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Chembiochem : a European Journal of... Dec 2022SARS-CoV-2 infects human epithelial cells through specific interaction with angiotensin-converting enzyme 2 (ACE2). In addition, heparan sulfate proteoglycans act as the...
SARS-CoV-2 infects human epithelial cells through specific interaction with angiotensin-converting enzyme 2 (ACE2). In addition, heparan sulfate proteoglycans act as the attachment factor to promote the binding of viral spike protein receptor binding domain (RBD) to ACE2 on host cells. Though the rapid development of vaccines has contributed significantly to preventing severe disease, mutated SARS-CoV-2 strains, especially the SARS-CoV-2 Omicron variant, show increased affinity of RBD binding to ACE2, leading to immune escape. Thus, there is still an unmet need for new antiviral drugs. In this study, we constructed pharmacophore models based on the spike RBD of SARS-CoV-2 and SARS-CoV-2 Omicron variant and performed virtual screen for best-hit compounds from our disaccharide library. Screening of 96 disaccharide structures identified two disaccharides that displayed higher binding affinity to RBD in comparison to reported small molecule antiviral drugs. Further, screening PharmMapper demonstrated interactions of the disaccharides with a number of inflammatory cytokines, suggesting a potential for disaccharides with multiple-protein targets.
Topics: Humans; Angiotensin-Converting Enzyme 2; Antiviral Agents; Binding Sites; COVID-19; Disaccharides; Protein Binding; Receptors, Virus; SARS-CoV-2; Spike Glycoprotein, Coronavirus; High-Throughput Screening Assays
PubMed: 36265004
DOI: 10.1002/cbic.202200461 -
Biochemistry Sep 20203--Sulfation on the glucosamine sugar unit in heparan sulfate (HS) is linked to various biological functions, including the anticoagulant activity to treat thrombotic...
3--Sulfation on the glucosamine sugar unit in heparan sulfate (HS) is linked to various biological functions, including the anticoagulant activity to treat thrombotic disorders in hospitals. The 3--sulfated glucosamine is biosynthesized by heparan sulfate glucosamine 3-sulfotransferases. Because of its biological significance, there is a need for 3--sulfated oligosaccharide standards to facilitate the compositional analysis of HS. These oligosaccharides must contain a Δ-unsaturated uronic acid (ΔUA) residue at the nonreducing end, which is due to the depolymerization reaction catalyzed by heparin lyases used during the compositional analysis procedure. Here, we describe a protocol for the preparation of one 3--sulfated disaccharide (compound ) and three 3--sulfated tetrasaccharides (compound -) in a milligram scale. The synthesis of 3--sulfated disaccharide and tetrasaccharide standards was completed by degrading synthetic octasaccharides using heparin lyases. Further analysis revealed that 3--sulfated oligosaccharide standards are labile under basic conditions, confirming the findings from a previous study. The unwanted degradation was reduced by decreasing the pH in the presence of phosphate buffer. The 3--sulfated oligosaccharide standards are reagents to characterize 3--sulfation in HS derived from biological sources.
Topics: Chemistry Techniques, Synthetic; Disaccharides; Heparitin Sulfate; Reference Standards
PubMed: 31608625
DOI: 10.1021/acs.biochem.9b00838 -
Glycoconjugate Journal Feb 2010The whole tissue of the earthworm (Eisenia andrei) was lyophilized and extracted to purify glycosaminoglycans. Fractions, eluting from an anion-exchange column at 1.0 M...
The whole tissue of the earthworm (Eisenia andrei) was lyophilized and extracted to purify glycosaminoglycans. Fractions, eluting from an anion-exchange column at 1.0 M and 2.0 M NaCl, showed the presence of acidic polysaccharides on agarose gel electrophoresis. Monosaccharide compositional analysis showed that galactose and glucose were most abundant monosaccharides in both fractions. Depolymerization of the polysaccharide mixture with glycosaminoglycan-degrading enzymes confirmed the presence of chondroitin sulfate/dermatan sulfate and heparan sulfate in the 2.0 M NaCl fraction. The content of GAGs (uronic acid containing polysaccharide) in the 2.0 M NaCl fraction determined by carbazole assay was 2%. Disaccharide compositional analysis using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis after chondroitinase digestion (ABC and ACII), showed that the chondroitin sulfate/dermatan sulfate contained a 4-O-sulfo (76%), 2,4-di-O-sulfo (15%), 6-O-sulfo (6%), and unsulfated (4%) uronic acid linked N-acetylgalactosamine residues. LC-ESI-MS analysis of heparin lyase I/II/III digests demonstrated the presence of N-sulfo (69%), N-sulfo-6-O-sulfo (25%) and 2-O-sulfo-N-sulfo-6-O-sulfo (5%) uronic acid linked N-acetylglucosamine residues.
Topics: Animals; Chromatography, Ion Exchange; Chromatography, Liquid; Disaccharides; Electrophoresis, Agar Gel; Glycosaminoglycans; Monosaccharides; Oligochaeta; Spectrometry, Mass, Electrospray Ionization
PubMed: 20013352
DOI: 10.1007/s10719-009-9273-6 -
International Journal of Molecular... Sep 2020The level of human natural antibodies of immunoglobulin M isotype against Le in patients with breast cancer is lower than in healthy women. The epitope specificity of...
The level of human natural antibodies of immunoglobulin M isotype against Le in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors' blood using Le-Sepharose (Le is Galβ1-3GlcNAcβ). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with Le, which implies the impossibility of binding to regular glycoproteins of non-malignant cells. The avidity (as dissociation constant value) of antibodies probed with a multivalent disaccharide is 10 M; the nanomolar level indicates that the concentration is sufficient for physiological binding to the cognate antigen. Testing of several breast cancer cell lines showed the strongest binding to ZR 75-1. Interestingly, only 7% of the cells were positive in a monolayer with a low density, increasing up to 96% at highest density. The enhanced interaction (instead of the expected inhibition) of antibodies with ZR 75-1 cells in the presence of Galβ1-3GlcNAcβ disaccharide, indicates that the target epitope of anti-Le antibodies is a molecular pattern with a carbohydrate constituent rather than a glycan.
Topics: Animals; Antibodies; Breast Neoplasms; Cell Line, Tumor; Disaccharides; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Galactans; Humans; Immunoglobulin M; Mice; Mice, Knockout; Polysaccharides; Protein Binding
PubMed: 32899593
DOI: 10.3390/ijms21186511 -
Archives of Biochemistry and Biophysics May 2023The lactate dehydrogenase from rabbit skeletal muscle (rbLDH) is a tetrameric enzyme, known to undergo dissociation when exposed to acidic pH conditions. Moreover, it...
The lactate dehydrogenase from rabbit skeletal muscle (rbLDH) is a tetrameric enzyme, known to undergo dissociation when exposed to acidic pH conditions. Moreover, it should be mentioned that this dissociation translates into a pronounced loss of enzyme activity. Notably, among the compounds able to stabilize proteins and enzymes, the disaccharide trehalose represents an outperformer. In particular, trehalose was shown to efficiently counteract quite a number of physical and chemical agents inducing protein denaturation. However, no information is available on the effect, if any, exerted by trehalose against the dissociation of protein oligomers. Accordingly, we thought it of interest to investigate whether this disaccharide is competent in preventing the dissociation of rbLDH induced by acidic pH conditions. Further, we compared the action of trehalose with the effects triggered by maltose and cellobiose. Surprisingly, both these disaccharides enhanced the dissociation of rbLDH, with maltose being responsible for a major effect when compared to cellobiose. On the contrary, trehalose was effective in preventing enzyme dissociation, as revealed by activity assays and by Dynamic Light Scattering (DLS) experiments. Moreover, we detected a significant decrease of both K and V when the rbLDH activity was tested (at pH 7.5 and 6.5) as a function of pyruvate concentration in the presence of trehalose. Further, we found that trehalose induces a remarkable increase of V when the enzyme is exposed to pH 5. Overall, our observations suggest that trehalose triggers conformational rearrangements of tetrameric rbLDH mirrored by resistance to dissociation and peculiar catalytic features.
Topics: Animals; Rabbits; Trehalose; Maltose; Cellobiose; L-Lactate Dehydrogenase; Disaccharides; Hydrogen-Ion Concentration
PubMed: 37001749
DOI: 10.1016/j.abb.2023.109584 -
The FEBS Journal Apr 2020Carbohydrate (or sugar) molecules are extremely diverse regarding their length, linkage and epimeric state. Selective acquisition of these molecules inside the cell is...
Carbohydrate (or sugar) molecules are extremely diverse regarding their length, linkage and epimeric state. Selective acquisition of these molecules inside the cell is achieved by the substrate (or solute)-binding protein of ATP-binding cassette (ABC) transport system. However, the molecular mechanism underlying the selective transport of diverse carbohydrates remains unclear mainly owing to their structural complexity and stereochemistry. This study reports crystal structures of an α-glycoside-binding protein (αGlyBP, ORF ID: TTHA0356 from Thermus thermophilus HB8) in complex with disaccharide α-glycosides namely trehalose (α-1,1), sucrose (α-1,2), maltose (α-1,4), palatinose (α-1,6) and glucose within a resolution range of 1.6-2.0 Å. Despite transporting multiple types of sugars, αGlyBP maintains its stereoselectivity for both glycosidic linkage as well as an epimeric hydroxyl group. Out of the two subsites identified in the active-site pocket, subsite B which accommodates the glucose and glycosyl unit of disaccharide α-glycosides is highly conserved. In addition, structural data confirms the paradoxical behavior of glucose, where it replaces the high-affinity ligand(s) (disaccharide α-glycosides) from the active site of the protein. Comparative assessment of open and closed conformations of αGlyBP along with mutagenic and thermodynamic studies identifies the hinge region as the first interaction site for the ligands. On the other hand, encapsulation of ligand inside the active site is achieved through the N-terminal domain (NTD) movement, whereas the C-terminal domain (CTD) of αGlyBP is identified to be rigid and postulated to be responsible for maintaining the interaction with the transmembrane domain (TMD) during substrate translocation. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession number(s) 6J9W, 6J9Y, 6JAD, 6JAG, 6JAH, 6JAI, 6JAL, 6JAM, 6JAN, 6JAO, 6JAP, 6JAQ, 6JAR, 6JAZ, 6JB0, 6JB4, 6JBA, 6JBB and 6JBE.
Topics: ATP-Binding Cassette Transporters; Bacterial Proteins; Computational Biology; Crystallography, X-Ray; Disaccharides; Glycosides; Models, Molecular; Protein Conformation; Thermodynamics; Thermus thermophilus
PubMed: 31608555
DOI: 10.1111/febs.15093 -
Journal of Biochemistry Feb 1998Compositional analyses of heparin (Hep) and heparan sulfate (HS) have been undertaken with disaccharide units obtained by either enzymatic digestion with heparitinases...
Compositional analyses of heparin (Hep) and heparan sulfate (HS) have been undertaken with disaccharide units obtained by either enzymatic digestion with heparitinases or hydrazinolysis/deamination reaction of polysaccharides. Unsaturated disaccharide units generated by the enzymatic method are detectable on HPLC with a uv detector recording absorbance at 230 nm. On the other hand, disaccharide units generated by the chemical method possess a component of 2,5-anhydromannose (AnMan) bearing aldehyde groups in addition to intact iduronic acid (IdoA) or glucuronic acid (GlcA). The aldehyde groups of the disaccharide units are usually reduced with sodium borotritide, and detected by radiochromatography. Both of them, however, involve inevitable experimental problems, such as the use of costly enzymes and radioisotopes. In the present study, we have established a novel composition analysis system for Hep and HS essentially based on the chemical method. After hydrazinolysis/deamination treatment of Hep and HS, the aldehyde groups of AnMan in the disaccharide units generated were coupled with paranitrophenyl (PNP-) hydrazine instead of reduction with sodium borotritide, AnMan-CH=N-NH-PNP (AnMan-PNP) being formed. Then, the PNP-labeled disaccharides were pre-treated on a SepPak C-18 cartridge column, and subsequently separated and detected on ion-pairing reversed-phase HPLC with a detector recording absorbance at 390 nm. With the present system, the order of elution was GlcA-AnMan-PNP (GM), IdoA-AnMan-PNP (IM), IdoA(2S)-AnMan-PNP (ISM), IdoA-AnMan(6S)-PNP (IMS), and IdoA(2S)-AnMan(6S)-PNP (ISMS). As an application, the disaccharide compositions of heparin from bovine intestine and heparan sulfate from bovine kidney were analyzed by the present method, and the results were comparable to those obtained by a well-established enzymatic method. The present compositional analysis was demonstrated to be reliable and economical.
Topics: Acetylation; Animals; Cattle; Chromatography, High Pressure Liquid; Deamination; Disaccharides; Heparin; Heparitin Sulfate; Indicators and Reagents; Intestinal Mucosa; Intestines; Kidney; Nitrous Acid; Phenylhydrazines; Sulfates
PubMed: 9538198
DOI: 10.1093/oxfordjournals.jbchem.a021928 -
Journal of Bacteriology May 2009Neisseria sicca 4320 expresses two carbohydrate-containing components with sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities that resemble those of...
Neisseria sicca 4320 expresses two carbohydrate-containing components with sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities that resemble those of lipooligosaccharide and lipopolysaccharide. Using matrix-assisted laser desorption ionization--time of flight and electrospray ionization mass spectrometry, we characterized a disaccharide carbohydrate repeating unit expressed by this strain. Gas chromatography identified the sugars composing the unit as rhamnose and N-acetyl-D-glucosamine. Glycosidase digestion confirmed the identity of the nonreducing terminal sugar of the disaccharide and established its beta-anomeric configuration. Mass spectrometry analysis and lectin binding were used to verify the linkages within the disaccharide repeat. The results revealed that the disaccharide repeat is [-4) beta-L-rhamnose (1-3) beta-N-acetyl-D-glucosamine (1-] with an N-acetyl-D-glucosamine nonreducing terminus. This work is the first structural characterization of a molecule that possesses rhamnose in the genus Neisseria.
Topics: Acetylglucosamine; Blotting, Western; Chromatography, Gas; Disaccharides; Electrophoresis, Polyacrylamide Gel; Gas Chromatography-Mass Spectrometry; Molecular Structure; Neisseria sicca; Oligosaccharides; Rhamnose; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 19270085
DOI: 10.1128/JB.01433-08 -
Journal of Bacteriology Mar 2022The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for -acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of...
The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for -acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-β--acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves nonreducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc--acetylglucosamine (MurNAc-GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His-fusions and characterized as exo-lytic β--acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially -deacetylated PGN glycans from B. subtilis were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic β--acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-β--acetylmuramidases. Two exo--acetylmuramidases from belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the nonreducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic -acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.
Topics: Acetylglucosamine; Bacillus subtilis; Cell Wall; Disaccharides; Peptidoglycan; Substrate Specificity; Tannerella forsythia
PubMed: 35129368
DOI: 10.1128/jb.00597-21 -
International Journal of Molecular... Jul 2022Disaccharide complexes have been shown experimentally to be useful for drug delivery or as an antifouling surface biofilm, and are promising drug-encapsulation and...
Disaccharide complexes have been shown experimentally to be useful for drug delivery or as an antifouling surface biofilm, and are promising drug-encapsulation and delivery candidates. Although such complexes are intended for medical applications, to date no studies at the molecular level have been devoted to the influence of complexation on the enzymatic decomposition of polysaccharides. A theoretical approach to this problem has been hampered by the lack of a suitable computational tool for binding such non-covalent complexes to enzymes. Herein, we combine quantum-mechanical calculations of disaccharides complexes with a nonstandard docking GaudiMM engine that can perform such a task. Our results on four different complexes show that they are mostly stabilized by electrostatic interactions and hydrogen bonds. This strong non-covalent stabilization demonstrates the studied complexes are some excellent candidates for self-assembly smart materials, useful for drug encapsulation and delivery. Their advantage lies also in their biocompatible and biodegradable character.
Topics: Disaccharides; Hydrogen Bonding; Static Electricity
PubMed: 35887053
DOI: 10.3390/ijms23147705