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BACKGROUND: In sharp contrast with analysis of N-glycan that can be prepared by PNGase F, O-glycan analysis remains challenging due to a lack of versatile and simple procedures, especially those mediating cleavage of O-glycans from proteins. Most N-glycans and O-glycans are modified with sialic acids at the non-reducing end and their glycosidic linkages are labile, making it difficult to measure glycans by mass spectrometric analysis. In addition, sialic acid residues present on glycan chains via α2,3-, α2,6-, and α2,8-linkages as structural isomers. RESULTS: In this study, we firstly established a direct and linkage-specific derivatization method for sialylated O-glycans on proteins via linkage-specific lactone-opening aminolysis. In this procedure, labile sialylated glycans were not only stabilized, but also allowed distinguishing between sialyl linkages. Furthermore, we revealed that general reductive ß-elimination was not useful for O-glycan cleavages with undesirable degradations of resulting methyl amides. Using ß-elimination in the presence of pyrazolone (PMP), with low pH despite alkali base concentration, SALSA-derivatized O-glycans could be cleaved with minimal degradations. Cleaved and PMP-labeled O-glycans could be efficiently prepared in an open reaction system at high temperature (evaporative BEP reaction) and detected by simple liquid-phase extraction. Moreover, in the evaporative BEP reaction by changing the alkali solution with LiOH, the lithiated O-glycans could be observed and provided a lot of fragment information reflecting the complex structure of the O-glycans. SIGNIFICANCE: Direct sialic acid linkage-specific derivatization of O-glycans on glycoproteins is simple protocol containing in-solution aminolysis-SALSA and acetonitrile precipitation for removal of excess reagents. Evaporative ß-elimination with pyrazolone makes possible intact O-linked glycan analysis just by liquid-phase extraction. These analytical methods established by the appropriate combination of direct-SALSA and evaporative ß-elimination will facilitate O-glycomic studies in various biological samples.
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Polisacáridos , Ácidos Siálicos , Polisacáridos/química , Ácidos Siálicos/químicaRESUMEN
Sialylation is a terminal glycosylated modification of glycoproteins that regulates critical biological events such as cell adhesion and immune response. Our previous study showed that integrin α3ß1 plays a crucial role in regulating the sialylation of N-glycans. However, the underlying mechanism for the regulation remains unclear. This study investigated how sialylation is affected by focal adhesion kinase (FAK), which is a critical downstream signal molecule of integrin ß1. We established a stable FAK knockout (KO) cell line using the CRISPR/Cas9 system in HeLa cells. The results obtained from lectin blot, flow cytometric analysis, and MS showed that the sialylation levels were significantly decreased in the KO cells compared with that in wild-type (WT) cells. Moreover, phosphatidylinositol 4-phosphate (PI4P) expression levels were also reduced in the KO cells due to a decrease in the stability of phosphatidylinositol 4-kinase-IIα (PI4KIIα). Notably, the decreased levels of sialylation, PI4P, and the complex formation between GOLPH3 and ST3GAL4 or ST6GAL1, which are the main sialyltransferases for modification of N-glycans, were significantly restored by the re-expression of FAK. Furthermore, the decreased sialylation and phosphorylation of Akt and cell migration caused by FAK deficiency all were restored by overexpressing PI4KIIα, which suggests that PI4KIIα is one of the downstream molecules of FAK. These findings indicate that FAK regulates sialylation via the PI4P synthesis pathway and a novel mechanism is suggested for the integrin-FAK-PI4KIIα-GOLPH3-ST axis modulation of sialylation in N-glycans.
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Quinasa 1 de Adhesión Focal , Polisacáridos , Transducción de Señal , Humanos , Quinasa 1 de Adhesión Focal/metabolismo , Células HeLa , Proteínas de la Membrana/metabolismo , Fosforilación , Polisacáridos/metabolismoRESUMEN
Metaproteomics is a relatively young field that has only been studied for approximately 15 years. Nevertheless, it has the potential to play a key role in disease research by elucidating the mechanisms of communication between the human host and the microbiome. Although it has been useful in developing an understanding of various diseases, its analytical strategies remain limited to the extended application of proteomics. The sequence databases in metaproteomics must be large because of the presence of thousands of species in a typical sample, which causes problems unique to large databases. In this review, we demonstrate the usefulness of metaproteomics in disease research through examples from several studies. Additionally, we discuss the challenges of applying metaproteomics to conventional proteomics analysis methods and introduce studies that may provide clues to the solutions. We also discuss the need for a standard false discovery rate control method for metaproteomics to replace common target-decoy search approaches in proteomics and a method to ensure the reliability of peptide spectrum match.
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This study determined the effect of brefeldin A (BFA) on the free N-glycomic profile of HepG2 cells to better understand the effect of blocking intracellular vesicle formation and transport of proteins from the endoplasmic reticulum to the Golgi apparatus. A series of exoglycosidase- and endoglycosidase-assisted analyses clarified the complex nature of altered glycomic profiles. A key feature of BFA-mediated alterations in Gn2-type glycans was the expression of unusual hybrid-, monoantennary- and complex-type free N-glycans (FNGs). BFA-mediated alterations in Gn1-type glycans were characterized by the expression of unusual hybrid- and monoantennary-FNGs, without significant expression of complex-type FNGs. A time course analysis revealed that sialylated hybrid- and complex-type Gn2-type FNGs were generated later than asialo-Gn2-type FNGs, and the expression profiles of Gn2-type FNGs and N-glycans were found to be similar, suggesting that the metabolic flux of FNGs is the same as that of protein-bound N-glycans. Subcellular glycomic analysis revealed that almost all FNGs were detected in the cytoplasmic extracts. Our data suggest that hybrid-, monoantennary- and complex-type Gn2-type FNGs were cleaved from glycoproteins in the cytosol by cytosolic PNGase, and subsequently digested by cytosolic endo-ß-N-acetylglucosaminidase (ENGase) to generate Gn1-type FNGs. The substrate specificity of ENGase explains the limited expression of complex Gn1 type FNGs.
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Glicósido Hidrolasas , Polisacáridos , Humanos , Brefeldino A/farmacología , Células Hep G2 , Polisacáridos/metabolismo , Manosil-Glicoproteína Endo-beta-N-AcetilglucosaminidasaRESUMEN
Glycans are involved in many fundamental cellular processes such as growth, differentiation, and morphogenesis. However, their broad structural diversity makes analysis difficult. Glycomics via mass spectrometry has focused on the composition of glycans, but informatics analysis has not kept pace with the development of instrumentation and measurement techniques. We developed Toolbox Accelerating Glycomics (TAG), in which glycans can be added manually to the glycan list that can be freely designed with labels and sialic acid modifications, and fast processing is possible. In the present work, we improved TAG for large-scale analysis such as cohort analysis of serum samples. The sialic acid linkage-specific alkylamidation (SALSA) method converts differences in linkages such as α2,3- and α2,6-linkages of sialic acids into differences in mass. Glycans modified by SALSA and several structures discovered in recent years were added to the glycan list. A routine to generate calibration curves has been implemented to explore quantitation. These improvements are based on redefinitions of residues and glycans in the TAG List to incorporate information on glycans that could not be attributed because it was not assumed in the previous version of TAG. These functions were verified through analysis of purchased sera and 74 spectra with linearity at the level of R2 > 0.8 with 81 estimated glycan structures obtained including some candidate of rare glycans such as those with the N,N'-diacetyllactosediamine structure, suggesting they can be applied to large-scale analyses.
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Glicómica , Ácido N-Acetilneuramínico , Humanos , Glicómica/métodos , Polisacáridos/química , Ácidos Siálicos/química , Espectrometría de MasasRESUMEN
Swainsonine (SWA), a potent inhibitor of class II α-mannosidases, is present in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants. The mechanisms underlying SWA-induced animal poisoning are not fully understood. In this study, we analyzed the alterations that occur in N- and free N-glycomic upon addition of SWA to HepG2 cells to understand better SWA-induced glycomic alterations. After SWA addition, we observed the appearance of SWA-specific glycomic alterations, such as unique fucosylated hybrid-type and fucosylated M5 (M5F) N-glycans, and a remarkable increase in all classes of Gn1 FNGs. Further analysis of the context of these glycomic alterations showed that (fucosylated) hybrid type N-glycans were not the precursors of these Gn1 FNGs and vice versa. Time course analysis revealed the dynamic nature of glycomic alterations upon exposure of SWA and suggested that accumulation of free N-glycans occurred earlier than that of hybrid-type N-glycans. Hybrid-type N-glycans, of which most were uniquely core fucosylated, tended to increase slowly over time, as was observed for M5F N-glycans. Inhibition of swainsonine-induced unique fucosylation of hybrid N-glycans and M5 by coaddition of 2-fluorofucose caused significant increases in paucimannose- and fucosylated paucimannose-type N-glycans, as well as paucimannose-type free N-glycans. The results not only revealed the gross glycomic alterations in HepG2 cells induced by swainsonine, but also provide information on the global interrelationships between glycomic alterations.
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Glicómica , Swainsonina , Animales , Glicosilación , Células Hep G2 , Humanos , Polisacáridos , Swainsonina/toxicidadRESUMEN
BACKGROUND: Glycan-related genes play a fundamental role in various processes for energy acquisition and homeostasis maintenance while adapting to the environment in which the organism exists; however, their role in the microbiome in the environment is unclear. METHODS: Sequence alignment was performed between known glycan-related genes and complete genomes of microorganisms, and optimal parameters for identifying glycan-related genes were determined based on the alignments. Using the constructed scheme (> 90% of identity and > 25 aa of alignment length), glycan-related genes in various environments were identified from 198 different metagenome data. RESULTS: As a result, we identified 86.73 million glycan-related genes from the metagenome data. Among the 12 environments classified in this study, the percentage of glycan-related genes was high in the human-associated environment, suggesting that these environments utilize glycan metabolism better than other environments. On the other hand, the relative abundances of both glycoside hydrolases and glycosyltransferases surprisingly had a coverage of over 80% in all the environments. These glycoside hydrolases and glycosyltransferases were classified into two groups of (1) general enzyme families identified in various environments and (2) specific enzymes found only in certain environments. The general enzyme families were mostly from genes involved in monosaccharide metabolism, and most of the specific enzymes were polysaccharide degrading enzymes. CONCLUSION: These findings suggest that environmental microorganisms could change the composition of their glycan-related genes to adapt the processes involved in acquiring energy from glycans in their environments. Our functional glyco-metagenomics approach has made it possible to clarify the relationship between the environment and genes from the perspective of carbohydrates, and the existence of glycan-related genes that exist specifically in the environment.
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Metagenoma , Metagenómica , Adaptación Fisiológica , Glicósido Hidrolasas , Humanos , PolisacáridosRESUMEN
Background & Aims: Periodontitis increases the risk of nonalcoholic fatty liver disease (NAFLD); however, the underlying mechanisms are unclear. Here, we show that gut dysbiosis induced by oral administration of Porphyromonas gingivalis, a representative periodontopathic bacterium, is involved in the aggravation of NAFLD pathology. Methods: C57BL/6N mice were administered either vehicle, P. gingivalis, or Prevotella intermedia, another periodontopathic bacterium with weaker periodontal pathogenicity, followed by feeding on a choline-deficient, l-amino acid-defined, high-fat diet with 60 kcal% fat and 0.1% methionine (CDAHFD60). The gut microbial communities were analyzed by pyrosequencing the 16S ribosomal RNA genes. Metagenomic analysis was used to determine the relative abundance of the Kyoto Encyclopedia of Genes and Genomes pathways encoded in the gut microbiota. Serum metabolites were analyzed using nuclear magnetic resonance-based metabolomics coupled with multivariate statistical analyses. Hepatic gene expression profiles were analyzed via DNA microarray and quantitative polymerase chain reaction. Results: CDAHFD60 feeding induced hepatic steatosis, and in combination with bacterial administration, it further aggravated NAFLD pathology, thereby increasing fibrosis. Gene expression analysis of liver samples revealed that genes involved in NAFLD pathology were perturbed, and the two bacteria induced distinct expression profiles. This might be due to quantitative and qualitative differences in the influx of bacterial products in the gut because the serum endotoxin levels, compositions of the gut microbiota, and serum metabolite profiles induced by the ingested P. intermedia and P. gingivalis were different. Conclusions: Swallowed periodontopathic bacteria aggravate NAFLD pathology, likely due to dysregulation of gene expression by inducing gut dysbiosis and subsequent influx of gut bacteria and/or bacterial products.
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Microbioma Gastrointestinal , Enfermedad del Hígado Graso no Alcohólico/microbiología , Porphyromonas gingivalis , Prevotella intermedia , Administración Oral , Animales , Deficiencia de Colina , Dieta Alta en Grasa , Heces/microbiología , Células Hep G2 , Humanos , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/patología , ARN Ribosómico 16SRESUMEN
Some bacteria are symbiotic in tumor tissues, and metabolites of several bacterial species have been found to cause DNA damage. However, to date, the association between bacteria and host genetic alterations in colorectal cancer (CRC) has not been fully investigated. We evaluated the association between the intra-tumor microbiome and host genetic alterations in 29 Japanese CRC patients. The tumor and non-tumor tissues were extracted from the patients, and 16S rRNA genes were sequenced for each sample. We identified enriched bacteria in tumor and non-tumor tissues. Some bacteria, such as Fusobacterium, which is already known to be enriched in CRC, were found to be enriched in tumor tissues. Interestingly, Bacteroides, which is also known to be enriched in CRC, was enriched in non-tumor tissues. Furthermore, it was shown that certain bacteria that often coexist within tumor tissue were enriched in the presence of a mutated gene or signal pathway with mutated genes in the host cells. Fusobacterium was associated with many mutated genes, as well as cell cycle-related pathways including mutated genes. In addition, the patients with a high abundance of Campylobacter were suggested to be associated with mutational signature 3 indicating failure of double-strand DNA break repairs. These results suggest that CRC development may be partly caused by DNA damage caused by substances released by bacterial infection. Taken together, the identification of distinct gut microbiome patterns and their host specific genetic alterations might facilitate targeted interventions, such as modulation of the microbiome in addition to anticancer agents or immunotherapy.
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Obesity is a risk factor for periodontal disease (PD). Initiation and progression of PD are modulated by complex interactions between oral dysbiosis and host responses. Although obesity is associated with increased susceptibility to bacterial infection, the detailed mechanisms that connect obesity and susceptibility to PD remain elusive. Using fecal microbiota transplantation and a ligature-induced PD model, we demonstrated that gut dysbiosis-associated metabolites from high-fat diet (HFD)-fed mice worsen alveolar bone destruction. Fecal metabolomics revealed elevated purine degradation pathway activity in HFD-fed mice, and recipient mice had elevated levels of serum uric acid upon PD induction. Furthermore, PD induction caused more severe bone destruction in hyperuricemic than normouricemic mice, and the worsened bone destruction was completely abrogated by allopurinol, a xanthine oxidase inhibitor. Thus, obesity increases the risk of PD by increasing production of uric acid mediated by gut dysbiosis. IMPORTANCE Obesity is an epidemic health issue with a rapid increase worldwide. It increases the risk of various diseases, including periodontal disease, an oral chronic infectious disease. Although obesity increases susceptibility to bacterial infection, the precise biological mechanisms that link obesity and susceptibility to periodontal disease remain elusive. Using fecal microbial transplantation, experimental periodontitis, and metabolomics, our study demonstrates uric acid as a causative substance for greater aggravation of alveolar bone destruction in obesity-related periodontal disease. Gut microbiota from obese mice upregulated the purine degradation pathway, and the resulting elevation of serum uric acid promoted alveolar bone destruction. The effect of uric acid was confirmed by administration of allopurinol, an inhibitor of xanthine oxidase. Overall, our study provides new insights into the pathogenic mechanisms of obesity-associated periodontal disease and the development of new therapeutic options for the disease.
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Pérdida de Hueso Alveolar/etiología , Microbioma Gastrointestinal , Obesidad/microbiología , Periodontitis/microbiología , Ácido Úrico/metabolismo , Pérdida de Hueso Alveolar/patología , Animales , Dieta Alta en Grasa , Disbiosis , Trasplante de Microbiota Fecal , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/complicaciones , Periodontitis/etiología , Factores de Riesgo , Ácido Úrico/análisisRESUMEN
ABO blood antigens on the human red blood cell membrane as well as different cells in various human tissues have been thoroughly studied. Anti-A and -B antibodies of IgM are present in serum/plasma, but blood group-specific glyco-antigens have not been extensively described. In this study, we performed comprehensive and quantitative serum glycomic analyses of various glycoconjugates and free oligosaccharides in all blood groups. Our comprehensive glycomic approach revealed that blood group-specific antigens in serum/plasma are predominantly present on glycosphingolipids on lipoproteins rather than glycoproteins. Expression of the ABO antigens on glycosphingolipids depends not only on blood type but also on secretor status. Blood group-specific glycans in serum/plasma were classified as type I, whereas those on RBCs had different structures including hexose and hexosamine residues. Analysis of free oligosaccharides revealed that low-molecular-weight blood group-specific glycans, commonly containing lacto-N-difucotetraose, were expressed in serum/plasma according to blood group. Furthermore, comprehensive glycomic analysis in human cerebrospinal fluid showed that many kinds of free oligosaccharides were highly expressed, and low-molecular-weight blood group-specific glycans, which existed in plasma from the same individuals, were present. Our findings provide the first evidence for low-molecular-weight blood group-specific glycans in both serum/plasma and cerebrospinal fluid.
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Antígenos de Grupos Sanguíneos , Glicómica , Glicoproteínas , Humanos , Oligosacáridos , PolisacáridosRESUMEN
Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.
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Autofagosomas/metabolismo , Estrés Oxidativo , Proteína Sequestosoma-1/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagosomas/ultraestructura , Autofagia , Línea Celular , Geles , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Hígado/lesiones , Hígado/patología , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Unión Proteica , Liposomas UnilamelaresRESUMEN
Glycans present extraordinary structural diversity commensurate with their involvement in numerous fundamental cellular processes including growth, differentiation, and morphogenesis. Unlike linear DNA and protein sequences, glycans have heterogeneous structures that differ in composition, branching, linkage, and anomericity. These differences pose a challenge to developing useful software for glycomic analysis. To overcome this problem, we developed the novel Toolbox Accelerating Glycomics (TAG) program. TAG consists of three units: 'TAG List' creates a glycan list that is used for database searching in TAG Expression; 'TAG Expression' automatically annotates and quantifies glycan signals and draws graphs; and 'TAG Pathway' maps the obtained expression information to biosynthetic pathways. Herein, we discuss the concepts, outline the TAG process, and demonstrate its potential using glycomic expression profile data from Chinese hamster ovary (CHO) cells and mutants lacking a functional Npc1 gene (Npc1 knockout (KO) CHO cells). TAG not only drastically reduced the amount of time and labor needed for glycomic analysis but also detected and quantified more glycans than manual analysis. Although this study was limited to the analysis of N-glycans and free oligosaccharides, the glycomic platform will be expanded to facilitate the analysis of O-glycans and glycans of glycosphingolipids.
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Glicómica/métodos , Polisacáridos/análisis , Programas Informáticos , Animales , Células CHO , Cricetinae , Cricetulus , Técnicas de Inactivación de Genes , Glicoproteínas/metabolismo , Glicoesfingolípidos/metabolismo , Proteína Niemann-Pick C1/deficiencia , Proteína Niemann-Pick C1/genética , Oligosacáridos/análisis , Polisacáridos/biosíntesis , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Sialic acid attached to nonreducing ends of glycan chains via different linkages is associated with specific interactions and physiological events. Linkage-specific derivatization of sialic acid is of great interest for distinguishing sialic acids by mass spectrometry, specifically for events governed by sialyl linkage types. In the present study, we demonstrate that α-2,3/8-sialyl linkage-specific amidation of esterified sialyloligosaccharides can be achieved via an intramolecular lactone. The method of lactone-driven ester-to-amide derivatization for sialic acid linkage-specific alkylamidation, termed LEAD-SALSA, employs in-solution ester-to-amide conversion to directly generate stable and sialyl linkage-specific glycan amides from their ester form by mixing with a preferred amine, resulting in the easy assignments of sialyl linkages by comparing the signals of esterified and amidated glycan. Using this approach, we demonstrate the accumulation of altered N-glycans in cardiac muscle tissue during mouse aging. Furthermore, we find that the stability of lactone is important for ester-to-amide conversion based on experiments and density functional theory calculations of reaction energies for lactone formation. By using energy differences of lactone formation, the LEAD-SALSA method can be used not only for the sialyl linkage-specific derivatization but also for distinguishing the branching structure of galactose linked to sialic acid. This simplified and direct sialylglycan discrimination will facilitate important studies on sialylated glycoconjugates.
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The susceptibility to glycation of all d-glucose-containing reducing disaccharides (kojibiose, sophorose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, and gentiobiose) was evaluated by Maillard browning and the percentages of their acyclic forms estimated using a novel method to evaluate reactivity toward oxime formation were compared for the first time. This new method is facile and applicable to non-labeled carbohydrates, and it is extremely sensitive, more so than any other previously reported methods. The disaccharides linked by 1-6 bonds displayed both high browning intensity and oxime formation reactivity, and they had the greatest amount of the acyclic form. On the other hand, the proportion of acyclic form was generally very low when glucoses were linked by 1-2, 1-3 and 1-4 bonds. The stability of the 1-3 linkage was drastically reduced when basicity was increased due to ß-elimination and the production of a highly reactive dehydrated hexose. The 1-4-linked structures, involved in the formation of amylose and cellulose, respectively, were found to be advantageous due to their relatively low susceptibility to glycation.
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Disacáridos/química , Glucosa/química , Reacción de Maillard , Oximas/síntesis química , Conformación de Carbohidratos , Glicosilación , Oxidación-Reducción , Oximas/químicaRESUMEN
Potential inhibitors of a target biomolecule, NAD-dependent deacetylase Sirtuin 1, were identified by a contest-based approach, in which participants were asked to propose a prioritized list of 400 compounds from a designated compound library containing 2.5 million compounds using in silico methods and scoring. Our aim was to identify target enzyme inhibitors and to benchmark computer-aided drug discovery methods under the same experimental conditions. Collecting compound lists derived from various methods is advantageous for aggregating compounds with structurally diversified properties compared with the use of a single method. The inhibitory action on Sirtuin 1 of approximately half of the proposed compounds was experimentally accessed. Ultimately, seven structurally diverse compounds were identified.
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Sialic acids form the terminal sugars in glycan chains on glycoproteins via α2,3, α2,6, or α2,8 linkages, and structural isomers of sialyl linkages play various functional roles in cell recognition and other physiological processes. We recently developed a novel procedure based on sialic acid linkage-specific alkylamidation via lactone ring opening (aminolysis-SALSA). Herein, we have investigated an isotope labeling of α2,3-linked sialic acid residues (iSALSA) using amine hydrochloride salts. One limitation of SALSA using amine hydrochloride salts may be solved by adding only tert-butylamine (t-BA) as an acid scavenger, and comparative and quantitative glycomic analyses can be performed using iSALSA. We also developed quantitative glycomic analysis using dual isotope-labeled glycans by derivatizing with aminooxy-functionalized tryptophanylarginine methyl ester (aoWR) and iSALSA at the reducing and nonreducing end, respectively. Furthermore, we demonstrate that the amount of α2,3-linked sialoglycans in serum are altered during liver fibrosis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography MS (LC/MS) analyses. We revealed that the ratio of A33,6,6 to A3F3,6,6 was gradually decreased along with liver fibrosis progression. Therefore, these glycan alterations are potential diagnostic markers of nonalcoholic steatohepatitis (NASH) fibrosis progression.
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Glicómica/métodos , Ácido N-Acetilneuramínico/química , Polisacáridos/química , Aminas/química , Biomarcadores , Glicoproteínas/química , Humanos , Marcaje Isotópico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Polisacáridos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Sialic acids occur widely as glycoconjugates at the nonreducing ends of glycans. Glycosphingolipids (GSLs) include a large number of sialyl-linked glycan isomers with α2,3-, α2,6-, and α2,8-linked polysialic acids. Thus, it is difficult to distinguish structural isomers with the same mass by mass spectrometry. The sialic acid linkage specific alkylamidation (SALSA) method has been developed for discriminating between α2,3- and α2,6-linked isomers, but sequential amidation of linkage-specific sialic acids is generally complicated and time-consuming. Moreover, analysis of GSL-glycans containing α2,8-linked polysialic acids using solid-phase SALSA has not been reported. Herein, we report a novel SALSA method focused on ring-opening aminolysis (aminolysis-SALSA), which shortens the reaction time and simplifies the experimental procedures. We demonstrate that aminolysis-SALSA can successfully distinguish serum GSL-glycan isomers by mass spectrometry. In addition, ring-opening aminolysis can easily be applied to amine and hydrazine derivatives.
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Gangliósidos/sangre , Glicómica/métodos , Lactonas/química , Polisacáridos/sangre , Ácidos Siálicos/química , Animales , Bovinos , Fenómenos Químicos , Gangliósidos/química , Isomerismo , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Elucidating pathways related to posttranslational modifications (PTMs) such as glycosylation is of growing importance in post-genome science and technology. Graphical networks describing the relationships among glycan-related molecules, including genes, proteins, lipids, and various biological events, are considered extremely valuable and convenient tools for the systematic investigation of PTMs. Glyco-Net (http://bibi.sci.hokudai.ac.jp/functions/) can dynamically make network figures among various biological molecules and biological events. A certain molecule or event is expressed with a node, and the relationship between the molecule and the event is indicated by arrows in the network figures. In this chapter, we mention the features and current status of the Glyco-Net and a simple example of the search with the Glyco-Net.