Basic and Applied Research

Every living cell in nature generates a complex and diverse array of glycans that is critical for the evolution, development, functioning, and survival of all natural biological systems (Chapter 1). A strong basic knowledge base regarding the genomics, chemistry, biochemistry, biosynthesis, and biological roles of these ubiquitous and diverse molecules is now well established (Chapters 2–19). The broad outlines of their natural occurrence and evolution (Chapters 20–27) and their recognition by glycan-binding proteins (Chapters 28–38) is becoming clear, as is their important role in normal and abnormal physiology and disease (Chapters 39–47). Facile methods for their analysis, manipulation, and synthesis of glycans have also been established (Chapters 48–56), and their significance in the biotechnology and pharmaceutical industries, nanotechnology, and bioenergy and materials science is obvious (Chapters 57–59). Given all these major advances in basic and applied research on glycans, there is no reason why this major class of biomolecules should continue to be the “dark matter of the biological universe.” However, an entire generation of scientists has been largely trained without much exposure to, or knowledge of, glycans. Thus, it will still be a while before these molecules take their rightful place in the mainstream of thinking in conventional molecular and cellular biology and medicine. Continued advances in basic and applied research on glycans will occur, but this needs to be coupled with the training of a new generation of scientists, engineers, and physicians, for whom these molecules will be an obvious aspect of their understanding of living systems, in health and disease.

Health and Development

As emphasized in a recent report from the National Research Council of the National Academy of Sciences, nearly every disease process that affects humans and other animals involves glycans (Chapters 39–47). In past decades, it has been realized that most functions of complex glycans are required at the multicellular (organismic) level. In contrast, cycling monosaccharides (e.g., O-GlcNAc in the nucleus and cytoplasm) serve regulatory functions at the single cell level. The critical roles of complex glycans in the biology of intact organisms has been dramatically illustrated by the contrast between the viability of glycosyltransferase mutant cell lines in culture (Chapter 49) with the often lethal outcome caused by inactivation of the same enzymes in living organisms (Chapter 41). Transgenic mouse studies and the severe phenotypes of human congenital diseases of glycosylation (Chapter 45) have dramatically revealed the critical importance of glycans in development, physiology, and disease.

Most major diseases also involve disordered inflammation and immunity, in which the glycan-binding selectins, Siglecs, galectins, and other glycan-binding proteins (Chapters 34–36) play critical roles. Most pathogens, viruses, bacteria, and parasites gain entry to cells by binding to glycans on the cell surface (Chapters 42 and 43). In addition, many vaccines against infectious agents are directed against microbial glycans. Proteoglycans play critical roles in development, tissue morphogenesis, and cardiovascular disease and in regulating the actions of cytokines and growth factors. The glycosaminoglycan heparin is one of the oldest and most commonly used “drugs” in the clinic. Notch signaling, which plays a major role in controlling morphogenesis in development and cell fate decisions, is controlled by glycans (Chapter 13), and glycans on the surface of tumor cells play critical roles in tumor progression and metastasis (Chapter 47). Many of the current therapeutics in use or under development are glycoproteins, like monoclonal antibodies, which often require particular N-glycans for functional efficacy. Defects in the synthesis of glycan chains on dystroglycan underlie many types of congenital muscular dystrophy (Chapter 45). Dysregulated O-GlcNAcylation contributes to the etiologies of diabetes, neurodegeneration, cardiovascular disease, and cancer (Chapter 19). Although the few scientists and physicians well educated in glycobiology are acutely aware of the importance of glycans in disease, most others have not yet learned much about this major class of molecules. However, it is now clear that studies of glycans will be essential for understanding the pathophysiology of most diseases and the development of effective therapies. For the great potential of glycobiology to be realized, efforts need to be directed toward education of all graduate and postgraduate students, clinicians, and basic scientists and to advancing technologies that will allow ease of experimentation.

Renewable Energy

Clearly, in the not too distant future, we will run out of fossil fuels, which are not renewable resources. Plants are by far the most efficient source of renewable energy because they efficiently use photosynthesis to trap the sun's energy, mostly in the form of glycans. The challenge in using plants as a source of fuel is the difficulty in degrading plant cell walls into smaller glycans that can be converted into usable fuels at a low cost (Chapter 59). In the future, glycoscience will play a critical role in the evolution of our society from one based on burning fossil fuels to one based on the use of sustainable energy, in part derived from rapidly growing plants and algae.

Industry and Materials

Carbon sequestration by photosynthesis to produce glycans is the major process preventing carbon dioxide from building up to levels that would cause severe “greenhouse” warming of the planet, with unpredictable climate disruption as a major consequence. Wood, which is mostly made up of complex glycans, is already a major building material. Paper, textiles, cellophane, and rayon are others examples of everyday materials made from glycans. As our supply of petroleum runs out, glycans will increasingly be used to provide materials for the manufacture of plastics and a myriad of polymers (Chapter 59). Various forms of modified cellulose, for example, will be critical for the manufacturing of exotic materials and as carbon sources for various chemicals.

Analytical Methods

Advances in mass spectrometry instrumentation and methods in recent years have been remarkable (Chapters 50 and 51). The ability to use mass spectrometry to map glycan attachment sites, to profile glycan structural variations, and to determine detailed fine structures of glycans has progressed rapidly because of technological advances in instrumentation, including electron capture and electron transfer dissociation fragmentation methods, and the development of ion traps with very high mass accuracy and sensitivity. In the future, ion-mobility separations in the gas phase will allow the identification of glycan isomers that have identical molecular masses in mixtures. In addition, there have been major advances in the chromatographic isolation and separation of complex glycans. New ultra-high-pressure pumps and very small particulate high-pressure liquid chromatography (HPLC) resins that can withstand high pressures will also greatly increase the resolving power of chromatography. Advances in ion mobility and high-resolution mass spectrometric methods are, for the first time, allowing us to begin to decode the information content of glycosaminoglycans (Chapter 17). The development of induced hyperpolarization methods shows great promise in greatly increasing the sensitivity of nuclear magnetic resonance (NMR) analyses of glycans (Chapter 50), which has been a major limitation in the analyses of biological samples. Bio-orthogonal labeling methods continue to be developed that will allow the study of changes in glycans in living cells in real time. Currently, for atomic structural analyses of glycoproteins, including X-ray crystallography, it is usually necessary to remove glycans to allow for crystallization. In the future, as electron imaging methods, such as cryo-electron microscopy (cryo-EM), continue to reach higher resolution approaching that achieved by crystallographic analyses, there is hope to be able to obtain images of glycoconjugates at atomic resolution, without the need to remove the glycans. As NMR becomes more sensitive and can handle larger molecules, one will also be able to visualize glycoconjugates in physiologically relevant solvents and at appropriate temperatures. These developments will allow a much more accurate view of the 3D structures of glycoconjugates. Eventually, one will need to define the population of molecular species (glycoforms of glycoproteins and glycolipids) at an organismal and cellular level to fully understand structure/function relationships of glycoconjugates in response to extracellular stimuli. Existing technologies that show great promise include top-down glycoproteomics and high-throughput, high-resolution imaging methods. In theory, if these methods live up to their promise, one day it may be possible to define the complete set of molecular species of glycoconjugates in a population and thus elucidate how they collectively contribute to the many individual functions of a gene product. However, all these methods still result in loss of information about labile modifications of glycans and also involve breaking apart the intact glycome into pieces before analysis. Ultimately, one will need to understand the actual structure of the intact glycome in situ, in living systems.

In parallel with advances in such sophisticated methods, it is also important to “democratize” the practical approach to glycobiology by developing simple methods that can be used by the average biologist working at the bench, without sophisticated instrumentation. In this regard, one should be able to take advantage of the fact that millions of years of pathogen and symbiont interactions with hosts have already generated a large number of highly specific glycan-binding proteins, which could be harnessed to interrogate glycosylation at multiple levels of resolution.

Chemical and Enzymatic Synthesis

Because of their stereochemistry and water solubility, organic synthesis of glycans has proven to be one of the most challenging areas of synthetic organic chemistry (Chapter 53). The combined use of purified glycosyltransferases, with chemically synthesized glycan precursors, has proven to be invaluable in the stereoselective synthesis of complex glycans (Chapter 54). Automated synthesis of glycans is rapidly becoming a reality, and even the synthesis of the most complex glycans, such as glycosaminoglycans, is becoming possible. In the future, it seems likely that the synthesis of glycans will become nearly as facile and widely available as the synthesis of nucleic acids and proteins. It should be possible for biologists to easily obtain glycoconjugates that have homogeneously uniform glycans, to further explore the structural and functional roles of the glycans. The availability of nearly all of the major glycosyltransferases and glycosidases in recombinant form should also greatly increase our ability to modify glycoconjugates for investigations of structure/function relationships. In addition, glycoengineering of proteins and lipids will make it possible to generate molecules with novel properties, for a wide variety of applications.

Genomics and Enzymology of Glycans

The complete mapping of multiple genomes continues to have a huge impact on the advancement of glycoscience. The identification, sequencing, and classification of genes important to glycosciences have already allowed us to understand the evolution and functions of genes that affect glycosylation (“glycogenes”) at an unprecedented rate (Chapters 8 and 52). The wealth of information in these databases has only just begun to be mined. These studies will lead to the identification of novel glycosylation enzymes and should shed light on how various species have evolved to produce their glycoforms and glycotypes. Current projects to make cDNAs, mRNA, and proteins for most of the common glycoenzymes available to the wider research community will greatly facilitate the study of glycoconjugates by all investigators. The combination of organic synthesis and chemoenzymatic synthesis will allow the production of well-defined glycan arrays that contain the various binding epitopes for glycan-binding proteins. Recombinant glycosyltransferases will become common tools to generate specific glycans on glycoproteins and living cells, and these enzymes will greatly enhance the many studies that now rely on the use of lectins (Chapter 48) whose specificity is often not well defined. Finally, the rapidly expanding analytical toolkit will be instrumental to decipher the regulatory mechanisms that mediate homeostasis of glycoconjugates at a cellular level. As with analytical methods, it is also important to “democratize” the approaches by developing simple methods for the average biologist working at the bench.

High-Throughput Analyses

In recent years, great advancements in the glycomic profiling of glycans from cells, tissues, or organisms have occurred. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has allowed the rapid profiling of the majority of N- and O-glycans in a sample (Chapter 51). As matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instruments also gain the ability to perform tandem mass spectrometry, confidence in the structure of profiled glycans will continue to improve. However, detailed structural analysis of individual glycans still remains a low-throughput method that requires considerable expertise and expensive instrumentation. The development of glycan arrays presenting hundreds of different glycans of defined structure is perhaps one of the most impactful recent developments in glycobiology (Chapter 56). These arrays have allowed for rapid, high-throughput analysis of the binding specificities of numerous biologically important glycan-binding proteins, including lectins, antibodies, viruses, and bacteria. It has been estimated that these arrays will need to contain between 10,000 and 20,000 different glycans to cover most possible glycotopes (this very conservative estimate does not take into account various common and labile modifications of glycans). In addition, more specialized arrays will be required to study glycan-binding proteins made by prokaryotes or to study proteins that bind to glycosaminoglycans, for example. Fortunately, the ability to make such large and diverse glycan arrays is on the horizon, and the remaining challenge will be to better understand the parameters of the glycan arrays, besides the glycans themselves, that influence binding, such as linkers, spacing, and density of glycans on solid supports.

Need for More Facile Methods to Explore Glycobiology

At the present time, the study of glycan structure/function relationships requires highly specialized expertise in synthetic and analytical chemistry. The inherent difficulty in studying glycans remains a major challenge preventing glycobiology—the study of glycan function—from entering mainstream conceptual frameworks of biology. In response to this challenge, strong emphasis has recently been placed on the development of facile technologies that will allow nonglycobiologists, without specialized analytical chemistry skills, to study glycans in a meaningful way. In fact, the use of glycosyltransferase and glycosidase probes to manipulate glycans is not any more difficult than the use of restriction enzymes and endonucleases to analyze DNA. The use of antibodies and lectins specific for glycans also exemplifies methods that can be readily applied by biologists lacking expensive analytical equipment or extensive glycobiology expertise (Chapter 49). In fact, several kits for the study of glycans have emerged, as companies recognize the future market potential of glycobiology. In addition, companies are offering highly purified enzymes, antibodies, and chemistries, making it much easier to study glycans. In contrast, few glycans are available for experimental purposes and most enzymes that act on glycans are not commercially available. Equally important, most investigators are not trained to know what questions to ask regarding the roles of glycans attached to their favorite glycoconjugate. In addition to new and facile methods to study glycans, education of the next generation of cell biologists and biochemists with respect to the importance of glycans will be key to advancing glycoscience to the next level.

What Are the Roles of Glycans in the Organization of the Components in the Plasma Membrane, Glycocalyx, and Extracellular Matrix?

Glycans affect the stability and turnover of cell-surface resident molecules and many can self-associate in the plane of the membrane. Do they play a role in the organization and regional concentration of molecules at the cell surface? How do the various glycoconjugates on cell surfaces collaborate among themselves and with glycan-binding proteins to organize the glycocalyx of a given cell or its extracellular matrix? After all, most living cells in a multicellular organism exist within a glycan-enriched gel, not in the artificially fluid medium of the tissue culture dish.

How Do Glycans Regulate Cellular Signaling from the Cell Surface to the Nucleus?

Complex glycans clearly regulate the function, stability, and residence time of receptors at the cell surface, and the addition of O-GlcNAc to nuclear and cytoplasmic proteins regulates many of the signaling pathways downstream from these receptors. Yet, still very little is known about the mechanistic roles of glycans in these pathways.

Is There a Biological Significance for Site-Specific Glycan Diversity on Glycoproteins?

Glycoproteins typically contain an array of many glycans with different structures at individual sites in the polypeptide. Evidence suggests that these arrays of glycans are site-specific and cell type–specific, and not random in distribution. How does site-specific diversity arise and how is it controlled? Does each glycoform have different biological functions or variable strengths, and/or does the site-specific diversity control molecular interactions? Or is the complexity the natural outcome of evolutionary arms races between hosts and their pathogens, which exploit glycans in various ways?

Can Specific Glycoforms of Glycoproteins Be Used to Improve Biomarkers for Disease?

Many investigators are trying to discover biomarkers to help diagnose disease, such as cancer. It has been proposed that identification of specific glycoforms of a glycoprotein might improve the specificity and sensitivity of these biomarkers.

How Can the Marked Mobility of Glycans in Biological Systems Be Taken into Account?

Unlike most other biomolecules, glycans have a substantial degree of mobility in aqueous solution. This further increases the potential information content of these molecules but also raises further challenges for their analysis and biological exploration.

How Do Cell-Surface Glycans and Matrix Glycans Contribute to Cancer Progression and Metastasis?

Data from glycan analysis, animal models, and correlations from the clinic strongly indicate that cell-surface glycans are critical to tumor progression and metastasis. Yet mechanistically, relatively little is known with respect to the roles glycans play in these processes.

The Glycosciences Should Have a Huge Impact on Antiviral, Antibacterial, and Antifungal Diagnostics and Therapeutics—How Does One Make This Happen?

It is known that glycans are critical to infectious disease and are key antigens in the generation of several vaccines. As conventional antibiotics become less effective, how does one exploit glycans to fight infectious disease?

Can Glycan Analytical Methods Be Improved to Avoid Loss of Labile Modifications?

Most current analytical methods for glycans result in a marked loss of labile modifications, particularly ester groups of various kinds. Given their location and potential biological importance, there is likely a large unexplored area of glycobiology that can only be approached with new techniques.

What Is the True Extent of Diversity of the Nonulosonic Acids and What Are the Functions of this Diversity?

The discovery that the highly diverse sialic acids are just the proverbial tip of the iceberg of the ancestral family of nonulosonic acids has raised many new questions. It appears that investigators are just beginning to scratch the surface of this enormous diversity, and its potential biological significance.

How Can One Decode the Information Content in Glycosaminoglycans and Proteoglycans at the Molecular Level?

The potential information content of glycosaminoglycans and proteoglycans is enormous. Yet, investigators are only just beginning to be able to understand how the arrangement of sulfate esters and uronic acids affects affinity and protein-binding selectivity. Elucidating how such diversity is regulated temporally and spatially is also a major challenge, going forward.

What Are the Molecular Mechanisms by Which O-GlcNAcylation Regulates Transcription and Signaling in Response to Nutrients?

Current data indicate that O-GlcNAc serves as a nutrient sensor to regulate signaling and transcription. Yet, mechanistically, almost nothing is known with respect to how the cycling of this sugar on and off proteins regulates these processes at the molecular level.

Has Nature Only Made Use of a Small Subset of All the Possible Monosaccharides?

There are hundreds of known monosaccharides and many can be present in different ring and/or modified forms. So far, it appears that natural systems have only incorporated a fraction of this diversity. Is this because of rate limits on genomic and enzymatic evolution in the opportunity to incorporate new monosaccharides, or is this an ascertainment bias, owing to the fact that only a small fraction of species on the planet has been sampled. Is it possible that every possible monosaccharide has been used somewhere in nature?

What Are the Complete Structures of All the Glycans within the Human Glycome or Any Animal or Plant Glycome?

Every day witnesses new glycan structures being identified, with previously unknown and novel sugar linkages, along with unique and unanticipated modifications, such as acetylation, methylation, phosphodiesters, and sulfation. How many such glycan structures exist within a glycome and to what glycoproteins or lipids are they attached, and where are they attached? Much of our knowledge of the human glycome comes from studying serum glycoproteins and several cell lines, but there is little information about the overall complexity of the human glycome either at the glycan level or the glycoprotein level.

What Is the True Extent of Diversity of Glycans, Glycan-Binding Proteins, and Glycan-Degrading Enzymes in the Microbiome?

It is clear that investigators are just beginning to scratch the surface of the organismal diversity within the complex diverse microbiomes that occupy various niches in nature. Each time a new species within a given microbiome is sequenced, the genomes are found to predict hundreds of “carbohydrate-active” enzymes. Understanding the mechanisms and functions of all these molecules is a major challenge for the future, as is the deciphering of the structures of the complex glycans they generate, bind, or degrade.

Do More Complex Biological Systems Have Simpler Glycosylation?

Of the hundreds of known monosaccharides in nature, less than a dozen are found in vertebrates, and many more tend to be found in simpler, earlier evolved systems. Is this an ascertainment bias, or does increasing biological complexity place limits on the fraction of the genome that can be committed to controlling glycosylation?

What Is the Significance of Lineage-Specific Gains and Losses of Specific Glycans during Evolution?

Current data indicate that glycans are the most rapidly diverging components of life-forms, with distinct gains and losses that sometimes become (or stay) polymorphic in various lineages and clades. It seems likely that this is a consequence of the intimate involvement of glycans in the most rapidly evolving aspects of biological systems (i.e., host–pathogen interactions, immunity, and reproduction). Further studies are needed to ascertain if glycans are indeed involved in the process of speciation.

When Will the Integration of Glyosciences into the Mainstream of Bioinformatics Be Achieved?

Currently, searches of the main genome and protein databases used by essentially all biologists yield almost no information on the glycosylation state of glycoproteins. Indeed, there is as yet no central comprehensive database of glycan structures that an investigator can mine to determine the occurrence, provenance, or phylogeny of a specific glycan of interest. An international effort led by nearly every major country involved in the glycosciences has come together to agree on uniform standards for describing glycans in computer databases, and to develop methods to curate and maintain glycan structural data. Together with the expanding use of uniform symbol nomenclature (Chapter 1), this is the first step toward integrating glycomic data into mainstream protein databases (Chapter 52).

When Will Glycobiology Completely Merge into a Holistic Approach to Biology?

As with other fields like protein sciences, there will always be a small cadre of investigators whose primary interest is in the structure, chemistry, biochemistry, and biology of glycans. But a combination of advances in methodology and education will eventually integrate glycosciences into the general awareness of biologists who are currently not trained to understand the significance of glycans. As our understanding of the functions of glycans increases, biochemists and biologists will come to recognize that glycan modifications are no less important than the amino acids that make up the polypeptide backbone or the nucleotides and nucleotide derivatives that make up DNA and RNA. In the long run, glycobiology will merge into a holistic approach to biological systems. When that intellectual singularity is eventually achieved, further editions of this book may no longer be necessary.