RESUMEN
Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through proteolysis of an exposed reactive center loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localized Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterization of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we first performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344-Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 glycosylation was experimentally observed and supported in silico by modeling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and GalNAc-T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialoglycans (disialyl T) and asialoglycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on biologically relevant CBG RCL glycosylation events, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.
Asunto(s)
Elastasa de Leucocito , Transcortina , Glicosilación , Hidrocortisona/metabolismo , Elastasa de Leucocito/metabolismo , Polisacáridos , Proteolisis , Transcortina/genética , Transcortina/química , Transcortina/metabolismo , HumanosRESUMEN
In the meningitis belt of sub-Saharan Africa, there are cyclic meningococcal epidemics that coincide with clonal waves of Neisseria meningitidis carriage and invasive disease. In the framework of longitudinal colonization and disease studies in Ghana and Burkina Faso, meningococcal isolates belonging to the closely related hypervirulent A:ST-5, A:ST-7, and A:ST-2859 clones have been collected from 1998 to 2011 during meningococcal outbreaks. A comparative whole-genome sequencing study with 100 of these isolates identified the pilin glycosylation (pgl) locus as one hot spot of recombination. Frequent exchange of pgl genes in N. meningitidis by lateral gene transfer results in differences in the glycosylation patterns of pilin and other cell surface glycoproteins. In this study, we looked at both recombination and phase variation of the pgl genes of these clinical isolates and analyzed the glycan structures resulting from different pgl alleles and their variable expression. Our results indicate that the basal O-linked sugar of the glycans expressed by these isolates is masked by various additional mono- or disaccharide structures whose expression is highly variable due to the phase-variable expression of pgl genes. We also observed a distinct glycoform in two isolates with pgl loci that were modified by recombination. These data suggest that variation in N. meningitidis protein glycosylation could be crucial for bacterial adaptation to evade herd immunity in semi-immune populations. Investigating pilin glycosylation in N. meningitidis can shed light on the mechanisms by which this pathogen evades the host immune response, and may help identify potential targets for novel therapies and vaccines.
Asunto(s)
Meningitis , Neisseria meningitidis , Humanos , Neisseria meningitidis/genética , Proteínas Fimbrias/genética , Glicosilación , Serogrupo , Brotes de Enfermedades , PolisacáridosRESUMEN
BACKGROUND: Normal human tissues do not express glycans terminating with the sialic acid N-glycolylneuraminic acid (Neu5Gc), yet Neu5Gc-containing glycans have been consistently found in human tumor tissues, cells and secretions and have been proposed as a cancer biomarker. We engineered a Neu5Gc-specific lectin called SubB2M, and previously reported elevated Neu5Gc biomarkers in serum from ovarian cancer patients using a Surface Plasmon Resonance (SPR)-based assay. Here we report an optimized SubB2M SPR-based assay and use this new assay to analyse sera from breast cancer patients for Neu5Gc levels. METHODS: To enhance specificity of our SPR-based assay, we included a non-sialic acid binding version of SubB, SubBA12, to control for any non-specific binding to SubB2M, which improved discrimination of cancer-free controls from early-stage ovarian cancer. We analysed 96 serum samples from breast cancer patients at all stages of disease compared to 22 cancer-free controls using our optimized SubB2M-A12-SPR assay. We also analysed a collection of serum samples collected at 6 monthly intervals from breast cancer patients at high risk for disease recurrence or spread. RESULTS: Analysis of sera from breast cancer cases revealed significantly elevated levels of Neu5Gc biomarkers at all stages of breast cancer. We show that Neu5Gc serum biomarker levels can discriminate breast cancer patients from cancer-free individuals with 98.96% sensitivity and 100% specificity. Analysis of serum collected prospectively, post-diagnosis, from breast cancer patients at high risk for disease recurrence showed a trend for a decrease in Neu5Gc levels immediately following treatment for those in remission. CONCLUSIONS: Neu5Gc serum biomarkers are a promising new tool for early detection and disease monitoring for breast cancer that may complement current imaging- and biopsy-based approaches.
Asunto(s)
Neoplasias de la Mama , Biomarcadores de Tumor , Neoplasias de la Mama/diagnóstico , Femenino , Humanos , Recurrencia Local de Neoplasia , Ácidos Neuramínicos/metabolismoRESUMEN
Infections by blood flukes (Cardicola spp.) are considered the most significant health issue for ranched bluefin tuna, a major aquaculture industry in Japan and Australia. The host-parasite interfaces of trematodes, namely their teguments, are particularly rich in carbohydrates, which function both in evasion and modulation of the host immune system, while some are primary antigenic targets. In this study, histochemistry and mass spectrometry techniques were used to profile the glycans of Cardicola forsteri. Fluorescent lectin staining of adult flukes indicates the presence of oligomannose (Concanavalin A-reactive) and fucosylated (Pisum sativum agglutinin-reactive) N-glycans. Additionally, reactivity of succinylated wheat germ agglutinin (s-WGA) was localised to several internal organs of the digestive and monoecious reproductive systems. Glycan structures were further investigated with tandem mass spectrometry, which revealed structures indicated by lectin reactivity. While O-glycans from these adult specimens were not detectable by mass spectrometry, several oligomannose, paucimannosidic, and complex-type N-glycans were identified, including some carrying hexuronic acid and many carrying core xylose. This is, to our knowledge, the first glycomic characterisation of a marine platyhelminth, with broader implications for research into other trematodes.
Asunto(s)
Enfermedades de los Peces , Parásitos , Schistosomatidae , Infecciones por Trematodos , Animales , Enfermedades de los Peces/parasitología , Lectinas , Polisacáridos , Schistosoma , Infecciones por Trematodos/parasitología , Atún/parasitologíaRESUMEN
GlycoStore ( http://www.glycostore.org ) is an open access chromatographic and electrophoretic retention database of glycans characterized from glycoproteins, glycolipids, and biotherapeutics. It is a continuation of the GlycoBase project (Oxford Glycobiology Institute and National Institute for Bioprocessing Research and Training, Ireland) but addresses many of the technological limitations that impacted the growth of GlycoBase, in particular, improvements to the bioinformatics architecture, enhancing data annotations and coverage, and improving connectivity with external resources. The first release of GlycoStore (October 2017) contains over 850 glycan entries accompanied by 8500+ retention positions including data from: (1) fluorescently labelled released glycans determined using hydrophilic interaction chromatography (HILIC) ultrahigh-performance liquid chromatography (U/HPLC) and reversed phase (RP)-U/HPLC; (2) porous graphitized carbon chromatography (PGC) interfaced with ESI-MS/MS; and (3) capillary electrophoresis with laser induced fluorescence detection (CE-LIF). In this chapter, we outline the objectives of GlycoStore, and describe a selection of step-by-step workflows for navigating and browsing the information available. We also provide a short description of informatics tools available to query the database using Semantic technologies. The information presented in this chapter supplements our documentation knowledge base that describes interface improvements, new features and tools, and content updates ( https://unicarbkb.freshdesk.com/ ).
Asunto(s)
Electroforesis Capilar , Glicómica , Cromatografía Líquida de Alta Presión , Glicoproteínas , Polisacáridos , Espectrometría de Masas en TándemRESUMEN
N-linked glycosylation is a ubiquitous protein modification that is capable of modulating protein structure, function and interactions. Many proteins in the brain associated with the synapse and important for synaptic transmission are highly glycosylated and their glycosylation could be important for learning and memory related molecular processes and synaptic plasticity. In the present study, we extend the knowledge of the synaptic glycome and glycoproteome by performing glycan- and intact glycopeptide-focused analyses of isolated rat nerve terminals (synaptosomes) by LC-MS/MS. Overall, glycomics identified a total of 41 N-glycans in isolated synaptosomes. Sialylated N-glycans represented only 7% of the total abundance of the rat synaptosome N-glycome with oligomannose, neutral hybrid and complex type N-glycans being the most abundant structures. Using detergent extraction of the active zone proteins from the synaptosomes revealed a change in the active zone glycan abundance in comparison with the rest of the synaptosome glycan content. Characterization of intact sialylated N-linked glycopeptides enriched by titanium dioxide chromatography revealed more than 85% selectivity of sialylated species and the presence of NeuGc on active zone proteins. In addition, both disialic and trisialic acid modified glycans were present on synaptic glycoproteins, although oxonium ion profiling revealed that trisialic units were only present on glycoproteins in the detergent soluble fraction. However, correct identification of intact sialylated N-linked glycopeptides using the Byonic program failed, most likely due to the lack of peptide backbone fragmentation during tandem mass spectrometry.
Asunto(s)
Glicopéptidos , Espectrometría de Masas en Tándem , Animales , Cromatografía Liquida , Glicómica , Glicopéptidos/metabolismo , Glicosilación , RatasRESUMEN
Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases, suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development, but our molecular understanding of the precise glycans, catalytic enzymes, and lectins involved remains only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown digalactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labeling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins, most notably the upregulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation, suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the upregulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.
Asunto(s)
Galectina 1/metabolismo , Glicopéptidos/metabolismo , Desarrollo de Músculos , Animales , Línea Celular , Galectina 1/genética , Glicómica , Glicosilación , Masculino , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Procesamiento Proteico-Postraduccional , Proteómica , RatasRESUMEN
Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77-81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fucosylation mediated more efficient ADCC and clearance than anti-D Ig. Galactosylation of B mAb-Ds was 57-83% but 15-58% for rodent mAb-Ds. HH mAb-Ds had non-human sugars. These data reveal high galactosylation like anti-D Ig (>60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Eritroblastosis Fetal/terapia , Inmunoglobulina G/inmunología , Globulina Inmune rho(D)/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Línea Celular , Cricetulus , Fucosa/metabolismo , Galactosa/metabolismo , Glicosilación , Humanos , Hibridomas/inmunología , Inmunoglobulina G/metabolismo , Ratones , Ácido N-Acetilneuramínico/metabolismo , Ratas , Globulina Inmune rho(D)/metabolismo , Globulina Inmune rho(D)/uso terapéutico , Resultado del TratamientoRESUMEN
Protein glycosylation is the most complex and prevalent post-translation modification in terms of the number of proteins modified and the diversity generated. To understand the functional roles of glycoproteins it is important to gain an insight into the repertoire of oligosaccharides present. The comparison and relative quantitation of glycoforms combined with site-specific identification and occupancy are necessary steps in this direction. Computational platforms have continued to mature assisting researchers with the interpretation of such glycomics and glycoproteomics data sets, but frequently support dedicated workflows and users rely on the manual interpretation of data to gain insights into the glycoproteome. The growth of site-specific knowledge has also led to the implementation of machine-learning algorithms to predict glycosylation which is now being integrated into glycoproteomics pipelines. This short review describes commercial and open-access databases and software with an emphasis on those that are actively maintained and designed to support current analytical workflows.
Asunto(s)
Bases de Datos de Proteínas , Glicómica/métodos , Glicoproteínas/química , Proteómica/métodos , Programas Informáticos , Animales , Bacterias/química , Biología Computacional , Glicosilación , Humanos , Aprendizaje Automático , Plantas/química , Procesamiento Proteico-PostraduccionalRESUMEN
While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.
Asunto(s)
Manosa/metabolismo , Neoplasias/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Progresión de la Enfermedad , Glicosilación , Humanos , Espectrometría de Masas en TándemRESUMEN
The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative created by experts in the fields of glycobiology, glycoanalytics and glycoinformatics to design guidelines that improve the reporting and reproducibility of glycoanalytical methods. Previously, the MIRAGE Commission has published guidelines for describing sample preparation methods and the reporting of glycan array and mass spectrometry techniques and data collections. Here, we present the first version of guidelines that aim to improve the quality of the reporting of liquid chromatography (LC) glycan data in the scientific literature. These guidelines cover all aspects of instrument setup and modality of data handling and manipulation and is cross-linked with other MIRAGE recommendations. The most recent version of the MIRAGE-LC guidelines is freely available at the MIRAGE project website doi:10.3762/mirage.4.
Asunto(s)
Glicómica , Polisacáridos/análisis , Cromatografía Liquida , HumanosRESUMEN
Summary: GlycoStore is a curated chromatographic, electrophoretic and mass-spectrometry composition database of N-, O-, glycosphingolipid (GSL) glycans and free oligosaccharides associated with a range of glycoproteins, glycolipids and biotherapeutics. The database is built on publicly available experimental datasets from GlycoBase developed in the Oxford Glycobiology Institute and then the National Institute for Bioprocessing Research and Training (NIBRT). It has now been extended to include recently published and in-house data collections from the Bioprocessing Technology Institute (BTI) A*STAR, Macquarie University and Ludger Ltd. GlycoStore provides access to approximately 850 unique glycan structure entries supported by over 8500 retention positions determined by: (i) hydrophilic interaction chromatography (HILIC) ultra-high performance liquid chromatography (U/HPLC) and reversed phase (RP)-U/HPLC with fluorescent detection; (ii) porous graphitized carbon (PGC) chromatography in combination with ESI-MS/MS detection; and (iii) capillary electrophoresis with laser induced fluorescence detection (CE-LIF). GlycoStore enhances many features previously available in GlycoBase while addressing the limitations of the data collections and model of this popular resource. GlycoStore aims to support detailed glycan analysis by providing a resource that underpins current workflows. It will be regularly updated by expert annotation of published data and data obtained from the project partners. Availability and implementation: http://www.glycostore.org. Supplementary information: Supplementary data are available at Bioinformatics online.
Asunto(s)
Bases de Datos de Compuestos Químicos , Glicómica/métodos , Oligosacáridos/química , Polisacáridos/química , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , Glucolípidos , Glicoproteínas , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Oligosacáridos/metabolismo , Polisacáridos/metabolismo , Espectrometría de Masas en TándemRESUMEN
Porous graphitised carbon-liquid chromatography (PGC-LC) has been proven to be a powerful technique for the analysis and characterisation of complex mixtures of isomeric and isobaric glycan structures. Here we evaluate the elution behaviour of N-glycans on PGC-LC and thereby provide the potential of using chromatographic separation properties, together with mass spectrometry (MS) fragmentation, to determine glycan structure assignments more easily. We used previously reported N-glycan structures released from the purified glycoproteins Immunoglobulin G (IgG), Immunoglobulin A (IgA), lactoferrin, α1-acid glycoprotein, Ribonuclease B (RNase B), fetuin and ovalbumin to profile their behaviour on capillary PGC-LC-MS. Over 100 glycan structures were determined by MS/MS, and together with targeted exoglycosidase digestions, created a N-glycan PGC retention library covering a full spectrum of biologically significant N-glycans from pauci mannose to sialylated tetra-antennary classes. The resultant PGC retention library ( http://www.glycostore.org/showPgc ) incorporates retention times and supporting fragmentation spectra including exoglycosidase digestion products, and provides detailed knowledge on the elution properties of N-glycans by PGC-LC. Consequently, this platform should serve as a valuable resource for facilitating the detailed analysis of the glycosylation of both purified recombinant, and complex mixtures of, glycoproteins using established workflows.
Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Polisacáridos/química , Cromatografía Liquida/instrumentación , Glicoproteínas/química , Glicoproteínas/metabolismo , Grafito/química , Espectrometría de Masas/instrumentación , Polisacáridos/análisisRESUMEN
RATIONALE: High protein production and secretion with eukaryotic glycosylation machinery make T. reesei RUT-C30 a suitable expression host for recombinant proteins. The N-glycosylation of secreted proteins of RUT-C30 is known to vary depending on culture nutrients but O-glycosylation has been less extensively studied. METHODS: O-Glycans and glycopeptides from secreted proteins were separated by porous graphitised carbon and C-18 liquid chromatography, respectively. O-Glycans were analysed in negative ion mode by electrospray ionisation linear ion trap mass spectrometry and glycopeptides in positive ion mode by electrospray ionisation hybrid quadrupole-orbitrap mass spectrometry. Tandem mass spectrometry was used on O-glycans and glycopeptides including ion trap higher energy collision-induced dissociation (tHCD) to detect glycan fragments not detectable with standard ion trap fragmentation. tHCD allowed targeted MS3 experiments to be performed on structures containing hexuronic acid, which was not possible with ion trap CID, validating this novel O-glycan composition. Positive mode C18-LC/ESI-MS/MS was used to identify and characterise glycopeptides found to be modified with this class of O-glycans, identifying cellobiohydrolase I as a carrier of these novel O-glycans. RESULTS: Negative mode ion trap higher energy collision-induced dissociation allowed detection and targeted MS3 experiments to be performed on the hexuronic acid substituent of O-glycan structures, which was not possible with ion trap CID, validating the novel O-glycan composition to include hexuronic acid. Using glycopeptide analysis, this novel O-glycan composition was found to be present on the catalytic domain of cellobiohydrolase I, the most abundant secreted protein by T. reesei. CONCLUSIONS: These are the first reported O-glycans to contain acidic sugars in fungi and they could have significant implications for cellobiohydrolase I structure and activity as well as the activity of recombinant proteins expressed in this host system. Copyright © 2017 John Wiley & Sons, Ltd.
RESUMEN
The access to biodatabases for glycomics and glycoproteomics has proven to be essential for current glycobiological research. This chapter presents available databases that are devoted to different aspects of glycobioinformatics. This includes oligosaccharide sequence databases, experimental databases, 3D structure databases (of both glycans and glycorelated proteins) and association of glycans with tissue, disease, and proteins. Specific search protocols are also provided using tools associated with experimental databases for converting primary glycoanalytical data to glycan structural information. In particular, researchers using glycoanalysis methods by U/HPLC (GlycoBase), MS (GlycoWorkbench, UniCarb-DB, GlycoDigest), and NMR (CASPER) will benefit from this chapter. In addition we also include information on how to utilize glycan structural information to query databases that associate glycans with proteins (UniCarbKB) and with interactions with pathogens (SugarBind).
Asunto(s)
Glicómica/métodos , Glicoproteínas/química , Polisacáridos/química , Proteómica/métodos , Animales , Conformación de Carbohidratos , Cromatografía Líquida de Alta Presión/métodos , Bases de Datos de Compuestos Químicos , Bases de Datos de Proteínas , Humanos , Espectrometría de Masas/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Programas InformáticosRESUMEN
Glycan structures attached to proteins are comprised of diverse monosaccharide sequences and linkages that are produced from precursor nucleotide-sugars by a series of glycosyltransferases. Databases of these structures are an essential resource for the interpretation of analytical data and the development of bioinformatics tools. However, with no template to predict what structures are possible the human glycan structure databases are incomplete and rely heavily on the curation of published, experimentally determined, glycan structure data. In this work, a library of 45 human glycosyltransferases was used to generate a theoretical database of N-glycan structures comprised of 15 or less monosaccharide residues. Enzyme specificities were sourced from major online databases including Kyoto Encyclopedia of Genes and Genomes (KEGG) Glycan, Consortium for Functional Glycomics (CFG), Carbohydrate-Active enZymes (CAZy), GlycoGene DataBase (GGDB) and BRENDA. Based on the known activities, more than 1.1 million theoretical structures and 4.7 million synthetic reactions were generated and stored in our database called UniCorn. Furthermore, we analyzed the differences between the predicted glycan structures in UniCorn and those contained in UniCarbKB (www.unicarbkb.org), a database which stores experimentally described glycan structures reported in the literature, and demonstrate that UniCorn can be used to aid in the assignment of ambiguous structures whilst also serving as a discovery database.
Asunto(s)
Biología Computacional/métodos , Bases de Datos Genéticas , Glicosiltransferasas/genética , Polisacáridos/química , Vías Biosintéticas , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Humanos , Estructura Molecular , Polisacáridos/biosíntesisRESUMEN
RATIONALE: Negative ion collision-induced dissociation (CID) spectra of released N-glycans provide very informative structural information relating to branching patterns and location of residues such as fucose. For some structural studies, particularly those involving chromatography, glycans are often reduced to avoid production of multiple peaks from α- and ß-anomers. We examined the effect of reduction on the production of diagnostic fragment ions and on the ion mobility properties of N-glycans. METHODS: Released N-glycans from the glycoproteins bovine fetuin, ribonuclease B, chicken ovalbumin, and porcine thyroglobulin were reduced with sodium cyanoborohydride and both negative ion CID spectra and ion mobility properties of their phosphate adducts were examined with a Waters Synapt G2Si travelling-wave ion mobility mass spectrometer with electrospray sample introduction. Estimated collisional cross sections were measured with dextran as the calibrant, RESULTS: Fragment ions were similar to those from the unreduced glycans with the exception that the prominent (2,4) A cleavage ion from the reducing terminus was replaced by a prominent [M-H3 PO4](-) ion. Other ions arising from the chitobiose core were of lower relative abundance than those from the unreduced glycans. Estimated collisional cross sections were similar to those of the unreduced compounds but with symmetrical arrival time distribution (ATD) profiles, unlike those of the unreduced glycans whose peaks often contained prominent asymmetry. This observation showed that this asymmetry was due to anomer separation. CONCLUSIONS: Reduction of the reducing terminal GlcNAc residue resulted in fewer diagnostic ions from the chitobiose core but fragmentation of the remainder of the molecules generally paralleled that of the unreduced glycans. Thus, most structural information, with the exception of the linkage position of fucose on the core GlcNAc, was available. ATD peaks were symmetrical with the result that cross sections were more appropriate for data-base searching than those from the non-reduced compounds where asymmetry produced lower precision in the measurement.
Asunto(s)
Glicoproteínas/química , Polisacáridos/análisis , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Bovinos , Pollos , Iones/química , Manosa/análisis , Espectrometría de Masas , Oxidación-Reducción , PorcinosRESUMEN
UNLABELLED: Sequencing oligosaccharides by exoglycosidases, either sequentially or in an array format, is a powerful tool to unambiguously determine the structure of complex N- and O-link glycans. Here, we introduce GlycoDigest, a tool that simulates exoglycosidase digestion, based on controlled rules acquired from expert knowledge and experimental evidence available in GlycoBase. The tool allows the targeted design of glycosidase enzyme mixtures by allowing researchers to model the action of exoglycosidases, thereby validating and improving the efficiency and accuracy of glycan analysis. AVAILABILITY AND IMPLEMENTATION: http://www.glycodigest.org.
Asunto(s)
Glicósido Hidrolasas , Oligosacáridos/química , Programas Informáticos , Bases del ConocimientoRESUMEN
Glycosylation of proteins is one of the most important PTMs, with more than half of all human proteins estimated to be glycosylated. It is widely known that aberrant glycosylation has been implicated in many different diseases due to changes associated with biological function and protein folding. In cancer, there is increasing evidence pertaining to the role of glycosylation in tumour formation and metastasis. Alterations in cell surface glycosylation, particularly terminal motifs, can promote invasive behaviour of tumour cells that ultimately lead to the progression of cancer. While a majority of studies have investigated protein glycosylation changes in cancer cell lines and tumour tissue for individual cancers, the review presented here represents a comprehensive, in-depth overview of literature on the structural changes of glycosylation and their associated synthetic enzymes in five different cancer types originating from the breast, colon, liver, skin and ovary. More importantly, this review focuses on key similarities and differences between these cancers that reflect the importance of structural changes of cell surface N- and O-glycans, such as sialylation, fucosylation, degree of branching and the expression of specific glycosyltransferases for each cancer. It is envisioned that the understanding of these biologically relevant glycan alterations on cellular proteins will facilitate the discovery of novel glycan-based biomarkers which could potentially serve as diagnostic and prognostic indicators of cancer.
Asunto(s)
Membrana Celular/metabolismo , Glicosiltransferasas/genética , Proteínas de la Membrana/metabolismo , Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Glucanos/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Humanos , Proteínas de la Membrana/genética , Neoplasias/diagnóstico , Neoplasias/patología , PronósticoRESUMEN
OBJECTIVE: Glycosylation is the most common post-translational modification and is altered in disease. The typical glycosylation change in patients with inflammatory arthritis (IA) is a decrease in galactosylation levels on IgG. The aim of this study is to evaluate the effect of anti-TNF therapy on whole serum glycosylation from IA patients and determine whether these alterations in the glycome change upon treatment of the disease. METHODS: Serum samples were collected from 54 IA patients before treatment and at 1 and 12 months after commencing anti-TNF therapy. N-linked glycans from whole serum samples were analysed using a high-throughput hydrophilic interaction liquid chromatography-based method. RESULTS: Glycosylation on the serum proteins of IA patients changed significantly with anti-TNF treatment. We observed an increase in galactosylated glycans from IgG, also an increase in core-fucosylated biantennary galactosylated glycans and a decrease in sialylated triantennary glycans with and without outer arm fucose. This increase in galactosylated IgG glycans suggests a reversing of the N-glycome towards normal healthy profiles. These changes are strongly correlated with decreasing CRP, suggesting a link between glycosylation changes and decreases in inflammatory processes. CONCLUSION: Glycosylation changes in the serum of IA patients on anti-TNF therapy are strongly associated with a decrease in inflammatory processes and reflect the effect of anti-TNF on the immune system.