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.
•Recent advancements in glycoinformatics tools to support analysis of glycan and glycopeptide data sets are summarised.•Review summarises the growth of machine learning applications to facilitate the ...prediction of glycosylation sites.•Review provides an update on international efforts to improve data interoperability in the glycosciences.
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.
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.
Abstract
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.
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.
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•Proteomic, glycomic and glycoproteomic analysis of myogenesis.•Mechanistic insights into site-specific glycoproteome regulation via discrete glycosidases and glycosyltransferases.•Quantification and validation of glycan-binding proteins.•Functional analysis of LGALS1 reveals a role in myogenesis and muscle development.
The proteome, N-glycome, and N-glycoproteome have been determined during myotube formation. This revealed a complex regulation of N-glycosylation including a switch in sialylation linkages, digalactosylation, and paucimannosylation on receptors and adhesion molecules. The data also revealed regulation of several glycan-binding proteins including LGALS1, and functional validation revealed a role of this protein in myogenesis and skeletal muscle development.
Abstract
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.
Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure–function relationships. Owing ...to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200
N
- and
O
-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data.
Figure
ᅟ
Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein ...function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α) and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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