Editorial overview: Protein–carbohydrate complexes and glycosylation: A new age of discovery in the glycosciences

Age of Discovery was a period where sea-faring European nations explored regions across the globe, discovered new lands (at least new to them), and founded international trade and cross-cultural relationships. Today, the field of Glycoscience is engaged in its own Age of Discovery, charting new paths through the complex world of sugars. Technological breakthroughs that were important to the Age of Exploration were the adoption of the magnetic compass and advances in ship design. Continuing the metaphor, the modern Age of Discovery in the Glycosciences is underpinned by new technologies and techniques. The cartographers of old sought to make maps to shine a light on newly discovered lands; the modern glycoscientist seeks to uncover the mysteries of carbohydrates and their roles in biology and create maps to document their discoveries. These scientific maps describe the molecular structure of carbohydrates and their biosynthetic proteins and interaction partners and establish their pathways of metabolism and help to build our understanding of the roles of glycans. In this issue of Current Opinion in Structural Biology, we receive dispatches from dedicated glycoscientist explorers and map-makers who are working tirelessly to secure a new understanding of the structures of glycans and glycoconjugates, and the roles of protein-carbohydrate complexes and glycosylation.

Rapid advances in analytical methods are driving new discoveries in the structure, dynamics, and distributions of glycans, and several reviews in this issue highlight new techniques and technologies that are revolutionizing our knowledge of glycan structures. Determining the complete chemical structure of a glycan is the first step in understanding its role, biosynthesis, and metabolism. The incredible structural diversity of carbohydrates arises from their variable linkages, assorted decorations, and occurrence in conjugates, which collectively means that there is no universal workflow to determine the structure. Alba Silipo et al. present an authoritative overview of the modern techniques that can be brought to bear on this problem (including the central role of modern nuclear magnetic resonance (NMR) spectroscopy), and how by tactical application of a variety of techniques we can slowly and methodically chip away and solve complex structural challenges. Once information about connectivity and structure is resolved, decoding the function of the sugar code can be assisted by knowledge of its 3D shape, and by a better understanding of the heterogeneity of glycans present in natural samples, such as the assorted glycans present on glycoproteins. For many years the study of the structure and function of glycoproteins using NMR spectroscopy has been hindered by technical barriers, including limited access to materials with the appropriate isotope labeling and by incomplete methodology to define conformational heterogeneity and composition. Oscar Millet et al. summarize how advances in sample preparation and NMR spectroscopy are combining to provide an increasingly powerful toolbox to quantify glycoforms, determine the preferred conformation of sugars in solution, and define protein-glycan interactions. Because NMR provides time-averaged conformations in solution, it is well-matched with molecular dynamics (MD) calculations. Topically, MD has been used to determine the extent to which glycan microheterogeneity impacts the antigenicity of the SARS-CoV-2 virus spike (S) glycoprotein. Moving beyond the molecular level, glycoproteomics allows surveillance and identification of carbohydrates at the scale of the proteome, primarily through the application of mass spectrometry. Thomas and Scott describe new technologies, enrichment systems, and analysis strategies in this rapidly advancing field. When applied to mucin-O-glycosylation, O-GlcNAc glycosylation, and N-linked glycosylation these approaches now enable the identification/quantification of hundreds and even thousands of glycosylation sites in a single experiment supporting the use of glycoproteomics to identify system-level glycoproteome changes that occur during disease.

Glycans and their glycoconjugates are not static species; they are built and degraded by a complex repertoire of enzymes that have evolved to process the diverse structures in our glycome. An especially complex environment exists within our gut where more than 1 trillion bacteria act on nondigestible polysaccharides, mainly from plant-based foods in our diets. Tamura and Brumer provide an authoritative review on glycan utilization systems of gut bacteria. These systems have proven to be a veritable gold mine for the discovery of carbohydrate-active enzymes and have led to new structural discoveries in how nature processes glycans. One of the (many) reasons why glycoproteins bear glycans is to act as quality control and traffic signals to choreograph the folding, trafficking, and degradation of glycoproteins in the cell. Studies of this fascinating system are delivering new insights and the review by Kuribara and Totani describes how innovative chemical approaches are being used to uncover intricate details of these systems.

The O-linked N-acetylglucosamine (O-GlcNAc) modification is the most abundant intracellular protein glycosylation and dynamically regulates processes such as transcription, translation, and signaling. Aberrant O-GlcNAc modifications have been detected in cancer, diabetes, Alzheimer’s, and other diseases. However, our understanding of the precise roles of O-GlcNAc has been hampered by several challenges, including the fact that O-GlcNAc modification on thousands of proteins is dynamically regulated by just two opposing enzymes in humans (O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA)), and the paucity of tools to probe O-GlcNAc functions in a more specific manner. In the last few years, significant efforts have been devoted to tackling these challenges and led to a series of exciting new discoveries. In this issue, Pratt et al. review enzymatic and chemical methods for synthetically ‘encoding’ the O-GlcNAc modification into peptides and proteins that in turn enable decoding of the site-specific effects of protein O-GlcNAc modification for structural and functional studies, and even can be exploited to improve the therapeutic potential of non-natively O-GlcNAc modified peptides. Vocadlo et al. beautifully highlight recent advances in innovative assays for high-throughput screening of OGT and OGA modulators, as well as new developments of inhibitors for more effective and specific targeting of O-GlcNAc cycling enzymes in vitro and in cells. In addition to serving as powerful tools to dissect O-GlcNAc functions, several OGA inhibitors have entered into translational preclinical or clinical stages as potential therapeutic or diagnostic agents. These exciting advances accelerate molecular understanding of O-GlcNAc functions in physiology and pathology and should bring significant therapeutic benefits. Interestingly, there are two OGT-like genes in the model plant Arabidopsis thaliana: SECRET AGENT (SEC) and SPINDLY (SPY), and they are predicted to share similar 3D protein structures with human OGT. However, only SEC can catalyze O-GlcNAc transfer to modify plant proteins, while the enzymatic activity of SPY remained a mystery until recently. Tai-ping Sun describes the unexpected discovery and characterization of the O-fucosyltransferase function of SPY, revealing an important role of this unique nucleocytoplasmic enzyme in regulating diverse plant developmental processes.

In pathogenic bacteria, glycosylation allows adhesion to host cells and manipulation of eukaryotic host function. Hyun-Soo Cho highlights recently discovered bacterial glycosylation with unexpected glycosidic linkages, including tyrosine and arginine side chains. Structural and mechanistic studies provide fundamental insights into the glycosylation on the atypical acceptor amino acids of host proteins and will facilitate the development of new drugs targeting the glycosylating bacterial pathogens. Glycans play important roles in mediating host–pathogen interactions yet research has been mainly focused on pathogen glycans. Qin and Mahal outline the latest findings in the host glycome showing that it is dynamically responsive to infection. Mechanistic studies are uncovering a wealth of implications in host immunity, leading to a scientific revolution in this field. New method developments have been fundamental to enable glycome analyses. Vakhrushev et al. summarise quantitative strategies in the analysis of O-GalNAc type of glycosylation that is important in a broad range of biological samples and glycoprotein therapeutics.

Huge strides have been made in our ability to map proteins, through the now mature technique of single-crystal protein X-ray crystallography, and the rapidly advancing technology of high-resolution cryo-EM. Bai and Li review how cryo-EM is driving breakthroughs in the study of large-membrane-bound glycosyltransferases of the GT-C superfamily that have been challenging to solve by using NMR or X-ray protein crystallography. These enzymes use lipid-linked donor substrates to build N-linked glycans in eukaryotes, O-linked mannans in yeast, and complex cell wall glycans in mycobacteria. These structures have provided new mechanistic insights and greatly enriched our understanding of this important family of glycosyltransferases. The peptidoglycan layer in the cell wall of gram-positive bacterial pathogens such as Staphylococcus aureus is permeated with anionic glycopolymers known as wall teichoic acids (WTAs) and lipoteichoic acids (LTAs). Stehle et al. describe how X-ray crystallography has allowed the determination of the structures of several glycosyltransferases that help build these phosphoglycans, and how they recognize their substrates. Glycosyltransferases act cooperatively to build the defining structure of bacteria, the cell wall. Liston and Willis review how glycoconjugates are assembled in gramnegative and gram-positive bacteria to build the wall. In this area, structural biology has cast light on the structural basis of initial glycosyltransferase steps in the cytoplasm, the transport of lipid-linked glycoconjugates across the inner membrane, the polymerization steps in the periplasm, and the export of large polymeric glycoconjugates from the cell into the wall.

From the perspective of the current day, the Age of Discovery can be viewed as a period when technological advances and geographic discoveries laid an early foundation for the globalized world in which we live today. Likewise, the new discoveries reviewed in this issue will broaden our appreciation for the world of biology and support efforts to improve human health.

Biography

Jiaoyang Jiang obtained her Ph.D. degree in Chemistry under the supervision of Prof. David Cane at Brown University in 2009. Following that, she performed postdoctoral research at Harvard Medical School working with Prof. Suzanne Walker. Jiang started her appointment as an Assistant Professor at the University of Wisconsin–Madison in 2013 and became Associate Professor in 2019. Her group is interested in the substrate recognition and functional regulation of protein glycosylation in physiological and pathological processes.

Spencer Williams graduated from the University of Western Australia with a BSc(Hons) (1994) and studied for his PhD (1998) in Carbohydrate Chemistry at the same institution with Prof Robert (Bob) Stick. He received postdoctoral training in the laboratories of Prof Stephen Withers at the University of British Columbia and Prof Carolyn Bertozzi at the University of California at Berkeley, where he gained a solid grounding in enzyme mechanism and glycobiology. He was appointed in 2002 at the University of Melbourne, where he is now a Professor of Chemistry. His interests include carbohydrate chemistry and biochemistry, pathways of carbohydrate metabolism, medicinal chemistry, enzyme mechanism, and glycoimmunology.