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Talanta Nov 2022Glycans are the most abundant organic polymers in nature. They are essential to living organisms and regulate a wide range of biological functions. However, mass...
Glycans are the most abundant organic polymers in nature. They are essential to living organisms and regulate a wide range of biological functions. However, mass spectrometry-based identification of glycan isomers remains challenging due to the complexity of their structures including their complex compositions, linkages, and anomeric configurations. In this study, two novel complex ions, the mononuclear copper-bound dimeric ions [(Cu)(A)(L-His)-H] and the mononuclear copper-bound quaternary ions [(Cu)(A)(L-Ser)-H] (where A denotes a disaccharide, and L-Ser/His denotes l-serine/histidine), were designed for the collision-induced dissociation-based identification and relative quantification of 14 disaccharide isomers. When the unique fragmentation patterns of the above two types of complex ions were mapped into a three-dimensional vector, all the isomers were completely distinguished. Of note, the established method is able to identify mixtures of linkage isomers only using tandem mass spectrometry based on linkage-specific fragment ions of histidine-based complex ions. Finally, the method was successfully applied to the identification and relative quantification of two disaccharide isomers (lactose and sucrose) in dairy beverages. In conclusion, the established method is sensitive to subtle structural differences in disaccharide isomers and has the potential to be used for the differentiation of various glycans.
Topics: Copper; Disaccharides; Histidine; Ions; Isomerism; Polysaccharides; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry
PubMed: 35717753
DOI: 10.1016/j.talanta.2022.123674 -
Biochemical and Biophysical Research... Jul 2022Chondroitin sulfate (CS) and heparan sulfate (HS) are sulfated glycosaminoglycan (GAG) chains that consist of repeating disaccharide units composed of hexosamine and...
Chondroitin sulfate (CS) and heparan sulfate (HS) are sulfated glycosaminoglycan (GAG) chains that consist of repeating disaccharide units composed of hexosamine and hexuronic acid. GAG chains exhibit diverse bioactivities in a structure-specific manner. Marine invertebrates are a rich source of highly sulfated and rare structures of GAG chains. Here, we isolated GAGs from the green-lipped mussel Perna canaliculus, an aquaculture species that is produced on a large scale. We separated GAGs based on the degree of negative charges and analyzed their disaccharide compositions. CS and HS both exhibited characteristic compositions of differently sulfated disaccharides. CS chains showed a higher degree of sulfation than HS chains and contained a high percentage of the E unit disaccharide GlcA-GalNAc(4,6-O-disulfate). Furthermore, CS chains rich in the E unit stimulated the neurite outgrowth of primary cultured neurons. The present results indicate the potential of P. canaliculus GAGs as biomaterials to study the structure-function relationships of GAGs.
Topics: Animals; Chondroitin Sulfates; Disaccharides; Glycosaminoglycans; Heparitin Sulfate; Perna; Sulfates
PubMed: 35504089
DOI: 10.1016/j.bbrc.2022.04.095 -
PloS One 2022O-GlcNAcylation is the only sugar modification for proteins present in the cytoplasm and nucleus and is thought to be involved in the regulation of protein function and...
O-GlcNAcylation is the only sugar modification for proteins present in the cytoplasm and nucleus and is thought to be involved in the regulation of protein function and localization. Currently, several methods are known for detecting O-GlcNAcylated proteins using monoclonal antibodies or wheat germ agglutinin, but these methods have some limitations in their sensitivity and quantitative comparison. We developed a new disaccharide-tag method to overcome these problems. This is a method in which a soluble GalNAc transferase is expressed intracellularly, extended to a disaccharide of GalNAc-GlcNAc, and detected using a Wisteria japonica agglutinin specific to this disaccharide. We verified the method using human c-Rel protein and also highly sensitively compared the difference in O-GlcNAc modification of intracellular proteins associated with differentiation from embryonic stem cell (ESC) to epiblast-like cells (EpiLC). As one example of such a modification, a novel O-GlcNAc modification was found in the transcription factor Sox2 at residue Ser263, and the modification site could be identified by nano liquid chromatography-mass spectrometry.
Topics: Acetylglucosamine; Animals; Disaccharides; Glycosylation; Humans; Mammals; Mass Spectrometry; N-Acetylglucosaminyltransferases; Protein Processing, Post-Translational; Proteins
PubMed: 35604954
DOI: 10.1371/journal.pone.0267804 -
Biomacromolecules Jun 2021Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for...
Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP's unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs. Using nuclear magnetic resonance, two-dimensional infrared spectroscopy, and circular dichroism, the expected modifications were confirmed as well as their effect on AFGPs solution structure. We find that the presence of all the hydroxyls on the disaccharides is a requirement for the native AFGP hysteresis as well as the maximal inhibition of ice recrystallization. The saccharide hydroxyls are apparently as important as the acetyl group on the galactosamine, the α-linkage between the disaccharide and threonine, and the methyl groups on the threonine and alanine. We conclude that the use of hydrogen-bonding through the hydroxyl groups of the disaccharide and hydrophobic interactions through the polypeptide backbone are equally important in promoting the antifreeze activities observed in the native AFGPs. These important criteria should be considered when designing synthetic mimics.
Topics: Antifreeze Proteins; Disaccharides; Glycoproteins; Hydrogen Bonding; Ice; Magnetic Resonance Spectroscopy
PubMed: 33957041
DOI: 10.1021/acs.biomac.1c00313 -
Angewandte Chemie (International Ed. in... Mar 2020C-Glycosides are both a common motif in many bioactive natural products and important glycoside mimetics. We demonstrate that activating a hemiacetal with a sulfonyl...
C-Glycosides are both a common motif in many bioactive natural products and important glycoside mimetics. We demonstrate that activating a hemiacetal with a sulfonyl chloride, followed by treating the resultant glycosyl sulfonate with an enolate results in the stereospecific construction of β-linked C-glycosides. This reaction tolerates a range of acceptors and donors, including disaccharides. The resulting products can be readily derivatized into C-glycoside analogues of β-glycoconjugates, including C-disaccharide mimetics.
Topics: Alkylation; Disaccharides; Glycoconjugates; Glycosides; Glycosylation; Molecular Structure; Stereoisomerism; Sulfinic Acids
PubMed: 31880395
DOI: 10.1002/anie.201914221 -
Critical Reviews in Food Science and... 2022Production and applications of difructose anhydride III (DFA-III) have attracted considerable attention because of its versatile physiological functions. Recently,... (Review)
Review
Production and applications of difructose anhydride III (DFA-III) have attracted considerable attention because of its versatile physiological functions. Recently, large-scale production of DFA-III has been continuously explored, which opens a horizon for applications in the food and pharmaceutical industries. This review updates recent advances involving DFA-III, including: biosynthetic strategies, purification, and large-scale production of DFA-III; physiological functions of DFA-III and related mechanisms; DFA-III safety evaluations; present applications in food systems, existing problems, and further research prospects. Currently, enzymatic synthesis of DFA-III has been conducted both industrially and in academic research. Two biosynthetic strategies for DFA-III production are summarized: single- and double enzyme-mediated. DFA-III purification is achieved via yeast fermentation. Enzyme membrane bioreactors have been applied to meet the large-scale production demands for DFA-III. In addition, the primary physiological functions of DFA-III and their underlying mechanisms have been proposed. However, current applications of DFA-III are limited. Further research regarding DFA-III should focus on commercial production and purification, comprehensive study of physiological properties, extensive investigation of large-scale human experiments, and expansion of industrial applications. It is worthy to dig deep into potential application and commercial value of DFA-III.
Topics: Disaccharides; Fermentation; Humans; Saccharomyces cerevisiae
PubMed: 33775189
DOI: 10.1080/10408398.2021.1904823 -
Organic & Biomolecular Chemistry May 2022Heparan sulfate (HS), a glycosaminoglycan related to heparin, is a linear polysaccharide, consisting of repeating disaccharide units. This compound is involved in...
Heparan sulfate (HS), a glycosaminoglycan related to heparin, is a linear polysaccharide, consisting of repeating disaccharide units. This compound is involved in multiple biological processes such as inflammation, coagulation, angiogenesis and viral infections. Our work focuses on the synthesis of simple HS analogs for the study of structure-activity relationships, with the aim of modulating these biological activities. Thioglycoside analogs, in which the interglycosidic oxygen is replaced by a sulfur atom, are very interesting compounds in terms of therapeutic applications. Indeed, the thioglycosidic bond leads to an improvement of their stability and can allow the inhibition of enzymes involved in physiological and pathological processes. In our previous work, we developed a synthetic sequence which led to a non-sulfated thiodisaccharide analog of HS. In this paper, we report our results of the development of a new synthetic method allowing access to the novel sulfated -disaccharide, as well as to their oxygenated analogues (-disaccharide and sulfated -disaccharide). These 4 compounds were also tested for the inhibition of heparanase, an enzyme involved in biological processes like tumor growth and inflammation. The obtained IC values in the micromolar range showed the impact of the interglycosidic sulfur atom and the 6-sulfate group.
Topics: Disaccharides; Glucuronidase; Heparitin Sulfate; Humans; Inflammation; Sulfur
PubMed: 35388870
DOI: 10.1039/d2ob00250g -
Glycobiology Feb 2021Chondroitin sulfate (CS)and dermatan sulfate (DS) are negatively charged polysaccharides found abundantly in animal tissue and have been extensively described to play...
Chondroitin sulfate (CS)and dermatan sulfate (DS) are negatively charged polysaccharides found abundantly in animal tissue and have been extensively described to play key roles in health and disease. The most common method to analyze their structure is by digestion into disaccharides with bacterial chondroitinases, followed by chromatography and/or mass spectrometry. While studying the structure of oncofetal CS, we noted a large variation in the activity and specificity of commercially available chondroitinases. Here studied the kinetics of the enzymes and used high-performance liquid chromatography-mass spectrometry to determine the di- and oligosaccharide products resulting from the digestion of commercially available bovine CS A, shark CS C and porcine DS, focusing on chondroitinases ABC, AC and B from different vendors. Application of a standardized assay setup demonstrated large variations in the enzyme-specific activity compared to the values provided by vendors, large variation in enzyme specific activity of similar enzymes from different vendors and differences in the extent of cleavage of the substrates and the generated products. The high variability of different chondroitinases highlights the importance of testing enzyme activity and monitoring product formation in assessing the content and composition of chondroitin and DSs in cells and tissues.
Topics: Animals; Carbohydrate Conformation; Cattle; Chondroitin Sulfates; Chondroitinases and Chondroitin Lyases; Dermatan Sulfate; Disaccharides; Substrate Specificity; Swine
PubMed: 32573715
DOI: 10.1093/glycob/cwaa056 -
Methods in Molecular Biology (Clifton,... 2023Chondroitin sulfate proteoglycans (CSPGs) are polyanionic extra/pericellular matrix macromolecules that surround almost all cell types and create microenvironmental...
Chondroitin sulfate proteoglycans (CSPGs) are polyanionic extra/pericellular matrix macromolecules that surround almost all cell types and create microenvironmental niches to support miscellaneous cellular events. In general, the multifunctional properties of CSPGs are attributable to the structural divergence of the CS glycosaminoglycan (GAG) moieties. Because the expression profiles of the GAG chains of CSPGs change with developmental stage, aging, and disease progression, characterization of the GAG chains is essential to understand the functional roles of CSPGs. This chapter describes the basic protocols for GAG moiety-based immunochemical detection of CSPGs in biological samples in conjunction with CS disaccharide composition analysis.
Topics: Chondroitin Sulfate Proteoglycans; Glycosaminoglycans; Disaccharides; Chondroitin Sulfates; Chondroitin
PubMed: 36662459
DOI: 10.1007/978-1-0716-2946-8_2 -
Electrophoresis Jan 2023The feasibility of on-capillary derivatization of saccharides by aromatic amine-based fluorescent labeling agents was tested. To avoid the problematic evolution of...
The feasibility of on-capillary derivatization of saccharides by aromatic amine-based fluorescent labeling agents was tested. To avoid the problematic evolution of gaseous hydrogen cyanide, the Schiff base reduction by sodium cyanoborohydride, as the second step of the standard reductive amination protocol, was omitted. Glucose was used as a model analyte and 7-amino-1,3-naphthalenedisulfonic acid as the labeling agent. Our experiments showed that the direct reaction of the saccharide with the labeling agent in 2.5-M acetic acid yields a labeled product that is sufficiently stable to be separated from the labeling agent in 20-mM phosphate buffer, pH 3.5, and detected using UV detection. The glucose and label zones were introduced separately into the capillary and mixed using a negative voltage. Mixing voltage, its duration, the concentration of acetic acid in the reaction zone, and the waiting time between mixing and separation were optimized. To show the applicability of the procedure to a broader range of analytes, a mixture of different types of saccharides, that is, xylose (pentose), fucose (hexose), glucose (hexose), N-acetylglucosamine (N-acetylaminosaccharide), and lactose (disaccharide), was subjected to derivatization and analysis under the optimal conditions. The linearity and repeatability of the process were evaluated as critical parameters for its analytical applications. Six-point calibration dependences in the 1-50 mM range showed excellent determination coefficients of 0.9992 or higher for all five saccharides tested. The repeatability of the labeled saccharide peak areas was between 2.2% and 4.3%.
Topics: Glucose; Acids; Coloring Agents; Electrophoresis, Capillary; Disaccharides
PubMed: 35699059
DOI: 10.1002/elps.202200136