Oligosaccharide Fragments Research Articles

Stimulation of adventitious root formation by the oligosaccharin OSRG at the transcriptome level

ABSTRACTOligosaccharins, which are biologically active oligosaccharide fragments of cell wall polysaccharides, may regulate the processes of growth and development as well as the response to stress factors. We characterized the effect of the oligosaccharin that stimulates rhizogenesis (OSRG) on the gene expression profile in the course of IAA-induced formation of adventitious roots in hypocotyl explants of buckwheat (Fagopyrum esculentum Moench.). The transcriptomes at two stages of IAA-induced root primordium formation (6 h and 24 h after induction) were compared after either treatment with auxin alone or joint treatment with auxin and OSRG. The set of differentially expressed genes indicated the special importance of oligosaccharin at the early stage of auxin-induced adventitious root formation. The list of genes with altered mRNA abundance in the presence of oligosaccharin included those, which Arabidopsis homologs encode proteins directly involved in the response to auxin as well as proteins that contribute to redox regulation, detoxification of various compounds, vesicle trafficking, and cell wall modification. The obtained results contribute to understanding the mechanism of adventitious root formation and demonstrate that OSRG is involved in fine-tuning of ROS and auxin regulatory modes involved in root development.

Mechanism by which β-glucanase improves the quality of fermented barley flour-based food products

The enrichment of β-glucan in barley significantly decreases the quality of dough and baked bread, and β-glucanase can improve dough and bread quality. Nevertheless, the mechanism by which β-glucanase improves the quality of fermented barley flour-based products is still poorly understood. The gluten microcosmic structure, molecular structure and yeast gas production capacity were investigated using Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy, Scanning electron microscopy (SEM) and F3 rheological fermentation techniques. The results showed that β-Glucanase can degrade the high-molecular-weight β-glucan to low-molecular-weight oligosaccharide fragments, which reduces the viscosity of the β-glucans, promotes cross-linking between the gluten molecules, and indirectly improves the gluten network structure, thereby alleviating the negative effects of β-glucans. The β-glucosidase produced during yeast metabolism can further hydrolyse low-molecular-weight oligosaccharides into reducing sugars that can be used by yeast, increasing the carbon sources available to yeasts and the gas production capability of yeasts.

Structural characteristics of carrageenans of red alga Mastocarpus pacificus from sea of Japan

The sulfated polysaccharide from sterile alga Mastocarpus pacificus was investigated. Partial reductive hydrolysis and NMR spectroscopy showed that the extracted polysaccharides were only carrageenans. According to FT-IR- and NMR spectroscopy this polysaccharide was a hybrid kappa/iota-carrageenan with a predominance of kappa-type units. According to MALDI-TOFMS, oligosaccharide fragments obtained by mild acid hydrolysis had a polymerization degree of 1–9, while chains built up of galactose residues were up to 3. Tandem ESI mass spectrometry together with innovative 18O-labelling method showed that the polymer chain of the carrageenan included kappa-carrabiose, kappa-carratetraose, iota-carrabiose, hybrid kappa/iota oligosaccharide units and contained minor insertions of mu-carrageenan (the precursor of kappa-carrageenan). Parallel artificial membrane permeability assay shown that the studied carrageenan inhibited bile salts permeation through an artificial membrane imitating the gastrointestinal barrier by 50 % on average compared to negative control independent of incubation time. However, its action was less pronounced than the hindering ability of cholestyramine.

A top-down chemo-enzymatic approach towards N-acetylglucosamine-N-acetylmuramic oligosaccharides: Chitosan as a reliable template

An unprecedented approach towards oligosaccharides containing N-acetylglucosamine-N-acetylmuramic (NAG-NAM) units was developed. These novel bacterial cell wall surrogates were obtained from chitosan via a top down approach involving both chemical and enzymatic reactions. The chemical modification of chitosan using a molecular clamp based strategy, allowed obtaining N-acetylglucosamine-N-acetylmuramic (NAG-NAM) containing oligomers. Intercalation of NAM residues was confirmed through the analysis of oligosaccharide fragments from enzymatic digestion and it was found that this route affords NAG-NAM containing oligosaccharides in 33% yield. These oligosaccharides mimic the carbohydrate basic skeleton of most bacterial cell surfaces. The oligosaccharides prepared are biologically relevant and will serve as a platform for further molecular recognition studies with different receptors and enzymes of both bacterial cell wall and innate immune system. This strategy combining both chemical modification and enzymatic digestion provides a novel and simple route for an easy access to bacterial cell wall fragments – biologically important targets.

Research progress and secondary development ideas of Astragali Radix polysaccharides for injection

Astragali Radix( AR) polysaccharide for injection( Guoyao Zhunzi Z20040086) is a traditional Chinese medicine for intravenous powder injection developed by Shanxi Academy of Traditional Chinese Medicine in early 1990 s by taking advantage of AR resources in Shanxi province. The effective parts of AR polysaccharides were obtained by advanced technology. The hemogram of patients with radiotherapy and chemotherapy showed alleviations in clinic. However,due to the technical bottleneck in separation of the complex polysaccharides mixture and the difficulties in accurate measurement of the polysaccharide structures,the pharmacodynamic mechanism of the drug remained unclear,and the side effect was hard to control. In recent years,the theoretical studies for polysaccharide receptors have indicated that when polysaccharides bound to protein receptors,only the oligosaccharide fragments of the polysaccharide molecule bound to the receptors,and one or more active sites of oligosaccharide fragments may existed in the polysaccharide molecule.Therefore,the active center of polysaccharides can be studied based on the level of oligosaccharides through degradation of the polysaccharides,which provided a new strategy for breaking through the bottleneck in polysaccharide structure determination. Therefore,this paper reviews the current status of studies for AR polysaccharides for injection,the polysaccharide receptors theory and successful cases,in order to propose the secondary development ideas of AR polysaccharides for injection. The study results will lay a material foundation for the development of new drugs of polysaccharides from traditional Chinese medicine,and provide a basis for the resolution of international difficulties in quality control of polysaccharide drugs and molecular models,so as to further study of glycobiology,and enrich the polysaccharide receptors theory.

Gas-Phase Fragmentation of Cyclic Oligosaccharides in Tandem Mass Spectrometry.

Modern mass spectrometry, including electrospray and MALDI, is applied for analysis and structure elucidation of carbohydrates. Cyclic oligosaccharides isolated from different sources (bacteria and plants) have been known for decades and some of them (cyclodextrins and their derivatives) are widely used in drug design, as food additives, in the construction of nanomaterials, etc. The peculiarities of the first- and second-order mass spectra of cyclic oligosaccharides (natural, synthetic and their derivatives and modifications: cyclodextrins, cycloglucans, cyclofructans, cyclooligoglucosamines, etc.) are discussed in this minireview.

Traceless Photolabile Linker Expedites the Chemical Synthesis of Complex Oligosaccharides by Automated Glycan Assembly.

Automated glycan assembly (AGA) aims at accelerating access to synthetic oligosaccharides to meet the demand for defined glycans as tools for molecular glycobiology. The linkers used to connect the growing glycan chain to the solid support play a pivotal role in the synthesis strategy as they determine all chemical conditions used during the synthesis and the form of the glycan obtained at the end of it. Here, we describe a traceless photolabile linker used to prepare carbohydrates with a free reducing end. Modification of the o-nitrobenzyl scaffold of the linker is key to high yields and compatibility with the AGA workflow. The assembly of an asymmetrical biantennary N-glycan from oligosaccharide fragments prepared by AGA and linear as well as branched β-oligoglucans is described to illustrate the power of the method. These substrates will serve as standards and biomarkers to examine the unique specificity of glycosyl hydrolases.

Differentiation of Intracellular Hyaluronidase Isoform by Degradable Nanoassembly Coupled with RNA-Binding Fluorescence Amplification.

Hyaluronidase has two cruical isoforms, hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2), which are essential for cellular hyaluronic acid (HA) catabolism to generate different-sized oligosaccharide fragments for performing different physiological functions. In particular, Hyal-1 is the major tumor-derived hyaluronidase. Thus, specific detection of one hyaluronidase isoform, especially Hyal-1, in live cells is of scientific significance but remains challenging. Herein, by use of differentiated tolerance capability of an amphiphilic HA-based nanoassembly to Hyal-1 and Hyal-2, we rationally design a Hyal-1 specific nanosensor, consisting of cholesterylamine-modified HA nanoassembly (CHA) and RNA-binding fluorophores (RBF). The RBF molecules were entrapped in CHA to switch off their fluorescence via aggregation caused quenching. However, CHA can be disassembled by Hyal-1 to release RBF, resulting in fluorescence activation. Moreover, the fluorescence of the released RBF is further enhanced by cytoplasm RNA. Owing to this cascade signal amplification, this nanosensor RBF@CHA displays a significant change of signal-to-background-noise ratio (120-fold) toward 16 μg/mL Hyal-1 in cellular lysates. In contrast, it is resistant to Hyal-2. By virtue of its selective and sensitive characteristics under a complicated matrix, RBF@CHA had been successfully applied for specifically visualizing Hyal-1 over Hyal-2 inside live cells for the first time, detecting a low level of intracellular Hyal-1 and distinguishing normal and cancer cells with different expressions of Hyal-1. This approach would be useful to better understand biological functions and related diseases of intracellular Hyal-1.

Precise Structure and Anticoagulant Activity of Fucosylated Glycosaminoglycan from Apostichopus japonicus: Analysis of Its Depolymerized Fragments.

Apostichopus japonicus is one of the most economically important species in sea cucumber aquaculture in China. Fucosylated glycosaminoglycan from A. japonicus (AjFG) has shown multiple pharmacological activities. However, results from studies on the structure of AjFG are still controversial. In this study, the deaminative depolymerization method that is glycosidic bond-selective was used to prepare the depolymerized products from AjFG (dAjFG), and then a series of purified oligosaccharide fragments such as tri-, hexa-, nona-, and dodecasaccharides were obtained from dAjFG by gel permeation chromatography. The 1D/2D NMR and ESI-MS spectrometry analyses showed that these oligosaccharides had the structural formula of l-FucS-α1,3-d-GlcA-β1,3-{d-GalNAc4S6S-β1,4-[l-FucS-α1,3-]d-GlcA-β1,3-}n-d-anTal-diol4S6S (n = 0, 1, 2, 3; FucS represents Fuc2S4S, Fuc3S4S, or Fuc4S). Thus, the unambiguous structure of native AjFG can be rationally deduced: it had the backbone of {-4-d-GlcA-β1,3-d-GalNAc4S6S-β1-}n, which is similar to chondroitin sulfate E, and each d-GlcA residue in the backbone was branched with a l-FucS monosaccharide at O-3. Bioactivity assays confirmed that dAjFG and nonasaccharides and dodecasaccharides from AjFG had potent anticoagulant activity by intrinsic FXase inhibition while avoiding side effects such as FXII activation and platelet aggregation.

An Arabidopsis berberine bridge enzyme-like protein specifically oxidizes cellulose oligomers and plays a role in immunity.

The plant cell wall is the barrier that pathogens must overcome to cause a disease, and to this end they secrete enzymes that degrade the various cell wall components. Due to the complexity of these components, several types of oligosaccharide fragments may be released during pathogenesis and some of these can act as damage-associated molecular patterns (DAMPs). Well-known DAMPs are the oligogalacturonides (OGs) released upon degradation of homogalacturonan and the products of cellulose breakdown, i.e. the cellodextrins (CDs). We have previously reported that four Arabidopsis berberine bridge enzyme-like (BBE-like) proteins (OGOX1-4) oxidize OGs and impair their elicitor activity. We show here that another Arabidopsis BBE-like protein, which is expressed coordinately with OGOX1 during immunity, specifically oxidizes CDs with a preference for cellotriose (CD3) and longer fragments (CD4-CD6). Oxidized CDs show a negligible elicitor activity and are less easily utilized as a carbon source by the fungus Botrytis cinerea. The enzyme, named CELLOX (cellodextrin oxidase), is encoded by the gene At4g20860. Plants overexpressing CELLOX display an enhanced resistance to B. cinerea, probably because oxidized CDs are a less valuable carbon source. Thus, the capacity to oxidize and impair the biological activity of cell wall-derived oligosaccharides seems to be a general trait of the family of BBE-like proteins, which may serve to homeostatically control the level of DAMPs to prevent their hyperaccumulation.

Heteromultivalent Glycooligomers as Mimetics of Blood Group Antigens.

Precision glycomacromolecules have proven to be important tools for the investigation of multivalent carbohydrate-lectin interactions by presenting multiple glycan epitopes on a highly-defined synthetic scaffold. Herein, we present a new strategy for the versatile assembly of heteromultivalent glycomacromolecules that contain different carbohydrate motifs in proximity within the side chains. A new building block suitable for the solid-phase polymer synthesis of precision glycomacromolecules was developed with a branching point in the side chain that bears a free alkyne and a TIPS-protected alkyne moiety, which enables the subsequent attachment of different carbohydrate motifs by on-resin copper-mediated azide-alkyne cycloaddition reactions. Applying this synthetic strategy, heteromultivalent glycooligomers presenting fragments of histo-blood group antigens and human milk oligosaccharides were synthesized and tested for their binding behavior towards bacterial lectin LecB.

Cell wall traits that influence plant development, immunity, and bioconversion.

The architecture of the plant cell wall is highly dynamic, being substantially re-modeled during growth and development. Cell walls determine the size and shape of cells and contribute to the functional specialization of tissues and organs. Beyond the physiological dynamics, the wall structure undergoes changes upon biotic or abiotic stresses. In this review several cell wall traits, mainly related to pectin, one of the major matrix components, will be discussed in relation to plant development, immunity and industrial bioconversion of biomass, especially for energy production. Plant cell walls are a source of oligosaccharide fragments with a signaling function for both development and immunity. Sensing cell wall damage, sometimes through the perception of released damage-associated molecular patterns (DAMPs), is crucial for some developmental and immunity responses. Methodological advances that are expected to deepen our knowledge of cell wall (CW) biology will also be presented.

Desiccation tolerance in plants: Structural characterization of the cell wall hemicellulosic polysaccharides in three Selaginella species

Drought-induced dehydration of vegetative tissues in lycopods affects growth and survival. Different species of Selaginella have evolved a series of specialized mechanisms to tolerate desiccation in vegetative tissues in response to water stress. In the present study, we report on the structural characterization of the leaf cell wall of the desiccation-tolerant species S. involvens and two desiccation-sensitive species, namely S. kraussiana and S. moellendorffii. Isolated cell walls from hydrated and desiccated leaves of each species were fractionated and the resulting oligosaccharide fragments were analyzed to determine their structural features. Our results demonstrate that desiccation induces substantial modifications in the cell wall composition and structure. Altogether, these data highlight the fact that structural remodeling of cell wall hemicellulosic polysaccharides including XXXG-rich xyloglucan, arabinoxylan and acetylated galactomannan is an important process in order to mitigate desiccation stress in Selaginella.

Novel Dextran-Supported Biological Probes Decorated with Disaccharide Entities for Investigating the Carbohydrate-Protein Interactions of Gal-3.

The quest for novel natural‐like biomolecular probes that can be used to gain information on biological recognition events is of topical interest to several scientific areas. In particular, the recognition of carbohydrates by proteins modulates a number of important biological processes. These molecular recognition events are, however, difficult to study by the use of naturally occurring oligosaccharides and polysaccharides owing to their intrinsic structural heterogeneity and to the many technical difficulties encountered during the isolation of sufficient quantities of pure material for detailed structural and biological studies. Therefore, the construction of homogenous biomolecular probes that can mimic both the biophysical properties of polysaccharide backbones and the properties of bioactive oligosaccharide fragments are highly sought after. Herein, synthetic methodology for the construction of well‐defined bioconjugates consisting of biologically relevant disaccharide fragments grafted onto a dextran backbone is presented, and a preliminary NMR spectroscopy study of their interactions with galectin‐3 as a model lectin is conducted.

A mechanistic microbial underpinning for the size-reactivity continuum of dissolved organic carbon degradation

The reservoir of dissolved organic carbon (DOC) in the ocean is modified by multiple input and removal processes. Incubation experiments as well as measurements of oceanic DOC have demonstrated that the high molecular weight (HMW) fraction of DOC typically has a younger radiocarbon age and is more reactive biologically than the low molecular weight (LMW) fraction of DOC. These observations have been summarized as a ‘size-reactivity continuum’ of DOC reactivity, but mechanistic explanations for these observations have been lacking. Here we describe how our recent discovery of ‘selfish’ HMW organic matter uptake among bacteria in surface ocean waters may help explain the rapid removal of HMW DOC. ‘Selfish’ substrate uptake by bacteria encompasses rapid binding and partial hydrolysis of intact polysaccharides on the outer membrane of bacteria, seamlessly followed by the transport of large oligosaccharide fragments into the periplasm with little to no loss of LMW hydrolysis products. ‘Selfish’ bacteria therefore process HMW substrates in a manner distinct from bacteria that carry out extracellular hydrolysis that yields LMW hydrolysis products in the environment. Recognition of the presence and prevalence of selfish bacteria in the ocean has profound implications for carbon flow – the source and quantity of LMW substrates made available to non-extracellular-enzyme producing bacteria – as well as for efforts to model and measure bacterial interactions during organic matter degradation. This discovery also highlights the importance of targeted substrate binding and uptake as key (often understudied) factors in geochemical investigations of microbially driven carbon cycling in the ocean. We conclude with some speculative thoughts about the factors that may determine the prevalence of selfish substrate uptake in the environment.

Application of a Janus aglycon with dual function in benzyl-free synthesis of spacer-armed oligosaccharide fragments of polysaccharides from rhizobacterium Azospirillum brasilense sp7

Both protective and pre-spacer features of 4-(2-chloroethoxy)phenyl (CEP) aglycon, which belong to the class of Janus aglycons, were engaged in a benzyl-free synthesis of oligosaccharide fragments of polysaccharides from rhizobacterium Azospirillum brasilense sp7. Introduction of α-1,4-linked L-fucose residue was performed using 3,4-di-O-benzoyl-2-O-triisopropylsilyl-α-L-fucopyranosyl N-phenyltrifluoroacetimidate in excellent stereoselectivity and high yields. The obtained deprotected di-, tri- and tetrasaccharides contain 4-(2-azidoethoxy)phenyl (AEP) spacer aglycon, which allows straightforward preparation of neoglycoconjugates that will be used for the study of the role of lipopolysaccharide of rhizobacterium A. brasilense sp7 in plant–microbe symbiosis. The intermediate protected oligosaccharide building blocks with cleavable CEP/AEP aglycons have a strong potential for further application in the synthesis of more complex oligosaccharides.

Study of the Conformational Variety of the Oligosaccharide Substrates of Neuraminidases from Pathogens using Molecular Modeling

Analysis of the conformational variety of the oligosaccharide fragments of the human glycan receptors LSTa (α-D-Neu5Ac-(2-3)-β-D-Gal-(1-3)-β-D-GlcNAc-(1-3)-β-D-Gal(1-4)-D-Glc) and LSTc (α-D-Neu5Ac-(2-6)-β-D-Gal-(1-3)-β-D-GlcNAc-(1-3)-β-D-Gal(1-4)-D-Glc) in aqueous solution has been performed with the comprehensive use of molecular modeling and statistical data processing followed by determination of major and minor stabilized conformers and selection of relevant topologies. The sialic acid ring conformational free energy landscape for both pentasaccharides has been reconstructed and analyzed giving a specification of the most probable distorted ring conformations of the basic chair 1C4 structure. The obtained results are in a good agreement with experimental data generated by nuclear magnetic resonance spectroscopy and X-ray crystallography.

Structural characterization of Glehniae Radix polysaccharides using partial acid hydrolysis-hydrophilic interaction liquid chromatography-mass spectrometry

Water-soluble polysaccharides from traditional Chinese medicine have properties of complex structure and high molecular, resulting in hardly complete their structural characterization.However, a "bottom-up" approach could solve this problem.Glehniae Radix extract was extracted with hot water and then precipitated by 40% ethanol to obtain Glehniae Radix polysaccharides (RGP). Subsequently, a partial acid hydrolysis method was carried out and the effects of acid concentration, time and temperature on hydrolysis were investigated. Under the optimum hydrolysis condition (1.5 mol•L⁻¹ trifluoroacetic acid, 4 h, and 80 ℃), RGP were hydrolyzed to characteristic oligosaccharide fragments. Futher, a hydrophilic liquid chromatography- mass spectrometry method was used for the separation and structural characterization of the polysaccharide hydrolysates. According to MS and MS/MS analysis of several standard disaccharides, a method for determining the type of polysaccharide glycosidic linkage by mass spectrometry was established. The results showed that the polysaccharide hydrolysates were linear glucan containing 1, 4-glycosidic bonds. And gluco-oligosaccharides with the degrees of polymerization (DP) of 4-11 were obtained after partial acid hydrolysis.

25 負イオンMALDI質量分析法による中性糖鎖の構造解析(口頭発表の部)

25 負イオンMALDI質量分析法による中性糖鎖の構造解析(口頭発表の部)

Chemical Synthesis of Oligosaccharides Related to the Cell Walls of Plants and Algae.

Plant cell walls are composed of an intricate network of polysaccharides and proteins that varies during the developmental stages of the cell. This makes it very challenging to address the functions of individual wall components in cells, especially for highly complex glycans. Fortunately, structurally defined oligosaccharides can be used as models for the glycans, to study processes such as cell wall biosynthesis, polysaccharide deposition, protein-carbohydrate interactions, and cell-cell adhesion. Synthetic chemists have focused on preparing such model compounds, as they can be produced in good quantities and with high purity. This Review contains an overview of those plant and algal polysaccharides that have been elucidated to date. The majority of the content is devoted to detailed summaries of the chemical syntheses of oligosaccharide fragments of cellulose, hemicellulose, pectin, and arabinogalactans, as well as glycans unique to algae. Representative synthetic routes within each class are discussed in detail, and the progress in carbohydrate chemistry over recent decades is highlighted.