Glucuronic Acid Units Research Articles

Characterization of carboxylated cellulose nanofibrils and oligosaccharides from Kraft pulp fibers and their potential elicitor effect on the gene expression of Capsicum annuum

Biomass-derived oligo- and polysaccharides may act as elicitors, i.e., bioactive molecules that trigger plant immune responses. This is particularly important to increase the resistance of plants to abiotic and biotic stresses. In this study, cellulose nanofibrils (CNF) gels were obtained by TEMPO-mediated oxidation of unbleached and bleached kraft pulps. The molecular structures were characterized with ESI and MALDI MS. Analysis of the fine sequences was achieved by MS and MS/MS of the water-soluble oligosaccharides obtained by acid hydrolysis of the CNF gels. The analysis revealed the presence of two families: one corresponding to homoglucuronic acid sequences and the other composed by alternating glucose and glucuronic acid units. The CNF gels, alone or with the addition of the water-soluble oligosaccharides, were tested on Chili pepper (Capsicum annuum). Based on the characterization of the gene expression with Next Generation Sequencing (NGS) of the C. annuum's total messenger RNA, the differences in growth of the C. annuum seeds correlated well with the downregulation of the pathways regulating photosynthesis. A downregulation of the response to abiotic factors was detected, suggesting that these gels would improve the resistance of the C. annuum plants to abiotic stress due to, e.g., water deprivation and cold temperatures.

Multi-step semi-synthesis, structural characterization and growth factor interaction study of regiochemically sulfated diabolican polysaccharides

Diabolican is an exopolysaccharide (EPS) produced by Vibrio diabolicus HE800, a mesophilic bacterium firstly isolated from a deep-sea hydrothermal field. Its glycosaminoglycan (GAG)-like structure, consisting of a tetrasaccharide repeating unit composed of two aminosugars (N-acetyl-glucosamine and N-acetyl-galactosamine) and two glucuronic acid units, suggested to subject it to regioselective sulfation processes, in order to obtain some sulfated derivatives potentially acting as GAG mimics. To this aim, a multi-step semi-synthetic approach, relying upon tailored sequence of regioselective protection, sulfation and deprotection steps, was employed in this work. The chemical structure of the obtained sulfated diabolican derivatives was characterized by a multi-technique analytic approach, in order to define both degree of sulfation (DS) and sulfation pattern within the polysaccharide repeating unit, above all. Finally, binding affinity for some growth factors relevant for biomedical applications was measured for both starting diabolican and sulfated derivatives thereof. Collected data suggested that sulfation pattern could be a key structural element for the selective interaction with signaling proteins not only in the case of native GAGs, as already known, but also for GAG-like structures obtained by regioselective sulfation of naturally unsulfated polysaccharides.

Enhanced Hyaluronic Acid Production from Priestia flexa N7 Isolates

Background: Hyaluronic acid (HA) is a gel-like substance made up of glucuronic acid and N-acetylglucosamine units, capable of absorbing and retaining water, present in hydrated gel form across human and animal tissues. It aids in joint lubrication and moisture retention and acts as a cushion for shock absorption. HA has unique biological properties, promoting fibroblast cell growth, aiding wound healing, and exhibiting low solubility and viscosity, making it an organic ingredient in tissue culture techniques. It is utilized in eye drops and skin ointments and plays a vital role in the extracellular matrix, rendering it invaluable in medical and cosmetic applications, such as treating osteoarthritis and enhancing skin wound recovery. Methods: The methods employed in this study involve isolating microorganisms, screening bacterial strains capable of synthesizing HA, identifying bacteria using molecular biological methods, and researching optimal conditions to select bacterial strains that produce the highest HA concentrations. Results: In this study, strain Priestia flexa N7 was studied for suitable conditions for HA biosynthesis. Bacterial strains were fermented for 48 h on medium containing the following ingredients: glucose (60 g/L); yeast extract (5.0 g/L); peptone (20 g/L); K2HPO4 (2.0 g/L); Na2HPO4 (1.0 g/L); NaCl (2.0 g/L); FeSO4 (1.0 g/l); sodium glutamate (9.0 g/L); and MgSO4.7H2O (2.0 g/L) and pH 8.0 at 37°C under the condition of continuous shaking at 150 rpm. The maximum HA production achieved was 1105 mg/L. Conclusions: The mentioned bacterial strain exhibits significant potential for HA synthesis and is extensively employed in producing items across the health care, medical, food, and cosmetic industries. These findings revealed the most effective HA acid manufacturing strategy for achieving maximum output.

Fabrication of cell-laden microbeads and microcapsules composed of bacterial polyglucuronic acid

In the past decades, the microencapsulation of mammalian cells into microparticles has been extensively studied for various in vitro and in vivo applications. The aim of this study was to demonstrate the viability of bacterial polyglucuronic acid (PGU), an exopolysaccharide derived from bacteria and composed of glucuronic acid units, as an effective material for cell microencapsulation. Using the method of dropping an aqueous solution of PGU-containing cells into a Ca2+-loaded solution, we produced spherical PGU microbeads with >93 % viability of the encapsulated human hepatoma HepG2 cells. Hollow-core microcapsules were formed via polyelectrolyte complex layer formation of PGU and poly-l-lysine, after which Ca2+, a cross-linker of PGU, was chelated, and this was accomplished by sequential immersion of microbeads in aqueous solutions of poly-l-lysine and sodium citrate. The encapsulated HepG2 cells proliferated and formed cell aggregates within the microparticles over a 14-day culture, with significantly larger aggregates forming within the microcapsules. Our results provide evidence for the viability of PGU for cell microencapsulation for the first time, thereby contributing to advancements in tissue engineering.

Total Synthesis of Starfish Cyclic Steroid Glycosides. Part 3, Formal Total Synthesis of Luzonicosides A and D

Comprehensive SummaryLuzonicosides A (1) and D (2) are the major saponins isolated from pacific starfish Echinaster Luzonicus, featuring a sterically congested 16‐membered ring which is formed by bridging the C3 and C6 of a 3β,6β‐dihydroxy‐Δ7‐ene steroid with a 1,2‐trans‐linked linear trisaccharide via glycosidic and ether bonds, respectively. Here, we describe a formal total synthesis of starfish cyclic steroid glycosides 1 and 2. The synthetic route is highly modular and versatile, involving construction of the 16‐membered macrocycles via Au(I)‐catalyzed intramolecular glycosylation, highly efficient installation of unsaturated sugar moieties with ortho‐hexynylbenzoates, elaboration of glucuronic acid unit via post‐oxidation, and a de novo synthesis of the ether‐linked pyranose unit.

Identification of Matriglycan by Dual Exoglycosidase Digestion of α-Dystroglycan.

Matriglycan is a linear polysaccharide of alternating xylose and glucuronic acid units [-Xyl-α1,3-GlcA-β1,3]n that is uniquely synthesized on α-dystroglycan (α-DG) and is essential for neuromuscular function and brain development. It binds several extracellular matrix proteins that contain laminin-globular domains and is a receptor for Old World arenaviruses such as Lassa Fever virus. Monoclonal antibodies such as IIH6 are commonly used to detect matriglycan on α-DG. However, endogenous expression levels are not sufficient to detect and analyze matriglycan by mass spectrometry approaches. Thus, there is a growing need to independently confirm the presence of matriglycan on α-DG and possibly other proteins. We used an enzymatic approach to detect matriglycan, which involved digesting it with two thermophilic exoglycosidases: β-Glucuronidase from Thermotoga maritima and α-xylosidase from Sulfolobus solfataricus. This allowed us to identify and categorize matriglycan on α-DG by studying post-digestion changes in the molecular weight of α-DG using SDS-PAGE followed by western blotting with anti-matriglycan antibodies, anti-core α-DG antibodies, and/or laminin binding assay. In some tissues, matriglycan is capped by a sulfate group, which renders it resistant to digestion by these dual exoglycosidases. Thus, this method can be used to determine the capping status of matriglycan. To date, matriglycan has only been identified on vertebrate α-DG. We anticipate that this method will facilitate the discovery of matriglycan on α-DG in other species and possibly on other proteins. Key features • Analysis of endogenous matriglycan on dystroglycan from any animal tissue. • Matriglycan is digested using thermophilic enzymes, which require optimum thermophilic conditions. • Western blotting is used to assay the success and extent of digestion. • Freshly purified enzymes work best to digest matriglycan.

Structure of the human heparan sulfate polymerase complex EXT1-EXT2

Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains. A combination of in vitro and in cellulo mutational studies is used to dissect the functional role of the four catalytic sites. While EXT1 can catalyze both glycosyltransferase reactions, our results indicate that EXT2 might only have N-acetylglucosamine transferase activity. Our findings provide mechanistic insight into heparan sulfate chain elongation as a nonprocessive process and lay the foundation for future studies on EXT1-EXT2 function in health and disease.

Structural Effect on Adjuvanticity of Saponins.

We have prepared a number of saponin-based vaccine adjuvant candidates. These unnatural saponins have a different terminal-functionalized side chain incorporated into the glucuronic acid unit that is attached to a triterpenoid core at its C3 position. The semisynthetic saponin adjuvants have shown significantly different immunostimulatory activities, suggesting that the structure of the side chain, triterpenoid core, and oligosaccharide domain together orchestrate saponin adjuvant's potentiation of immune responses. Among these new adjuvant candidates, VSA-2 (5b), a derivative of Momordica saponin (MS) II, showed consistent enhancement of immunoglobulin G2a (IgG2a) production when it was in formulation with either ovalbumin or recombinant hemagglutinin B (rHagB) antigen. With rHagB antigen, it induced a significantly higher IgG2a response than the positive control GPI-0100, a well-studied semisynthetic saponin adjuvant mixture derived from Quillaja saponaria Molina saponins, known for its ability to induce a balanced Th1/Th2 immunity. These results confirm that Momordica saponins are a viable natural source to provide potent saponin adjuvants after simple chemical derivatization and identify VSA-2 (5b) as another MS-based promising immunostimulant lead owing to its distinctive ability in potentiating the IgG2a response.

Effect of adding galactomannans on some physical and chemical properties of hyaluronic acid

Hyaluronic acid (HA) and galactomannans (such as guar gum - GG and locust bean gum - LBG) are biomacromolecules with various applications due to their physicochemical characteristics in solution, especially their ability to form films or hydrogels in some conditions. Some of these applications are possible because they are nontoxic and biocompatible. Mixtures of HA and GG or LBG, at pH 7.4, were prepared at 25 g·L−1 of HA:LBG or HA:GG with percentages of 50:50, 60:40, 70:30 and 80:20 (w/w) and evaluated by rheology and NMR experiments regarding the interactions. The best synergism observed was between HA:LBG, most pronounced at 50% (w/w), where the dynamic modulus values G′ and G″ indicated the formation of physical gels with viscoelastic behavior close to that of HA. 2D NOESY spectra revealed hydrogen interconnectivity of H-1 glucuronic acid units of HA with H-5 mannose units of the main galactomannan chains. This synergism, besides producing a hydrogel with interesting properties, also can decrease the cost by partially replacing HA in many applications, such as in nutraceutical foods, support for tissue engineering and cosmetics.

Photosynthetic conversion of CO2 to hyaluronic acid by engineered strains of the cyanobacterium Synechococcus sp. PCC 7002

Hyaluronic acid (HA), consisting of alternating N-acetylglucosamine and glucuronic acid units, is a natural polymer with diverse cosmetic and medical applications. Currently, HA is produced by overexpressing HA synthases from gram-negative Pasteurella multocida (encoded by pmHAS) or gram-positive Streptococcus equisimilis (encoded by seHasA) in various heterotrophic microbial production platforms. Here we introduced these two different types of HA synthase into the fast-growing cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) to explore the capacity for producing HA in a photosynthetic system. Our results show that both HA synthases enable Syn7002 to produce HA photoautotrophically, but that overexpression of the soluble HA synthase (PmHAS) is less deleterious to cell growth and results in higher production. Genetic disruption of the competing cellulose biosynthetic pathway increased the HA titer by over 5-fold (from 14mg/L to 80mg/L) and the relative proportion of HA with molecular mass greater than 2MDa. Introduction of glmS and glmU, coding for enzymes involved in the biosynthesis of the precursor UDP-N-acetylglucosamine, in combination with partial glycogen depletion, allowed photosynthetic production of 112mg/L of HA in 5 days, an 8-fold increase in comparison to the initial PmHAS expressing strain. Addition of tuaD and gtaB (coding for genes involved in UDP-glucuronic acid biosynthesis) also improved the HA yield, albeit to a lesser extent. Overall our results have shown that cyanobacteria hold promise for the sustainable production of pharmaceutically important polysaccharides from sunlight and CO2.

Mechanism research on catalytic pyrolysis of sulfated polysaccharide using ZSM-5 catalysts by Py-GC/MS and density functional theory studies

The mechanisms of primary product formation on catalytic pyrolysis of seaweed sulfated polysaccharide with ZSM-5 were examined in detail by using Py-GC/MS analysis and subsequent density functional theory. Results showed that furfurals were the main product (50.3%) in bio-oil during the catalytic pyrolysis of sulfated polysaccharides. Furfural (9.1%), 5-(Hydroxymethyl)-2(5 H)-furanone (3.1%) and acetone (3.2%) were defined as typical products of Glucuronic acid unit. 5-methyl furfural (41.2%), 5-methyl-2(3 H)-Furanone (0.5%) and 5-methyl furan (5.4%) can be converted from rhamnose unit. The subsequent density functional theory calculation indicated that the catalytic pyrolysis of sulfated polysaccharides by ZSM-5 catalyst could boost the pyrolysis and upgrade the bio-oil quality since the promotion of hydrodeoxygenation on catalytic reactions and ZSM-5 could promote the formation of 5-methyl furfural. The hydrogen bonding and van der Waals force interactions improved the pyrolysis pathways and decreased the energy barrier, which promoted the pyrolysis. This study confirmed that ZSM-5 had a catalytic influence on pyrolysis of sulfated polysaccharides through theoretical and experimental analysis, which is helpful to understand the formation mechanism of seaweed catalytic pyrolysis products.

Synthesis and Immunological Studies of Oligosaccharides that Consist of the Repeating Unit of Streptococcus pneumoniae Serotype 3 Capsular Polysaccharide.

A highly convergent and efficient strategy was developed for the chemical synthesis of complex oligosaccharides of Streptococcus pneumoniae type 3 capsular polysaccharides that contain multiple glucuronic acid units. Once the oligoglucosides were efficiently and stereoselectively assembled, the designated glucose units were regioselectively oxidized to glucuronic acid in one step at the final synthetic stage, which helped avoid difficult glycosylations that involved glucuronic acid. The target oligosaccharides had a free amino group at the reducing terminus and varied caps at the non-reducing terminus to enable further modification and structure-activity relationship studies. Immunological evaluations of the oligosaccharide-tetanus toxoid conjugates showed that they elicited robust T-cell-dependent immunoglobulin G antibody responses and that the sugar chain length had a major impact on their immunological properties. In particular, the penta- and hexasaccharides were identified as promising antigens for vaccine development.

Distinct reaction mechanisms for hyaluronan biosynthesis in different kingdoms of life.

Hyaluronan (HA) is an acidic high molecular weight cell surface polysaccharide ubiquitously expressed by vertebrates, some pathogenic bacteria and even viruses. HA modulates many essential physiological processes and is implicated in numerous pathological conditions ranging from autoimmune diseases to cancer. In various pathogens, HA functions as a non-immunogenic surface polymer that reduces host immune responses. It is a linear polymer of strictly alternating glucuronic acid and N-acetylglucosamine units synthesized by HA synthase (HAS), a membrane-embedded family-2 glycosyltransferase. The enzyme synthesizes HA and secretes the polymer through a channel formed by its own membrane-integrated domain. To reveal how HAS achieves these tasks, we determined the biologically functional units of bacterial and viral HAS in a lipid bilayer environment by co-immunoprecipitation, single molecule fluorescence photobleaching, and site-specific cross-linking analyses. Our results demonstrate that bacterial HAS functions as an obligate homo-dimer with two functional HAS copies required for catalytic activity. In contrast, the viral enzyme, closely related to vertebrate HAS, functions as a monomer. Using site-specific cross-linking, we identify the dimer interface of bacterial HAS and show that the enzyme uses a reaction mechanism distinct from viral HAS that necessitates a dimeric assembly.

Hyaluronate effect on bacterial biofilm in ENT district infections: a review.

Bacterial resistance is a growing phenomenon which led the scientific community to search for new therapeutic targets, such as biofilm. A bacterial biofilm is a surface-associated agglomerate of microorganisms embedded in a self-produced extracellular polymeric matrix made of polysaccharides, nucleic acids, and proteins. Scientific literature offers several reports on a biofilm's role in infections regarding various body districts. The presence of a bacterial biofilm is responsible for poor efficacy of antibiotic therapies along with bacterial infections in ear, nose, and throat (ENT) districts such as the oral cavity, ear, nasal cavities, and nasal sinuses. In particular, bacterial biofilms are associated with recalcitrant and symptomatically more severe forms of chronic rhinosinusitis. As of today, there are no therapeutic options for the eradication of bacterial biofilm in ENT districts. Hyaluronic acid is a glycosaminoglycan composed of glucuronic acid and N-acetylglucosamine disaccharide units. Its efficacy in treating rhinosinusitis, whether or not associated with polyposis, is well documented, as well as results from its effects on mucociliary clearance, free radical production and mucosal repair. This review's aim is to evaluate the role of bacterial biofilms and the action exerted on it by hyaluronic acid in ENT pathology, with particular attention to the rhinosinusal district. In conclusion, this paper underlines how the efficacy of hyaluronate as an anti-bacterial biofilm agent is well demonstrated by in vitro studies; it is, however, only preliminarily demonstrated by clinical studies.

Decoration of Chondroitin Polysaccharide with Threonine: Synthesis, Conformational Study, and Ice-Recrystallization Inhibition Activity.

Several threonine (Thr)- and alanine (Ala)-rich antifreeze glycoproteins (AFGPs) and polysaccharides act in nature as ice recrystallization inhibitors. Among them, the Thr-decorated capsular polysaccharide (CPS) from the cold-adapted Colwellia psychrerythraea 34H bacterium was recently investigated for its cryoprotectant activity. A semisynthetic mimic thereof was here prepared from microbial sourced chondroitin through a four-step strategy, involving a partial protection of the chondroitin polysaccharide as a key step for gaining an unprecedented quantitative amidation of its glucuronic acid units. In-depth NMR and computational analysis suggested a fairly linear conformation for the semisynthetic polysaccharide, for which the antifreeze activity by a quantitative ice recrystallization inhibition assay was measured. We compared the structure-activity relationships for the Thr-derivatized chondroitin and the natural Thr-decorated CPS from C. psychrerythraea.

Confirmation of the configuration of two glucuronic acid units in glycyrrhizic acid

Confirmation of the configuration of two glucuronic acid units in glycyrrhizic acid

Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides.

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1–35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation.

Effect of Different Heat Treatment Temperatures on the Chemical Composition and Structure of Chinese Fir Wood

The properties of wood can be improved after heat-treatment. There have been many studies dealing with mechanical properties and chemical modifications of heat-treated wood. The purpose of this paper is to provide a new analysis method, to give better insights on the hemicelluloses, cellulose, and lignin contents of heat-treated wood by using Fourier transform infrared (FT-IR) and two-dimensional infrared (2D-IR) spectroscopies. The FT-IR spectra results showed progressive degradation in the carbonyl groups of the glucuronic acid units and degradation of the pyranose of hemicelluloses. These changes were measured as the percentage decrease of crystalline cellulose and the loss of C=O and C=C groups linking together the aromatic skeleton of lignin. The 2D-IR spectra showed the appearance of 5 × 5 peak clusters in the 1000 to 1200 cm-1 region, which could account for the hemicellulose degradation. In addition, changes in the degree of sensitivity at 1627 and 1509 cm-1 coincided with cross-linking reactions among the aromatic units in the lignin molecules.

Uncovering the Catalytic Direction of Chondroitin AC Exolyase: FROM THE REDUCING END TOWARDS THE NON-REDUCING END

Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin AC lyase (ChnAC) (EC 4.2.2.5) catalyzes the degradation of various GAGs, including chondroitin sulfate and hyaluronic acid, to give the corresponding disaccharides containing an Δ(4)-unsaturated uronic acid at their non-reducing terminus. ChnAC has been isolated from various bacteria and utilized as an enzymatic tool for study and evaluating the sequencing of GAGs. Despite its substrate specificity and the fact that its crystal structure has been determined to a high resolution, the direction in which ChnAC catalyzes the cleavage of oligosaccharides remain unclear. Herein, we have determined the structural cues of substrate depolymerization and the cleavage direction of ChnAC using model substrates and recombinant ChnAC protein. Several structurally defined oligosaccharides were synthesized using a chemoenzymatic approach and subsequently cleaved using ChnAC. The degradation products resulting from this process were determined by mass spectrometry. The results revealed that ChnAC cleaved the β1,4-glycosidic linkages between glucuronic acid and glucosamine units when these bonds were located on the reducing end of the oligosaccharide. In contrast, the presence of a GlcNAc-α-1,4-GlcA unit at the reducing end of the oligosaccharide prevented ChnAC from cleaving the GalNAc-β1,4-GlcA moiety located in the middle or at the non-reducing end of the chain. These interesting results therefore provide direct proof that ChnAC cleaves oligosaccharide substrates from their reducing end toward their non-reducing end. This conclusion will therefore enhance our collective understanding of the mode of action of ChnAC.

Medical Gains of Chondroitin Sulfate Upon Fucosylation.

Chondroitin sulfate (CS) is a glycosaminoglycan (GAG) composed of alternating N-acetyl galactosamine and glucuronic acid units within disaccharide building blocks. CS is a key functional component in proteoglycans of cartilaginous tissues. Owing to its numerous biological roles, CS is widely explored in the pharmaceutical market as nutraceutical ingredient commonly utilized against arthritis, osteoarthrosis, and sometimes osteoporosis. Tissues like shark cartilage and bovine trachea are common sources of CS. Nonetheless, a new CS type has been introduced and investigated in the last few decades in what regards its medical potentials. It is named fucosylated chondroitin sulfate (FucCS). This less common CS type is isolated exclusively from the body wall of sea cucumbers. The presence of fucosyl branching units in the holothurian FucCS gives to this unique GAG, therapeutic properties in various pathophysiological systems which are inexistent in the common CS explored in the market. Examples of these systems are coagulation, thrombosis, hemodialysis, atherosclerosis, cellular growth, angiogenesis, fibrosis, tumor growth, inflammation, viral and protozoan infections, hyperglycemia, diabetes-related pathological events and tissue damage. This report aims at describing the medical benefits gained upon fucosylation of CS. Clinical prospects of these medical benefits are also discussed herein.