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Recent technological advances in glycobiology have resulted in a large influx of data and the publication of many papers describing discoveries in glycoscience. However, the terms used in describing glycan structural features are not standardized, making it difficult to harmonize data across biomolecular databases, hampering the harvesting of information across studies and hindering text mining and curation efforts. To address this shortcoming, the Glycan Structure Dictionary has been developed as a reference dictionary to provide a standardized list of widely used glycan terms that can help in the curation and mapping of glycan structures described in publications. Currently, the dictionary has 190 glycan structure terms with 297 synonyms linked to 3,332 publications. For a term to be included in the dictionary, it must be present in at least 2 peer-reviewed publications. Synonyms, annotations, and cross-references to GlyTouCan, GlycoMotif, and other relevant databases and resources are also provided when available. The purpose of this effort is to facilitate biocuration, assist in the development of text mining tools, improve the harmonization of search, and browse capabilities in glycoinformatics resources and help to map glycan structures to function and disease. It is also expected that authors will use these terms to describe glycan structures in their manuscripts over time. A mechanism is also provided for researchers to submit terms for potential incorporation. The dictionary is available at https://wiki.glygen.org/Glycan_structure_dictionary. [ABSTRACT FROM AUTHOR]

Aberrant glycosylation is a key mechanism employed by cancer cells to evade immune surveillance, induce angiogenesis and metastasis, among other hallmarks of cancer. Sialic acids, distinctive terminal glycan structures located on glycoproteins or glycolipids, are prominently upregulated across various tumor types, including colorectal cancer (CRC). Sialylated glycans modulate anti-tumor immune responses through their interactions with Siglecs, a family of glycan-binding receptors with specificity for sialic acid-containing glycoconjugates, often resulting in immunosuppression. In this paper, we investigated the immunomodulatory function of ST3Gal5, a sialyltransferase that catalyzes the addition of α2-3 sialic acids to glycosphingolipids, since lower expression of ST3Gal5 is associated with better survival of CRC patients. We employed CRISPR/Cas9 to knock out the ST3Gal5 gene in two murine CRC cell lines MC38 and CT26. Glycomics analysis confirmed the removal of sialic acids on glycolipids, with no discernible impact on glycoprotein sialylation. Although knocking out ST3Gal5 in both cell lines did not affect in vivo tumor growth, we observed enhanced levels of regulatory T cells in CT26 tumors lacking ST3Gal5. Moreover, we demonstrate that the absence of ST3Gal5 affected size and blood vessel density only in MC38 tumors. In summary, we ascertain that sialylation of glycosphingolipids has a limited influence on the anti-tumor immune response in CRC, despite detecting alterations in the tumor microenvironment, possibly due to a shift in ganglioside abundance. [ABSTRACT FROM AUTHOR]

Glycans play a vital role in health, disease, bioenergy, biomaterials and bio-therapeutics. As a result, there is keen interest to identify and increase glycan data in bioinformatics databases like ChEBI and PubChem, and connecting them to resources at the EMBL-EBI and NCBI to facilitate access to important annotations at a global level. GlyTouCan is a comprehensive archival database that contains glycans obtained primarily through batch upload from glycan repositories, glycoprotein databases and individual laboratories. In many instances, the glycan structures deposited in GlyTouCan may not be fully defined or have supporting experimental evidence and citations. Databases like ChEBI and PubChem were designed to accommodate complete atomistic structures with well-defined chemical linkages. As a result, they cannot easily accommodate the structural ambiguity inherent in glycan databases. Consequently, there is a need to improve the organization of glycan data coherently to enhance connectivity across the major NCBI, EMBL-EBI and glycoscience databases. This paper outlines a workflow developed in collaboration between GlyGen, ChEBI and PubChem to improve the visibility and connectivity of glycan data across these resources. GlyGen hosts a subset of glycans (~29,000) from the GlyTouCan database and has submitted valuable glycan annotations to the PubChem database and integrated over 10,500 (including ambiguously defined) glycans into the ChEBI database. The integrated glycans were prioritized based on links to PubChem and connectivity to glycoprotein data. The pipeline provides a blueprint for how glycan data can be harmonized between different resources. The current PubChem, ChEBI and GlyTouCan mappings can be downloaded from GlyGen (https://data.glygen.org). [ABSTRACT FROM AUTHOR]

Glycosyltransferases (GTs) are carbohydrate-active enzymes (CAZy) involved in the synthesis of natural glycan structures. The application of CAZy is highly demanded in biotechnology and pharmaceutics. However, it is being hindered by the lack of high-quality and comprehensive repositories of the research data accumulated so far. In this paper, we describe a new curated Carbohydrate Structure Glycosyltransferase Database (CSDB_GT). Currently, CSDB_GT provides ca. 780 activities exhibited by GTs, as well as several other CAZy, found in Arabidopsis thaliana and described in ca. 180 publications. It covers most published data on A. thaliana GTs with evidenced functions. CSDB_GT is linked to the Carbohydrate Structure Database (CSDB), which stores data on archaeal, bacterial, fungal and plant glycans. The CSDB_GT data are supported by experimental evidences and can be traced to original publications. CSDB_GT is freely available at http://csdb.glycoscience.ru/gt.html. [ABSTRACT FROM AUTHOR]

Carbohydrate structures in the Carbohydrate Structure Database have been referenced to glycoepitopes from the Immune Epitope Database allowing users to explore the glycan structures and contained epitopes. Starting with an epitope, one can figure out the glycans from other organisms that share the same structural determinant, and retrieve the associated taxonomical, medical, and other data. This database mapping demonstrates the advantages of the integration of immunological and glycomic databases. [ABSTRACT FROM AUTHOR]

Immunofluorescence microscopy (IFM) and immunogold transmission electron microscopy (TEM) are the two main techniques commonly used to detect polysaccharides in plant cell walls. Both are important in localizing cell wall polysaccharides, but both have major limitations, such as low resolution in IFM and restricted sample size for immunogold TEM. In this study, we have developed a robust technique that combines immunocytochemistry with scanning electron microscopy (SEM) to study cell wall polysaccharide architecture in xylem cells at high resolution over large areas of sample. Using multiple cell wall monoclonal antibodies (mAbs), this immunogold SEM technique reliably localized groups of hemicellulosic and pectic polysaccharides in the cell walls of five different xylem structures (vessel elements, fibers, axial and ray parenchyma cells, and tyloses). This demonstrates its important advantages over the other two methods for studying cell wall polysaccharide composition and distribution in these structures. In addition, it can show the three-dimensional distribution of a polysaccharide group in the vessel lateral wall and the polysaccharide components in the cell wall of developing tyloses. This technique, therefore, should be valuable for understanding the cell wall polysaccharide composition, architecture and functions of diverse cell types. [ABSTRACT FROM AUTHOR]

Motivation We have previously designed and implemented a tree-based ontology to represent glycan structures with the aim of searching these structures with a glyco-driven syntax. This resulted in creating the GlySTreeM knowledge-base as a linchpin of the structural matching procedure and we now introduce a query language, called GlycoQL, for the actual implementation of a glycan structure search. Results The methodology is described and illustrated with a use-case focused on Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike protein glycosylation. We show how to enhance site annotation with federated queries involving UniProt and GlyConnect, our glycoprotein database. Availability and implementation https://glyconnect.expasy.org/glycoql/. [ABSTRACT FROM AUTHOR]

Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3) and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637 and SW780 express high levels of the fucosylated Lewis-X antigen (Lex, CD15) (Galβ1–4(Fucα1–3)GlcNAcβ1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N -glycans between these cell types. O -glycans are highly differentiated, as RT4 cells synthesize core 2-based O -glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1–3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers. [ABSTRACT FROM AUTHOR]

The extent of liver fibrosis predicts prognosis and is important for determining treatment strategies for chronic hepatitis. During the fibrosis progression, serum levels of Mac2 binding protein (M2BP) increase and the N -glycan structure changes to enable binding to Wisteria floribunda agglutinin (WFA) lectin. As a novel diagnostic marker, glycosylation isomer of M2BP (M2BPGi) has been developed. However, its glycan structures recognized by WFA are unclear. In this study, we analyzed site-specific N -glycan structures of serum M2BP using Glyco-RIDGE (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile) method. We evaluated five sample types: (1) M2BP immunoprecipitated from normal healthy sera (NHS-IP(+)), (2) M2BP immunoprecipitated from sera of patients with liver cirrhosis (stage 4; F4-IP(+)), (3) M2BP captured with WFA from serum of patients with liver cirrhosis (stage 4; F4-WFA(+)), (4) recombinant M2BP produced by HEK293 cells (rM2BP) and (5) WFA-captured rM2BP (rM2BP-WFA(+)). In NHS-IP(+) M2BP, bi-antennary N -glycan was the main structure, and LacNAc extended to its branches. In F4-IP(+) M2BP, many branched structures, including tri-antennary and tetra-antennary N -glycans, were found. F4-WFA(+) showed a remarkable increase in branched structures relative to the quantity before enrichment. In recombinant M2BP, both no sialylated-LacdiNAc and -branched LacNAc structures were emerged. The LacdiNAc structure was not found in serum M2BP. Glycosidase-assisted HISCL assays suggest that reactivity with WFA of both serum and recombinant M2BP depends on unsialylated and branched LacNAc and in part of recombinant depends on LacdiNAc. On M2BPGi, the highly branched LacNAc, probably dense cluster of LacNAc, would be recognized by WFA. [ABSTRACT FROM AUTHOR]

Recent years have seen great advances in the development of glycoproteomics protocols and methods resulting in a sustainable increase in the reporting proteins, their attached glycans and glycosylation sites. However, only very few of these reports find their way into databases or data repositories. One of the major reasons is the absence of digital standard to represent glycoproteins and the challenging annotations with glycans. Depending on the experimental method, such a standard must be able to represent glycans as complete structures or as compositions, store not just single glycans but also represent glycoforms on a specific glycosylation side, deal with partially missing site information if no site mapping was performed, and store abundances or ratios of glycans within a glycoform of a specific site. To support the above, we have developed the GlycoConjugate Ontology (GlycoCoO) as a standard semantic framework to describe and represent glycoproteomics data. GlycoCoO can be used to represent glycoproteomics data in triplestores and can serve as a basis for data exchange formats. The ontology, database providers and supporting documentation are available online (https://github.com/glycoinfo/GlycoCoO). [ABSTRACT FROM AUTHOR]

Alterations to glycans in cancer patients have been used to identify novel tumor biomarkers. Most of these studies have focused on protein glycosylation but less attention has been paid to free-glycans. Here, we analyzed acidic free-glycans in the urine of cancer patients to identify novel tumor marker candidates. Specifically, urine samples were collected from patients with gastric cancer, pancreatic cancer and cholangiocarcinoma as well as normal controls. The free-glycans were extracted from creatinine-adjusted urine and fluorescently labeled with 2-aminopyridine. Initially, we performed profiling of urinary free-glycans by high-performance liquid chromatography and mass spectrometry with enzymatic and chemical degradation. More than 100 glycans, including novel structures, were identified. The chromatographic peaks suggested some of these glycans were present at elevated levels in cancer patients. To verify cancer-associated alterations, we compared the glycan levels between cancer patients and normal controls by selected reaction monitoring. Representative structures of glycans with elevated levels in cancer patients included the following: small glycans related to sialyllactose; sialyl Lewis X; lactose- and N-acetyllactosamine (LacNAc) type-II-core glycans with LacNAc (type-I or II)-extensions and modifications of α1,3/4-fucose and/or 6-sulfate on the Glc/GlcNAc; free- N -glycans containing sialylation or β1,6-branch of 6-sulfo Lewis X; novel NeuAcα2-3Galβ1-4(+/−Fucα1-3) Xylα1-3Glc glycans. Our results provide further insight into urinary free-glycans and suggest the potential utility of these compounds as tumor markers. [ABSTRACT FROM AUTHOR]

Vertebrates are estimated to have arisen over 500 million years ago in the Cambrian Period. Species that survived the Big Five extinction events at a global scale underwent repeated adaptive radiations along with habitat expansions from the sea to the land and sky. The development of the endoskeleton and neural tube enabled more complex body shapes. At the same time, vertebrates became suitable for the invasion and proliferation of foreign organisms. Adaptive immune systems were acquired for responses to a wide variety of pathogens, and more sophisticated systems developed during the evolution of mammals and birds. Vertebrate glycans consist of common core structures and various elongated structures, such as Neu5Gc, Galα1–3Gal, Galα1–4Gal, and Galβ1–4Gal epitopes, depending on the species. During species diversification, complex glycan structures were generated, maintained or lost. Whole-genome sequencing has revealed that vertebrates harbor numerous and even redundant glycosyltransferase genes. The production of various glycan structures is controlled at the genetic level in a species-specific manner. Because cell surface glycans are often targets of bacterial and viral infections, glycan structural diversity is presumed to be protective against infections. However, the maintenance of apparently redundant glycosyltransferase genes and investment in species-specific glycan structures, even in higher vertebrates with highly developed immune systems, are not well explained. This fact suggests that glycans play important roles in unknown biological processes. [ABSTRACT FROM AUTHOR]

Motivation Glycans are biomolecules that take an important role in the biological processes of living organisms. They form diverse, complicated structures such as branched and cyclic forms. Web3 Unique Representation of Carbohydrate Structures (WURCS) was proposed as a new linear notation for uniquely representing glycans during the GlyTouCan project. WURCS defines rules for complex glycan structures that other text formats did not support, and so it is possible to represent a wide variety glycans. However, WURCS uses a complicated nomenclature, so it is not human-readable. Therefore, we aimed to support the interpretation of WURCS by converting WURCS to the most basic and widely used format IUPAC. Results In this study, we developed GlycanFormatConverter and succeeded in converting WURCS to the three kinds of IUPAC formats (IUPAC-Extended, IUPAC-Condensed and IUPAC-Short). Furthermore, we have implemented functionality to import IUPAC-Extended, KEGG Chemical Function (KCF) and LinearCode formats and to export WURCS. We have thoroughly tested our GlycanFormatConverter and were able to show that it was possible to convert all the glycans registered in the GlyTouCan repository, with exceptions owing only to the limitations of the original format. The source code for this conversion tool has been released as an open source tool. Availability and implementation https://github.com/glycoinfo/GlycanFormatConverter.git Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Motivation: A glycan consists of monosaccharides linked by glycosidic bonds, has branches and forms complex molecular structures. Databases have been developed to store large amounts of glycan-binding experiments, including glycan arrays with glycan-binding proteins. However, there are few bioinformatics techniques to analyze large amounts of data for glycans because there are few tools that can handle the complexity of glycan structures. Thus, we have developed the MCAW (Multiple Carbohydrate Alignment with Weights) tool that can align multiple glycan structures, to aid in the understanding of their function as binding recognition molecules. Results: We have described in detail the first algorithm to perform multiple glycan alignments by modeling glycans as trees. To test our tool, we prepared several data sets, and as a result, we found that the glycan motif could be successfully aligned without any prior knowledge applied to the tool, and the known recognition binding sites of glycans could be aligned at a high rate amongst all our datasets tested. We thus claim that our tool is able to find meaningful glycan recognition and binding patterns using data obtained by glycan-binding experiments. The development and availability of an effective multiple glycan alignment tool opens possibilities for many other glycoinformatics analysis, making this work a big step towards furthering glycomics analysis. [ABSTRACT FROM AUTHOR]

Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(β3)GlcNAc, Gal(β4)GlcNAc and Gal(β3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl- Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and β4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(β4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior. [ABSTRACT FROM AUTHOR]

Rice α-fucosidase (α-fucosidase Os, 58 kDa) that is active for α1-4 fucosyl linkage in Lewis a unit of plantN-glycans was purified to homogeneity. α-fucosidase Os showed activity against α1-3 fucosyl linkage in Lacto-N-fucopentaose III but not α1-3 fucosyl linkage in the core of plantN-glycans. The N-terminal sequence of α-fucosidase Os was identified as A-A-P-T-P-P-P-L-, and this sequence was found in the amino acid sequence of the putative rice α-fucosidase 1 (Os04g0560400). [ABSTRACT FROM PUBLISHER]

We have previously reported that oyster hepatopancreas contained three unusual a-ketoside hydrolases: (i) a 3-deoxy-D-manno-oct-2-ulosonic acid a-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-D-glycero-Dgalacto- non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed α-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant α-Kdo-ase (R-α-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S277PDDGKTW and S328TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-α-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo2-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS fromNeisseriameningitides A1 andM992 that contained extra 5-6 sugar units at the outer corewere refractory to R-α-Kdo-ase. This R-α-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans. [ABSTRACT FROM AUTHOR]

Motivation: Glycans play critical roles in many biological processes, and their structural diversity is key for specific protein-glycan recognition. Comparative structural studies of biological molecules provide useful insight into their biological relationships. However, most computational tools are designed for protein structure, and despite their importance, there is no currently available tool for comparing glycan structures in a sequence order- and size-independent manner. Results: A novel method, GS-align, is developed for glycan structure alignment and similarity measurement. GS-align generates possible alignments between two glycan structures through iterative maximum clique search and fragment superposition. The optimal alignment is then determined by the maximum structural similarity score, GS-score, which is size-independent. Benchmark tests against the Protein Data Bank (PDB) N-linked glycan library and PDB homologous/non-homologous N-glycoprotein sets indicate that GS-align is a robust computational tool to align glycan structures and quantify their structural similarity. GS-align is also applied to template-based glycan structure prediction and monosaccharide substitution matrix generation to illustrate its utility. [ABSTRACT FROM AUTHOR]

Most currently available glycan structure databases use their own proprietary structure representation schema and contain numerous annotation errors. These cause problems when glycan databases are used for the annotation or mining of data generated in the laboratory. Due to the complexity of glycan structures, curating these databases is often a tedious and labor-intensive process. However, rigorously validating glycan structures can be made easier with a curation workflow that incorporates a structure-matching algorithm that compares candidate glycans to a canonical tree that embodies structural features consistent with established mechanisms for the biosynthesis of a particular class of glycans. To this end, we have implemented Qrator, a web-based application that uses a combination of external literature and database references, user annotations and canonical trees to assist and guide researchers in making informed decisions while curating glycans. Using this application, we have started the curation of large numbers of N-glycans, O-glycans and glycosphingolipids. Our curation workflow allows creating and extending canonical trees for these classes of glycans, which have subsequently been used to improve the curation workflow. [ABSTRACT FROM PUBLISHER]