Surface Density of Ligands Controls In-Plane and Aggregative Modes of Multivalent Glycovesicle-Lectin Recognitions.

Glycovesicles are ideal tools to delineate finer mechanisms of the interactions at the biological cell membranes. Multivalency forms the basis which, in turn, should surpass more than one mechanism in order to maintain multiple roles that the ligand-lectin interactions encounter. Ligand densities hold a prime control to attenuate the interactions. In the present study, mannose trisaccharide interacting with a cognate receptor, namely, Con A, is assessed at the vesicle surface. Synthetic (1→3)(1→6)-branched mannose trisaccharides tethered with a diacetylene monomer and glycovesicles of varying sugar densities were prepared. The polydiacetylene vesicles were prepared by maintaining uniform lipid concentrations. The interactions of the glycovesicles with the lectin were probed through dynamic light scattering and UV-Vis spectroscopy techniques. Binding efficacies were assessed by surface plasmon resonance. Aggregative and in-plane modes of interactions show ligand-density dependence at the vesicle surface. Vesicles with sparsely populated ligands engage lectin in an aggregative mode (trans-), leading to a cross-linked complex formation. Whereas glycovesicles embedded with dense ligands engage lectin interaction in an in-plane mode intramolecularly (cis-). Sub-nanomolar dissociation constants govern the intramolecular interaction occurring within the plane of the vesicle, and are more efficacious than the aggregative intermolecular interactions.

Surface Ligand Density Switches Glycovesicles between Monomeric and Multimeric Lectin Recognition.

Carbohydrate-protein interactions define a multitude of cellular recognition events. We present herein synthetic glycovesicles as cell-surface mimics in order to switch the nature of lectin recognition. The covalent glycovesicles, constituted with diacetylene monomers of various ligand densities at their surfaces, are prepared through photo-polymerization. Vesicles with sparsely imbedded ligands engage in a lectin interaction leading to the formation of a dense, crosslinked multimeric complex. On the other hand, vesicles with many ligands, or completely covered with them, switch the lectin interaction to form a fully soluble monomeric complex, without crosslinking. Nanomolar dissociation constants govern these interactions, as assessed by a ligand-displacement assay. The study demonstrates the switching nature - between monomeric and multimeric - of the interaction as a function of ligand density in the vesicles; the results are directly relevant to understanding such a phenomenon occurring at cell surfaces.

Synthetic arabinomannan glycolipids impede mycobacterial growth, sliding motility and biofilm structure.

Mycobacterium has evolved distinct cell wall and strategies such as biofilm formation, which helps it to survive in hostile conditions. We have reported previously that arabinofuranoside containing glycolipids exhibit inhibition activities against the above functions of the mycobacterial species M. smegmatis. In search for activities mediated by oligosaccharide glycolipids, we report herein the inhibitory activities of a linear and a branched pentasaccharides having arabinan and mannan moieties. In the presence of the pentasaccharide glycolipids, a significant reduction in mycobacterial growth is observed, concomitant with reductions in sliding motility and colonization through biofilm formation, at the optimal glycolipid concentrations of 50-100 µg mL(-1). Especially the biofilm coat is ruptured by ~80-85 % in the presence of glycolipids. Pentasaccharides alone without the lipidic chain show only a weak effect. The glycolipids are non-toxic, as evaluated through their effect on RBCs. Analysis of the mycolic acid profile of glycolipid treated biofilm shows that α- and epoxy mycolic acids are downregulated significantly, in comparison to glycolipid untreated biofilms. Lipidomics profile analysis through mass spectrometry further reveals profound downregulation of phosphatidylinositol mannosides, acylatedphosphoglycerols and mycolic acid family, namely, keto-, alpha- and methoxymycolic acids.

Multivalent glycoliposomes and micelles to study carbohydrate-protein and carbohydrate-carbohydrate interactions.

This tutorial review describes multivalent carbohydrate-protein and carbohydrate-carbohydrate interaction studies that utilize self-assembled aggregates of thermodynamically stable liposomes and micelles. Strategies to prepare multivalent glycoliposomes and micelles include: (i) insertion of synthetic glycolipids into matrix lipids; (ii) preparation of glycolipids that aggregate to liposomes and micelles and (iii) modification of the hydrophilic surfaces with desired sugars. Several design strategies have been developed in order to obtain constituent glycolipids, having multivalent sugar moieties and their subsequent interactions with proteins were assessed in relation to the type of linkers that connect the hydrophilic and lipophilic segments. Lipophilic segments other than alkyl chains have also been developed. Polymer based glycoliposomes and micelles form an emphasis. Further, glycoliposomes facilitate studies of carbohydrate-carbohydrate interactions. An overview of the various types of glycoliposomes and micelles used to study carbohydrate-protein and carbohydrate-carbohydrate recognition phenomena is presented.

Synthesis of ß-arabinofuranoside glycolipids, studies of their binding to surfactant protein-A and effect on sliding motilities of M. smegmatis.

Surfactant protein A (SP-A), which is a lung innate immune system component, is known to bind glycolipids present at the cell surface of a mycobacterial pathogen. Lipoarabinomannan (LAM), a component of mycobacterial thick, waxy cell wall, is one of the glycolipid ligands for SP-A. In order to assess binding of synthetic glycolipids with SP-A and the glycosidic linkage preferences for the interaction, ß-arabinofuranoside trisaccharide glycolipids constituted with ß-(1→2), ß-(1→3) and ß-(1→2), ß-(1→5) linkages relevant to LAM were synthesized through chemical glycosylations. The efficacies of synthetic glycolipids to interact with SP-A were assessed by using the surface plasmon resonance (SPR) technique, from which association-dissociation rate constants and equilibrium binding constants were derived. The equilibrium binding constants of the interaction of two constitutionally varying ß-arabinofuranoside glycolipids with SP-A were found to be in the millimolar range. A comparison of the results with few α-anomeric arabinofuranoside glycolipids showed that glycolipids with ß-anomeric linkages were having relatively lower equilibrium binding constants than those with α-anomeric linkages in binding to the protein, whereas oligosaccharides alone, without lipidic chains, exhibited higher equilibrium binding constants. Further, the synthetic compounds inhibited the growth of mycobacteria and affected sliding motilities of the bacteria, although to an extent relatively lesser than that of synthetic compounds constituted with α-anomeric linkages.

Synthesis, biological studies of linear and branched arabinofuranoside-containing glycolipids and their interaction with surfactant protein A.

Oligoarabinofuranoside-containing glycolipids relevant to mycobacterial cell wall components were synthesized in order to understand the functional roles of such glycolipids. A series of linear tetra-, hexa-, octa- and a branched heptasaccharide oligoarabinofuranosides, with 1 â†’ 2 and 1 â†’ 5 α-linkages between the furanoside residues, were synthesized by chemical methods from readily available monomer building blocks. Upon the synthesis of glycolipids, constituted with a double alkyl chain-substituted sn-glycerol core and oligosaccharide fragments, biological studies were performed to identify the effect of synthetic glycolipids on the biofilm formation and sliding motilities of Mycobacterium smegmatis. Synthetic glycolipids and arabinofuranosides displayed an inhibitory effect on the growth profile, but mostly on the biofilm formation and maturation. Similarly, synthetic compounds also influenced the sliding motility of the bacteria. Further, biophysical studies were undertaken, so as to identify the interactions of the glycolipids with a pulmonary surfactant protein, namely surfactant protein A (SP-A), with the aid of the surface plasmon resonance technique. Specificities of each glycolipid interacting with SP-A were thus evaluated. From this study, glycolipids were found to exhibit higher apparent association constants than the corresponding oligosaccharide portion alone, without the double alkyl group-substituted glycerol core.

Synthetic arabinomannan glycolipids and their effects on growth and motility of the Mycobacterium smegmatis.

Arabinomannan-containing glycolipids, relevant to the mycobacterial cell-wall component lipoarabinomannan, were synthesized by chemical methods. The glycolipids were presented with tri- and tetrasaccharide arabinomannans as the sugar portion and a double alkyl chain as the lyophilic portion. Following synthesis, systematic biological and biophysical studies were undertaken in order to identify the effects of the glycolipids during mycobacterium growth. The studies included mycobacterial growth, biofilm formation and motility assays. From the studies, it was observed that the synthetic glycolipid with higher arabinan residues inhibited the mycobacterial growth, lessened the biofilm formation and impaired the motility of mycobacteria. A surface plasmon resonance study involving the immobilized glycan surface and the mycobacterial crude lysates as analytes showed specificities of the interactions. Further, it was found that cell lysates from motile bacteria bound oligosaccharide with higher affinity than non-motile bacteria.

Synthesis and mycobacterial growth inhibition activities of bivalent and monovalent arabinofuranoside containing alkyl glycosides.

Arabinofuranosides constitute one of the important components of cell wall structures of mycobacteria. With this importance of arabinofuranosides in mind, alkyl glycosides bearing arabinofuranoside trisaccharides were prepared, wherein the sugars were presented either in the monovalent or bivalent forms. Following the synthesis, the monovalent and bivalent alkyl glycosides were tested for their activities in a mycobacterial growth assay. The growth of the mycobacterial strain M. smegmatis was assessed in the presence of the alkyl glycosides and it was realized that the alkyl glycosides acted as inhibitors of the mycobacterial growth. The inhibition of the growth, caused by the above alkyl glycosides, was not observed for the arabinofuranose trisaccharide alone, without the alkyl groups, and for an alkyl glycoside bearing maltose as the sugar component.