Sweet spots in functional glycomics

Information contained in the mammalian glycome is decoded by glycan-binding proteins (GBPs) that mediate diverse functions including host-pathogen interactions, cell trafficking and transmembrane signaling. Although information on the biological roles of GBPs is rapidly expanding, challenges remain in identifying the glycan ligands and their impact on GBP function. Protein-glycan interactions are typically low affinity, requiring multivalent interactions to achieve a biological effect. Though many glycoproteins can carry the glycan structure recognized by the GBP, other factors, such as recognition of protein epitopes and microdomain localization, may restrict which glycoproteins are functional ligands in situ. Recent advances in development of glycan arrays, synthesis of multivalent glycan ligands, bioengineering of cell-surface glycans and glycomics databases are providing new tools to identify the ligands of GBPs and to elucidate the mechanisms by which they participate in GBP function.     

Advancing glycomics: implementation strategies at the consortium for functional glycomics

Glycomics-an integrated approach to study structure-function relationships of complex carbohydrates (or glycans)-is an emerging field in this age of post-genomics. Realizing the importance of glycomics, many large scale research initiatives have been established to generate novel resources and technologies to advance glycomics. These initiatives are generating and cataloging diverse data sets necessitating the development of bioinformatic platforms to acquire, integrate, and disseminate these data sets in a meaningful fashion. With the consortium for functional glycomics (CFG) as the model system, this review discusses databases and the bioinformatics platform developed by this consortium to advance glycomics.     

Human disease glycomics: technology advances enabling protein glycosylation analysis – part 1

Introduction: Protein glycosylation is recognized as an important post-translational modification, with specific substructures having significant effects on protein folding, conformation, distribution, stability and activity. However, due to the structural complexity of glycans, elucidating glycan structure-function relationships is demanding. The fine detail of glycan structures attached to proteins (including sequence, branching, linkage and anomericity) is still best analysed after the glycans are released from the purified or mixture of glycoproteins (glycomics). The technologies currently available for glycomics are becoming streamlined and standardized and many features of protein glycosylation can now be determined using instruments available in most protein analytical laboratories.

Areas covered: This review focuses on the current glycomics technologies being commonly used for the analysis of the microheterogeneity of monosaccharide composition, sequence, branching and linkage of released N- and O-linked glycans that enable the determination of precise glycan structural determinants presented on secreted proteins and on the surface of all cells.

Expert commentary: Several emerging advances in these technologies enabling glycomics analysis are discussed. The technological and bioinformatics requirements to be able to accurately assign these precise glycan features at biological levels in a disease context are assessed.     

Glycan reductive isotope labeling for quantitative glycomics

Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed glycan reductive isotope labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [¹²C₆]aniline and [¹³C₆]aniline. These dual-labeled aniline-tagged glycans can be recovered by reverse-phase chromatography and can be quantified based on ultraviolet (UV) absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins, using this method. This technique allows linear relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of glycomics.     

A two-dimensional array for simultaneous sequencing of N- and o-glycans and their glycoforms on specific glycosylation sites

Glycans play major roles in living organisms. Thus, essential information is required on diverse glycans, their location, and moieties in proteins, as well as for technology in a high-throughput manner, for improving functional glycomics. In the present study, we describe a new approach involving a 2-D array, which has the potential to fulfill both requirements. The first dimension of the array is composed of various lectins immobilized to a MALDI plate. The second dimension consists of initial proteolysis, then sequential exoglycosidase digestion using highly specific enzymes. The products of such digestions are peptide/glycopeptide mixtures conjugating different glycan fragments from which the exoglycosidase has removed specific terminal residues. Consequently, a series of spectra are obtained when lectin-attached products are analyzed by MALDI-TOF MS. By using well-known glycoproteins and NKp46D2-Ig, a recombinant fusion natural killer receptor with unknown glycans produced in CHO cells, we proved the usefulness of the method, demonstrating rapid and simultaneous determination of N- and O-glycan sequences, their glycan moieties, and subtypes on each of the determined glycosylation sites. This strategy provides a tool that can rapidly explore glycan structures and might contribute to a better understanding of process- and disease-related glycoproteins.     

GlycReSoft: A Software Package for Automated Recognition of Glycans from LC/MS Data

Glycosylation modifies the physicochemical properties and protein binding functions of glycoconjugates. These modifications are biosynthesized in the endoplasmic reticulum and Golgi apparatus by a series of enzymatic transformations that are under complex control. As a result, mature glycans on a given site are heterogeneous mixtures of glycoforms. This gives rise to a spectrum of adhesive properties that strongly influences interactions with binding partners and resultant biological effects. In order to understand the roles glycosylation plays in normal and disease processes, efficient structural analysis tools are necessary. In the field of glycomics, liquid chromatography/mass spectrometry (LC/MS) is used to profile the glycans present in a given sample. This technology enables comparison of glycan compositions and abundances among different biological samples, i.e. normal versus disease, normal versus mutant, etc. Manual analysis of the glycan profiling LC/MS data is extremely time-consuming and efficient software tools are needed to eliminate this bottleneck. In this work, we have developed a tool to computationally model LC/MS data to enable efficient profiling of glycans. Using LC/MS data deconvoluted by Decon2LS/DeconTools, we built a list of unique neutral masses corresponding to candidate glycan compositions summarized over their various charge states, adducts and range of elution times. Our work aims to provide confident identification of true compounds in complex data sets that are not amenable to manual interpretation. This capability is an essential part of glycomics work flows. We demonstrate this tool, GlycReSoft, using an LC/MS dataset on tissue derived heparan sulfate oligosaccharides. The software, code and a test data set are publically archived under an open source license.     

Glycomics and Proteomics Approaches to Investigate Early Adenovirus–Host Cell Interactions

Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Quantitation by isobaric labeling: applications to glycomics

The study of glycosylation patterns (glycomics) in biological samples is an emerging field that can provide key insights into cell development and pathology. A current challenge in the field of glycomics is to determine how to quantify changes in glycan expression between different cells, tissues, or biological fluids. Here we describe a novel strategy, quantitation by isobaric labeling (QUIBL), to facilitate comparative glycomics. Permethylation of a glycan with (13)CH 3I or (12)CH 2DI generates a pair of isobaric derivatives, which have the same nominal mass. However, each methylation site introduces a mass difference of 0.002922 Da. As glycans have multiple methylation sites, the total mass difference for the isobaric pair allows separation and quantitation at a resolution of approximately 30000 m/Delta m. N-Linked oligosaccharides from a standard glycoprotein and human serum were used to demonstrate that QUIBL facilitates relative quantitation over a linear dynamic range of 2 orders of magnitude and permits the relative quantitation of isomeric glycans. We applied QUIBL to quantitate glycomic changes associated with the differentiation of murine embryonic stem cells to embryoid bodies.     

Glycoscience aids in biomarker discovery

The glycome consists of all glycans (or carbohydrates) within a biological system, and modulates a wide range of important biological activities, from protein folding to cellular communications. The mining of the glycome for disease markers represents a new paradigm for biomarker discovery; however, this effort is severely complicated by the vast complexity and structural diversity of glycans. This review summarizes recent developments in analytical technology and methodology as applied to the fields of glycomics and glycoproteomics. Mass spectrometric strategies for glycan compositional profiling are described, as are potential refinements which allow structure-specific profiling. Analytical methods that can discern protein glycosylation at a specific site of modification are also discussed in detail. Biomarker discovery applications are shown at each level of analysis, highlighting the key role that glycoscience can play in helping scientists understand disease biology.     

Glycosylation in cellular mechanisms of health and disease

Glycosylation produces an abundant, diverse, and highly regulated repertoire of cellular glycans that are frequently attached to proteins and lipids. The past decade of research on glycan function has revealed that the enzymes responsible for glycosylation-the glycosyltransferases and glycosidases-are essential in the development and physiology of living organisms. Glycans participate in many key biological processes including cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. This review discusses the increasingly sophisticated molecular mechanisms being discovered by which mammalian glycosylation governs physiology and contributes to disease.     

From glycomics to functional glycomics of sugar chains: Identification of target proteins with functional changes using gene targeting mice and knock down cells of FUT8 as examples

Comprehensive analyses of proteins from cells and tissues are the most effective means of elucidating the expression patterns of individual disease-related proteins. On the other hand, the simultaneous separation and characterization of proteins by 1-DE or 2-DE followed by MS analysis are one of the fundamental approaches to proteomic analysis. However, these analyses do not permit the complete structural identification of glycans in glycoproteins or their structural characterization. Over half of all known proteins are glycosylated and glycan analyses of glycoproteins are requisite for fundamental proteomics studies. The analysis of glycan structural alterations in glycoproteins is becoming increasingly important in terms of biomarkers, quality control of glycoprotein drugs, and the development of new drugs. However, usual approach such as proteoglycomics, glycoproteomics and glycomics which characterizes and/or identifies sugar chains, provides some structural information, but it does not provide any information of functionality of sugar chains. Therefore, in order to elucidate the function of glycans, functional glycomics which identifies the target glycoproteins and characterizes functional roles of sugar chains represents a promising approach. In this review, we show examples of functional glycomics technique using alpha 1,6 fucosyltransferase gene (Fut8) in order to identify the target glycoprotein(s). This approach is based on glycan profiling by CE/MS and LC/MS followed by proteomic approaches, including 2-DE/1-DE and lectin blot techniques and identification of functional changes of sugar chains.     

Emerging glycomics technologies

The nascent field of glycomics is currently undergoing rapid development, largely as a result of advances in technologies for analyzing glycan structure, unraveling glycan-protein interactions and establishing the functional significance of glycans. A meeting was held in November 2006 to explore the challenges and opportunities ahead for this emerging 'omics' domain.     

Integrated mass spectrometric strategy for characterizing the glycans from glycosphingolipids and glycoproteins: direct identification of sialyl Le(x) in mice

The current interest in applying systems biology approaches to studying an organism's form or function promises to reveal further insights into the role of glycosylation in cells and whole organisms. This has prompted the development of a rapid, sensitive method of profiling the glycan component of both glycosphingolipids and glycoproteins from a single sample. Here we report a new mass spectrometric screening strategy for characterizing glycosphingolipid-derived oligosaccharides, which can be integrated into an existing highly sensitive glycoprotein glycomics strategy. Using ceramide glycanase to release the glycans from glycosphingolipids, this method provides a reliable profile of the glycosphingolipid-derived glycans present in a sample and has revealed new glycan structures. Glycoproteins are also efficiently recovered using this method, allowing the subsequent analysis of glycoprotein-derived glycans by mass spectrometry. The high sensitivity of this glycomic screening method allowed us to directly characterize the sialyl Le(x) epitope from mouse brain for the first time, where it was observed on an O-mannose structure. Thus, we present a mass spectrometric method that allows glycomic screening of N- and O-glycans as well as glycosphingolipid-derived glycans from a single tissue.     

Bioinformatics for glycomics: status, methods, requirements and perspectives

The term 'glycomics' describes the scientific attempt to identify and study all the glycan molecules - the glycome - synthesised by an organism. The aim is to create a cell-by-cell catalogue of glycosyltransferase expression and detected glycan structures. The current status of databases and bioinformatics tools, which are still in their infancy, is reviewed. The structures of glycans as secondary gene products cannot be easily predicted from the DNA sequence. Glycan sequences cannot be described by a simple linear one-letter code as each pair of monosaccharides can be linked in several ways and branched structures can be formed. Few of the bioinformatics algorithms developed for genomics/proteomics can be directly adapted for glycomics. The development of algorithms, which allow a rapid, automatic interpretation of mass spectra to identify glycan structures is currently the most active field of research. The lack of generally accepted ways to normalise glycan structures and exchange glycan formats hampers an efficient cross-linking and the automatic exchange of distributed data. The upcoming glycomics should accept that unrestricted dissemination of scientific data accelerates scientific findings and initiates a number of new initiatives to explore the data.     

Clinical application of quantitative glycomics

ABSTRACT

Introduction: Aberrant glycosylation has been associated with many diseases. Decades of research activities have reported many reliable glycan biomarkers of different diseases which enable effective disease diagnostics and prognostics. However, none of the glycan markers have been approved for clinical diagnosis. Thus, a review of these studies is needed to guide the successful clinical translation.

Area covered: In this review, we describe and discuss advances in analytical methods enabling clinical glycan biomarker discovery, focusing only on studies of released glycans. This review also summarizes the different glycobiomarkers identified for cancers, Alzheimer’s disease, diabetes, hepatitis B and C, and other diseases.

Expert commentary: Along with the development of techniques in quantitative glycomics, more glycans or glycan patterns have been reported as better potential biomarkers of different diseases and proved to have greater diagnostic/diagnostic sensitivity and specificity than existing markers. However, to successfully apply glycan markers in clinical diagnosis, more studies and verifications on large biological cohorts need to be performed. In addition, faster and more efficient glycomic strategies need to be developed to shorten the turnaround time. Thus, glycan biomarkers have an immense chance to be used in clinical prognosis and diagnosis of many diseases in the near future.     

Mammalian O-mannosylation: unsolved questions of structure/function

Post-translational modification of polypeptides with glycans increases the diversity of the structures of proteins and imparts increased functional diversity. Here, we review the current literature on a relatively new O-glycosylation pathway, the mammalian O-mannosylation pathway. The importance of O-mannosylation is illustrated by the fact that O-mannose glycan structures play roles in a variety of processes including viral entry into cells, metastasis, cell adhesion, and neuronal development. Furthermore, mutations in the enzymes of this pathway are causal for a variety of congenital muscular dystrophies. Here we highlight the protein substrates, glycan structures, and enzymes involved in O-mannosylation as well as our gaps in understanding structure/function relationships in this biosynthetic pathway.     

糖生物信息学数据库

糖生物信息学是在糖生物学和糖组学发展的基础上,结合计算机技术,对生命活动过程中,参与糖链及与其相互作用的蛋白质等分子研究所产生的数据进行获取、储存、解析、模拟以及预测等内容的综合学科.糖生物信息学数据库是糖生物信息学发展到一定阶段,对糖组学等研究中产生的数据进行专门储藏与查询的应用工具.目前国际互联网中存在近百个糖生物信息学相关数据库,涉及内容包括糖链结构、参与糖链合成的基因或者蛋白质、糖结合蛋白、代谢通路、糖链或相互作用蛋白质等分子三维结构,或糖组学实验结果等领域.本文将归纳总结糖生物信息学数据库,为现有研究提供帮助.     

Mass spectrometry-based shotgun glycomics for discovery of natural ligands of glycan-binding proteins

The non-covalent associations of complex carbohydrates (glycans) with glycan-binding proteins mediate many important physiological and pathophysiological processes. Identifying these interactions is essential to understanding their diverse biological functions and enables the development of new disease treatments and diagnostics. Knowledge of the repertoire of glycans recognized by most glycan-binding proteins and their affinities is incomplete. Mass spectrometry-based screening of natural glycan libraries has emerged as a promising approach to defining the glycan interactome of glycan-binding proteins. Here, we review recent advances in mass spectrometry-based natural library screening that have led to the discovery of glycan ligands of endogenous and exogenous proteins and illuminated their binding specificities.