Sepsis-trained macrophages promote antitumoral tissue-resident T cells.

Sepsis induces immune alterations, which last for months after the resolution of illness. The effect of this immunological reprogramming on the risk of developing cancer is unclear. Here we use a national claims database to show that sepsis survivors had a lower cumulative incidence of cancers than matched nonsevere infection survivors. We identify a chemokine network released from sepsis-trained resident macrophages that triggers tissue residency of T cells via CCR2 and CXCR6 stimulations as the immune mechanism responsible for this decreased risk of de novo tumor development after sepsis cure. While nonseptic inflammation does not provoke this network, laminarin injection could therapeutically reproduce the protective sepsis effect. This chemokine network and CXCR6 tissue-resident T cell accumulation were detected in humans with sepsis and were associated with prolonged survival in humans with cancer. These findings identify a therapeutically relevant antitumor consequence of sepsis-induced trained immunity.

Synthesis of galactomannan fragments to help NMR assignment of polysaccharides extracted from lichens.

The synthesis of six model trisaccharides representative of galactomannans produced by lichens was performed through stereoselective glycosylation. These standards include linear and branched galactomannans bearing either galactofuranosyl or galactopyranosyl entities. The complete assignment of 1H and 13C signals for both forms of synthetically reduced oligosaccharides was performed. The resulting NMR data were used to quickly demonstrate the structural characteristics of minor polysaccharides within different extracts of three representative lichens.

Galf-Specific Neolectins: Towards Promising Diagnostic Tools.

In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.

Ring-Size Memory of Galactose-Containing MS/MS Fragments: Application to the Detection of Galactofuranose in Oligosaccharides and Their Sequencing.

Analysis of glycans remains a difficult task due to their isomeric complexity. Despite recent progress, determining monosaccharide ring size, a type of isomerism, is still challenging due to the high flexibility of the five-membered ring (also called furanose). Galactose is a monosaccharide that can be naturally found in furanose configuration in plant and bacterial polysaccharides. In this study, we used the coupling of tandem mass spectrometry and infrared ion spectroscopy (MS/MS-IR) to investigate compounds containing galactofuranose and galactopyranose. We report the IR fingerprints of monosaccharide fragments and demonstrate for the first time galactose ring-size memory upon collision-induced dissociation (CID) conditions. The linkage of the galactose unit is further obtained by analyzing disaccharide fragments. These findings enable two possible applications. First, labeled oligosaccharide patterns can be analyzed by MS/MS-IR, yielding full sequence information, including the ring size of the galactose unit; second, MS/MS-IR can be readily applied to unlabeled oligosaccharides to rapidly identify the presence of a galactofuranose unit, as a standalone analysis or prior to further sequencing.

Conformational preferences of the flexible galactofuranose sugar in gas-phase.

In contrast with the predominant pyranose form of galactose, galactofuranose is known to be highly flexible. Such flexibility poses a remarkable challenge in terms of structural studies, thus hindering the in depth understanding of the structure/function relationship in this rare sugar. A thorough computational study based on molecular dynamics and density functional theory supported by vibrational spectroscopy in the gas phase was carried out to provide a better understanding of the instrinsic conformational preferences of galactofuranose. Based on energetic and spectroscopic criteria, we report a subtantially reduced conformational landscape: methyl α-D-galactofuranose adopts E2/1E conformations and methyl ß-D-galactofuranose adopts 1T2/1E conformations.

Distinguishing Galactoside Isomers with Mass Spectrometry and Gas-Phase Infrared Spectroscopy.

Sequencing glycans is demanding due to their structural diversity. Compared to mammalian glycans, bacterial glycans pose a steeper challenge because they are constructed from a larger pool of monosaccharide building blocks, including pyranose and furanose isomers. Though mammalian glycans incorporate only the pyranose form of galactose (Galp), many pathogens, including Mycobacterium tuberculosis and Klebsiella pneumoniae, contain galactofuranose (Galf) residues in their cell envelope. Thus, glycan sequencing would benefit from methods to distinguish between pyranose and furanose isomers of different anomeric configurations. We used infrared multiple photon dissociation (IRMPD) spectroscopy with mass spectrometry (MS-IR) to differentiate between pyranose- and furanose-linked galactose residues. These targets pose a challenge for MS-IR because the saccharides lack basic groups, and galactofuranose residues are highly flexible. We postulated cationic groups that could complex through hydrogen bonding would offer a solution. Here, we present the first MS-IR analysis of hexose ammonium adducts. We compared their IR fingerprints with those of lithium adducts. We determined the diagnostic MS-IR signatures of the α- and ß-anomers of galactose in furanose and pyranose forms. We also showed these signatures could be applied to disaccharides to assign galactose ring size. Our findings highlight the utility of MS-IR for analyzing the unique substructures that occur in bacterial glycans.

Synthesis of an Exhaustive Library of Naturally Occurring Galf-Manp and Galp-Manp Disaccharides. Toward Fingerprinting According to Ring Size by Advanced Mass Spectrometry-Based IM-MS and IRMPD.

Nature offers a huge diversity of glycosidic derivatives. Among numerous structural modulations, the nature of the ring size of hexosides may induce significant differences on both biological and physicochemical properties of the glycoconjugate of interest. On this assumption, we expect that small disaccharides bearing either a furanosyl entity or a pyranosyl residue would give a specific signature, even in the gas phase. On the basis of the scope of mass spectrometry, two analytical techniques to register those signatures were considered, i.e., the ion mobility (IM) and the infrared multiple photon dissociation (IRMPD), in order to build up cross-linked databases. d-Galactose occurs in natural products in both tautomeric forms and presents all possible regioisomers when linked to d-mannose. Consequently, the four reducing Galf-Manp disaccharides as well as the four Galp-Manp counterparts were first synthesized according to a highly convergent approach, and IM-MS and IRMPD-MS data were second collected. Both techniques used afforded signatures, specific to the nature of the connectivity between the two glycosyl entities.

Arabinogalactan-like Glycoproteins from Ulva lactuca (Chlorophyta) Show Unique Features Compared to Land Plants AGPs.

Arabinogalactan proteins (AGPs) encompass a diverse group of plant cell wall proteoglycans, which play an essential role in plant development, signaling, plant-microbe interactions, and many others. Although they are widely distributed throughout the plant kingdom and extensively studied, they remain largely unexplored in the lower plants, especially in seaweeds. Ulva species have high economic potential since various applications were previously described including bioremediation, biofuel production, and as a source of bioactive compounds. This article presents the first experimental confirmation of AGP-like glycoproteins in Ulva species and provides a simple extraction protocol of Ulva lactuca AGP-like glycoproteins, their partial characterization and unique comparison to scarcely described Solanum lycopersicum AGPs. The reactivity with primary anti-AGP antibodies as well as Yariv reagent showed a great variety between Ulva lactuca and Solanum lycopersicum AGP-like glycoproteins. While the amino acid analysis of the AGP-like glycoproteins purified by the ß-d-glucosyl Yariv reagent showed a similarity between algal and land plant AGP-like glycoproteins, neutral saccharide analysis revealed unique glycosylation of the Ulva lactuca AGP-like glycoproteins. Surprisingly, arabinose and galactose were not the most prevalent monosaccharides and the most outstanding was the presence of 3-O-methyl-hexose, which has never been described in the AGPs. The exceptional structure of the Ulva lactuca AGP-like glycoproteins implies a specialized adaptation to the marine environment and might bring new insight into the evolution of the plant cell wall.

Modulation of the Activity and Regioselectivity of a Glycosidase: Development of a Convenient Tool for the Synthesis of Specific Disaccharides.

The synthesis of disaccharides, particularly those containing hexofuranoside rings, requires a large number of steps by classical chemical means. The use of glycosidases can be an alternative to limit the number of steps, as they catalyze the formation of controlled glycosidic bonds starting from simple and easy to access building blocks; the main drawbacks are the yields, due to the balance between the hydrolysis and transglycosylation of these enzymes, and the enzyme-dependent regioselectivity. To improve the yield of the synthesis of ß-d-galactofuranosyl-(1→X)-d-mannopyranosides catalyzed by an arabinofuranosidase, in this study we developed a strategy to mutate, then screen the catalyst, followed by a tailored molecular modeling methodology to rationalize the effects of the identified mutations. Two mutants with a 2.3 to 3.8-fold increase in transglycosylation yield were obtained, and in addition their accumulated regioisomer kinetic profiles were very different from the wild-type enzyme. Those differences were studied in silico by docking and molecular dynamics, and the methodology revealed a good predictive quality in regards with the regioisomer profiles, which is in good agreement with the experimental transglycosylation kinetics. So, by engineering CtAraf51, new biocatalysts were enabled to obtain the attractive central motif from the Leishmania lipophosphoglycan core with a higher yield and regioselectivity.

Formation of Amphiphilic Molecules from the Most Common Marine Polysaccharides, toward a Sustainable Alternative?

Marine polysaccharides are part of the huge seaweeds resources and present many applications for several industries. In order to widen their potential as additives or bioactive compounds, some structural modifications have been studied. Among them, simple hydrophobization reactions have been developed in order to yield to grafted polysaccharides bearing acyl-, aryl-, alkyl-, and alkenyl-groups or fatty acid chains. The resulting polymers are able to present modified physicochemical and/or biological properties of interest in the current pharmaceutical, cosmetics, or food fields. This review covers the chemical structures of the main marine polysaccharides, and then focuses on their structural modifications, and especially on hydrophobization reactions mainly esterification, acylation, alkylation, amidation, or even cross-linking reaction on native hydroxyl-, amine, or carboxylic acid functions. Finally, the question of the necessary requirement for more sustainable processes around these structural modulations of marine polysaccharides is addressed, considering the development of greener technologies applied to traditional polysaccharides.

Identification and Quantification of Any Isoforms of Carbohydrates by 2D UV-MS Fingerprinting of Cold Ions.

Biological functionality of isomeric carbohydrates may differ drastically, making their identifications indispensable in many applications of life science. Because of the large number of isoforms, structural assignment of saccharides is challenging and often requires a use of different orthogonal analytical techniques. We demonstrate that isomeric carbohydrates of any isoforms can be distinguished and quantified using solely the library-based method of 2D ultraviolet fragmentation spectroscopy-mass spectrometry (2D UV-MS) of cold ions. The two-dimensional "fingerprint" identities of UV transparent saccharides were revealed by photofragmentation of their noncovalent complexes with aromatic molecules. We assess the accuracy of the method by comparing the known relative concentrations of isomeric carbohydrates mixed in solution with the concentrations that were mathematically determined from the measured in the gas-phase fingerprints of the complexes. For the tested sets with up to five isomers of di- to heptasaccharides, the root-mean-square deviation of 3-5% was typically achieved. This indicates the expected level of accuracy in analysis of unknown mixtures for isomeric carbohydrates of similar complexity.

6-Deoxy-6-fluoro galactofuranosides: regioselective glycosylation, unexpected reactivity, and anti-leishmanial activity.

Selective glycosylation of the C-6 fluorinated galactofuranosyl acceptor 2 was studied with four galactofuranosyl donors. It was highlighted that this electron-withdrawing atom strongly impacted the behavior of the acceptor, thus leading to unprecedented glycosylation pathways. Competition between expected glycosylation of 2, ring expansion of this acceptor and furanosylation, and intermolecular aglycon transfer was observed. Further investigation of the fluorinated synthetic compounds showed that the presence of fluorine atom contributed to increase the inhibition of the growth of Leishmania tarentolae, a non-pathogenic strain of Leishmania.

Spectroscopic diagnostic for the ring-size of carbohydrates in the gas phase: furanose and pyranose forms of GalNAc.

Hexoses are mainly found in nature in the pyranose form (6-membered ring). Yet, furanose forms (5-membered ring) are observed in some rare polysaccharides. Using IRMPD spectroscopy (InfraRed Multiple Photon Dissociation), we propose a straightforward diagnostic of the ring-size of N-acetyl galactosamine ions. The furanose form of N-acetyl galactosamine was synthesized and its protonated ion was isolated in an ion trap to measure its gas phase vibrational spectrum by IRMPD. Comparison with the IRMPD spectrum of its pyranose counterpart reveals that they have distinctive optical fingerprints. This new MS-based diagnostic opens the way to facile identification of the ring-size in oligosaccharides. Our experimental data also provide new insights to support the theoretical description of the conformational behavior of the furanose ring, which is notoriously more flexible than the pyranose form but remains difficult to assess.

Regioselective Galactofuranosylation for the Synthesis of Disaccharide Patterns Found in Pathogenic Microorganisms.

Koenigs-Knorr glycosylation of acceptors with more than one free hydroxyl group by 2,3,5,6-tetrabenzoyl galactofuranosyl bromide was performed using diphenylborinic acid 2-aminoethyl ester (DPBA) as inducer of regioselectivity. High regioselectivity for the glycosylation on the equatorial hydroxyl group of the acceptor was obtained thanks to the transient formation of a borinate adduct of the corresponding 1,2-cis diol. Nevertheless formation of orthoester byproducts hampered the efficiency of the method. Interestingly electron-withdrawing groups on O-6 or on C-1 of the acceptor displaced the reaction in favor of the desired galactofuranosyl containing disaccharide. The best yield was obtained for the furanosylation of p-nitrophenyl 6-O-acetyl mannopyranoside. Precursors of other disaccharides, found in the glycocalix of some pathogens, were synthesized according to the same protocol with yields ranging from 45 to 86%. This is a good alternative for the synthesis of biologically relevant glycoconjugates.

Synthesis and biological properties of galactofuranosyl-containing fluorescent dyes.

Two fluorescent galactofuranosides were synthesized and their biological activities evaluated on non-infected and Leishmania infected macrophages. Both tagged scaffolds were able to penetrate macrophages. Compared to the activity of the parent octyl galactofuranoside used as a reference, the fluorescein-conjugate showed altered biological properties while the rhodamine 6G one synergistically acted with the lipid chain to significantly increase antiparasitic activity.

Biocatalyzed synthesis of difuranosides and their ability to trigger production of TNF-α.

Transglycosylation reactions biocatalyzed by the native arabinofuranosidase Araf51 and using d-galactosyl, d-fucosyl and 6-deoxy-6-fluoro-D-galactosyl derivatives as donors and acceptors provided di-to pentahexofuranosides. The immunostimulatory potency of these compounds, and more especially their ability to induce production of TNF-α, was evaluated on the murine macrophage cell line, Raw 264.7. The results obtained showed concentration-dependent and most importantly, structure-dependent responses. Interestingly, oligoarabinofuranosides belonging to the oligopentafuranoside family displayed concentration-, chain length and aglycon-dependent bioactivities irrespective of their fine chemical variations. Thus, neo-oligofuranosides in D-Galf series, as well as their D-Fucf and 6-fluorinated counterparts are indeed potential sources of immunostimulating agents.

An ethoxylated surfactant enhances the penetration of the sulfated laminarin through leaf cuticle and stomata, leading to increased induced resistance against grapevine downy mildew.

Some ß-1,3-glucans and particularly sulfated laminarin (PS3) are known as resistance inducers (RIs) in grapevine against the downy mildew. However, their efficacy in vineyard is still often too low, which might be caused by a limited penetration through the leaf cuticle following spray application. We used (14) C-sucrose uptake experiments with grapevine leaves in order to select a surfactant as saccharide penetration enhancer. Our results showed that although sucrose foliar uptake was low, it was strongly enhanced by Dehscofix CO125 (DE), a highly ethoxylated surfactant. Fluorescent saccharides were then produced and laser scanning microscopy was used to analyze their foliar diffusion pattern in Arabidopsis thaliana and grapevine. Interestingly, sucrose and PS3 were seemingly able to penetrate the leaf cuticle only when formulated with DE. Diffusion could preferentially occur via stomata, anticlinal cell walls and trichomes. In grapevine, PS3 penetration rate was much higher on the stomateous abaxial surface of the leaf than on the adaxial surface. Finally, using DE allowed a higher level of downy mildew control by PS3, which corroborated diffusion observations. Our results have practical consequences for the improvement of treatments with saccharidic inducers on grape. That is, formulation of such RIs plays a critical role for their cuticular diffusion and consequently their efficacy. Also, spray application should preferentially target the abaxial surface of the leaves in order to maximize their penetration.

Semi-rational approach for converting a GH36 α-glycosidase into an α-transglycosidase.

A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions. In order to use them as catalysts for oligosaccharide synthesis, the balance between these two competing reactions has to be shifted toward transglycosylation. We previously designed a semi-rational approach to convert the Thermus thermophilus ß-glycosidases into transglycosidases by mutating highly conserved residues located around the -1 subsite. In an attempt to verify that this strategy could be a generic approach to turn glycosidases into transglycosidases, Geobacillus stearothermophilus α-galactosidase (AgaB) was selected in order to obtain α-transgalactosidases. This is of particular interest as, to date, there are no efficient α-galactosynthases, despite the considerable importance of α-galactooligosaccharides. Thus, by site-directed mutagenesis on 14 AgaB residues, 26 single mutants and 22 double mutants were created and screened, of which 11 single mutants and 6 double mutants exhibited improved synthetic activity, producing 4-nitrophenyl α-d-galactopyranosyl-(1,6)-α-d-galactopyranoside in 26-57% yields against only 22% when native AgaB was used. It is interesting to note that the best variant was obtained by mutating a second-shell residue, with no direct interaction with the substrate or a catalytic amino acid. As this approach has proved to be efficient with both α- and ß-glycosidases, it is a promising route to convert retaining glycosidases into transglycosidases.

Structural and biochemical characterization of the laminarinase ZgLamCGH16 from Zobellia galactanivorans suggests preferred recognition of branched laminarin.

Laminarin is a ß-1,3-D-glucan displaying occasional ß-1,6 branches. This storage polysaccharide of brown algae constitutes an abundant source of carbon for marine bacteria such as Zobellia galactanivorans. This marine member of the Bacteroidetes possesses five putative ß-1,3-glucanases [four belonging to glycosyl hydrolase family 16 (GH16) and one to GH64] with various modular architectures. Here, the characterization of the ß-glucanase ZgLamC is reported. The catalytic GH16 module (ZgLamCGH16) was produced in Escherichia coli and purified. This recombinant enzyme has a preferential specificity for laminarin but also a significant activity on mixed-linked glucan (MLG). The structure of an inactive mutant of ZgLamCGH16 in complex with a thio-ß-1,3-hexaglucan substrate unravelled a straight active-site cleft with three additional pockets flanking subsites -1, -2 and -3. These lateral pockets are occupied by a glycerol, an acetate ion and a chloride ion, respectively. The presence of these molecules in the vicinity of the O6 hydroxyl group of each glucose moiety suggests that ZgLamCGH16 accommodates branched laminarins as substrates. Altogether, ZgLamC is a secreted laminarinase that is likely to be involved in the initial step of degradation of branched laminarin, while the previously characterized ZgLamA efficiently degrades unbranched laminarin and oligo-laminarins.

Synthesis and evaluation of 1,2-trans alkyl galactofuranoside mimetics as mycobacteriostatic agents.

The simple octyl ß-D-galactofuranoside was previously described as a good bacteriostatic agent against Mycobacterium smegmatis, a non-pathogenic model of M. tuberculosis. In order to decipher its mechanism of action, STD NMR on whole M. smegmatis cells was implemented. It outlined the crucial role of the alkyl chain and the possibility of modulation on the furanosyl entity. Then, 16 new alkyl furanosides were synthesized in order to optimize the mycobacteriostatic activity. They all present the pending alkyl chain in a 1,2-trans configuration relative to the sugar ring. Three families were studied that differ by a substituent on the primary position of the galactofuranose ring, the series or the pending alkyl chain. Four of these neofuranosides showed growth inhibition inferior to the parent octyl ß-D-galactofuranoside. Double alkyl chains at C-1 and a polar substituent on the primary position of the furanoside significantly favored the activity. Finally, a mixed biantennary alkyl/aryl ß-D-galactofuranoside exhibited the best growth inhibition concentration at 90 µM.