RÉSUMÉ
Gut microbial ß-glucuronidases have drawn much attention due to their role as a potential therapeutic target to alleviate some drugs or their metabolites-induced gastrointestinal toxicity. In this study, fifteen 5-phenyl-2-furan derivatives containing 1,3-thiazole moiety (1-15) were synthesized and evaluated for their inhibitory effects against Escherichia coli ß-glucuronidase (EcGUS). Twelve of them showed satisfactory inhibition against EcGUS with IC50 values ranging from 0.25 µM to 2.13 µM with compound 12 exhibited the best inhibition. Inhibition kinetics studies indicated that compound 12 (Ki = 0.14 ± 0.01 µM) was an uncompetitive inhibitor for EcGUS and molecular docking simulation further predicted the binding model and capability of compound 12 with EcGUS. A preliminary structure-inhibitory activity relationship study revealed that the heterocyclic backbone and bromine substitution of benzene may be essential for inhibition against EcGUS. The compounds have the potential to be applied in drug-induced gastrointestinal toxicity and the findings would help researchers to design and develop more effective 5-phenyl-2-furan type EcGUS inhibitors.
Sujet(s)
Découverte de médicament , Escherichia coli/enzymologie , Furanes/pharmacologie , Glucuronidase/antagonistes et inhibiteurs , Glycoprotéines/pharmacologie , Thiazoles/pharmacologie , Relation dose-effet des médicaments , Furanes/synthèse chimique , Furanes/composition chimique , Glucuronidase/métabolisme , Glycoprotéines/synthèse chimique , Glycoprotéines/composition chimique , Simulation de docking moléculaire , Structure moléculaire , Relation structure-activité , Thiazoles/synthèse chimique , Thiazoles/composition chimiqueRÉSUMÉ
Glycosylation is a major modification of secreted and cell surface proteins, and the resultant glycans show considerable heterogeneity in their structures. To understand the biological processes arising from each glycoform, the preparation of homogeneous glycoproteins is essential for extensive biological experiments. To establish a more robust and rapid synthetic route for the synthesis of homogeneous glycoproteins, we studied several key reactions based on amino thioacids. We found that diacyl disulfide coupling (DDC) formed with glycosyl asparagine thioacid and peptide thioacid yielded glycopeptides. This efficient coupling reaction enabled us to develop a new glycoprotein synthesis method, such as the bifunctional thioacid-mediated strategy, which can couple two peptides with the N- and C-termini of glycosyl asparagine thioacid. Previous glycoprotein synthesis methods required valuable glycosyl asparagine in the early stage and subsequent multiple glycoprotein synthesis routes, whereas the developed concept can generate glycoproteins within a few steps from peptide and glycosyl asparagine thioacid. Herein, we report the characterization of the DDC of amino thioacids and the efficient ability of glycosyl asparagine thioacid to be used for robust glycoprotein semisynthesis.
Sujet(s)
Asparagine/analogues et dérivés , Cytokines/synthèse chimique , Glycoprotéines/synthèse chimique , Thiols/composition chimique , Escherichia coli/composition chimique , Escherichia coli/métabolisme , Glycopeptides/composition chimique , Glycosylation , Modèles moléculaires , Peptides/composition chimique , Peptides/métabolisme , Conformation des protéines , SulfuresRÉSUMÉ
The ß-glucuronidase, a lysosomal enzyme, catalyzes the cleavage of glucuronosyl-O-bonds. Its inhibitors play a significant role in different medicinal therapies as they cause a decrease in carcinogen-induced colonic tumors by reducing the level of toxic substances present in the intestine. Among those inhibitors, bisindole derivatives had displayed promising ß-glucuronidase inhibition activity. In the current study, hydrazone derivatives of bisindolymethane (1-30) were synthesized and evaluated for in vitro ß-glucuronidase inhibitory activity. Twenty-eight analogs demonstrated better activity (IC50 = 0.50-46.5 µM) than standard D-saccharic acid 1,4-lactone (IC50 = 48.4 ± 1.25 µM). Compounds with hydroxyl group like 6 (0.60 ± 0.01 µM), 20 (1.50 ± 0.10 µM) and 25 (0.50 ± 0.01 µM) exhibited the most potent inhibitory activity, followed by analogs with fluorine 21 (3.50 ± 0.10 µM) and chlorine 23 (8.20 ± 0.20 µM) substituents. The presence of hydroxyl group at the aromatic side chain was observed as the main contributing factor in the inhibitory potential. From the docking studies, it was predicted that the active compounds can fit properly in the binding groove of the ß-glucuronidase and displayed significant binding interactions with essential residues.
Sujet(s)
Glycoprotéines , Hydrazones , Indoles , Glucuronidase/antagonistes et inhibiteurs , Glucuronidase/composition chimique , Glycoprotéines/synthèse chimique , Glycoprotéines/composition chimique , Hydrazones/synthèse chimique , Hydrazones/composition chimique , Indoles/synthèse chimique , Indoles/composition chimique , Simulation de docking moléculaireRÉSUMÉ
Owing to the generation of heterogeneous glycoproteins in cells, it is highly difficult to study glycoprotein-mediated biological events and to develop biomedical agents. Thus, general and efficient methods to prepare homogeneous glycoproteins are in high demand. Herein, we report a general method for the efficient preparation of homogeneous glycoproteins that utilizes a combination of genetic code expansion and chemoselective ligation techniques. In the protocol to produce glycan-defined glycoproteins, an alkyne tag-containing protein, generated by genetic encoding of an alkynylated unnatural amino acid, was quantitatively coupled via click chemistry to versatile azide-appended glycans. The glycoproteins produced by the present strategy were found to recognize mammalian cell-surface lectins and enter the cells through lectin-mediated internalization. Also, cell studies exhibited that the glycoprotein containing multiple mannose-6-phosphate residues enters diseased cells lacking specific lysosomal glycosidases by binding to the cell-surface M6P receptor, and subsequently migrates to lysosomes for efficient degradation of stored glycosphingolipids.
Sujet(s)
Glycoprotéines/synthèse chimique , Glycoprotéines/métabolisme , Polyosides/composition chimique , Alcynes/composition chimique , Azotures/composition chimique , Biocatalyse , Chimie click , Fibroblastes/métabolisme , Ganglioside GM2/métabolisme , Glycoprotéines/génétique , Glycosylation , Humains , Lectines/métabolisme , Lysosomes/métabolisme , Mutation , Polyosides/génétique , Maturation post-traductionnelle des protéines , Cellules THP-1 , beta-N-Acetylhexosaminidases/synthèse chimique , beta-N-Acetylhexosaminidases/génétique , beta-N-Acetylhexosaminidases/métabolismeRÉSUMÉ
Site-specific glycosylation of a functional recombinant protein thioester is reported. The thioester functionalized protein sfGFP-Y151ThioD, prepared by genetic code expansion, underwent native chemical ligation with the cysteine-conjugated glycans H-Cys-NH-GlcNAc and H-Cys-NH-(GlcNAc)2(Man)3 to give the corresponding cysteine-bridged glycoproteins. The intact glycoproteins, which retained their fluorescence, were characterized by top-down mass spectrometry and gel electrophoresis. The bridging cysteine provided a convenient handle for affinity chromatography purification of the glycoproteins via a removable biotin tag. Given the influence that specific glycoforms can have on a protein's function, the ability to attach a homogeneous glycan to an intact protein in a functional group controlled yet sequon-independent manner could find widespread application. These preliminary results set the stage for development of the expressed protein glycoligation (EPG) concept.
Sujet(s)
Cystéine/composition chimique , Glycoprotéines/synthèse chimique , Biocatalyse , Cystéine/synthèse chimique , Escherichia coli/génétique , Glycoprotéines/composition chimique , Glycoprotéines/génétique , Modèles moléculaires , Techniques de synthèse en phase solideRÉSUMÉ
Calpain inhibitors have been proposed as drug candidates for neurodegenerative disorders, with ABT-957 entering clinical trials for Alzheimer's disease and mild cognitive impairment. The structure of ABT-957 was very recently disclosed, and trials were terminated owing to inadequate CNS concentrations to obtain a pharmacodynamic effect. The multistep synthesis of an α-ketoamide peptidomimetic inhibitor series potentially including ABT-957 was optimized to yield diastereomerically pure compounds that are potent and selective for calpain-1 over papain and cathepsins B and K. As the final oxidation step, with its optimized synthesis protocol, does not alter the configuration of the substrate, the synthesis of the diastereomeric pair (R)-1-benzyl-N-((S)-4-((4-fluorobenzyl)amino)-3,4-dioxo-1-phenylbutan-2-yl)-5-oxopyrrolidine-2-carboxamide (1 c) and (R)-1-benzyl-N-((R)-4-((4-fluorobenzyl)amino)-3,4-dioxo-1-phenylbutan-2-yl)-5-oxopyrrolidine-2-carboxamide (1 g) was feasible. This allowed the exploration of stereoselective inhibition of calpain-1, with 1 c (IC50 =78â nM) being significantly more potent than 1 g. Moreover, inhibitor 1 c restored cognitive function in amnestic mice.
Sujet(s)
Amnésie/traitement médicamenteux , Calpain/antagonistes et inhibiteurs , Glycoprotéines/pharmacologie , Neuroprotecteurs/pharmacologie , Pyrrolidines/pharmacologie , Amnésie/induit chimiquement , Amnésie/métabolisme , Animaux , Calpain/métabolisme , Glycoprotéines/synthèse chimique , Glycoprotéines/composition chimique , Souris , Structure moléculaire , Neuroprotecteurs/synthèse chimique , Neuroprotecteurs/composition chimique , Pyrrolidines/synthèse chimique , Pyrrolidines/composition chimique , Scopolamine , StéréoisomérieRÉSUMÉ
Glycoproteins are produced by the post-translational modification process of proteins and they play an important role in mediating various biological processes. Our understanding towards biochemical functions of individual glycoproteins has been seriously hampered due to the heterogeneous expression of carbohydrate parts in glycoproteins. Despite the advancement in recombinant expression and chromatographic techniques, the isolation of pure glycoforms remains nearly impossible. To obtain homogenous glycoproteins, tremendous efforts hves been spent in developing various ligation and glycosylation techniques. This minireview discusses selected methods for the preparation and ligation of glycopeptides. The importance of the development of new chemical synthesis method for glycoproteins has also been discussed, which would be one of the next directions in this field.
Sujet(s)
Glycopeptides/synthèse chimique , Glycoprotéines/synthèse chimique , Glycopeptides/composition chimique , Glycoprotéines/composition chimique , Glycosylation , Structure moléculaireRÉSUMÉ
The Thomsen-Friedenreich-antigen, Gal(ß1-3)GalNAc(α1-O-Ser/Thr (TF-antigen), is presented on the surface of most human cancer cell types. Its interaction with galectin 1 and galectin 3 leads to tumor cell aggregation and promotes cancer metastasis and T-cell apoptosis in epithelial tissue. To further explore multivalent binding between the TF-antigen and galectin-3, the TF-antigen was enzymatically synthesized in high yields with GalNAc(α1-EG3-azide as the acceptor substrate by use of the glycosynthase BgaC/Glu233Gly. Subsequently, it was coupled to alkynyl-functionalized bovine serum albumin via a copper(I)-catalyzed alkyne-azide cycloaddition. This procedure yielded neo-glycoproteins with tunable glycan multivalency for binding studies. Glycan densities between 2 and 53 glycan residues per protein molecule were obtained by regulated alkynyl-modification of the lysine residues of BSA. The number of coupled glycans was quantified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a trinitrobenzene sulfonic acid assay. The binding efficiency of the neo-glycoproteins with human galectin-3 and the effect of multivalency was investigated and assessed using an enzyme-linked lectin assay. Immobilized neo-glycoproteins of all modification densities showed binding of Gal-3 with increasing glycan density. However, multivalent glycan presentation did not result in a higher binding affinity. In contrast, inhibition of Gal-3 binding to asialofetuin was effective. The relative inhibitory potency was increased by a factor of 142 for neo-glycoproteins displaying 10 glycans/protein in contrast to highly decorated inhibitors with only 2-fold increase. In summary, the functionality of BSA-based neo-glycoproteins presenting the TF-antigen as multivalent inhibitors for Gal-3 was demonstrated.
Sujet(s)
Antigènes glycanniques associés aux tumeurs/composition chimique , Antigènes glycanniques associés aux tumeurs/métabolisme , Protéines du sang/métabolisme , Galectines/métabolisme , Glycoprotéines/synthèse chimique , Fixation compétitive , Protéines du sang/génétique , Catalyse , Cuivre/composition chimique , Réaction de cycloaddition , Galectines/génétique , Glycoprotéines/métabolisme , Protéines immobilisées/composition chimique , Protéines immobilisées/métabolisme , Techniques immunoenzymatiques/méthodes , Sérumalbumine bovine/composition chimique , beta-Galactosidase/métabolismeRÉSUMÉ
The calpains are a conserved family of cysteine proteases that includes several isoforms of which µ-calpain and m-calpain are the most widely distributed in mammalian cells. Calpains have been implicated in normal physiological processes as well as cellular abnormalities such as neurodegenerative disorders, cataract, and cancer. Therefore, calpain inhibitors are of interest as potential therapeutic agents. We have synthesized four new sulfonamide-based peptidomimetic compounds 2-5 as inhibitors of µ-calpain that incorporate (E)-1-(phenyl)-2-phenyldiazene and (E)-1-(phenyl)-2-phenylethene functionalities as the N-terminal capping groups of the inhibitors. Compound 5 with Ki value of 9 nM versus µ-calpain was the most potent member of the group. The compounds were predicted to be more lipophilic compared to MDL28170 based on CLogP estimation. They displayed moderate to good antiproliferative activity versus melanoma cell lines (A-375 and B-16F1) and PC-3 prostate cancer cells in vitro. Additionally, one member of the group (compound 3) inhibited DU-145 cell invasion by 80% at 2 µM concentration in the Matrigel cell invasion assay.
Sujet(s)
Antinéoplasiques/pharmacologie , Glycoprotéines/pharmacologie , Peptidomimétiques/pharmacologie , Sulfonamides/pharmacologie , Antinéoplasiques/synthèse chimique , Antinéoplasiques/composition chimique , Prolifération cellulaire/effets des médicaments et des substances chimiques , Relation dose-effet des médicaments , Tests de criblage d'agents antitumoraux , Glycoprotéines/synthèse chimique , Glycoprotéines/composition chimique , Humains , Structure moléculaire , Peptidomimétiques/synthèse chimique , Peptidomimétiques/composition chimique , Relation structure-activité , Sulfonamides/synthèse chimique , Sulfonamides/composition chimique , Cellules cancéreuses en cultureRÉSUMÉ
High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.
Sujet(s)
Protéines de transport/analyse , Protéines Escherichia coli/analyse , Glycoprotéines/analyse , Tests de criblage à haut débit/méthodes , Spectrométrie de masse MALDI/méthodes , Actinobacillus pleuropneumoniae/enzymologie , Protéines de transport/synthèse chimique , Protéines de transport/génétique , Escherichia coli/composition chimique , Protéines Escherichia coli/synthèse chimique , Protéines Escherichia coli/génétique , Glycoprotéines/synthèse chimique , Glycoprotéines/génétique , Glycosylation , Glycosyltransferase/composition chimique , Mutation , Banque de peptides , Étude de validation de principe , Protéines recombinantes/analyse , Protéines recombinantes/synthèse chimique , Protéines recombinantes/génétiqueRÉSUMÉ
Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.
Sujet(s)
Anticorps monoclonaux/immunologie , Dystroglycanes/immunologie , Glycoprotéines/immunologie , Mucines/immunologie , Syndrome de Walker-Warburg/diagnostic , Dystroglycanes/composition chimique , Glycoprotéines/synthèse chimique , Humains , Mucines/composition chimiqueRÉSUMÉ
Plant glycoproteins, especially allergenic glycoproteins such as pollen allergens, often carry antigenic N-glycans with α1-3 fucose and/or ß1-2 xylose residue(s) on the trimannosyl core structure. We previously reported that one of such antigenic free-form N-glycans, Man3Xyl1Fuc1GlcNAc2 (M3FX) suppressed IL-4 production from Th2 cells of pollinosis patients. For the molecular-level analysis of this immunoactivity, an effective and convenient procedure for large scale preparation of the immunoactive free-form N-glycan and a synthesis of glycopolymers bearing multivalent M3FX has been required. During the preparation of prebiotic oligosaccharides from several edible beans, we found that the free-form M3FX accumulates in relatively large amounts in white kidney beans. In this report, we describe a new procedure for preparation of M3FX from white kidney bean powders by a combination of ion-exchange method, gel-filtration, and hydrophilic partitioning. The high-purity of M3FX prepared by this procedure was confirmed by MS-analysis and 1H-NMR, suggesting that the free-form M3FX can be used for the synthesis of neoglycopolymer. Using this new procedure, the immunoactive oligosaccharide can be prepared without the chemical method such as hydrazinolysis and other purification steps required to exclude other type of N-glycans.
Sujet(s)
Allergènes/composition chimique , Glycoprotéines/composition chimique , Glycoprotéines/synthèse chimique , Oligosaccharides/composition chimique , Phaseolus/composition chimique , Allergènes/immunologie , Techniques de chimie synthétique , Glycoprotéines/immunologie , PoudresRÉSUMÉ
Asymmetrically branched precision glycooligomers are synthesized by solid-phase polymer synthesis for studying multivalent carbohydrate-protein interactions. Through the stepwise assembly of Fmoc-protected oligo(amidoamine) building blocks and Fmoc/Dde-protected lysine, straightforward variation of structural parameters such as the number and length of arms, as well as the number and position of carbohydrate ligands, is achieved. Binding of 1-arm and 3-arm glycooligomers toward lectin receptors langerin and concanavalin A (ConA) was evaluated where the smallest 3-arm glycooligomer shows the highest binding toward langerin, and stepwise elongation of one, two, or all three arms leads to decreased binding. When directly comparing binding toward langerin and ConA, we find that structural variation of the scaffold affects glycomimetic ligand binding differently for the different targets, indicating the potential to tune such ligands not only for their avidity but also for their selectivity toward different lectins.
Sujet(s)
Antigènes CD/composition chimique , Glucides/composition chimique , Glycoprotéines/composition chimique , Lectines de type C/composition chimique , Lectines liant le mannose/composition chimique , Protéines/composition chimique , Antigènes CD/génétique , Glucides/synthèse chimique , Glucides/génétique , Concanavaline A/composition chimique , Concanavaline A/génétique , Concanavaline A/métabolisme , Glycoprotéines/synthèse chimique , Glycoprotéines/ultrastructure , Humains , Lectines de type C/génétique , Ligands , Lectines liant le mannose/génétique , Liaison aux protéines/génétique , Conformation des protéines , Protéines/génétique , Protéines/ultrastructure , Récepteur mitogène/composition chimique , Récepteur mitogène/génétiqueRÉSUMÉ
Recent advances have demonstrated the feasibility and robustness of chemical synthesis for the production of homogeneously glycosylated protein forms (glycoforms). By taking advantage of the unmatchable flexibility and precision provided by chemical synthesis, the quantitative effects of glycosylation were obtained using chemical glycobiology approaches. These findings greatly advanced our fundamental knowledge of glycosylation. More importantly, analysis of these findings has led to the development of glycoengineering guidelines for rationally improving the properties of peptides and proteins. In this chapter, we present the key experimental steps for chemical biology studies of protein glycosylation, with the aim of facilitating and promoting research in this important but significantly underexplored area of biology.
Sujet(s)
Glycopeptides/synthèse chimique , Glycoprotéines/synthèse chimique , Animaux , Techniques de chimie synthétique/méthodes , Glycopeptides/composition chimique , Glycoprotéines/composition chimique , Glycosylation , Humains , Modèles moléculaires , Conformation des protéines , Pliage des protéines , Stabilité protéique , Biologie synthétique/méthodesRÉSUMÉ
Chromen-4-one substituted oxadiazole analogs 1-19 have been synthesized, characterized and evaluated for ß-glucuronidase inhibition. All analogs exhibited a variable degree of ß-glucuronidase inhibitory activity with IC50 values ranging in between 0.8 ± 0.1-42.3 ± 0.8 µM when compared with the standard d-saccharic acid 1,4 lactone (IC50 = 48.1 ± 1.2 µM). Structure activity relationship has been established for all compounds. Molecular docking studies were performed to predict the binding interaction of the compounds with the active site of enzyme.
Sujet(s)
Benzopyranes/synthèse chimique , Benzopyranes/pharmacologie , Glycoprotéines/synthèse chimique , Glycoprotéines/pharmacologie , Oxadiazoles/synthèse chimique , Oxadiazoles/pharmacologie , Benzopyranes/composition chimique , Glucuronidase/composition chimique , Glucuronidase/métabolisme , Glycoprotéines/composition chimique , Concentration inhibitrice 50 , Ligands , Simulation de docking moléculaire , Oxadiazoles/composition chimiqueRÉSUMÉ
Chagas disease (ChD), caused by the protozoan parasite Trypanosoma cruzi, affects millions of people worldwide. Chemotherapy is restricted to two drugs, which are partially effective and may cause severe side effects, leading to cessation of treatment in a significant number of patients. Currently, there are no biomarkers to assess therapeutic efficacy of these drugs in the chronic stage. Moreover, no preventive or therapeutic vaccines are available. In this chapter, we describe the purification of Trypanosoma cruzi trypomastigote-derived glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins) for their use as antigens for the reliable primary or confirmatory diagnosis and as prognostic biomarkers for early assessment of cure following ChD chemotherapy. We also describe, as an example, the synthesis of a potential tGPI-mucin-derived α-Gal-terminating glycan and its coupling to a carrier protein for use as diagnostic and prognostic biomarker in ChD.
Sujet(s)
Maladie de Chagas/diagnostic , Protéines liées au GPI/isolement et purification , Glycoprotéines/composition chimique , Mucines/isolement et purification , Protéines de protozoaire/isolement et purification , Trypanosoma cruzi/composition chimique , Animaux , Lignée cellulaire , Test ELISA/méthodes , Protéines liées au GPI/composition chimique , Glycoprotéines/synthèse chimique , Humains , Macaca mulatta , Modèles moléculaires , Mucines/composition chimique , Protéines de protozoaire/composition chimique , Spectrométrie de masse MALDI/méthodesRÉSUMÉ
The effects of pH on the freeze-thaw stability of glycated soy protein isolate (SPI) and soy protein isolate hydrolysate (SPH) were studied. The covalent compounds were prepared by conjugating SPI, SPH and dextran under heated Maillard reaction, which the macromolecules were named SPI-D and SPH-D. SDS-PAGE analysis verified that SPI-D and SPH-D form a covalent bond through the Maillard reaction. Afterwards, the effects of pH on the freeze-thaw stability of SPI, SPI-D and SPH-D emulsions were evaluated. The covalent conjugate stabilized emulsions improved the stability of the emulsions to pH stress. After freeze-thaw cycles, SPH-D revealed the lowest particle size, degree of coalescence (CD) and oiling off. The results above were also supported by optical microscopy analysis.
Sujet(s)
Dextrane/composition chimique , Glycoprotéines/composition chimique , Fragments peptidiques/composition chimique , Protéines de soja/composition chimique , Dextrane/synthèse chimique , Émulsions/synthèse chimique , Émulsions/composition chimique , Congélation , Glycoprotéines/synthèse chimique , Concentration en ions d'hydrogène , Taille de particule , Stabilité protéique , ProtéolyseRÉSUMÉ
A large number of children in the autism spectrum disorder suffer from gastrointestinal (GI) conditions, such as constipation and diarrhea. Clostridium bolteae is a part of a set of pathogens being regularly detected in the stool samples of hosts affected by GI and autism symptoms. Accompanying studies have pointed out the possibility that such microbes affect behaviour through the production of neurotoxic metabolites in a so-called, gut-brain connection. As an extension of our Clostridium difficile polysaccharide (PS)-based vaccine research, we engaged in the discovery of C. bolteae surface carbohydrates. So far, studies revealed that C. bolteae produces a specific immunogenic PS capsule comprised of disaccharide repeating blocks of mannose (Manp) and rhamnose (Rhap) units: α-D-Manp-(1â[-4)-ß-D-Rhap- (1â3)-α-D-Manp-(1â]n. For vaccinology and further immunogenic experiments, a method to produce C. bolteae PS conjugates has been developed, along with the chemical syntheses of the PS non-reducing end linkage, with D-Rha or L-Rha, α-D-Manp-(1â4)-α-D-Rhap- (1âO(CH2)5NH2 and α-D-Manp-(1â4)-α-L-Rhap-(1âO(CH2)5NH2, equipped with an aminopentyl linker at the reducing end for conjugation purposes. The discovery of C. bolteae PS immunogen opens the door to the creation of non-evasive diagnostic tools to evaluate the frequency and role of this microbe in autistic subjects and to a vaccine to reduce colonization levels in the GI tract, thus impeding the concentration of neurotoxins.
Sujet(s)
Trouble autistique/microbiologie , Clostridiales/composition chimique , Polyosides bactériens/composition chimique , Vaccins antibactériens/synthèse chimique , Vaccins antibactériens/composition chimique , Séquence glucidique , Glycoprotéines/synthèse chimique , Glycoprotéines/composition chimique , Humains , Oligosaccharides/synthèse chimique , Vaccins conjugués/composition chimiqueRÉSUMÉ
Chemical synthesis is a well-established method for the preparation in the research laboratory of multiple-tens-of-milligram amounts of correctly folded, high purity protein molecules. Chemically synthesized proteins enable a broad spectrum of novel protein science. Racemic mixtures consisting of d-protein and l-protein enantiomers facilitate crystallization and determination of protein structures by X-ray diffraction. d-Proteins enable the systematic development of unnatural mirror image protein molecules that bind with high affinity to natural protein targets. The d-protein form of a therapeutic target can also be used to screen natural product libraries to identify novel small molecule leads for drug development. Proteins with novel polypeptide chain topologies including branched, circular, linear-loop, and interpenetrating polypeptide chains can be constructed by chemical synthesis. Medicinal chemistry can be applied to optimize the properties of therapeutic protein molecules. Chemical synthesis has been used to redesign glycoproteins and for the a priori design and construction of covalently constrained novel protein scaffolds not found in nature. Versatile and precise labeling of protein molecules by chemical synthesis facilitates effective application of advanced physical methods including multidimensional nuclear magnetic resonance and time-resolved FTIR for the elucidation of protein structure-activity relationships. The chemistries used for total synthesis of proteins have been adapted to making artificial molecular devices and protein-inspired nanomolecular constructs. Research to develop mirror image life in the laboratory is in its very earliest stages, based on the total chemical synthesis of d-protein forms of polymerase enzymes.
Sujet(s)
Glycoprotéines/composition chimique , Glycoprotéines/synthèse chimique , Modèles moléculaires , Cristallographie aux rayons X , Spectroscopie infrarouge à transformée de FourierRÉSUMÉ
The advancement of glycoscience is critically dependent on the access to a large number of glycans for their functional study. Naturally occurring glycans are considered a viable source for diverse and biologically relevant glycan libraries. A mixture of free reducing glycans released from natural sources can be fluorescently tagged and separated by chromatography to produce a natural glycan library. Anthranilic acid (AA) has been widely used to fluorescently tag reducing glycans for HPLC or LC/MS analysis. However, AA conjugated glycans are not efficiently immobilized on microarray slides due to the lack of a primary alkylamine functional group. In this study, we have developed simple and efficient chemistry for bioconjugation and further functionalization of glycan-AA conjugates. This new approach enables quick preparation of glycan microarrays and neoglycoproteins from glycan-AA conjugates, which can be separated by weak anion exchange (WAX) and C18 reversed-phase HPLC.