Sugar Headgroup Research Articles

Synthesis of a Versatile Building Block for the Preparation of 6-N-Derivatized α-Galactosyl Ceramides: Rapid Access to Biologically Active Glycolipids

A concise route to the 6-azido-6-deoxy-α-galactosyl-phytosphingosine derivative 9 is reported. Orthogonal protection of the two amino groups allows elaboration of 9 into a range of 6-N-derivatized α-galactosyl ceramides by late-stage introduction of the acyl chain of the ceramide and the 6-N-group in the sugar headgroup. Biologically active glycolipids 6 and 8 have been synthesized to illustrate the applicability of the approach.

Glycocluster Design for Improved Avidity and Selectivity in Blocking Human Lectin/Plant Toxin Binding to Glycoproteins and Cells

Blocking lectin/toxin binding to human cells by suitable inhibitors can therapeutically protect them from harmful effects. Clustered design of ligand presentation holds the promise of affinity increase relative to the free sugar and inherent selectivity among lectin targets. Using first a solid-phase assay with a glycoprotein presenting N-glycans as lectin-reactive probe, we assessed the inhibitory potency of bi- to tetravalent clusters on a plant toxin and three human adhesion/growth-regulatory lectins. Enhanced avidity relative to the free sugar was detected together with lectin-type selectivity. These effects were confirmed on the level of cells in vitro, also for two leguminous lectins. The lack of toxicity in cell proliferation assays excluded concerns to further work on these compounds. The given cluster design and the strategic combination of the two assay systems of increasing biorelevance will thus be helpful to take the next steps in drug development, e.g. tailoring the sugar headgroup.

New Unsymmetrical Bolaamphiphiles: Synthesis, Assembly with DNA, and Application for Gene Delivery

The success in gene therapy relies strongly on new efficient gene delivery vectors. Nonviral vectors based on lipids and polymers constitute an important alternative to the viral vectors. However, the key problem with these vectors is the poor structural control of their DNA complexes. In the present work, following new design we synthesized unsymmetrical bolaamphiphiles, molecules bearing neutral sugar (gluconic acid) and dicationic ornithine head groups connected by different long hydrophobic spacers. Within this design, a positively charged headgroup is expected to bind DNA, the hydrophobic spacer is to drive the formation of a monolayer membrane shell around DNA, while the neutral group is to be exposed outside of the complex. Our fluorescence and gel electrophoresis data showed that self-assembly of bolas and their interaction with DNA depend strongly on the bola structure. The size of bola/DNA complexes (bolaplexes) estimated from dynamic light scattering data was ∼100 nm at low N/P (cationic nitrogen/DNA phosphate molar ratio), while at higher N/Ps it was significantly larger due to neutralization of their surface charge. Atomic force microscopy studies revealed nanostructural rod-shaped or spherical morphology of the bolaplexes. Transfection efficiency of the bolaplexes in vitro was significant when either DOPE or chloroquine were used as helping agents, suggesting that the key barrier for their internalization is the endosomal escape. Finally, all bolas showed low cytotoxicity (cell viability >80%). The present results show that bolas are prospective candidates for construction of nonviral gene delivery vectors. We believe that further optimization of polar head groups and a hydrophobic spacer in the bolas will lead to vectors with controlled small size and high transfection efficiency.

Intramolecular stable carbon isotopic analysis of archaeal glycosyl tetraether lipids

Glycolipids are prominent constituents in the membranes of cells from all domains of life. For example, diglycosyl-glycerol dibiphytanyl glycerol tetraethers (2Gly-GDGTs) are associated with methanotrophic ANME-1 archaea and heterotrophic benthic archaea, two archaeal groups of global biogeochemical importance. The hydrophobic biphytane moieties of 2Gly-GDGTs from these two uncultivated archaeal groups exhibit distinct carbon isotopic compositions. To explore whether the isotopic compositions of the sugar headgroups provide additional information on the metabolism of their producers, we developed a procedure to analyze the δ(13)C values of glycosidic headgroups. Successful determination was achieved by (1) monitoring the contamination from free sugars during lipid extraction and preparation, (2) optimizing the hydrolytic conditions for glycolipids, and (3) derivatizing the resulting sugars into aldononitrile acetate derivatives, which are stable enough to withstand a subsequent column purification step. First results of δ(13)C values of sugars cleaved from 2Gly-GDGTs in two marine sediment samples, one containing predominantly ANME-1 archaea and the other benthic archaea, were obtained and compared with the δ(13)C values of the corresponding biphytanes. In both samples the dominant sugar headgroups were enriched in (13)C relative to the corresponding major biphytane. This (13)C enrichment was significantly larger in the putative major glycolipids from ANME-1 archaea (∼15‰) than in those from benthic archaea (<7‰). This method opens a new analytical window for the examination of carbon isotopic relationships between sugars and lipids in uncultivated organisms.

Structural Determination and Tryptophan Fluorescence of Heterokaryon Incompatibility C2 Protein (HET-C2), a Fungal Glycolipid Transfer Protein (GLTP), Provide Novel Insights into Glycolipid Specificity and Membrane Interaction by the GLTP Fold

HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 A) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp(66), Asn(70), Lys(73), Trp(109), and His(147)) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by alpha-helices and a cooperative thermal unfolding transition of 49 degrees C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UV CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at approximately 355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (approximately 6 nm) permitting determination of binding affinities. The unique positioning of Trp(208) at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.

Beta-mannosylceramide represents a new class of NKT agonists that induces tumor immunity by a novel IFN-γ-independent mechanism. (89.7)

Abstract Type I NKT cells are a unique lymphocyte population which recognize lipid antigens presented by the non-classical MHC-like molecule CD1d. Activation of type I NKT cells by α-galactosylceramide (α-GC) induces tumor immunity by IFN-γ dependent mechanisms. In this study, we characterized how modifications of the sugar head group of α-GC impacted tumor protection in an in vivo mouse model of lung metastasis. β-mannosylceramide (β-ManCer), but not α-mannosylceramide or α-fucosylceramide, induced strong type I NKT cell-dependent tumor immunity. Because this is the first report of a β-linked glycosylcermide capable of inducing significant type I NKT cell-mediated tumor protection, we further characterized the mechanism by which β-ManCer induced tumor elimination. Interestingly, unlike α-GC and its analogs which protected through IFN-γ dependent mechanisms, β-ManCer induced very little cytokine production both in vitro and in vivo and retained protection in the absence of IFN-γ. Rather, β-ManCer protected through a TNF-α and nitric oxide synthase-dependent mechanism novel for NKT cells. Simultaneous administration of sub-therapeutic doses of β-ManCer and α-GC resulted in synergistic reduction in tumor burden, supporting the conclusion that these glycolipids work through different mechanisms. The distinct mechanism of action of β-ManCer and its ability to synergize with α-GC suggest that β-ManCer is the first representative of a new class of type I NKT cell agonists.

Small-Molecule Ligands of GD2 Ganglioside, Designed from NMR Studies, Exhibit Induced-Fit Binding and Bioactivity

Ganglioside GD2 is a cell surface glycosphingolipid. Targeting of GD2, i.e., by anti-GD2 mAb 3F8, is used clinically for cancer diagnosis, prognosis, and therapy. Here, the conformations of free GD2, and of GD2 bound to mAb 3F8, were resolved by saturation transfer difference NMR and molecular modeling. Then, three small-molecule cyclic peptide ligands that bind to GD2 selectively were designed. Transferred nuclear Overhauser enhancement of the GD2-bound conformation of the peptide ligands showed an induced-fit binding mechanism. The mAb 3F8 and the peptidic GD2 ligands mediate similar biological functions in cell-based assays of calcium fluxes and src activation. Thus, small molecules can selectively and functionally interact with a sugar head group. This work furthers the concept of rationally designing ligands for carbohydrate targets, and may be expanded to other clinically relevant gangliosides.

Glycolipid Acquisition by Human Glycolipid Transfer Protein Dramatically Alters Intrinsic Tryptophan Fluorescence: INSIGHTS INTO GLYCOLIPID BINDING AFFINITY

Glycolipid transfer proteins (GLTPs) are small, soluble proteins that selectively accelerate the intermembrane transfer of glycolipids. The GLTP fold is conformationally unique among lipid binding/transfer proteins and serves as the prototype and founding member of the new GLTP superfamily. In the present study, changes in human GLTP tryptophan fluorescence, induced by membrane vesicles containing glycolipid, are shown to reflect glycolipid binding when vesicle concentrations are low. Characterization of the glycolipid-induced "signature response," i.e. approximately 40% decrease in Trp intensity and approximately 12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine vesicles, prepared by sonication or extrusion and containing embedded glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate that major conformational alterations are not responsible for the glycolipid-induced GLTP signature response. Instead, glycolipid binding alters the local environment of Trp-96, which accounts for approximately 70% of total emission intensity of three Trp residues in GLTP and provides a stacking platform that aids formation of a hydrogen bond network with the ceramide-linked sugar of the glycolipid headgroup. The changes in Trp signal were used to quantitatively assess human GLTP binding affinity for various lipids including glycolipids containing different sugar headgroups and homogenous acyl chains. The presence of the glycolipid acyl chain and at least one sugar were essential for achieving a low-to-submicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity.

Detergent Localization In Model Proteo-bicelles

Several methods for crystallization of membrane proteins for structure determination have been published, including those which use discoidal membranes called bicelles. The bicelle-based method has proven to be a stable platform resulting in well-diffracting crystals of G-protein coupled receptors, and other proteins including bacteriorhodopsin. In typical ‘empty’ bicelles - those devoid of protein - long chain phospholipids make up the core of the disk, and micelle-forming detergents “cap” the disk by forming the rim. Short chain phospholipid and cholate analog detergents (e.g. DiC6PC, DHPC and CHAPSO) are included in this “capping” category having been shown to associate preferentially with the bicelle rim. In proteo-bicelles, formed by the mixture of protein-detergent complexes with preformed bicelles, a second type of detergent containing a sugar headgroup (e.g. octylglucoside and maltoside) is introduced. This second type of detergent has proven to be effective in membrane protein purification and stabilization. In this study, we use small angle neutron and x-ray scattering to explore the structure and phase-behavior changes induced by sugar-headgroup type detergents on bicelles and their influence on bicelle-based membrane protein crystallization. Preliminary results suggest that these sugar headgroup amphiphiles partition more heavily into the core of bicelles than their short chain phospholipid and cholate counterparts. An understanding of the roles of these amphiphiles in modifying the meso-structures which eventually lead to crystallization is a critical next step in furthering our understanding of the membrane protein crystallization process in these systems.

Spectral Characterization Of Het-C2, A Glycolipid-transfer Protein

Het-C2 is a small 23 kDa protein, isolated from the fungi Podospora anserina, homologous to mammalian GLTP, and capable of transferring glycospingolipids in vitro. The crystal structure of Het-C2 is unknown, but molecular models suggest conservation of the GLTP-fold. Here, the locations of the Trp residues in Het-C2 have been investigated to gain further insights into their function. Sequence homology shows one of Het-C2’s two Trp residues aligned with GLTP Trp96 which resides in the sugar headgroup liganding site. The other Het-C2 Trp did not align with either of GLTP's other two Trp residues. The Trp fluorescence spectrum of native Het-C2 exhibited an emission maximum at 355nm which red shifted 2nm upon denaturation with 8M urea, indicating Trp localization to a more polar environment. Acrylamide and KI quenched >90% of the average Trp fluorescence confirming that Het-C2 Trp residues are not buried in hydrophobic environments but reside in exposed polar regions. The linearity of Stern-Volmer plots for native Het-C2 and urea-denatured (8M) Het-C2 suggested dynamic quenching at physiological pH and ionic strength. The Stern-Volmer constants were higher for native protein than denatured protein. Upon interaction with probe-sonicated POPC vesicles, the Trp emission maximum blue shifted (∼2nm) and decreased in intensity (∼13.5%). Including glycolipid in the vesicles slightly enhanced the blue shift (∼3nm) and significantly decreased Trp intensity (∼21%). Far-UV-CD of Het-C2 showed secondary structure dominated by alpha-helices and with a highly cooperative, thermally induced melting transition near 43°C. Near-UV-CD indicated the induced optical activity of Trp/Tyr residues was unaffected by interaction with vesicles lacking or containing glycolipid. The results are analyzed and discussed within the context of the known locations and functions of human GLTP's three Trp residues. [Support: NIH/NIGMS GM45928 & GM34847, NIH/NCI CA121493, The Hormel & Mayo Foundations]

Computational Structure Activity Relationship Studies on the CD1d/Glycolipid/TCR Complex Using AMBER and AUTODOCK

The human CD1d protein presents a wide range of lipids to the TCR of invariant natural killer T cells (iNKT). Alpha-GalCer is one of the most potent iNKT stimulatory ligands presented by CD1d. The lipid portion of this ligand has been extensively investigated over the course of the past few years; however, the sugar portion of the ligand has received minimal attention. The following research focuses on computationally analyzing the recently crystallized CD1d/alpha-GalCer/TCR tertiary complex by molecular dynamics simulations using AMBER along with studying the structure activity relationship of the sugar headgroup also by simulation and docking using Autodock for a variety of alpha-GalCer analogs. The results show that the crystal structure is stable under simulation making it an accurate representation of the CD1d/alpha-GalCer/TCR complex and that modifications to the C2' and C3' positions of the sugar are not tolerated by the tertiary complex, whereas modifications to the C4' position are tolerated.

NMR spectral mapping of Lipid A molecular patterns affected by interaction with the innate immune receptor CD14

Soluble CD14 (sCD14) is a serum glycoprotein that binds to the Lipid A moiety of lipopolysaccharides (LPS) with high affinity as part of the innate immune response to bacterial endotoxins. In order to investigate structural interactions of Lipid A with sCD14, we have prepared an isotopically labeled form of a fully active and chemically defined endotoxin, Kdo 2–Lipid A, which allowed us to carry out detailed NMR spectral mapping of this agonist ligand bound to sCD14 and identify for the first time structural regions that are strongly affected during complex formation with sCD14. These map to two adjacent areas comprising the lower portions of the sugar headgroup and upper half of the acyl chains I, III, and V, which are spatially proximal to the 1- and 4′-phosphate ends. Additionally, we have detected for the first time, presence of differential dynamic behavior for the affected resonances, suggesting a likely role for dynamics in the mechanism of Lipid A pattern recognition by sCD14.

Calix[n]arene‐Based Glycoclusters: Bioactivity of Thiourea‐Linked Galactose/Lactose Moieties as Inhibitors of Binding of Medically Relevant Lectins to a Glycoprotein and Cell‐Surface Glycoconjugates and Selectivity among Human Adhesion/Growth‐Regulatory Galectins

Growing insights into the functionality of lectin-carbohydrate interactions are identifying attractive new targets for drug design. As glycan recognition is regulated by the structure of the sugar epitope and also by topological aspects of its presentation, a suitable arrangement of ligands in synthetic glycoclusters has the potential to enhance their avidity and selectivity. If adequately realized, such compounds might find medical applications. This is why we focused on lectins of clinical interest, acting either as a potent biohazard (a toxin from Viscum album L. akin to ricin) or as a factor in tumor progression (human galectins-1, -3, and -4). Using a set of 14 calix[n]arenes (n=4, 6, and 8) with thiourea-linked galactose or lactose moieties, we first ascertained the lectin-binding properties of the derivatized sugar head groups conjugated to the synthetic macrocycles. Despite their high degree of flexibility, the calix[6,8]arenes proved especially effective for the plant AB-toxin, in the solid-phase model system with a single glycoprotein (asialofetuin) and with human tumor cells in vitro. The bioactivity of the calix[n]arenes was also proven for human galectins. Notably, selectivity for the tested tandem-repeat-type galectin-4 among the three subgroups was determined at the level of solid-phase and cell assays, the large flexible macrocycles again figuring prominently as inhibitors. Alternate and cone versions of calix[4]arene with lactose units distinguished between galectins-1 and -4 versus galectin-3 in cell assays. The results thus revealed bioactivity of galactose-/lactose-presenting calix[n]arenes for medically relevant lectins and selectivity within the family of adhesion/growth-regulatory human galectins.

Manifestation of a Chiral Smectic C Phase in Diphenylbutadiene‐Cored Bolaamphiphilic Sugars

AbstractA series of symmetrical bolaamphiphiles possessing a diphenylbutadiene core and glucopyranoside head groups linked together by oligomethylene spacers, were synthesized and their thermotropic liquid crystalline properties investigated by polarized light optical microscopy, differential scanning calorimetry, X‐ray diffraction and electro‐optic switching. In spite of the presence of chiral centers, amphiphilic sugars in general do not exhibit macroscopic chirality and this phenomenon is attributed to strong hydrogen bonding between sugar head groups resulting in microphase‐segregated layer like arrangements. In the present study all the molecules investigated exhibited the smectic C* phase, i.e., tilted lamellar phase with macroscopic chiral ordering of the molecules. The stability of this phase increased with increase in the length of the oligomethylene spacers. Whereas for derivatives with spacers containing ≤4 methylene groups, the smectic C* phase was observed only in the cooling phase, for those containing spacers with ≥5 methylene groups this phase was observed both in the heating and cooling cycles. The absorption and fluorescence spectra of these materials suggest that the unusual observation of macroscopic chirality in these bolaamphiphiles containing free hydroxyl groups could be attributed to self‐aggregating behavior of the diphenylbutadiene core.

Condensing and Fluidizing Effects of Ganglioside GM1 on Phospholipid Films

Mixed monolayers of the ganglioside GM1 and the lipid dipalmitoylphosphatidlycholine (DPPC) at air-water and solid-air interfaces were investigated using various biophysical techniques to ascertain the location and phase behavior of the ganglioside molecules in a mixed membrane. The effects induced by GM1 on the mean molecular area of the binary mixtures and the phase behavior of DPPC were followed for GM1 concentrations ranging from 5 to 70mol %. Surface pressure isotherms and fluorescence microscopy imaging of domain formation indicate that at low concentrations of GM1 (<25mol %), the monolayer becomes continually more condensed than DPPC upon further addition of ganglioside. At higher GM1 concentrations (>25mol %), the mixed monolayer becomes more expanded or fluid-like. After deposition onto a solid substrate, atomic force microscopy imaging of these lipid monolayers showed that GM1 and DPPC pack cooperatively in the condensed phase domain to form geometrically packed complexes that are more ordered than either individual component as evidenced by a more extended total height of the complex arising from a well-packed hydrocarbon tail region. Grazing incidence x-ray diffraction on the DPPC/GM1 binary mixture provides evidence that ordering can emerge when two otherwise fluid components are mixed together. The addition of GM1 to DPPC gives rise to a unit cell that differs from that of a pure DPPC monolayer. To determine the region of the GM1 molecule that interacts with the DPPC molecule and causes condensation and subsequent expansion of the monolayer, surface pressure isotherms were obtained with molecules modeling the backbone or headgroup portions of the GM1 molecule. The observed concentration-dependent condensing and fluidizing effects are specific to the rigid, sugar headgroup portion of the GM1 molecule.

Controlled aggregation of adenine by sugars: physicochemical studies, molecular modelling simulations of sugar–aromatic CH–π stacking interactions, and biological significance

CH-Pi stacking interactions between carbohydrates and aromatic compounds play a central role in biomolecular recognition, especially in lectin-sugar and protein-glycolipid systems. In the present study, we have measured the solubility of the sparingly soluble aromatic base adenine in presence of various saccharides as an approach to investigate the interaction between adenine and sugars. Above 82.5 mM, adenine solutions gradually formed a crystalline precipitate which could be quantified by spectrophotometric turbidity measurements. Precipitation of adenine was increased by salts (NaCl and NaF) whereas it was prevented by DMSO, in agreement with the involvement of hydrophobic interactions (pi-pi stacking) in the vertical stacking of adenine molecules. Several monosaccharides and disaccharides were found to increase adenine solubility, with the following order: D-galactose = D-lactose > D-sucrose > D-glucose = D-maltose > D-ribose > D-fructose. Molecular mechanics simulations indicated that the potent cosolvent effect of beta-D-galactopyranose was probably mediated by CH-pi stacking interactions between its apolar surface and the aromatic structure of adenine. The polar OH groups of the sugars interacted with surrounding water molecules, ensuring the solubility of sugar-adenine complexes. In contrast, beta-D-fructofuranose, which has two polar faces, did not stack onto adenine and had a weak cosolvent effect. CH-pi stacking interactions were also demonstrated between 6-methylpurine and the sugar head group of glycolipids (glucosyl-, galactosyl- and lactosylceramide) but not with the charged head group of phosphatidylinositol-4,5-diphosphate. These data indicate that galactose-containing molecules have a high stacking propensity for aromatic compounds such as adenine, due to the specific structure of the galactose cycle.

Structural preferences of dioleoyl glycolipids with mono- and disaccharide head groups

The structural preferences of 1,2-dioleoyl- sn-glycerol glycolipids with glucose, galactose, maltose, and cellobiose as sugar head group were investigated under near physiological conditions with Fourier-transform infrared spectroscopy (FT-IR) and synchrotron radiation small-angle X-ray scattering (SAXS). Whereas all glycolipids have a very high fluidity at temperatures above 0 °C, the mono- and disaccharide compounds differ considerably in their aggregate structures. The monosaccharide compounds adopt only inverted hexagonal (H II) structures in the temperature range 5–70 °C, while the disaccharide compounds adopt only multilamellar structures. Since these and similar glycolipids are frequently found in nature, these data should be of relevance for the function of their host cell membranes.

Studies of the mesomorphic behavior of bivalent carbohydrate amphiphiles

A homologous series of bivalent glycolipids constituted of 1,3-bis-O-(β-D-glucopyranosyl)-2-O-alkyl glycerol, bearing two sugar head groups and one alkyl chain on a glycerol backbone, was synthesized. The mesomorphic behaviour was investigated with the aid of polarizing optical microscopy, differential scanning calorimetry and X-ray diffraction methods. The homologous members exhibit a smectic A phase, consistently with a partial bilayer structure. From experimental d-spacing and computationally derived length of the molecules, it is suggested that a partial interdigitation of the molecules is present in the bilayer structure.

Sophorolipids Having Enhanced Antibacterial Activity

Sophorolipids Having Enhanced Antibacterial Activity

Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation

OmpG, a monomeric pore-forming protein from Escherichia coli outer membranes, was refolded from inclusion bodies and crystallized in two different conformations. The OmpG channel is a 14-stranded beta-barrel, with short periplasmic turns and seven extracellular loops. Crystals grown at neutral pH show the channel in the open state at 2.3 A resolution. In the 2.7 A structure of crystals grown at pH 5.6, the pore is blocked by loop 6, which folds across the channel. The rearrangement of loop 6 appears to be triggered by a pair of histidine residues, which repel one another at acidic pH, resulting in the breakage of neighbouring H-bonds and a lengthening of loop 6 from 10 to 17 residues. A total of 151 ordered LDAO detergent molecules were found in the 2.3 A structure, mostly on the hydrophobic outer surface of OmpG, mimicking the outer membrane lipid bilayer, with three LDAO molecules in the open pore. In the 2.7 A structure, OmpG binds one OG and one glucose molecule as sugar substrates in the closed pore.