization of the OM is essential to understand how antibiotics penetrate across the lipid barrier of Gram-negative bacteria as it can lead to new therapeutic strategies and rational design of novel compounds. We have used both all-atom and coarse-grained molecular dynamics simulations to characterize the fully assembled OM bilayer of Pseudomonas aeruginosa, a human pathogen resistant to most clinical antibiotics. In our model, we represent the most abundant LPS of P. aeruginosa corresponding to the A band O-antigen. Our results show that the LPS behaves differently from normal glycerophospholipids, affecting several physical properties of the LPS-containing bilayers. The extensive hydrogen bond network of the LPS decreases the lateral mobility of the lipid bilayer. In addition, the highly charged condensation closer to water-membrane interface, and the massive and extended carbohydrate head groups increase water permeation, which propagates to the aliphatic tails of the inner leaflet. This effect contributes to the low ordering of the lipids in the inner leaflet and leads to expanded area per lipid. Thermodynamic data suggest that the outer membrane by itself prevents the translocation of drugs. However, this barrier is dramatically decreased when assisted by porins and other transport channels in OM.

Analysis of Sphingolipid Synthesis and Transport by Metabolic Labeling of Cultured Cells with [³H]Serine.

Analysis of lipid biosynthesis by radioactive precursor incorporation provides information on metabolic rates and the identity of rate-limiting enzymes and transporters. The biosynthesis of sphingolipids in cultured cells is initiated in the endoplasmic reticulum (ER) by the formation of a sphingoid base from serine and palmitoyl-CoA. N-acylation of the sphingoid base produces ceramide, which is transported to the Golgi apparatus where phosphocholine or carbohydrate headgroups are added to form sphingomyelin (SM) and complex glycosphingolipids (GSLs), respectively. Herein is described a protocol to measure ceramide and SM biosynthesis in cultured cells based on [(3)H]serine incorporation at the first step in the pathway. The method can be used to assay the effect of pharmacological and genetic manipulations on ceramide synthesis and transport to the Golgi apparatus.

Increased Emulsion Stability for Reverse Y‐Shaped Sugar‐Based Surfactants

AbstractA series of reverse Y‐shaped surfactants containing aromatic and aliphatic linkers to combine two short hydrocarbon chains and one carbohydrate head group was prepared. Liquid crystalline behavior, air–water interfacial properties, and efficiency as an emulsifier was investigated for each reverse Y‐shaped surfactant. All reverse Y‐shaped surfactants mediated higher emulsion stabilities for water‐in‐oil compared to common typical reference surfactants, reflecting an improved ability to cope with a curvature towards water. The introduction of a benzene ring into the linker substantially increased the affinity of the surfactant for hydrophobic media, resulting in improved emulsion stability for both water‐in‐oil and oil‐in‐water.