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.

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.

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.

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.

Gas-Phase Behavior of Galactofuranosides upon Collisional Fragmentation: A Multistage High-Resolution Ion Mobility Study.

Carbohydrates are ubiquitous in nature but are among the least conserved biomolecules in life. These biopolymers pose a particular challenge to analytical chemists because of their high diversity and structural heterogeneity. In addition, they contain many isomerisms that complicate their structural characterization, notably by mass spectrometry. The tautomerism of the constitutive subunits is of particular interest. A given cyclized monosaccharide unit can take two forms: a most common 6-membered ring (pyranose, p) and a more flexible 5-membered ring (furanose, f). The tautomers impact the biological properties of polysaccharides, resulting in interesting properties of the derived oligosaccharides. From an analytical point of view, the impact of tautomerism on the gas-phase behavior of ions has scarcely been described in the literature. In this work, we study the behavior of Galf-containing oligosaccharides, ionized as [M+Li]+ species, under collisional dissociation (CID) conditions using high-resolution and multistage ion mobility (IMS) on a Cyclic IMS platform. In the first part of this work, we studied whether disaccharidic fragments released from Galf-containing (Gal)1(Man)2 trisaccharides (and their Galp counterpart) would match the corresponding disaccharide standards, and─despite the fragments generally being a good match─we showed the possibility of Galf migrations and other unidentified alterations in the IMS profile. Next, we expanded on these unknown features using multistage IMS and molecular dynamics, unveiling the contributions of additional gas-phase conformers in the profile of fragments from a Galf-containing trisaccharide compared with the corresponding disaccharides.

Arabinogalactan Protein-Like Proteins From Ulva lactuca Activate Immune Responses and Plant Resistance in an Oilseed Crop.

Natural compounds isolated from macroalgae are promising, ecofriendly, and multifunctional bioinoculants, which have been tested and used in agriculture. Ulvans, for instance, one of the major polysaccharides present in Ulva spp. cell walls, have been tested for their plant growth-promoting properties as well as their ability to activate plant immune defense, on a large variety of crops. Recently, we have characterized for the first time an arabinogalactan protein-like (AGP-like) from Ulva lactuca, which exhibits several features associated to land plant AGPs. In land plant, AGPs were shown to play a role in several plant biological functions, including cell morphogenesis, reproduction, and plant-microbe interactions. Thus, isolated AGP-like proteins may be good candidates for either the plant growth-promoting properties or the activation of plant immune defense. Here, we have isolated an AGP-like enriched fraction from Ulva lactuca and we have evaluated its ability to (i) protect oilseed rape (Brassica napus) cotyledons against Leptosphaeria maculans, and (ii) its ability to activate immune responses. Preventive application of the Ulva AGP-like enriched fraction on oilseed rape, followed by cotyledon inoculation with the fungal hemibiotroph L. maculans, resulted in a major reduction of infection propagation. The noticed reduction correlated with an accumulation of H2O2 in treated cotyledons and with the activation of SA and ET signaling pathways in oilseed rape cotyledons. In parallel, an ulvan was also isolated from Ulva lactuca. Preventive application of ulvan also enhanced plant resistance against L. maculans. Surprisingly, reduction of infection severity was only observed at high concentration of ulvan. Here, no such significant changes in gene expression and H2O2 production were observed. Together, this study indicates that U. lactuca AGP-like glycoproteins exhibit promising elicitor activity and that plant eliciting properties of Ulva extract, might result not only from an ulvan-originated eliciting activities, but also AGP-like originated.

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.

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.

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.

In vitro and in vivo immunomodulatory properties of octyl-ß-D-galactofuranoside during Leishmania donovani infection.

BACKGROUND: The chemotherapeutic arsenal available to treat visceral leishmaniasis is currently limited, in view of many drawbacks such as high cost, toxicity or emerging resistance. New therapeutic strategies are particularly needed to improve the management and the outcome in immunosuppressed patients. The combination of an immunomodulatory drug to a conventional anti-Leishmania treatment is an emerging concept to reverse the immune bias from Th2 to Th1 response to boost healing and prevent relapses. METHODS: Here, immunostimulating and leishmanicidal properties of octyl-ß-D-galactofuranose (Galf) were assessed in human monocyte-derived macrophages (HM) and in a murine model, after challenge with Leishmania donovani promastigotes. We recorded parasite loads and expression of various cytokines and immune effectors in HM and mouse organs (liver, spleen, bone marrow), following treatment with free (Galf) and liposomal (L-Galf) formulations. RESULTS: Both treatments significantly reduced parasite proliferation in HM, as well as liver parasite burden in vivo (Galf, P < 0.05). Consistent with in vitro results, we showed that Galf- and L-Galf-treated mice displayed an enhanced Th1 immune response, particularly in the spleen where pro-inflammatory cytokines TNF-α, IL-1ß and IL-12 were significantly overexpressed compared to control group. The hepatic recruitment of myeloid cells was also favored by L-Galf treatment as evidenced by the five-fold increase of myeloperoxidase (MPO) induction, which was associated with a higher number of MPO-positive cells within granulomas. By contrast, the systemic level of various cytokines such as IL-1ß, IL-6, IL-17A or IL-27 was drastically reduced at the end of treatment. CONCLUSIONS: Overall, these results suggest that Galf could be tested as an adjuvant in combination with current anti-parasitic drugs, to restore an efficient immune response against infection in a model of immunosuppressed mice.

Hydrophobized laminarans as new biocompatible anti-oomycete compounds for grapevine protection.

Laminaran, a ß-(1→3)-glucan extracted from Laminaria digitata, is a known elicitor of plant defenses, but provides only low level of disease control in vineyard trials. In this context, laminaran was partly hydrophobized by grafting from 1.6 to 7.6 lauryl chains to the native saccharidic chain and the impact of sulfation of the hydrophobized glucans was studied. The activity of the different synthetized laminaran derivatives as antimicrobial agents against Plasmopara viticola, the causal agent of grape downy mildew, and as elicitors of defense reactions in planta, was evaluated. Our results showed that acylation imparts an antimicrobial activity to laminaran which is related to the degree of acylation, AL3, with 7.6 lauryl chains, being the most effective derivative. Sulfation of the acylated laminarans did not further increase the antimicrobial activity. Our results also demonstrated that the efficacy of AL3 against Plasmopara viticola was most likely due to the direct antimicrobial activity of the lauryl chains rather than to an elicitation of plant defenses.

Pseudomonas aeruginosa resistance of monosaccharide-functionalized glass surfaces.

Preventing microorganism colonization on a surface is a great challenge in the conception of medical, food and marine devices. Here, we describe the formation of carbohydrate functionalized glass surfaces with D-glucose, D-galactose and D-mannose and how they efficiently affected the bacterial attachment. The carbohydrate entities were covalently attached to the pre-functionalized surface by click chemistry thanks the copper catalysed alkyl-azide cycloaddition. Water contact angle and X-ray photoelectron spectroscopy characterisations showed a homogeneous and quantitative cycloaddition at the scale of microorganisms. The adhesion assays with Pseudomonas aeruginosa, used as model of opportunistic pathogen, indicated a significant diminution of almost 40% of the bacterial accumulation on glycosidic surfaces with respect to initial surface. This activity was further compared with a surface presenting a simple hydroxyl residue. Exploration of specific interactions through Lectin A deficient Pseudomonas aeruginosa mutant strain provided new evidences that Lectin A was involved in biofilm maturation, rather than bacterial attachment. Subsequently, the determination of surface free energy and the adhesion free energy between surfaces and bacterial cell wall showed that the adhesion was thermodynamically unfavourable.

Galactofuranosidase from JHA 19 Streptomyces sp.: subcloning and biochemical characterization.

Despite the crucial role of the rare galactofuranose (Galf) in many pathogenic micro-organisms and our increased knowledge of its metabolism, there is still a lack of recombinant and efficient galactofuranoside hydrolase available for chemo-enzymatic synthetic purposes of specific galactofuranosyl-conjugates. Subcloning of the Galf-ase from JHA 19 Streptomyces sp. and its further overexpression lead us to the production of this enzyme with a yield of 0.5 mg/L of culture. It exhibits substrate specificity exclusively towards pNP ß-d-Galf, giving a KM value of 250 µM, and the highest enzymatic efficiency ever observed of 14 mM-1  s-1. It proved to be stable to temperature up to 60 °C and to at least 4 freeze-thaw's cycles. Thus, Galf-ase demonstrated to be an efficient and stable biocatalyst with greatly improved specificity toward the galactofuranosyl entity, thus paving the way to the further development of transglycosylation and thioligation reactions.

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.