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Glycans are essential components of all living things because they function as key elements of cellular membranes and extracellular spaces by mediating cell-cell communication, transduction pathways, and cellular development, function, and survival. Because glycans are secondary gene products that depend on the availability of sub-cellular enzymes for synthesis, research on their structure, synthesis, and biological significance has lagged behind that of DNA and proteins due to both a lack of appreciation of their importance and the slow pace at which tools are being developed to study them. In this thesis, three projects focus on the development, application, and exploration of tools for glycan characterization. The first project resulted in the successful optimization of an analytical method to isolate and characterize O-linked glycans, on which relatively few research projects focus because of the limited availability of tools to isolate them and the need for specific analytical equipment to properly characterize them. Using this optimized method, the O-linked glycans of bovine mucin and fetuin were successfully profiled, and the analysis of the former provided the motivation for a second project focused on the significance of goblet cells and mucins in influenza infection. This project explored the potential benefits of a glycoprotein direct binding assay as a way to obtain quantitative information about lectin and influenza hemagglutinin specificities. Using mucins adsorbed to a polystyrene plate, it was possible to obtain quantitative binding constants for two commonly used lectins. The last project focused on the isolation and characterization of the cell surface N-linked glycans from chicken erythrocytes, turkey erythrocytes, and human tracheal epithelial (HTE) cells. Analysis of these cell types is warranted due to their importance as model systems to study influenza infection. The results of this project provide a context for future questions about the relevance of the erythrocyte model system for studying influenza binding specificities. All of these projects reiterate the importance of the study of glycobiology by showing how both the development and application of tools to study glycans can provide in new and interesting information about pathological processes related to human health and disease.
Sugar chains (glycans) are often attached to proteins and lipids and have multiple roles in the organization and function of all organisms. "Essentials of Glycobiology" describes their biogenesis and function and offers a useful gateway to the understanding of glycans.
This book provides current glycoinformatics methods and protocols used to support the determination of carbohydrate structures in biological samples as well as carbohydrate structure databases, the interaction of carbohydrates with proteins, and theoretical and experimental methods to study their three-dimensional structure and dynamics. Glycoinformatics explores this recently emerged field, which has come into being in order to address the needs of encoding, storing, and analyzing carbohydrate ‘sequences’ and their taxonomy using computers. Written in the highly successful Methods in Molecular Biology series format, chapters contain the kind of detailed description and key implementation advice to ensure successful results. Authoritative and timely, Glycoinformatics demonstrates the progress that has been achieved in glycoinformatics, which indicates that it is no longer a niche subject covered by only a few scientists but is truly coming of age.
As one of the most extensive and important protein post-translational modifications, glycosylation plays a vital role in regulating organisms and is associated with various physiological and pathological processes. Recently, researchers have focused on the need to characterize protein glycosylation sites, structures, and their degree of modification, to better understand their biological functions while also looking for potential biomarkers for diagnosis and treatment of disease. Mass spectrometry (MS) is one of the most powerful tools used to study biomolecules including glycoproteins and glycans. With the continuous development of glycoproteomics and glycomics based on MS analysis, more techniques have evolved and contribute to understanding the structure and function of glycoproteins and glycans. This book reviews advancements achieved in MS-based glycoproteomic analysis, including a wide range of analytical methodologies and strategies involved in selective enrichment; as well as qualitative, quantitative, and data analysis, together with their clinical applications. Significant examples are discussed to illustrate the principles, laboratory protocols, and advice for key implementation to ensure successful results. Mass Spectrometry–Based Glycoproteomics and Its Clinic Application will serve as a valuable resource to elucidate new techniques and their applications for students, postdocs, and researchers working in proteomics, glycoscience, analytical chemistry, biochemistry, and clinical medicine. Editor: Haojie Lu is a professor at Fudan University, specializing in proteomics based on mass spectrometry with particular emphasis on novel technologies for separation and identification of low-abundant proteins and post-translationally modified proteins (including glycosylation), as well as relative and absolute quantification methods for proteomics.
A new focus on glycoscience, a field that explores the structures and functions of sugars, promises great advances in areas as diverse as medicine, energy generation, and materials science, this report finds. Glycans-also known as carbohydrates, saccharides, or simply as sugars-play central roles in many biological processes and have properties useful in an array of applications. However, glycans have received little attention from the research community due to a lack of tools to probe their often complex structures and properties. Transforming Glycoscience: A Roadmap for the Future presents a roadmap for transforming glycoscience from a field dominated by specialists to a widely studied and integrated discipline, which could lead to a more complete understanding of glycans and help solve key challenges in diverse fields.
A Focused, State-of-the-Art Overview of This Evolving Field Presents Various Techniques for Glycoinformatics The development and use of informatics tools and databases for glycobiology and glycomics research have increased considerably in recent years. In addition to accumulating well-structured glyco-related data, researchers have now developed semi-automated methods for the annotation of mass spectral data and algorithms for capturing patterns in glycan structure data. These techniques have enabled researchers to gain a better understanding of how these complex structures affect protein function and other biological processes, including cancer. One of the few up-to-date books available in this important area, Glycome Informatics: Methods and Applications covers all known informatics methods pertaining to the study of glycans. It discusses the current status of carbohydrate databases, the latest analytical techniques, and the informatics needed for rapid progress in glycomics research. Providing an overall understanding of glycobiology, this self-contained guide focuses on the development of glycome informatics methods and current problems faced by researchers. It explains how to implement informatics methods in glycobiology. The author includes the required background material on glycobiology as well as the mathematical concepts needed to understand advanced mining and algorithmic techniques. She also suggests project themes for readers looking to begin research in the field.
This book is the first to be dedicated to the bioinformatics of carbohydrates and glycoproteins. It provides an introduction to this emerging field of science both for the experimentalist working in glycobiology and glycomics, and also for the computer scientist looking for background information for the development of highly sophisticated algorithmic approaches. The book provides an overview of the state-of-the-art in the field, with reviews on databases, and the tools in use for analysis, interpretation, and prediction of the structures of complex carbohydrates, and demonstrates the value of bioinformatics for glycobiology. The availability of comprehensive databases and corresponding bioinformatics tools, to access and analyse the large amounts of experimental data relating to the structure of carbohydrates, will be a prerequisite for the success of the large-scale glycomics projects that aim to decipher new, so far unknown, biological functions of glycans. Efficient bioinformatics descriptions and tools can considerably enhance the efficiency of glycomics research, in terms of data quality, analysis and experimental costs. For a complete understanding of the molecular processes in which carbohydrates are involved, such as protein–carbohydrate interactions and the impact of glycosylation on protein function, knowledge of the 3D structure of the carbohydrate, the protein–carbohydrate complex, or the glycoprotein is often indispensable. This book provides a thorough introduction into methods used for conformational analysis of carbohydrates. Key features: Describes bioinformatic approaches to handle carbohydrate-active enzymes and glycosylation. Provides an overview on bioinformatics tools that facilitate analysis of carbohydrate structures. Gives introduction into molecular modelling of carbohydrate 3D structure and carbohydrates contained in the Protein Databank. Assumes only a basic knowledge of biology and bioinformatics.
This volume provides a comprehensive understanding of the enigmatic identity of the glycome, a complex but important area of research that has been largely ignored due to its complexity. The authors thoroughly deal with almost all aspects of the glycome, i.e., elucidation of the glycan identity enigma and its role in regulation of the cellular process, and in disease etiology. The book bridges the knowledge gap in understanding the glycome, from being a cell signature to its applications in disease etiology. In addition, it details many of the major insights regarding the possible role of the glycome in various diseases as a therapeutic marker. The book systematically covers the major aspects of the glycome, including the significance of substituting the diverse monosaccharide units to glycoproteins, the role of glycans in disease pathologies, and the challenges and advances in glycobiology. The authors stress the significance and huge encoding power of carbohydrates as well as provide helpful insights in framing the bigger picture. The Glycome: Understanding the Diversity and Complexity of Glycobiology details state-of-the-art developments and emerging challenges of glycome biology, which are going to be key areas of future research, not only in the glycobiology field but also in pharmaceutics.
Mass spectrometry (MS) has evolved as a powerful tool for glycosylation analysis benefiting from its high speed, high resolution and sensitivity. It enables detailed structure characterization of glycans, glycosylation site and the peptide sequences of complex biological samples in a high-throughput. This dissertation is dedicated to developing MS-based analytical methods for the structure characterization and quantitative analysis of both released glycans and intact glycopeptides utilizing a bottom-up approach. We started by developing a hydrophilic interaction liquid chromatography (HILIC) coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) platform for improved quantitative analysis of N-glycans compared to MALDI-MS. Faced with the challenges of N-glycan structure complexity, capillary electrophoresis (CE) emerges as a robust separation technique affording very small amount of sample consumption, fast separation speed, and high resolving power. Therefore, we developed a CE-ESI-MS/MS based 6-plex aminoxy tandem mass tag (TMT) labeling method for quantitative analysis of protein N-glycosylation analysis from human serum, providing the ability of N-glycan quantitation across up to six samples. Improved resolution and quantification accuracy of the labeled human milk oligosaccharides (HMOs) isomers was also achieved by coupling CE with traveling wave ion mobility (TWIM)-CID-MS/MS. The advantage of the glycan-focused approach (glycomics) is that the wealthy structure information and glycan isomer separation could be achieved; however, this approach resulted in loss of the glycosylation site-specific information. Therefore, we developed a powerful glycoproteomics workflow for system-wide structure characterization and quantitation of intact glycopeptides. Improvements of this workflow include improved glycoprotein extraction, sequential enrichment strategies for improved N-/O- glycopeptide enrichment, hybrid novel fragmentation technique electron transfer and higher-energy collision dissociation (EThcD), multiplex quantitation enabled by our custom-made isobaric N, N-dimethyl leucine (DiLeu) tags, and automated FDR-based large-scale data analysis by Byonic. We have successfully applied this workflow to analyze complex biological samples for disease-related glyco-alteration study, including the glycosylation alteration in PKM2 knockout breast cancer cells vs. parental cells and glycosylation pattern alteration in AD patients.
Glycans are an important class of biological molecules that regulate a variety of physiological processes such as signal transduction, tissue development and microbial pathogenesis. However, due to the structural complexity of glycans and the unique intricacies of glycan-protein interactions, elucidating glycan structure-function relationships is challenging. Thus, uncovering the biological function of glycans requires an integrated approach, incorporating structural analysis of glycans, and glycan-proteins interactions with functional analysis. In this thesis, I develop new tools and implement integrated approaches to study glycans and glycan-binding proteins (GBPs). I apply these approaches to study glycans and GBPs in two areas: i) the role of hemagglutinin-glycan receptor specificity in human adaptation and pathogenesis of influenza and ii) the function of glycan regulation of cell-microenvironment interaction in cancer progression. Section 1: Influenza poses a significant public health threat and there is a constant looming threat of a pandemic. Pandemic viruses emerge when avian viruses acquire mutations that enable human adaptation, leading to infection of an antigenically naive host. Influenza Hemagglutinin (HA), and HA-glycan receptor interactions, play a central role in host tropism, transmissibility, and immune recognition. In section one, I develop and apply an integrated approach comprised of structural modeling, inter-amino acid network analysis, biochemical assays, and bioinformatics tools to study the hemagglutinin-glycan interaction and, in some cases, HA's antigenic properties. Using this approach, we i) identify the structural determinants required, and potential mutational paths, for H5N1 to quantitatively switch it's binding specificity to human glycans receptors, ii) identify the mutations that enable the 2013 outbreak H7N9 HA to improve binding to human glycan receptors in the upper respiratory tract, iii) uncover H3N2 strains that are currently circulating in birds and swine that possess features of a virus that could potentially re-emerge and cause a pandemic, and iv) characterize the glycan binding specificity of a novel 2011 Seal H3N8 HA. The approaches implemented here and the findings of these studies provide a framework for improved surveillance of influenza viruses circulating in non-human hosts that pose a pandemic threat. Section 2: Glycans are abundant on the cell surface, and at the cell-ECM interface where they mediate interactions between cells and their microenvironment. Despite this, the function of glycans in cancer progression remains largely understudied. Here, I develop an integrated approach to characterize the cell surface glycome, including N-linked, 0-linked glycans, and HSGAGs. This approach integrates glycogene expression data, analytical tools, and glycan binding protein reagents. I demonstrate that this platform enables rapid and efficient characterization of the N- and 0-linked glycome in a model cell system, representing metastatic versus non-metastatic cancer cells. Next, I apply this integrated approach to uncover new roles of glycans. I study the role that HSGAGs play in regulating cancer stem cell (CSC) activity in breast cancer. Here, we report that SULF1, an HSGAG modifying enzyme, is required for efficient tumor initiation, growth and metastasis of CSCs. Furthermore, we identify a putative mechanism by which SULF1 regulates interactions between CSCs and their microenvironment. The approaches implemented here and the finding of these studies Overall, this thesis provides important tools, approaches and insights to enable and improve the study of glycans and glycan binding proteins. Together the work here provides a framework for decoding structure-function relationship of glycans.