Mass Spectral Similarity Networking and Gas-Phase Fragmentation Reactions in the Structural Analysis of Flavonoid Glycoconjugates.

Flavonoids represent an important class of natural products with a central role in plant physiology and human health. Their accurate annotation using untargeted mass spectrometry analysis still relies on differentiating similar chemical scaffolds through spectral matching to reference library spectra. In this work, we combined molecular network analysis with rules for fragment reactions and chemotaxonomy to enhance the annotation of similar flavonoid glyconjugates. Molecular network topology progressively propagated the flavonoid chemical functionalization according to collision-induced dissociation (CID) reactions, as the following chemical attributes: aglycone nature, saccharide type and number, and presence of methoxy substituents. This structure-based distribution across the spectral networks revealed the chemical composition of flavonoids across intra- and interspecies and guided the putatively assignment of 64 isomers and isobars in the Chrysobalanaceae plant species, most of which are not accurately annotated by automated untargeted MS2 matching. These proof of concept results demonstrate how molecular networking progressively grouped structurally related molecules according to their product ion scans, abundances, and ratios. The approach can be extrapolated to other classes of metabolites sharing similar structures and diagnostic fragments from tandem mass spectrometry.

Identification of Metabolites of Eupatorin in Vivo and in Vitro Based on UHPLC-Q-TOF-MS/MS.

Eupatorin is the major bioactive component of Java tea (Orthosiphon stamineus), exhibiting strong anticancer and anti-inflammatory activities. However, no research on the metabolism of eupatorin has been reported to date. In the present study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) combined with an efficient online data acquisition and a multiple data processing method were developed for metabolite identification in vivo (rat plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora). A total of 51 metabolites in vivo, 60 metabolites in vitro were structurally characterized. The loss of CH2, CH2O, O, CO, oxidation, methylation, glucuronidation, sulfate conjugation, N-acetylation, hydrogenation, ketone formation, glycine conjugation, glutamine conjugation and glucose conjugation were the main metabolic pathways of eupatorin. This was the first identification of metabolites of eupatorin in vivo and in vitro and it will provide reference and valuable evidence for further development of new pharmaceuticals and pharmacological mechanisms.

Isomeric Separation and Characterisation of Glycoconjugates.

Individual monosaccharides can be linked in a variety of different combinations to form complex glycoconjugates. In contrast to DNA and proteins, glycoconjugate synthesis does not follow any template but is the consequence of the concerted action of various enzymes such as transferases and glycosidases . Thus, tools for glycoconjugate sequencing need to differentiate individual monosaccharide identity, linkage and anomericity to investigate and understand glycoconjugate function. In this chapter we provide a concise overview on the most commonly used and robust tools to separate and characterise glycoconjugate isomers.

Some Physical Properties of Protein Moiety of Alkali-Extracted Tea Polysaccharide Conjugates Were Shielded by Its Polysaccharide.

Polysaccharide conjugates were alkali-extracted from green tea (TPC-A). Although it contained 11.80% covalently binding proteins, TPC-A could not bind to the Coomassie Brilliant Blue dyes G250 and R250. TPC-A had no expected characteristic absorption peak of protein in the UV-vis spectrum scanning in the range of 200-700 nm. The UV-vis wavelength of 280 nm was not suitable to detect the presence of the protein portion of TPC-A. The zeta potential of TPC-A merely presented the negative charge properties of polysaccharides instead of the acid-base property of its protein section across the entire pH range. Furthermore, TPC-A was more stable when the pH of solution exceeded 4.0. In addition, no precipitation or haze was generated in the TPC-A/(-)-epigallocatechin gallate (EGCG) mixtures during 12 h storage. TPC-A has emulsifying activity, which indicated that its protein moiety formed hydrophobic groups. Thus, it was proposed that some physical properties of TPC-A protein were shielded by its olysaccharide, since the protein moiety was wrapped by its polysaccharide chains.

Thermophiles as potential source of novel endotoxin antagonists: the full structure and bioactivity of the lipo-oligosaccharide from Thermomonas hydrothermalis.

Thermomonas hydrothermalis is a Gram-negative thermophilic bacterium that is able to live at 50 °C. This ability is attributed to chemical modifications, involving those to bacterial cell-wall components, such as proteins and (glyco)lipids. As the main component of the outer membrane of Gram-negative bacteria, lipopolysaccharides (LPSs) are exposed to the environment, thus they can undergo structural chemical changes to allow thermophilic bacteria to live at their optimal growth temperature. Furthermore, as one of the major target of the eukaryotic innate immune system, LPS elicits host immune response in a structure-dependent mode; thus the uncommon chemical features of thermophilic bacterial LPSs might exert a different biological action on the innate immune system-an antagonistic effect, as shown in studies of LPS structure-activity relationship in the ongoing research into antagonist LPS candidates. Here, we report the complete structural and biological activity analysis of the lipo-oligosaccharide isolated from Thermomonas hydrothermalis, achieved by a multidisciplinary approach (chemical analysis, NMR, MALDI MS and cellular immunology). We demonstrate a tricky and interesting structure combined with a very interesting effect on human innate immunity.

Isolation and purification of glycoconjugates from complex biological sources by recycling high-performance liquid chromatography.

Among of the most urgent needs of the glycobiology community is to generate libraries of pure carbohydrate standards. While many oligosaccharides have recently been synthesized, some glycans of biomedical importance are still missing in existing collections or are available in only limited amounts. To address this need, we demonstrate the use of the relatively unexplored technique of recycling high-performance liquid chromatography (R-HPLC) to isolate and purify glycoconjugates from several natural sources. We were able to routinely achieve purities greater than 98%. In several cases, we were able to obtain isomerically pure substances, particularly for glycans with different positional isomerism. These purified substances can then be used in different analytical applications, for example, as standards for mass spectrometry (MS) and capillary-based separations. Moreover, using a bifunctional aromatic amine, the same derivatization agent can be used to enable UV detection of oligosaccharides during their purification and link the isolated molecules to functionalized surfaces and potentially create glycan arrays.

Identification of a novel tetrapeptide structure of the Mycobacterium avium glycopeptidolipid that functions as a specific target for the host antibody response.

Mycobacterium avium complex (MAC) is a group of non-tuberculous mycobacteria that cause tuberculosis-like diseases in humans. Unlike Mycobacterium tuberculosis, MAC expresses high levels of glycopeptidolipids (GPLs) containing a well-defined tetrapeptide-amino alcohol core, composed of D-phenylalanine, D-allo-threonine, D-alanine, and L-alaninol, that is modified with a fatty acid and sugar residues. Surprisingly, however, a careful scrutiny of the mass spectrum of MAC GPLs revealed the presence of ions that could not readily accountable for the known GPL structure. The magnitude of the ions was increased prominently when GPLs were isolated from the valine-supplemented culture, and the ions representing the authentic GPL species were diminished, suggesting the possibility that the basic structure of the peptide backbone might be altered in response to the exogenously added valine. Indeed, further mass spectrometry (MS)/MS and gas chromatography-MS analysis indicated a substitution of D-valine for the N-terminal D-phenylalanine of the tetrapeptide core, and the presence of D-valine and the absence of D-phenylalanine was confirmed by high-performance liquid chromatography, using the derivatized amino acid residues that were released from the tetrapeptide. Finally, specific antibodies to the purified valine-containing GPL species were detected in the serum of a MAC-infected guinea pig. Therefore, these results identify a new molecular species of MAC GPLs with immunogenic potential.

Natural glycoconjugates with antitumor activity.

Cancer is one of the major causes of death worldwide. As a consequence, many different therapeutic approaches, including the use of glycosides as anticancer agents, have been developed. Various glycosylated natural products exhibit high activity against a variety of microbes and human tumors. In this review we classify glycosides according to the nature of their aglycone (non-saccharidic) part. Among them, we describe anthracyclines, aureolic acids, enediyne antibiotics, macrolide and glycopeptides presenting different strengths and mechanisms of action against human cancers. In some cases, the glycosidic residue is crucial for their activity, such as in anthracycline, aureolic acid and enediyne antibiotics; in other cases, Nature has exploited glycosylation to improve solubility or pharmacokinetic properties, as in the glycopeptides. In this review we focus our attention on natural glycoconjugates with anticancer properties. The structure of several of the carbohydrate moieties found in these conjugates and their role are described. The structure­activity relationship of some of these compounds, together with the structural features of their interaction with the biological targets, are also reported. Taken together, all this information is useful for the design of new potential anti-tumor drugs.

Prefiltration enhances performance of sterile filtration for glycoconjugate vaccines.

Recent studies have reported very low capacity during sterile filtration of glycoconjugate vaccines due to rapid fouling of the sterile filter. The objective of this study was to explore the potential for significantly increasing the capacity of the sterile filter through the use of an appropriate prefilter. Data were obtained using prefilters with different pore size and chemistry, with the sterile filtration performed at constant filtrate flux using 0.22 µm nominal pore size Durapore® polyvinylidene difluoride membranes. Prefiltration through 5 µm pore size Durapore® or Nylon prefilters nearly eliminated the fouling of the sterile filter, leading to more than a 100-fold reduction in the rate of pressure increase for the sterile filter. This dramatic improvement in sterile filter performance was due to the removal of large components (greater than 1 µm in size) as confirmed by dynamic light scattering. These results demonstrate the potential of using large pore size prefilters to significantly enhance the performance of the sterile filtration process for the production of important glycoconjugate vaccines.

Exploitation of Capsule Polymerases for Enzymatic Synthesis of Polysaccharide Antigens Used in Glycoconjugate Vaccines.

The exploitation of recombinant enzymes for the synthesis of complex carbohydrates is getting increasing attention. Unfortunately, the analysis of the resulting products often requires advanced methods like nuclear magnetic resonance spectroscopy and mass spectrometry. Here, we use the capsule polymerases Cps4B and Cps11D from Actinobacillus pleuropneumoniae serotypes 4 and 11, respectively, as examples for the in vitro synthesis of capsule polymers similar to those used in glycoconjugate vaccine formulations. We demonstrate how substrate turnover in an enzymatic reaction can be analyzed by HPLC-based anion exchange chromatography and provide the protocol for separation and detection of UV-active polymer. Moreover, we describe how UV-inactive polymer can be separated and visualized using polyacrylamide gel electrophoresis followed by combined alcian blue-silver staining.

LC-MSMS profiling of flavonoid conjugates in wild Mexican lupine, Lupinus reflexus.

Profiles of flavonoid conjugates present in the root and leaf tissues of the Mexican wild lupine, Lupinus reflexus, were established using two LC-MSMS systems in the positive and negative ion modes. The ion trap mass spectrometer and quadrupole time-of flight instrument provided sequential MS(n) spectra and MSMS spectra with accurate m/z values of [M + H](+) and [M - H] (-) ions, respectively. Sixty-two flavone and isoflavone glycoconjugates were found and tentatively identified. Numerous isomeric or isobaric compounds with the same molecular mass could be differentiated. Isomeric di- and mono glucosides of biochanin A, genistein, 2'-hydroxygenistein, luteone, and 2,3-didehydrokievitone were distinguished on the basis of relative abundances of product ions. The studied flavonoid glycoconjugates were acylated with dicarboxylic aliphatic acids and their methyl esters at either the aglycone or glycosidic moiety.

A Rapid and Facile Purification Method for Glycan-Binding Proteins and Glycoproteins.

Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time-saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour Alternate Protocol 1: Preparation of crude extracts from Jack bean meal Alternate Protocol 2: Preparation of crude extracts from the corms of Colocasia esculenta, Xanthosoma sagittifolium, and from the bulbs of Allium sativum Alternate Protocol 3: Preparation of Escherichia coli cell lysates containing human galectin-3 Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin) Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension Basic Protocol 3: Detection of lectin activity of the crude extracts Basic Protocol 4: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays Basic Protocol 5: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target-TCA complex from other components of the crude extracts Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins).

Chondroitin sulfate isolated from the secretion of the venom-producing parotoid gland of Brazilian bufonid.

The parotoid gland of bufonids is characterized as a specialized integument region, formed by different gland types. The secretion elaborated by the largest glandular alveoli has been related to animal chemical defense and is constituted by granular protein content, associated with a basophilic and alcianophilic material with features of glycoconjugates. This study aimed to identify and characterize the glycoconjugates in the secretion of the largest granular gland of the parotoid gland of Rinella icterica by histochemical and immunohistochemical techniques at light microscopy, biochemical methods, and nuclear magnetic resonance spectroscopy. Our results showed that the glycoconjugate content contains a mixture of chondroitin­6­sulfate (C6S) and chondroitin-non-sulfate (C0S). Thus, chondroitin sulfate probably plays an important role in gland physiology, probably protecting the protein content while inside the secretory portion.

Effect of various extraction methods on the structure of polyphenolic-polysaccharide conjugates from Fragaria vesca L. leaf.

The purpose of this study was to compare the application of several extraction methods, including cold extraction (CAE), hot extraction (HAE), ultrasonic-assisted extraction (UAE), and microwave-assisted extraction (MAE), all carried out in 0.1 M NaOH, under their respective best parameters, for obtaining products from leaf of Fragaria vesca L. The extracts with the highest anticoagulant activity were purified by multi-step extraction procedure and separated by gel permeation chromatography, giving rise to the macromolecular complexes. They were subsequently structurally characterized using colorimetric methods, FT-IR, GC-MS, and NMR (1H and 1H/13C HSQC), as polyphenolic-polysaccharide conjugates with the anticoagulant activity. The polysaccharide parts of the conjugates obtained by different extraction procedures were found to vary significantly. The most selective in their activity were the glycoconjugates extracted in UAE and MAE processes, i.e. arabinogalactan and pectin-like conjugates, respectively. In terms of their anticoagulant activity all of them were non-direct factor Xa inhibitors mediated by antithrombin.

Improved conjugation and purification strategies for the preparation of protein-polysaccharide conjugates.

A glycoconjugate constituted by the Streptococcus pneumoniae serotype 14 capsular polysaccharide (CPS14) and bovine serum albumin (BSA) was prepared, and the unique properties of Sephadex LH-20 were used to separate the conjugate from the unconjugated material. The strength of this approach consists in its capacity to produce pure polysaccharide-protein conjugate in good yield and free from unconjugated material, a common residual contaminant of this type of immunobiologicals. The CPS14-BSA conjugate prepared via an improved 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)-activation technique was characterized chemically and its immunogenicity was evaluated in mice. The purified conjugate, unlike the corresponding polysaccharide, produced a T-cell-dependent response in this species.

Efficient cartridge purification for producing high molar activity [18F]fluoro-glycoconjugates via oxime formation.

INTRODUCTION: 18F-fluoroglycosylation via oxime formation is a chemoselective and mild radiolabeling method for sensitive molecules. Glycosylation can also improve the bioavailability, in vivo kinetics, and stability of the compound in blood, as well as accelerate clearance of biomolecules. A typical synthesis procedure for 18F-fluoroglycosylation with [18F]FDG (2-deoxy-2-[18F]fluoro-d-glucose) and [18F]FDR (5-deoxy-5-[18F]fluoro-d-ribose) involves two HPLC (high performance liquid chromatography) purifications: one after 18F-fluorination of the carbohydrate to remove its labeling precursor, and a second one after the oxime formation step to remove the aminooxy precursor. The two HPLC purifications can be time consuming and complicate the adaptation of the synthetic strategy in nuclear medicine applications and automated synthesis. We have developed a procedure in which SPE (solid phase extraction) and resin purification methods replace both of the needed HPLC purification steps. METHODS: We used [18F]FDR and [18F]FDG as prosthetic groups to radiolabel two aminooxy-modified model molecules, a tetrazine and a PSMA (prostate specific membrane antigen) inhibitor. After fluorination, the excess carbohydrate precursor was removed by derivatizing it with 4,4'-dimethoxytrityl chloride (DMT-Cl). The DMT moiety increases the hydrophobicity of the unreacted precursor making the separation from the fluorinated precursor possible with simple C18 Sep-Pak cartridge. For removal of the aminooxy precursor, we used a commercially available aldehyde resin (AminoLink, Thermo Fisher Scientific). C18 Sep-Pak SPE cartridge was used to separate [18F]FDR and [18F]FDG from the 18F-fluoroglycoconjugate end product. RESULTS: [18F]FDR and [18F]FDG were efficiently purified from their precursors, free fluorine-18, and other impurities. The aldehyde resin quantitatively removed the unreacted aminooxy precursors after the oxime formation. The fluorine-18 labeled oxime end products were obtained with high radiochemical purity (>99%) and molar activity (>600 GBq µmol-1). CONCLUSIONS: We have developed an efficient cartridge purification method for producing high molar activity 18F-glycoconjugates synthesized via oxime formation.

An arabinogalactan-glycoconjugate from Genipa americana leaves present anticoagulant, antiplatelet and antithrombotic effects.

Glycoconjugates extracted from Genipa americana leaves (PE-Ga) were separated into two fractions, denominated as PFI and PFII (total carbohydrate: 23-36%/uronic acid: 9-30%; protein:4-5%; polyphenols:0.776-0.812 mg/g), mainly composed by arabinose, galactose and uronic acid and presenting high (PFI) and low (PFII) molecular weight (based on polyacrylamide electrophoresis gel and gel permeation chromatography). Uronic acid was also detected by FT-IR (wavenumbers: 1410 and 1333 cm-1) and NMR (α-GalpA). Deproteinization of glycoconjugates showed reduced protein and polyphenol levels with loss of its biological effects. PE-Ga and PFII prolonged clotting time-aPTT (3.6 and 1.8x), while PE-Ga and PFI inhibited by 48% (100 µg/µL) the ADP-induced platelet aggregation. In vivo, these glycoconjugates at 1 mg/kg inhibited (37-53%) venous thrombus formation (4.7 ± 0.1 mg) and increased bleeding time (PE-Ga and PFI:3.0x; PFII:1.7x vs. PBS:906 ± 16.7 s). In conclusion, the arabinogalactan-rich glycoconjugate of G. americana leaves, containing uronic acid, present antiplatelet, anticoagulant (intrinsic/common pathway) and antithrombotic effects, with low hemorrhagic risk.

Immobilization of ß-galactosidase and α-mannosidase onto magnetic nanoparticles: A strategy for increasing the potentiality of valuable glycomic tools for glycosylation analysis and biological role determination of glycoconjugates.

Glycans present in biological glycoconjugates have several structural and functional roles. Elucidation of glycan structure and biological function is critical to understand their role in physiological and pathogenic process, enabling the development of diagnostic methods and disease treatment. Immobilized glycosidases are powerful tools for glycan analysis, as they are able to remove specific carbohydrates without altering the protein structure. Here we describe the individual immobilization of Aspergillus oryzae ß-galactosidase and Canavalia ensiformis α-mannosidase onto agarose and silica magnetic nanoparticles activated with cyanate ester groups. High immobilization yields (70-90%) were achieved, keeping above 60% of its original activity. Immobilized glycosidases were effective in the selective deglycosylation of model glycoproteins and a Fasciola hepatica lysate, evidenced by a decrease in specific lectin recognition of 40-50% after enzymatic deglycosylation. Immobilized glycosidases were reused for several deglycosylation cycles without loss of effectiveness. Their use was extended to the elucidation of the glycan role of native glycoconjugates. A decrease in the recognition of lactoferrin treated with α-mannosidase by a C-type lectin receptor, DC-SIGN was found. Also the specific deglycosylation of a F. hepatica lysate demonstrated the relevance of mannosylated glycans in the induction of Th2/Treg immune responses during the infection. Our results show successful immobilization of specific glycosidases in nano-supports and validate their utility to identify glycans biological functions.

Application of enzymes, sodium tripolyphosphate and cation exchange resin for the release of extracellular polymeric substances from sewage sludge. Characterization of the extracted polysaccharides/glycoconjugates by a panel of lectins.

The study describes extraction of extracellular polymeric substances (EPS) from sewage sludge by applying enzymes and enzymes combined with sodium tripolyphosphate (STPP). Additionally, a systematic study of two non-enzymatic extraction agents is described. The assessment of the released products is made by colorimetrical methods and polysaccharides/glycoconjugates identification by the interaction with four immobilized lectins. Bio-sludge from Helsingborg (Sweden) and Damhusåen (Denmark) were used as two case studies for testing enzymatic extractability and thereby to make useful prediction of sludge bio-digestibility. From Helsingborg sludge the enzymes extracted about 40% more of EPS than from Damhusåen. The polysaccharides/glycoconjugates in both sludges maintained the same level, and showed substantial different interaction motifs with lectins panel. Damhusåen enzymatic extracted EPS had an enhanced amount of suspended material that was post-hydrolysed by the use of polygalacturonase and lysozyme resulting in pectin like polymers and petiptidoglycans. Petiptidoglycan is a marker from bacterial cell debris. STPP and cation exchange resin (CER) released different quantities of EPS. The CER released polysaccharides/glycoconjugates had higher molecular weight and stronger affinity towards Concanavalin A than the one released by the action of STPP. Independent of the extraction conditions, STPP released elevated amounts of polyvalent cations and humic substances in contrast to the very low amounts of released by CER.

Fasciola hepatica glycoconjugates immuneregulate dendritic cells through the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin inducing T cell anergy.

Dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) expressed on a variety of DCs, is a C-type lectin receptor that recognizes glycans on a diverse range of pathogens, including parasites. The interaction of DC-SIGN with pathogens triggers specific signaling events that modulate DC-maturation and activity and regulate T-cell activation by DCs. In this work we evaluate whether F. hepatica glycans can immune modulate DCs via DC-SIGN. We demonstrate that DC-SIGN interacts with F. hepatica glycoconjugates through mannose and fucose residues. We also show that mannose is present in high-mannose structures, hybrid and trimannosyl N-glycans with terminal GlcNAc. Furthermore, we demonstrate that F. hepatica glycans induce DC-SIGN triggering leading to a strong production of TLR-induced IL-10 and IL-27p28. In addition, parasite glycans induced regulatory DCs via DC-SIGN that decrease allogeneic T cell proliferation, via the induction of anergic/regulatory T cells, highlighting the role of DC-SIGN in the regulation of innate and adaptive immune responses by F. hepatica. Our data confirm the immunomodulatory properties of DC-SIGN triggered by pathogen-derived glycans and contribute to the identification of immunomodulatory glyans of helminths that might eventually be useful for the design of vaccines against fasciolosis.