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PURPOSE: Rapid decision-making is essential in precision medicine for initiating molecular targeted therapy for patients with cancer. This study aimed to extract pathomorphologic features that enable the accurate prediction of genetic abnormalities in cancer from hematoxylin and eosin images using deep learning (DL). EXPERIMENTAL DESIGN: A total of 1,657 images (one representative image per patient) of thin formalin-fixed, paraffin-embedded tissue sections from either primary or metastatic tumors with next-generation sequencing-confirmed genetic abnormalities-including BRAFV600E and KRAS mutations, and microsatellite instability high (MSI-H)-that are directly relevant to therapeutic strategies for advanced colorectal cancer were obtained from the nationwide SCRUM-Japan GI-SCREEN project. The images were divided into three groups of 986, 248, and 423 images to create one training and two validation cohorts, respectively. Pathomorphologic feature-prediction DL models were first developed on the basis of pathomorphologic features. Subsequently, gene-prediction DL models were constructed for all possible combinations of pathomorphologic features that enabled the prediction of gene abnormalities based on images filtered by the combination of pathomorphologic feature-prediction models. RESULTS: High accuracies were achieved, with AUCs > 0.90 and 0.80 for 12 and 27, respectively, of 33 analyzed pathomorphologic features, with high AUCs being yielded for both BRAFV600E (0.851 and 0.859) and MSI-H (0.923 and 0.862). CONCLUSIONS: These findings show that novel next-generation pathology methods can predict genetic abnormalities without the need for standard-of-care gene tests, and this novel next-generation pathology method can be applied for colorectal cancer treatment planning in the near future.
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Neoplasias Colorrectales , Inestabilidad de Microsatélites , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Detección Precoz del Cáncer , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , MutaciónRESUMEN
To elucidate the relationship between the protein function and the diversity and heterogeneity of glycans conjugated to the protein, glycosylation sites, glycan variation, and glycan proportions at each site of the glycoprotein must be analyzed. Glycopeptide-based structural analysis technology using mass spectrometry has been developed; however, complicated analyses of complex spectra obtained by multistage fragmentation are necessary, and sensitivity and throughput of the analyses are low. Therefore, we developed a liquid chromatography/mass spectrometry (MS)-based glycopeptide analysis method to reveal the site-specific glycome (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile, Glyco-RIDGE). This method used accurate masses and retention times of glycopeptides, without requiring MS2, and could be applied to complex mixtures. To increase the number of identified peptide, fractionation of sample glycopeptides for reduction of sample complexity is required. Therefore, in this study, glycopeptides were fractionated into four fractions by hydrophilic interaction chromatography, and each fraction was analyzed using the Glyco-RIDGE method. As a result, many glycopeptides having long glycans were enriched in the highest hydrophilic fraction. Based on the monosaccharide composition, these glycans were thought to be poly-N-acetyllactosamine (polylactosamine [pLN]), and 31 pLN-carrier proteins were identified in HL-60 cells. Gene ontology enrichment analysis revealed that pLN carriers included many molecules related to signal transduction, receptors, and cell adhesion. Thus, these findings provided important insights into the analysis of the glycoproteome using our novel Glyco-RIDGE method. Graphical Abstract á .
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Glicoproteínas/química , Leucemia Promielocítica Aguda/patología , Polisacáridos/análisis , Cromatografía Liquida/métodos , Glicopéptidos/análisis , Células HL-60 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
BACKGROUND: Inherited mutations in glyco-related genes can affect the biosynthesis and degradation of glycans and result in severe genetic diseases and disorders. The Glyco-Disease Genes Database (GDGDB), which provides information about these diseases and disorders as well as their causative genes, has been developed by the Research Center for Medical Glycoscience (RCMG) and released in April 2010. GDGDB currently provides information on about 80 genetic diseases and disorders caused by single-gene mutations in glyco-related genes. Many biomedical resources provide information about genetic disorders and genes involved in their pathogenesis, but resources focused on genetic disorders known to be related to glycan metabolism are lacking. With the aim of providing more comprehensive knowledge on genetic diseases and disorders of glycan biosynthesis and degradation, we enriched the content of the GDGDB database and improved the methods for data representation. RESULTS: We developed the Genetic Glyco-Diseases Ontology (GGDonto) and a RDF/SPARQL-based user interface using Semantic Web technologies. In particular, we represented the GGDonto content using Semantic Web languages, such as RDF, RDFS, SKOS, and OWL, and created an interactive user interface based on SPARQL queries. This user interface provides features to browse the hierarchy of the ontology, view detailed information on diseases and related genes, and find relevant background information. Moreover, it provides the ability to filter and search information by faceted and keyword searches. CONCLUSIONS: Focused on the molecular etiology, pathogenesis, and clinical manifestations of genetic diseases and disorders of glycan metabolism and developed as a knowledge-base for this scientific field, GGDonto provides comprehensive information on various topics, including links to aid the integration with other scientific resources. The availability and accessibility of this knowledge will help users better understand how genetic defects impact the metabolism of glycans as well as how this impaired metabolism affects various biological functions and human health. In this way, GGDonto will be useful in fields related to glycoscience, including cell biology, biotechnology, and biomedical, and pharmaceutical research.
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Enfermedad/genética , Ontología de Genes , Bases del Conocimiento , Polisacáridos/metabolismo , Bases de Datos Genéticas , Internet , Mutación , Polisacáridos/biosíntesis , Interfaz Usuario-ComputadorRESUMEN
Glycans are known as the third major class of biopolymers, next to DNA and proteins. They cover the surfaces of many cells, serving as the 'face' of cells, whereby other biomolecules and viruses interact. The structure of glycans, however, differs greatly from DNA and proteins in that they are branched, as opposed to linear sequences of amino acids or nucleotides. Therefore, the storage of glycan information in databases, let alone their curation, has been a difficult problem. This has caused many duplicated efforts when integration is attempted between different databases, making an international repository for glycan structures, where unique accession numbers are assigned to every identified glycan structure, necessary. As such, an international team of developers and glycobiologists have collaborated to develop this repository, called GlyTouCan and is available at http://glytoucan.org/, to provide a centralized resource for depositing glycan structures, compositions and topologies, and to retrieve accession numbers for each of these registered entries. This will thus enable researchers to reference glycan structures simply by accession number, as opposed to by chemical structure, which has been a burden to integrate glycomics databases in the past.
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Bases de Datos de Compuestos Químicos , Polisacáridos/química , Estructura MolecularRESUMEN
The biological significance of glycans has been widely studied and reported in the past. However, most achievements of our predecessors are not readily available in existing databases. JCGGDB is a meta-database involving 15 original databases in AIST and 5 cooperative databases in alliance with JCGG: Japan Consortium for Glycobiology and Glycotechnology. It centers on a glycan structure database and accumulates information such as glycan preferences of lectins, glycosylation sites in proteins, and genes related to glycan syntheses from glycoscience and related fields. This chapter illustrates how to use three major search interfaces (Keyword Search, Structure Search, and GlycoChem Explorer) available in JCGGDB to search across multiple databases.
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Bases de Datos Factuales , Glicómica , Japón , Monosacáridos/análisis , Polisacáridos/química , Motor de Búsqueda , Programas InformáticosRESUMEN
Lectins are a large group of carbohydrate-binding proteins, having been shown to comprise at least 48 protein scaffolds or protein family entries. They occur ubiquitously in living organisms-from humans to microorganisms, including viruses-and while their functions are yet to be fully elucidated, their main underlying actions are thought to mediate cell-cell and cell-glycoconjugate interactions, which play important roles in an extensive range of biological processes. The basic feature of each lectin's function resides in its specific sugar-binding properties. In this regard, it is beneficial for researchers to have access to fundamental information about the detailed oligosaccharide specificities of diverse lectins. In this review, the authors describe a publicly available lectin database named "Lectin frontier DataBase (LfDB)", which undertakes the continuous publication and updating of comprehensive data for lectin-standard oligosaccharide interactions in terms of dissociation constants (Kd's). For Kd determination, an advanced system of frontal affinity chromatography (FAC) is used, with which quantitative datasets of interactions between immobilized lectins and >100 fluorescently labeled standard glycans have been generated. The FAC system is unique in its clear principle, simple procedure and high sensitivity, with an increasing number (>67) of associated publications that attest to its reliability. Thus, LfDB, is expected to play an essential role in lectin research, not only in basic but also in applied fields of glycoscience.
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Cromatografía de Afinidad/métodos , Bases de Datos como Asunto , Lectinas/metabolismo , Humanos , Cinética , Lectinas/clasificación , Oligosacáridos/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
In this study, we selected 181 nematode glycogenes that are orthologous to human glycogenes and examined their RNAi phenotypes. The results are deposited in the Caenorhabditis elegans Glycogene Database (CGGDB) at AIST, Tsukuba, Japan. The most prominent RNAi phenotypes observed are disruptions of cell cycle progression in germline mitosis/meiosis and in early embryonic cell mitosis. Along with the previously reported roles of chondroitin proteoglycans, glycosphingolipids and GPI-anchored proteins in cell cycle progression, we show for the first time that the inhibition of the functions of N-glycan synthesis genes (cytoplasmic alg genes) resulted in abnormal germline formation, ER stress and small body size phenotypes. The results provide additional information on the roles of glycoconjugates in the cell cycle progression mechanisms of germline and embryonic cells.
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Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Bases de Datos Genéticas , Genes de Helminto , Interferencia de ARN , Animales , Secuencia de Bases , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Secuencia de Carbohidratos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Estrés del Retículo Endoplásmico/genética , Regulación del Desarrollo de la Expresión Génica , Genotipo , Células Germinativas/citología , Células Germinativas/metabolismo , Glicoconjugados/química , Glicoconjugados/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Glicoesfingolípidos/química , Glicoesfingolípidos/metabolismo , Humanos , Meiosis/genética , Mitosis/genética , Datos de Secuencia Molecular , Fenotipo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
MOTIVATION: Over the last decades several glycomics-based bioinformatics resources and databases have been created and released to the public. Unfortunately, there is no common standard in the representation of the stored information or a common machine-readable interface allowing bioinformatics groups to easily extract and cross-reference the stored information. RESULTS: An international group of bioinformatics experts in the field of glycomics have worked together to create a standard Resource Description Framework (RDF) representation for glycomics data, focused on glycan sequences and related biological source, publications and experimental data. This RDF standard is defined by the GlycoRDF ontology and will be used by database providers to generate common machine-readable exports of the data stored in their databases. AVAILABILITY AND IMPLEMENTATION: The ontology, supporting documentation and source code used by database providers to generate standardized RDF are available online (http://www.glycoinfo.org/GlycoRDF/).
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Biología Computacional/métodos , Sistemas de Administración de Bases de Datos/normas , Bases de Datos Factuales/normas , Glicómica/métodos , Almacenamiento y Recuperación de la Información/métodos , Polisacáridos/química , Programas Informáticos , Documentación , Ontología de Genes , HumanosRESUMEN
In recent years, the Semantic Web has become the focus of life science database development as a means to link life science data in an effective and efficient manner. In order for carbohydrate data to be applied to this new technology, there are two requirements for carbohydrate data representations: (1) a linear notation which can be used as a URI (Uniform Resource Identifier) if needed and (2) a unique notation such that any published glycan structure can be represented distinctively. This latter requirement includes the possible representation of nonstandard monosaccharide units as a part of the glycan structure, as well as compositions, repeating units, and ambiguous structures where linkages/linkage positions are unidentified. Therefore, we have developed the Web3 Unique Representation of Carbohydrate Structures (WURCS) as a new linear notation for representing carbohydrates for the Semantic Web.
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Carbohidratos/química , Bases de Datos de Compuestos Químicos , Secuencia de Carbohidratos , Internet , Modelos Moleculares , Datos de Secuencia Molecular , Programas InformáticosRESUMEN
BACKGROUND: Glycoscience is a research field focusing on complex carbohydrates (otherwise known as glycans)a, which can, for example, serve as "switches" that toggle between different functions of a glycoprotein or glycolipid. Due to the advancement of glycomics technologies that are used to characterize glycan structures, many glycomics databases are now publicly available and provide useful information for glycoscience research. However, these databases have almost no link to other life science databases. RESULTS: In order to implement support for the Semantic Web most efficiently for glycomics research, the developers of major glycomics databases agreed on a minimal standard for representing glycan structure and annotation information using RDF (Resource Description Framework). Moreover, all of the participants implemented this standard prototype and generated preliminary RDF versions of their data. To test the utility of the converted data, all of the data sets were uploaded into a Virtuoso triple store, and several SPARQL queries were tested as "proofs-of-concept" to illustrate the utility of the Semantic Web in querying across databases which were originally difficult to implement. CONCLUSIONS: We were able to successfully retrieve information by linking UniCarbKB, GlycomeDB and JCGGDB in a single SPARQL query to obtain our target information. We also tested queries linking UniProt with GlycoEpitope as well as lectin data with GlycomeDB through PDB. As a result, we have been able to link proteomics data with glycomics data through the implementation of Semantic Web technologies, allowing for more flexible queries across these domains.
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Cholangiocarcinoma (CC) is a lethal malignancy because it exhibits asymptomatic growth infiltrating the surrounding structures and therefore is usually detected at an advanced stage. The mainstay of treatment for CC is complete resection with negative surgical margins. Therefore, its diagnosis at a relatively early stage is demanded for performing relevant surgical resection. Since the definitive CC diagnosis depends on invasive methods such as biliary cytology and biopsy, a noninvasive assay with high diagnostic accuracy is keenly required. We therefore developed a CC marker with high specificity by the Wisteria floribunda agglutinin (WFA)-assisted glycoproteomics approach. WFA-positive glycoproteins were enriched by the direct dissection of the WFA-stained CC tissue region and following WFA-agarose column chromatography. Subsequent analysis by mass spectrometry identified 71 proteins as candidate markers. Screening of these candidates by gene expression profiling and immunohistochemistry resulted in the selection of L1 cell adhesion molecule (L1CAM) as the most specific CC marker. We confirmed the importance of WFA-positivity for L1CAM using both bile and serum of CC and benign bile duct disease patients. Specifically, WFA-positive L1CAM was enriched from serum by the WFA-assisted affinity capturing, with which CC was efficiently distinguished from benign. In the primary verification study using bile from CC patients (n=29) and that of benign bile duct disease (n=29), WFA-positive L1CAM distinguished CC with high specificity (sensitivity=0.66, specificity=0.93, overall accuracy=0.79, area under the receiver operating curve [AUC]=0.82). The combined use of the WFA-positive L1CAM assay with the high sensitive assay detecting WFA-positive sialylated mucin 1 sufficiently improved the diagnostic accuracy of CC (overall accuracy=0.84, AUC=0.93). This combination will possibly be a precise procedure for CC diagnosis compared with conventional diagnostic techniques. BIOLOGICAL SIGNIFICANCE: In this study, we constructed the system for verification of the candidate molecules that exhibit disease specific glyco-alterations and discovered a useful CC marker by the glycoproteomics-assisted strategy for biomarker discovery. Based on the strategy, we previously found that WFA is the best probe to detect CC-specific glycosylation and WFA-positive sialyl MUC1 as a possible biomarker candidate. While the diagnostic specificity of WFA-positive sialyl MUC1 was not superb, we proposed a new biomarker candidate WFA-positive L1CAM with high specificity in bile and serum to complement the previous one. We proved that the novel combination assay of WFA-L1CAM and WFA-sialyl MUC1 selected based on our strategy has the possibility to become a reliable serological test. This study represents application of our strategy, which can be extrapolated to discovery of marker candidates for other diseases.
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Neoplasias de los Conductos Biliares , Biomarcadores de Tumor/metabolismo , Colangiocarcinoma , Glicoproteínas/metabolismo , Proteínas de Neoplasias/metabolismo , Lectinas de Plantas/química , Receptores N-Acetilglucosamina/química , Wisteria/química , Neoplasias de los Conductos Biliares/diagnóstico , Neoplasias de los Conductos Biliares/metabolismo , Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , Femenino , Humanos , Masculino , Proteómica/métodosRESUMEN
Protein glycosylation is a common post-translational modification that plays important roles in terms of protein function. However, analyzing the relationship between glycosylation and protein function remains technically challenging. This problem arises from the fact that the attached glycans possess diverse and heterogeneous structures. We believe that the first step to elucidate glycan function is to systematically determine the status of protein glycosylation under physiological conditions. Such studies involve analyzing differences in glycan structure on cell type (tissue), sex, and age, as well as changes associated with perturbations as a result of gene knockout of glycan biosynthesis-related enzyme, disease and drug treatment. Therefore, we analyzed a series of glycoproteomes in several mouse tissues to identify glycosylated proteins and their glycosylation sites. Comprehensive analysis was performed by lectin- or HILIC-capture of glycopeptide subsets followed by enzymatic deglycosylation in stable isotope-labeled water (H2¹8O, IGOT) and finally LC-MS analyses. In total, 5060 peptides derived from 2556 glycoproteins were identified. We then constructed a glycoprotein database, GlycoProtDB, using our experimental-based information to facilitate future studies in glycobiology.
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Bases de Datos de Proteínas , Glicoproteínas/análisis , Proteoma/análisis , Animales , Cromatografía Liquida , Glicómica , Glicoproteínas/sangre , Glicoproteínas/química , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Proteoma/química , Interfaz Usuario-ComputadorRESUMEN
Human zymogen granule protein 16 (ZG16p) contains a Jacalin-like lectin domain, although its glycan-binding properties are not fully understood. Here, we screened the glycan-binding specificity of ZG16p by recently developed glycoconjugate microarray. ZG16p appeared to exhibit selective binding to α- and ß-linked mannose-polyacrylamide-biotin probes. In more quantitative analysis using frontal affinity chromatography, dissociation constants to two types of polyvalent mannose, i.e. high-density mannose and yeast mannan, were determined to be 1.3 and 1.7 µM, respectively. Mutation of the evolutionarily conserved amino acid Asp151, which is involved in sugar binding among the Jacalin-related lectins (JRLs), abolished binding activity to mannose. By immunohistochemical staining, ZG16p was specifically detected in mucus-secreting cells of the digestive system such as serosanguineous acinar cells of the parotid gland, acinar cells of the pancreas and goblet cells of the intestine. Finally, we showed that ZG16p recognizes pathogenic Candida and Malassezia species in a polyvalent mannose-dependent manner. We propose that ZG16p is a novel member of mannose-specific JRLs, which recognizes pathogenic fungi through non-self polyvalent mannose in the digestive system.
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Candida/metabolismo , Lectinas/metabolismo , Malassezia/metabolismo , Manosa/metabolismo , Páncreas/metabolismo , Glándula Parótida/metabolismo , Sitios de Unión , Candida/patogenicidad , Cromatografía de Afinidad/métodos , Humanos , Mucosa Intestinal/metabolismo , Intestinos/química , Lectinas/genética , Malassezia/patogenicidad , Manosa/química , Análisis por Micromatrices/métodos , Mutación/genética , Páncreas/química , Glándula Parótida/química , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Distribución TisularRESUMEN
Human C21orf63 is a type-1 transmembrane protein of hitherto unknown function, with two repeats of putative 'galactose-binding lectin domains'. By using glycan microarray analysis and other assays, we found that human C21orf63 interacts with heparin and to a lesser extent with heparan sulphate. The C-terminal galactose-binding lectin domain of C21orf63 is necessary for heparin binding. The inability of other human proteins with galactose-binding lectin domains to interact with heparin suggests that heparin binding is a unique property of C21orf63. Results of real-time polymerase chain reaction and tissue immunostaining imply that C21orf63 is expressed on epithelia of various human tissues.
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Heparina/metabolismo , Lectinas/metabolismo , Proteínas de la Membrana/metabolismo , Estructura Terciaria de Proteína , Clonación Molecular , Bases de Datos de Proteínas , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Galectinas , Mucosa Gástrica/metabolismo , Expresión Génica , Glicosaminoglicanos/metabolismo , Heparina/análogos & derivados , Heparitina Sulfato/metabolismo , Humanos , Proteínas Inmovilizadas/metabolismo , Lectinas/química , Lectinas/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Análisis por Micromatrices , Microscopía Fluorescente , Proteínas Mutantes/química , Proteínas Mutantes/inmunología , Proteínas Mutantes/metabolismo , Especificidad de Órganos , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/metabolismoRESUMEN
The extensive involvement of glycan-binding proteins (GBPs) as regulators in diverse biological phenomena provides a fundamental reason to investigate their glycan-binding specificities. Here, we developed a glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of GBPs. Eighty-nine selected multivalent glycoconjugates comprising natural glycoproteins, neo-glycoproteins, and polyacrylamide (PAA)-conjugated glycan epitopes were immobilized on an epoxy-activated glass slide. The GBP binding was monitored by an evanescent-field fluorescence-assisted scanner at equilibrium without washing steps. The detection principle also allows direct application of unpurified GBPs with the aid of specific antibodies. Model experiments using plant lectins (RCA120, ConA, and SNA), galectins (3 and 8), a C-type lectin (DC-SIGN) and a siglec (CD22) provided data consistent with previous work within 4 h using less than 40 ng of GBPs per analysis. As an application, serum profiling of antiglycan antibodies (IgG and IgM) was performed with Cy3-labeled secondary antibodies. Moreover, novel carbohydrate-binding ability was demonstrated for a human IL-18 binding protein. Thus, the developed glycan array is useful for investigation of various types of GBPs, with the added advantage of wash-free analysis.
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Colorantes Fluorescentes/análisis , Glicoconjugados/análisis , Polisacáridos/análisis , Polisacáridos/metabolismo , Análisis por Matrices de Proteínas/métodos , Proteínas/análisis , Proteínas/metabolismo , Humanos , Unión Proteica , Especificidad por SustratoRESUMEN
Using C6-NBD-glucosylceramide (GlcCer) as a substrate, we detected the activity of a conduritol B epoxide-insensitive neutral glycosylceramidase in cytosolic fractions of zebrafish embryos, mouse and rat brains, and human fibroblasts. The candidates for the enzyme were assigned to the Klotho (KL), whose family members share a beta-glucosidase-like domain but whose natural substrates are unknown. Among this family, only the KL-related protein (KLrP) is capable of degrading C6-NBD-GlcCer when expressed in CHOP cells, in which Myc-tagged KLrP was exclusively distributed in the cytosol. In addition, knockdown of the endogenous KLrP by small interfering RNA increased the cellular level of GlcCer. The purified recombinant KLrP hydrolyzed 4-methylumbelliferyl-glucose, C6-NBD-GlcCer, and authentic GlcCer at pH 6.0. The enzyme also hydrolyzed the corresponding galactosyl derivatives, but each k(cat)/Km was much lower than that for glucosyl derivatives. The x-ray structure of KLrP at 1.6A resolution revealed that KLrP is a (beta/alpha)8 TIM barrel, in which Glu(165) and Glu(373) at the carboxyl termini of beta-strands 4 and 7 could function as an acid/base catalyst and nucleophile, respectively. The substrate-binding cleft of the enzyme was occupied with palmitic acid and oleic acid when the recombinant protein was crystallized in a complex with glucose. GlcCer was found to fit well the cleft of the crystal structure of KLrP. Collectively, KLrP was identified as a cytosolic neutral glycosylceramidase that could be involved in a novel nonlysosomal catabolic pathway of GlcCer.
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Citosol/enzimología , Glucosilceramidasa/química , Animales , Línea Celular , Cricetinae , Cricetulus , Cristalografía por Rayos X , Resistencia a Medicamentos/genética , Fibroblastos/enzimología , Expresión Génica , Glucosa/química , Glucosa/metabolismo , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Glucosilceramidas/biosíntesis , Glucosilceramidas/química , Glucuronidasa/genética , Glucuronidasa/metabolismo , Humanos , Inositol/análogos & derivados , Inositol/química , Inositol/farmacología , Proteínas Klotho , Lisosomas/enzimología , Ratones , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Interferente Pequeño , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.
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Retículo Endoplásmico/enzimología , Glucosiltransferasas/química , Trombospondina 1/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Clonación Molecular , Cricetinae , Cricetulus , ADN Complementario/genética , Escherichia coli/genética , Fucosa/metabolismo , Fucosiltransferasas/biosíntesis , Galactosiltransferasas , Glucosiltransferasas/análisis , Glucosiltransferasas/genética , Glicosilación , Humanos , Datos de Secuencia Molecular , Polisacáridos/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ADN , Especificidad por Sustrato , Trombospondina 1/químicaRESUMEN
Glycosylation is the most widespread posttranslational modification in eukaryotes; however, the role of oligosaccharides attached to proteins has been little studied because of the lack of a sensitive and easy analytical method for oligosaccharide structures. Recently, tandem mass spectrometric techniques have been revealing that oligosaccharides might have characteristic signal intensity profiles. We describe here a strategy for the rapid and accurate identification of the oligosaccharide structures on glycoproteins using only mass spectrometry. It is based on a comparison of the signal intensity profiles of multistage tandem mass (MSn) spectra between the analyte and a library of observational mass spectra acquired from structurally defined oligosaccharides prepared using glycosyltransferases. To smartly identify the oligosaccharides released from biological materials, a computer suggests which ion among the fragment ions in the MS/MS spectrum should yield the most informative MS3 spectrum to distinguish similar oligosaccharides. Using this strategy, we were able to identify the structure of N-linked oligosaccharides in immunoglobulin G as an example.
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Bases de Datos Factuales , Espectrometría de Masas/métodos , Oligosacáridos/análisis , Oligosacáridos/química , Secuencia de Aminoácidos , Glicoproteínas/análisis , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Inmunoglobulina G/química , Conformación Molecular , Datos de Secuencia MolecularRESUMEN
UNLABELLED: Bioinformatics resources for glycomics are very poor as compared with those for genomics and proteomics. The complexity of carbohydrate sequences makes it difficult to define a common language to represent them, and the development of bioinformatics tools for glycomics has not progressed. In this study, we developed a carbohydrate sequence markup language (CabosML), an XML description of carbohydrate structures. AVAILABILITY: The language definition (XML Schema) and an experimental database of carbohydrate structures using an XML database management system are available at http://www.phoenix.hydra.mki.co.jp/CabosDemo.html CONTACT: kikuchi@hydra.mki.co.jp.