ABSTRACT
Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelBSt catalyzed the symport of galactosides with Na+, Li+, or H+ but prefers the coupling with Na+. Previously, we determined the structures of the inward- and outward-facing conformation of MelBSt and the molecular recognition for galactoside and Na+. However, the molecular mechanisms for H+- and Na+-coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelBSt, the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na+-binding residues for the selection of Na+ and H+ by free energy simulations. Transport assays showed that the D55C mutant converted MelBSt to a solely H+-coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelBSt. Unexpectedly, the H+-coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H+-coupled symport mediated by MelBSt. This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelBSt.
Subject(s)
Sodium , Symporters , Symporters/chemistry , Symporters/metabolism , Symporters/genetics , Binding Sites , Crystallography, X-Ray , Sodium/metabolism , Sodium/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Salmonella typhimurium/metabolism , Salmonella typhimurium/genetics , Salmonella typhimurium/chemistry , Melibiose/metabolism , Melibiose/chemistry , Cations/metabolism , Cations/chemistry , Protein ConformationABSTRACT
Six oligosaccharides were discovered and isolated for the first time from Ziziphi Spinosae Semen. On the basis of spectroscopic analysis, their structures were determined to be verbascose (1), verbascotetraose (2), stachyose (3), manninotriose (4), raffinose (5), and melibiose (6). The prebiotic effect of the oligosaccharide fraction was assayed by eight gut bacterial growth in vitro, revealing a significant increase in cell density, up to 4-fold, for Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus johnsonii. The impact of six oligosaccharides with different degrees of polymerization (DPs) and structures on the growth of Lactobacillus acidophilus was evaluated. As a result, stachyose and raffinose demonstrated superior support for bacterial growth compared to the other oligosaccharides. This study explored the structure-activity relationship of raffinose family oligosaccharides (RFOs) and showed that the more the monosaccharide type, the more supportive the gut bacteria growth when oligosaccharides have the same molecular weight.
Subject(s)
Prebiotics , Semen , Raffinose/chemistry , Raffinose/metabolism , Semen/metabolism , Oligosaccharides/pharmacology , Oligosaccharides/metabolism , MelibioseABSTRACT
Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the Na+-coupled major facilitator superfamily transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well-studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelBSt by crystallography. To obtain other key kinetic states, here we raised camelid single-domain nanobodies (Nbs) and carried out a screening against the WT MelBSt under 4 ligand conditions. We applied an in vivo cAMP-dependent two-hybrid assay to detect interactions of Nbs with MelBSt and melibiose transport assays to determine the effects on MelBSt functions. We found that all selected Nbs showed partial to complete inhibitions of MelBSt transport activities, confirming their intracellular interactions. A group of Nbs (714, 725, and 733) was purified, and isothermal titration calorimetry measurements showed that their binding affinities were significantly inhibited by the substrate melibiose. When titrating melibiose to the MelBSt/Nb complexes, Nb also inhibited the sugar-binding. However, the Nb733/MelBSt complex retained binding to the coupling cation Na+ and also to the regulatory enzyme EIIAGlc of the glucose-specific phosphoenolpyruvate/sugar phosphotransferase system. Further, EIIAGlc/MelBSt complex also retained binding to Nb733 and formed a stable supercomplex. All data indicated that MelBSt trapped by Nbs retained its physiological functions and the trapped conformation is similar to that bound by the physiological regulator EIIAGlc. Therefore, these conformational Nbs can be useful tools for further structural, functional, and conformational analyses.
Subject(s)
Single-Domain Antibodies , Symporters , Single-Domain Antibodies/metabolism , Melibiose/metabolism , Symporters/metabolism , Ion Transport , Sodium/metabolismABSTRACT
The melibiose permease MelB is a well-studied Na+-coupled transporter of the major facilitator superfamily. However, the symport mechanism of galactosides and cations is still not fully understood, especially at structural levels. Here, we use single-molecule force spectroscopy to investigate substrate-induced structural changes of MelB from Salmonella typhimurium. In the absence of substrate, MelB equally populates two different states, from which one shows higher mechanical structural stability with additional stabilization of the cytoplasmic middle-loop C3. In the presence of either melibiose or a coupling Na+-cation, however, MelB increasingly populates the mechanically less stable state, which shows a destabilized middle-loop C3. In the presence of both substrate and co-substrate, this mechanically less stable state of MelB is predominant. Our findings describe how both substrates guide MelB transporters to populate two different mechanically stabilized states, and contribute mechanistic insights to the alternating-access action for the galactoside/cation symport catalyzed by MelB.
Subject(s)
Melibiose , Symporters , Melibiose/chemistry , Symporters/metabolism , Membrane Transport Proteins , Sodium/metabolism , Ion Transport , CationsABSTRACT
MAGE (melibiose-derived advanced glycation end-product) is the glycation product generated in the reaction of a model protein with melibiose. The in vivo analog accumulates in several tissues; however, its origin still needs explanation. In vitro MAGE is efficiently generated under dry conditions in contrast to the reaction carried in an aqueous solvent. Using liquid chromatography coupled with mass spectrometry, we analyzed the physicochemical properties and structures of myoglobin glycated with melibiose under different conditions. The targeted peptide analysis identified structurally different AGEs, including crosslinking and non-crosslinking modifications associated with lysine, arginine, and histidine residues. Glycation in a dry state was more efficient in the formation of structures containing an intact melibiose moiety (21.9%) compared to glycation under aqueous conditions (15.6%). The difference was reflected in characteristic fluorescence that results from protein structural changes and impact on a heme group of the model myoglobin protein. Finally, our results suggest that the formation of in vitro MAGE adduct is initiated by coupling melibiose to a model myoglobin protein. It is confirmed by the identification of intact melibiose moieties. The intermediate glycation product can further rearrange towards more advanced structures, including cross-links. This process can contribute to a pool of AGEs accumulating locally in vivo and affecting tissue biology.
Subject(s)
Glycation End Products, Advanced , Myoglobin , Myoglobin/chemistry , Glycation End Products, Advanced/metabolism , Melibiose , Lysine/metabolism , GlycosylationABSTRACT
Identifying the components of Camellia oleifera honey and pollen and conducting corresponding toxicological tests are essential to revealing the mechanism of Camellia oleifera toxicity to honey bees. In this research, we investigated the saccharides and alkaloids in honey, nectar, and pollen from Camellia oleifera, which were compared with honey, nectar, and pollen from Brassica napus, a widely planted flowering plant. The result showed that melibiose, manninotriose, raffinose, stachyose, and lower amounts of santonin and caffeine were found in Camellia oleifera nectar, pollen, and honey but not in B. napus nectar, pollen, and honey. Toxicological experiments indicated that manninotriose, raffinose, and stachyose in Camellia oleifera honey are toxic to bees, while alkaloids in Camellia oleifera pollen are not toxic to honey bees. The toxicity mechanism of oligosaccharides revealed by temporal metabolic profiling is that oligosaccharides cannot be further digested by honey bees and thus get accumulated in honey bees, disturbing the synthesis and metabolism of trehalose, ultimately causing honey bee mortality.
Subject(s)
Camellia , Santonin , Bees , Animals , Plant Nectar , Raffinose , Melibiose , Trehalose , Caffeine , Pollen , OligosaccharidesABSTRACT
Understanding the basis of biological diversity remains a central problem in evolutionary biology. Using microbial systems, adaptive diversification has been studied in (a) spatially heterogeneous environments, (b) temporally segregated resources, and (c) resource specialization in a homogeneous environment. However, it is not well understood how adaptive diversification can take place in a homogeneous environment containing a single resource. Starting from an isogenic population of yeast Saccharomyces cerevisiae, we report rapid adaptive diversification, when propagated in an environment containing melibiose as the carbon source. The diversification is driven due to a public good enzyme α-galactosidase, which hydrolyzes melibiose into glucose and galactose. The diversification is driven by mutations at a single locus, in the GAL3 gene in the S. cerevisiae GAL/MEL regulon. We show that metabolic co-operation involving public resources could be an important mode of generating biological diversity. Our study demonstrates sympatric diversification of yeast starting from an isogenic population and provides detailed mechanistic insights into the factors and conditions responsible for generating and maintaining the population diversity.
Subject(s)
Melibiose , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Melibiose/metabolism , Galactose/metabolism , Genotype , alpha-Galactosidase/genetics , alpha-Galactosidase/metabolismABSTRACT
Actinidia eriantha is a unique germplasm resource for kiwifruit breeding. Genetic diversity and nutrient content need to be evaluated prior to breeding. In this study, we looked at the metabolites of three elite A. eriantha varieties (MM-11, MM-13 and MM-16) selected from natural individuals by using a UPLC-MS/MS-based metabolomics approach and transcriptome, with a total of 417 metabolites identified. The biosynthesis and metabolism of phenolic acid, flavonoids, sugars, organic acid and AsA in A. eriantha fruit were further analyzed. The phenolic compounds accounted for 32.37% of the total metabolites, including 48 phenolic acids, 60 flavonoids, 7 tannins and 20 lignans and coumarins. Correlation analysis of metabolites and transcripts showed PAL (DTZ79_15g06470), 4CL (DTZ79_26g05660 and DTZ79_29g0271), CAD (DTZ79_06g11810), COMT (DTZ79_14g02670) and FLS (DTZ79_23g14660) correlated with polyphenols. There are twenty-three metabolites belonging to sugars, the majority being sucrose, glucose arabinose and melibiose. The starch biosynthesis-related genes (AeglgC, AeglgA and AeGEB1) were expressed at lower levels compared with metabolism-related genes (AeamyA and AeamyB) in three mature fruits of three varieties, indicating that starch was converted to soluble sugar during fruit maturation, and the expression level of SUS (DTZ79_23g00730) and TPS (DTZ79_18g05470) was correlated with trehalose 6-phosphate. The main organic acids in A. eriantha fruit are citric acid, quinic acid, succinic acid and D-xylonic acid. Correlation analysis of metabolites and transcripts showed ACO (DTZ79_17g07470) was highly correlated with citric acid, CS (DTZ79_17g00890) with oxaloacetic acid, and MDH1 (DTZ79_23g14440) with malic acid. Based on the gene expression, the metabolism of AsA acid was primarily through the L-galactose pathway, and the expression level of GMP (DTZ79_24g08440) and MDHAR (DTZ79_27g01630) highly correlated with L-Ascorbic acid. Our study provides additional evidence for the correlation between the genes and metabolites involved in phenolic acid, flavonoids, sugars, organic acid and AsA synthesis and will help to accelerate the kiwifruit molecular breeding approaches.
Subject(s)
Actinidia , Lignans , Actinidia/genetics , Actinidia/metabolism , Arabinose , Ascorbic Acid/metabolism , Chromatography, Liquid , Citric Acid/metabolism , Coumarins/metabolism , Fruit/genetics , Fruit/metabolism , Galactose/metabolism , Glucose/metabolism , Humans , Hydroxybenzoates , Lignans/metabolism , Melibiose/metabolism , Metabolomics , Oxaloacetates/metabolism , Phosphates/metabolism , Plant Breeding , Polyphenols/metabolism , Quinic Acid/metabolism , Starch/metabolism , Succinates/metabolism , Sucrose/metabolism , Tandem Mass Spectrometry , Tannins/metabolism , Transcriptome , Trehalose/metabolismABSTRACT
Melibiose-derived AGE (MAGE) is an advanced glycation end-product formed in vitro in anhydrous conditions on proteins and protein-free amino acids during glycation with melibiose. Our previous studies revealed the presence of MAGE antigen in the human body and tissues of several other species, including muscles, fat, extracellular matrix, and blood. MAGE is also antigenic and induces generation of anti-MAGE antibody. The aim of this paper was to identify the proteins modified by MAGE present in human body fluids, such as serum, plasma, and peritoneal fluids. The protein-bound MAGE formed in vivo has been isolated from human blood using affinity chromatography on the resin with an immobilized anti-MAGE monoclonal antibody. Using mass spectrometry and immunochemistry it has been established that MAGE epitope is present on several human blood proteins including serum albumin, IgG, and IgA. In serum of diabetic patients, mainly the albumin and IgG were modified by MAGE, while in healthy subjects IgG and IgA carried this modification, suggesting the novel AGE can impact protein structure, contribute to auto-immunogenicity, and affect function of immunoglobulins. Some proteins in peritoneal fluid from cancer patients modified with MAGE were also observed and it indicates a potential role of MAGE in cancer.
Subject(s)
Body Fluids , Melibiose , Body Fluids/metabolism , Glycation End Products, Advanced/metabolism , Humans , Immunoglobulin A , Immunoglobulin G , Melibiose/metabolism , Serum Albumin/analysisABSTRACT
Cation selectivity and coupling are important attributes of cation-coupled symporters. Salmonella typhimurium melibiose permease (MelBSt) catalyzes the co-transport of galactosides with cations (H+, Li+, or Na+). 3-D crystal structures of MelBSt have revealed the molecular recognition for sugar substrates, but the cation binding and coupling mechanisms have not been defined to atomic levels. In its human homolog MFSD2A, a lethal mutation was mapped at its Na+-binding pocket; however, none of the structures in this subfamily resolved its cation binding. In this study, molecular dynamics simulations reveal the binding interactions of Na+ and Li+ with MelBSt. Interestingly, Thr121, the lethal mutation position in MFSD2A, forms stable interaction with Na+ but is at a distance from Li+. Most mutations among 11 single-site Thr121 mutants of MelBSt exhibited little effects on the galactoside binding, but largely altered the cation selectivity with severe inhibitions on Na+ binding. Few mutants (Pro and Ala) completely lost the Na+ binding and Na+-coupled transport, but their Li+ or H+ modes of activity were largely retained. It can be concluded that Thr121 is necessary for Na+ binding, but not required for the binding of H+ or Li+, so a subset of the Na+-binding pocket is enough for Li+ binding. In addition, the protein stability for some mutants can be only retained in the presence of Li+, but not by Na+ due to the lack of affinity. This finding, together with other identified thermostable mutants, supports that the charge balance of the cation-binding site plays an important role in MelBSt protein stability.
Subject(s)
Bacterial Proteins , Salmonella typhimurium , Symporters , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cations/metabolism , Humans , Lithium/metabolism , Melibiose/metabolism , Salmonella typhimurium/enzymology , Salmonella typhimurium/genetics , Sodium/metabolism , Symporters/chemistry , Symporters/genetics , Symporters/metabolismABSTRACT
Enzymatic hydrolysis using ß-mannanase and α-galactosidase is necessary to produce low molecular weight galactomannan (LMW-GM) from galactomannans (GM) in the leguminous seeds. In this study, different ratios of avicel and melibiose were used as the inductors (carbon sources) for Trichoderma reesei to metabolize the enzyme cocktail containing ß-mannanase and α-galactosidase using one-pot fermentation technology. The obtained enzyme cocktail was used to efficiently produce LMW-GM from GM in Sesbania cannabina seeds. Results showed that 15 g/L avicel and 10 g/L melibiose were the best carbon sources to prepare enzyme cocktail containing ß-mannanase and α-galactosidase with activities of 3.69 ± 0.27 U/mL and 0.51 ± 0.02 U/mL, respectively. Specifically, melibiose could effectively induce the metabolite product of α-galactosidase by T. reesei, which showed good performance in degrading the galactose substituent from GM backbone. The degradation of galactose alleviated the spatial site-blocking effect for enzymatic hydrolysis by ß-mannanase and improved the yield of LMW-GM. This research can lay the foundation for the industrial technology amplification of LMW-GM production for further application.
Subject(s)
Sesbania , beta-Mannosidase , Carbon , Cellulose/metabolism , Fermentation , Galactose/analogs & derivatives , Hydrolysis , Mannans/metabolism , Melibiose , Molecular Weight , Seeds/metabolism , Technology , alpha-Galactosidase , beta-Mannosidase/metabolismABSTRACT
Enterohemorrhagic Escherichia coli (EHEC) O80:H2, belonging to sequence type ST301, is among the main causes of hemolytic and uremic syndrome in Europe, a major concern in young children. Aside from the usual intimin and Shiga toxin virulence factors (VFs), this emerging serotype possesses a mosaic plasmid combining extra-intestinal VF- and antibiotic resistance-encoding genes. This hybrid pathotype can be involved in invasive infections, a rare occurrence in EHEC infections. Here, we aimed to optimize its detection, improve its clinical diagnosis, and identify its currently unknown reservoir. O80:H2 EHEC strains isolated in France between 2010 and 2018 were phenotypically and genetically analyzed and compared with non-O80 strains. The specificity and sensitivity of a PCR test and a culture medium designed, based on the molecular and phenotypic signatures of O80:H2 EHEC, were assessed on a collection of strains and stool samples. O80:H2 biotype analysis showed that none of the strains (n = 137) fermented melibiose versus 5% of non-O80 EHEC (n = 19/352). This loss of metabolic function is due to deletion of the entire melibiose operon associated with the insertion of a 70-pb sequence (70mel), a genetic scar shared by all ST301 strains. This metabolic hallmark was used to develop a real-time PCR test (100% sensitivity, 98.3% specificity) and a melibiose-based culture medium including antibiotics, characterized by 85% specificity and sensitivity for clinical specimens. These new tools may facilitate the diagnosis of this atypical clone, help the food industry to identify the reservoir and improve our epidemiological knowledge of this threatening and emerging clone.
Subject(s)
Drug Resistance, Bacterial , Enterohemorrhagic Escherichia coli , Hemolytic-Uremic Syndrome , Anti-Bacterial Agents/pharmacology , Child , Child, Preschool , Culture Media , Drug Resistance, Bacterial/genetics , Enterohemorrhagic Escherichia coli/genetics , Enterohemorrhagic Escherichia coli/isolation & purification , Enterohemorrhagic Escherichia coli/metabolism , Fermentation , Hemolytic-Uremic Syndrome/diagnosis , Hemolytic-Uremic Syndrome/microbiology , Humans , Melibiose/metabolismABSTRACT
Major facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-D-galactoside or dodecyl-ß-D-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.
Subject(s)
Bacterial Proteins/chemistry , Crystallography, X-Ray , Melibiose/metabolism , Salmonella typhimurium/metabolism , Symporters/chemistryABSTRACT
An association between the cancer invasive activities of cells and their exposure to advanced glycation end-products (AGEs) was described early in some reports. An incubation of cells with BSA-AGE (bovine serum albumin-AGE), BSA-carboxymethyllysine and BSA-methylglyoxal (BSA-MG) resulted in a significant increase in DNA damage. We examined the genotoxic activity of new products synthesized under nonaqueous conditions. These were high molecular mass MAGEs (HMW-MAGEs) formed from protein and melibiose and low molecular mass MAGEs (LMW-MAGEs) obtained from the melibiose and N-α-acetyllysine and N-α-acetylarginine. We have observed by measuring of micronuclei in human lymphocytes in vitro that the studied HMW-MAGEs expressed the genotoxicity. The number of micronuclei (MN) in lymphocytes reached 40.22 ± 5.34 promille (MN/1000CBL), compared to 28.80 ± 6.50 MN/1000 CBL for the reference BSA-MG, whereas a control value was 20.66 ± 1.39 MN/1000CBL. However, the LMW-MAGE fractions did not induce micronuclei formation in the culture of lymphocytes and partially protected DNA against damage in the cells irradiated with X-ray. Human melanoma and all other studied cells, such as bronchial epithelial cells, lung cancer cells and colorectal cancer cells, are susceptible to the genotoxic effects of HMW-MAGEs. The LMW-MAGEs are not genotoxic, while they inhibit HMW-MAGE genotoxic activity. With regard to apoptosis, it is induced with the HMW-MAGE compounds, in the p53 independent way, whereas the low molecular mass product inhibits the apoptosis induction. Further investigations will potentially indicate beneficial apoptotic effect on cancer cells.
Subject(s)
Apoptosis , Glycation End Products, Advanced/toxicity , Micronuclei, Chromosome-Defective/drug effects , Arginine/analogs & derivatives , Cells, Cultured , DNA Damage , Glycation End Products, Advanced/chemical synthesis , Humans , Lymphocytes/drug effects , Lymphocytes/radiation effects , Lysine/analogs & derivatives , Melibiose/chemistry , Micronucleus Tests , X-RaysABSTRACT
MelB catalyzes the obligatory cotransport of melibiose with Na+, Li+, or H+. Crystal structure determination of the Salmonella typhimurium MelB (MelBSt) has revealed a typical major facilitator superfamily (MFS) fold at a periplasmic open conformation. Cooperative binding of Na+ and melibiose has been previously established. To determine why cotranslocation of sugar solute and cation is obligatory, we analyzed each binding in the thermodynamic cycle using three independent methods, including the determination of melting temperature by circular dichroism spectroscopy, heat capacity change (ΔCp), and regulatory phosphotransferase EIIAGlc binding with isothermal titration calorimetry (ITC). We found that MelBSt thermostability is increased by either substrate (Na+ or melibiose) and observed a cooperative effect of both substrates. ITC measurements showed that either binary formation yields a positive sign in the ΔCp, suggesting MelBSt hydration and a likely widening of the periplasmic cavity. Conversely, formation of a ternary complex yields negative values in ΔCp, suggesting MelBSt dehydration and cavity closure. Lastly, we observed that EIIAGlc, which has been suggested to trap MelBSt at an outward-open state, readily binds to the MelBSt apo state at an affinity similar to MelBSt/Na+. However, it has a suboptimal binding to the ternary state, implying that MelBSt in the ternary complex may be conformationally distant from the EIIAGlc-preferred outward-facing conformation. Our results consistently support the notion that binding of one substrate (Na+ or melibiose) favors MelBSt at open states, whereas the cooperative binding of both substrates triggers the alternating-access process, thus suggesting this conformational regulation could ensure the obligatory cotransport.
Subject(s)
Melibiose , Symporters , Protein Binding , Salmonella typhimurium/metabolism , Sodium/metabolism , Symporters/metabolismABSTRACT
The patho-mechanism of changes in the thyroid gland, including carcinogenesis, is a complex process, which involves oxidative stress. The goal of our investigation was to verify the extent of stress in the thyroid gland related to glycation. The study samples were comprised of blood sera, thyroid, and adipose tissue sections probed from 37 patients diagnosed with thyroid cancers and goiter. Using immuno-enzymatic and fluorometric assays we analyzed the content of advanced glycation end-products (AGEs), pentosidine, receptors for advanced glycation end-products (RAGE), scavenger receptor class (SR)-A, SR-B, glutathione, malondialdehyde and nitric oxide synthase. In addition to classic AGEs, a recent study detected the melibiose-derived glycation (MAGE) product. We demonstrated the presence of AGEs, MAGE and their receptors of the RAGE and SR-A. In addition, in the control samples of thyroid glands SR-B groups were detected as well as of pathological groups without noticeable tendency to antigen concentration in the area of carcinogenesis. Fluorescent AGEs correlate positively with glutathione, which supports the assumption that glycation stress leads to augmentation of oxidative stress and increase of the intensity of antioxidant mechanisms.
Subject(s)
Glycation End Products, Advanced/metabolism , Oxidative Stress , Thyroid Diseases/metabolism , Thyroid Gland/metabolism , Adult , Aged , Female , Humans , Male , Melibiose/metabolism , Middle Aged , Nitric Oxide Synthase/metabolism , Receptor for Advanced Glycation End Products/metabolism , Thyroid Diseases/pathology , Thyroid Gland/pathologyABSTRACT
Non-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.
Subject(s)
Diabetes Mellitus/immunology , Dietary Carbohydrates/immunology , Epitopes/immunology , Glycation End Products, Advanced/immunology , Melibiose/immunology , Animals , Autoantibodies/blood , Autoantibodies/immunology , Cross Reactions , Diabetes Mellitus/blood , Diabetes Mellitus/metabolism , Dietary Carbohydrates/metabolism , Glycation End Products, Advanced/metabolism , Glycosylation , Humans , Melibiose/metabolism , MiceABSTRACT
In many yeast species, the three genes at the centre of the galactose catabolism pathway, GAL1, GAL10 and GAL7, are neighbours in the genome and form a metabolic gene cluster. We report here that some yeast strains in the genus Torulaspora have much larger GAL clusters that include genes for melibiase (MEL1), galactose permease (GAL2), glucose transporter (HGT1), phosphoglucomutase (PGM1) and the transcription factor GAL4, in addition to GAL1, GAL10, and GAL7. Together, these eight genes encode almost all the steps in the pathway for catabolism of extracellular melibiose (a disaccharide of galactose and glucose). We show that a progenitor 5-gene cluster containing GAL 7-1-10-4-2 was likely present in the common ancestor of Torulaspora and Zygotorulaspora. It added PGM1 and MEL1 in the ancestor of most Torulaspora species. It underwent further expansion in the T. pretoriensis clade, involving the fusion of three progenitor clusters in tandem and the gain of HGT1. These giant GAL clusters are highly polymorphic in structure, and subject to horizontal transfers, pseudogenization and gene losses. We identify recent horizontal transfers of complete GAL clusters from T. franciscae into one strain of T. delbrueckii, and from a relative of T. maleeae into one strain of T. globosa. The variability and dynamic evolution of GAL clusters in Torulaspora indicates that there is strong natural selection on the GAL pathway in this genus.
Subject(s)
Galactose/metabolism , Genes, Fungal , Melibiose/metabolism , Metabolic Networks and Pathways/genetics , Multigene Family , Torulaspora/genetics , Torulaspora/metabolismABSTRACT
This paper investigates the solid-state behavior of two-phase solid dispersions involving small molecules. The effect of two sugars, trehalose and melibiose, on the recrystallization of amorphous paracetamol, and vice versa, was investigated. The solid dispersions were prepared via heating and quench-cooling, and then stored at a temperature of 38.5 ± 0.5 °C and relative humidities of 3 ± 1% and 75 ± 1%. X-ray powder diffraction (XRPD) confirmed that the solid dispersions were amorphous, while Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) revealed that the solid dispersions were two-phase systems with drug-rich and excipient-rich regions. XRPD was used to qualitatively and quantitatively study the crystallization of the components, and revealed that, despite the existence of two phases, the sugars hindered the crystallization of paracetamol. In contrast, once the paracetamol crystallization started, it accelerated the crystallization of the sugars. Overall, the study demonstrates that small-molecule solid-dispersions need not be single-phase to observe mutual influences between the components in crystallization behavior, and that these effects are likely mediated through interactions at the phase interfaces, as well as alterations in water sorption and mechanical effects.
Subject(s)
Melibiose , Trehalose , Acetaminophen , Calorimetry, Differential Scanning , Crystallization , Drug Stability , Solubility , Spectroscopy, Fourier Transform Infrared , X-Ray DiffractionABSTRACT
Parkinson's disease (PD) is a neurodegenerative disease characterized by selective dopaminergic (DAergic) neuronal degeneration in the substantia nigra (SN) and proteinaceous α-synuclein-positive Lewy bodies and Lewy neuritis. As a chemical chaperone to promote protein stability and an autophagy inducer to clear aggregate-prone proteins, a disaccharide trehalose has been reported to alleviate neurodegeneration in PD cells and mouse models. Its trehalase-indigestible analogs, lactulose and melibiose, also demonstrated potentials to reduce abnormal protein aggregation in spinocerebellar ataxia cell models. In this study, we showed the potential of lactulose and melibiose to inhibit α-synuclein aggregation using biochemical thioflavin T fluorescence, cryogenic transmission electron microscopy (cryo-TEM) and prokaryotic split Venus complementation assays. Lactulose and melibiose further reduced α-synuclein aggregation and associated oxidative stress, as well as protected cells against α-synuclein-induced neurotoxicity by up-regulating autophagy and nuclear factor, erythroid 2 like 2 (NRF2) pathway in DAergic neurons derived from SH-SY5Y cells over-expressing α-synuclein. Our findings strongly indicate the potential of lactulose and melibiose for mitigating PD neurodegeneration, offering new drug candidates for PD treatment.