Glycans are an underappreciated fourth class of macromolecules, which have recently been recognized as key players in almost all cellular processes. These sugar polymers are not static architectural decorations; rather, they serve as actively transcribed information molecules that tune immune responses, drive developmental patterning and spatiotemporally regulate protein function in response to environmental signals. Unlike nucleic acids or proteins, glycans are not synthesized using a template strand but rather are assembled by a complex, branched non-template driven biosynthetic process that is mediated by numerous enzymes. Glycan biosynthesis can produce a combinatorial array of branched and linear structures. This combinatorial diversity in glycan structures means that glycans can encode information at a higher density than a peptide of a similar size. As such, the cell surface is covered with a combinatorial "glyco-code" that can be sensed by receptors on the cell surface of neighboring cells, pathogens or soluble factors. As a result, glycobiology is at the forefront of research in the fields of immunology, cancer biology and stem cell biology, as many of these processes are found to be affected by small changes in glycan expression.
The glycome (the full complement of glycans made by a cell or organism) represents a dynamic language that converts physiological status into molecular syntax. The glycan structures that result from this language, which result from the integration of signals from metabolic flux, epigenetic circuitry, and stress-activated kinases, function as read-out biosensors of the intracellular and extracellular milieus. In multicellular settings, these glycan-displaying glycoproteins, glycolipids, and proteoglycans form tissue- and cell-type-specific "glycopatterns" that organize morphogen gradients, synaptic specificity, and angiogenic sprouting. Perturbation of glycopatterns by inherited defects in glycogenes, or acquired changes in nutrient milieu, can lead to a range of pathological phenotypes from congenital muscular dystrophy to metastatic progression. Glycans, therefore, are not mere accessory adornments, but represent an evolutionarily ancient regulatory layer that couples extrinsic cues to intrinsic reprogramming.
On a chemical level, a glycan is defined as a polymer composed of monosaccharide monomers that are linked together by glycosidic bonds. A monomer can be thought of as a poly-hydroxy carbonyl compound, which cyclises to a five- or six-membered hemiacetal, which then provides a reactive anomeric carbon, with α- or β-stereochemistry. The rings can then be linked through any of the hydroxyl groups. This can give rise to linear or branched chains, with the reducing end of the chain retaining a free hemiacetal and the non-reducing termini being capped. This potential for branching allows glycans to differ from nucleic acids and proteins in that a single hexamer may code for thousands of possible topological isomers. If the glycans are covalently attached to either proteins (N- or O-glycosylation) or lipids, it further increases this repertoire, as these result in hybrid molecules with glycan moieties that modulate folding, stability, half-life and receptor interactions.
The monosaccharide single sugars which are the monomers of glycans are in solution present as an equilibrium mixture of open-chain and cyclic hemiacetal forms (usually the cyclic form is the major one). Substituent effects along with temperature and ionic strength determine furanose and pyranose ratios within oligosaccharides which allows the glycan "face" available for enzyme action to adjust according to physiological conditions. Derivatized forms of common monosaccharides (amino sugars, uronic acids, deoxy sugars, etc.) serve to increase the diversity of potential chemical interactions with charged, hydrophobic or reactive groups, either by serving as recognition moieties or points of further post-polymerization modification.
The simplest glycans are disaccharides, which may be involved in metabolism as well as function as chemical signals. For instance, lactose, Galβ1,4Glc, is a reducing disaccharide, as the glucose terminus can freely interconvert between a cyclic form and an aldehyde, and may form glycation adducts by Maillard reaction in hyperglycemic stress. In contrast, sucrose, Glcα1,2βFru, is a non-reducing disaccharide (it has no free anomeric center), which chemically is much more stable, so it is used by plants as the main transport form of sugar. In addition to their nutritional functions, certain disaccharide motifs have been co-opted for other specific functions: for example, the human milk oligosaccharide 2'-fucosyllactose (Fucα1,2Galβ1,4Glc) can selectively promote growth of certain gut bifidobacteria and inhibit binding of pathogenic bacteria, and so very small glycan fragments can serve to control microbiome populations.
Longer oligosaccharides and polysaccharides are often found as insoluble structural polymers and information-rich ligands. Linear homopolymers, such as cellulose or chitin, can form fibers, cross-linked sheets or extended arrays that form tensile structures in plant cell walls or arthropod exoskeletons. Glycans with heteropolymeric backbones and periodic side chains, on the other hand, form hydrated gels that lubricate joints, and osmotic buffers. Branched N-glycans on membrane proteins form a glycocalyx that can serve simultaneously as kinetic barrier, mechanical sensor, and signaling platform, all within a single molecular layer. The combinatorial complexity increases dramatically with the number of monosaccharides, so that for instance a hexasaccharide has many more possible conformations than a hexapeptide, allowing a basis for high specificity without genetic number explosion.
The glycan structure does not follow a one-to-one script encoded in DNA but a set of permissive rules read by the shape of the catalytic pockets of glycosyl-transferases and the topography of the secretory organelles. Each sugar substrate is only added after being coupled to an appropriate carrier molecule, that is, as a nucleotide-sugar donor. The fate of each new sugar, as peripheral capping residue or side-branch progenitor, then depends on the local relative concentration of the alternative enzymes, the steric orientation of the acceptor, and the physicochemical environment within the Golgi cisternae. Glycan structures are a time-averaged property then, a time-integrated view of a kinetic flux rather than a solid object. The same peptide backbone can, therefore, show a diversity of glycoforms, each of which has a distinct charge, hydrodynamic radius, and lectin binding specificity just enough to qualify the same protein for multiple signaling pathways without any change to the amino-acid sequence. Structural plasticity also provides glycans with a degree of regulatory agility not seen in nucleic acids and proteins. They can be extended or trimmed, sulfated or acetylated, within minutes of a stimulus. Glycans provide a layer of post-translational code whose dynamic interpretation of environmental changes can have direct biochemical consequences. In addition to the covalent framework, glycans also exhibit higher-order conformational biases not dictated by the rules of secondary-structure elements but by stereo-electronic constraints. Exo-anomeric effects stabilize certain rotamers around the glycosidic oxygen. Steric repulsion between two axial substituents can force an otherwise extended chain to fold back on itself into a hairpin turn. Such local folds, rather than being a structural curiosity, can put hydroxyl groups in the right orientation for transient hydrogen-bonding with peptide loops to stabilize otherwise metastable protein conformations or shield protease recognition sites. In a membrane, the collective orientation of glycan dipoles can also create an electrostatic cloud that modulates cation partitioning and lateral diffusion of receptors. Glycan structure, therefore, needs to be treated not as a static pose but as a time-averaged ensemble whose population is constantly tuned by the metabolic state of the cell and mechanical stresses from the surrounding tissue.
Schematic representation of major classes of glycans found on mammalian cell surfaces.1,5
The specific glycosidic linkage has a stereochemical influence on the resulting bond rotation and hydrolytic stability of the bond as well as on the recognition profile of the whole glycan. An α-anomeric glycosidic oxygen (anti-orientation to C2 substituent) will often project the chain away from the protein backbone to avoid steric clash and to not interfere with the overall globular structure of the protein carrier. In contrast, a β-glycosidic linkage, especially when attached to serine or threonine, will keep the sugar closer to the protein surface which in turn allows close CH–π interactions that can lock local protein loops or cover kinase recognition motifs. The linkage position (e.g., 1→3, 1→4, or 1→6) further impacts the spatial arrangement of the chain with 1→4 linkages preferring an extended ribbon-like conformation that can be stabilized by hydrogen bonds between residues while 1→6 linkages contain a rotatable methylene spacer that often leads to globular clusters which are recognized by specific lectin families. These conformational preferences are further reinforced or overruled by other modifications: for example, the addition of a core fucose to N-glycans biases the whole antennae cluster into a more globular conformation. In turn, the underlying galactoses are less accessible to sialyltransferases, shifting the balance towards pro-inflammatory ligands. The glycosidic oxygen is also a center of stereoelectronic influence: the exo-anomeric effect favors gauche rotamers around the C1–O bond. On the other hand, so-called "rabbit-ear" lone-pair repulsion is unfavorable when the glycosidic bond is syn-periplanar with the adjacent oxygen. These quantum-mechanical effects are ultimately manifested as differences in bond length and vibration frequency that can be exploited by infrared or Raman spectroscopy to make inferences on linkage geometry in a native environment. The same linkage, which is kinetically stable at neutral pH of the extracellular space, can be susceptible to hydrolysis by glycoside hydrolases after the lowering of pH in the vesicles, emphasizing that this is a conditional bond, a switch, rather than a permanent weld.
Simple glycans (strings of repeating mono- or disaccharide units) can play structural or osmotic functions, but even these can possess nuanced strategies in their stereochemistry. The polysaccharide cellulose (a homopolymer of glucose units linked β1→4) uses cooperative, extended conformations of its pyranose rings to form semi-crystalline microfibrils, tensile in strength on a per-weight basis to that of steel. The linear (no branching) and diequatorial stereochemistry of each linkage allows maximal hydrogen-bonding between chains, creating a strong yet hydrated scaffold. In amylose, the backbone of α1→4-linked glucose units assumes a helical pitch that allows the hydrophobic collapse of inner residues, providing a binding pocket for iodine. This conformational ruse enables plants to store glucose as a dense, osmotically inactive mass. Thus, even "simple" glycans can use stereochemical information to signal mechanical or storage properties without the aid of heteromeric diversity. In complex glycans, on the other hand, this redundancy is used as an organizational principle. N-glycans can be initiated from a conserved trimannosyl-chitobiose platform but then proceed to diverge into hybrid, high-mannose, and multi-antennary types whose terminal compositions can be sialic acid, fucose, sulfate, or phosphate in various combinations. Branching thus expands the information storage capacity of a given chain length: Cells can create multiple distinct epitopes from one core structure by placing monosaccharide residues in various topological positions instead of only adding more monosaccharides. The biological cost/benefit of this increase in complexity is clear in the immune system: the presence of a single complex N-glycan on the Fc region of an antibody can modulate pro- vs anti-inflammatory signaling depending on whether α2,6- or α2,3-linked sialic acid is on its outer branches. Complexity, therefore, is not merely structural decoration but a regulatory grammar that enables a limited biosynthetic alphabet to generate a large repertoire of functional sentences.
The ultimate source of microheterogeneity is the natural variability of glycosidic bonding. In marked contrast to the peptide bond, which rigidly samples the single trans, planar conformation, each glycosidic linkage randomly samples a large conformational space that is defined by the two torsion angles (φ and ψ) about the C1–O and O–Cx bonds. Bulkiness of substituents, exo-anomeric effects, and even remote side groups shift the energy landscape of these angles such that a given pair of monosaccharides can occupy different low-energy rotamers when attached to different oligosaccharides. This conformational polymorphism can have direct functional effects: an extended disaccharide in solution may adopt a hairpin conformation once it is presented on the surface of a protein, thereby repositioning its hydroxyl groups for transient recognition by a lectin module. The rate of hydrolysis of a given bond is also exquisitely sensitive to these small conformational effects. A single modification from N-acetyl to N-glycolyl on the C2 substituent is enough to shift the transition-state energy barrier such that the linkage is no longer cleaved by a viral neuraminidase, leading to a mechanism for host-species restriction. Bond properties can also be altered by the environment. Divalent cations can coordinate with adjacent oxygens, thereby biasing the conformational ensemble toward more compact geometries that are higher in energy but become locally preferred. Temperature has a similar effect: high temperature shifts the Boltzmann distribution to populate more energetic rotamers, transiently unmasking otherwise hidden epitopes that can be probed by antibodies or lectins. The simple shear force generated at an endothelial surface is also enough to stretch out a GAG chain, thereby redefining the preferred dihedrals for 1→4 linkages and modulating the binding of chemokines. Glycosidic bond variability can thus be thought of as a point where chemistry and physics intersect, a tunable covalent linkage that can translate the thermal, ionic, and mechanical state of its environment into biochemical information without changing the underlying sequence.
A code that translates information in the extracellular environment into cellular responses. Glycans provide a highly flexible, non-genetic molecular interface by which extracellular signals can be transduced into intracellular responses. The flow of information can be encoded by an aspect as subtle as the structure of a single sugar monomer. Non-template glycan biosynthesis gives a single polypeptide the potential to be glycosylated in multiple patterns in response to distinct physiological conditions. Changes in branching or density, charge sequence or topology, or link position or angle can tune the half-life, avidity for a receptor, and even the phase behavior of a signaling complex. Glycans occupy a key position at the nexus of cellular regulation because they can function as chaperones, trafficking zip-codes, or passwords for immune activation. The repertoire of glycans can change on the time scale of hours to days in response to metabolic state, mechanical stress, or circadian rhythm, and so serve as an information source continuously replenished over an organism's lifetime to complement the more static information encoded in the genome. Glycans also contribute to, and often direct, the physicochemical environment of membranes and secreted fluids. The hydroxyl and carboxyl moieties of glycans can organize water structure to alter the diffusion rate of morphogens, clustering of receptors, or the energy barrier to phase separation of RNA–protein granules. In tissues, gradients of sulfated glycosaminoglycans can generate an electrostatic field that directs migration or locally concentrate growth factors in space and time. As such, glycans serve as structural fillers, signaling antennae, and metabolic buffers that are integrated into molecular tapestries that transduce biochemical, biomechanical, and bioelectrical information into biological responses.
A plethora of folding sensors await newly synthesized polypeptides in the ER lumen and folding states modulate their activity. Attachment of a preformed oligosaccharide to specific Asn side chains serves as a timer to start the quality-control clock: if the glycoprotein folds into its native structure swiftly, processing enzymes trim particular mannose markers and clear the molecule for transport. Chronic exposure of those mannose residues, however, leads to binding by lectin chaperones, sequestering the molecule for repeated attempts at refolding or marking it for degradation. The glycan acts as a sensor that differentiates between correctly folded proteins and aggregation-prone species, buffering the fidelity of the entire secretory pathway. In addition, the sheer volume of the glycan may also scaffold vulnerable regions against thermal fluctuations, reducing the entropy cost of folding and potentially enhancing resistance to proteases upon secretion. O-glycosylation in the cytosol provides a related mechanism of conformational regulation. Addition and removal of a single N-acetylglucosamine from Ser or Thr side chains directly competes with phosphorylation and serves as a binary switch between a flexible or rigid state. Modification of intrinsically disordered regions in this way can either drive liquid–liquid phase separation or inhibit pathological fibrillization, depending on the adjacent amino-acid sequence. Glycans can therefore serve as reversible allosteric ligands that rewire the energy landscape of their protein targets, tuning catalytic rates, recognition interfaces, and degradation rates in lieu of permanent covalent modification.
Fig. 2 Biological functions of glycosylation.2,5
Patterns of cell-surface glycans form an updatable barcode that advertises the differentiation status, metabolic activity, or stress history of a cell to patrolling lectins on adjacent cells. The branching may seem stochastic but is highly regulated: cytokines, mechanical stress, or diurnal hormones dial up or down the expression of glycosyltransferases, changing the glycan code within hours. Since the same lectin can bind several glycotopes with different affinities, the result of the interaction (activation, inhibition, or apoptosis) is dictated by the local concentration and nanoscale clustering of its ligands. This avidity-based logic enables glycans to serve as contextual filters that transduce graded extracellular cues into digital cellular responses, performing the function of an analog-to-digital converter at the cell surface. In development, for example, the ephemeral expression of fucosylated or sialylated motifs serve as molecular time-stamps that direct axon guidance or angiogenic branching. Selectins on endothelial cells only roll leukocytes along the vasculature when inflammatory cytokines expose the correct glycan password, ensuring spatially- and temporally-limited immune infiltration. Even the fate of stem-cells can be biased by glycan cues; a shift from high-mannose to complex N-glycans can bias stem-cell differentiation by altering the residence time of growth-factor receptors within lipid rafts. Glycans are thus an instructive code that converts extracellular context into intracellular instruction, in parallel to receptor tyrosine kinase signaling.
Immune cells interpret the glycan landscape to navigate homeostasis and danger. C-type lectin family pattern-recognition receptors (PRRs) have curved binding sites that have evolved to bind unique topologies of carbohydrates presented by fungal, bacterial or viral pathogens. This recognition event elicits phagocytosis or cytokine secretion, only if the PRR recognizes the pathogen-associated pattern at a certain density, to avoid attacking host glycans that may also present similar epitopes. In turn, sialylated host glycans can bind inhibitory Siglecs to provide a "do-not-eat" signal that increases the threshold for activation and limits bystander damage. Thus, the interplay of activating and tolerogenic glycan signals is crucial; a shift in this balance towards low sialylation or altered fucosylation can lead to chronic inflammation or autoimmunity. Antibody effector functions are also regulated by glycans. The structure of the Fc glycan determines the antibody's capacity for complement fixation or a more subdued anti-inflammatory function. Afucosylated antibodies bind activating Fc receptors with increased affinity, leading to potent natural-killer-cell degranulation against infected or tumor cells. In contrast, core-fucosylated antibodies bind inhibitory receptors, providing a molecular rheostat to prevent immune over-exuberance. Since the glycan profile of antibodies can be modulated by metabolic state or cytokine environment, the humoral immune system has a built-in feedback mechanism: the same antigen can lead to qualitatively different effector outcomes depending on the host's physiology. In this way, glycans serve as the immune system's internal editor, fine-tuning the tone and duration of responses to ensure protection without excessive injury.
Glycans are a metabolic palimpsest, a slate upon which the dynamics of physiological homeostasis and disease progression are etched in turn. In the healthy state, they cycle within a range that stabilizes membranes, calibrates receptors, and silences immune responses. But that range is also finely poised on a knife-edge of nutrient availability, inflammatory state, and oxidative stress. Sustained pressure from those sources will drive the glycome into new territory—shortened O-glycans, overly branched N-glycans, or hidden fucosylations that can no longer maintain cooperative cell–cell communication. Instead, they will sanction chronic signaling, immune suppression, and tissue repair that paves the way for disease. Crucially, this shift is often not a mere downstream effect of pathology. It may precede disease symptoms, acting as an early molecular readout of micro-environmental change. In this way, the glycome is both a recorder and a propagator of disease progression, providing a dynamic snapshot of health-to-disease trajectories that can be read long before organ failure sets in. And just as important, glycan changes are often reversible. Glycosylation is enzymatically malleable, and dietary changes, microbial metabolites, or pharmacological chaperones can re-calibrate glycan-processing enzymes to nudge the landscape back towards a health-compatible state. In this way, glycans are a promising target for preventive interventions that seek to restore immunological tolerance or limit tumor fitness. Overall, glycans are a two-way interface: they log the biochemical history of environmental exposures and, at the same time, offer a tractable substrate for corrective modulation, thus closing the conceptual loop between biomarker discovery and therapeutic intervention.
The glycan costumes of cancer cells are rewired to satisfy a multivariate optimization problem, composed of requirements for cell division, growth, immune evasion and metastasis. A common strategy is to truncate O-glycan extension early, giving rise to the cryptic antigens Tn and sialyl-Tn (see below). These simple structures can be densely displayed on mucins where they sterically inhibit maturation of immunological synapses while also promoting integrin binding to endothelial cells. Malignant cells may also increase expression of glycosyltransferases that add extra branches to N-glycans, thus expanding the binding surface for growth factor lectins and strengthening their survival signals. Core fucosylation can also be increased to enhance binding affinity of cytokine receptors, thereby reducing the amount of ligand needed for oncogenic activation. Crucially, these phenotypes are not epiphenomenal; knockdown of the glycosyltransferases responsible for these changes reverses the transformed phenotype in cell culture and reduces tumour formation in animal models, indicating a causal contribution of glycan remodeling to cancer progression. In the case of infectious agents, altered glycosylation of the host cell is often repurposed for entry and camouflage. Enveloped viruses like HIV-1 add an abnormally dense layer of oligomannose glycans (synthesized by the host cell) onto their surface proteins. This glycan shield hides conserved peptide epitopes that might be otherwise recognized by neutralizing antibodies, instead forcing the immune system to target more variable regions which can mutate to escape recognition. Bacteria use a different but related approach, often secreting glycosidases that trim glycans of the host cell to reveal otherwise hidden receptors that are needed for colonization. By modifying the local glycan landscape, microbes can create a nutrient-rich microenvironment for themselves while simultaneously inactivating immune lectins that depend on certain carbohydrate structures for pathogen recognition.
The dynamic nature of glycosylation to environmental changes, or, in the context of this review, disease-associated reprogramming, renders glycans highly attractive for monitoring disease development. Glycan antigens are typically readily accessible as shed or secreted species in biofluids, such as saliva, urine, or serum. In addition, since many glycan-epitopes that have altered abundances in disease are rarely to absent in healthy individuals, one can use orthogonal approaches to interrogate for these neo-antigens, such as the bisecting GlcNAc branch in neurodegeneration, or hypogalactosylated IgG Fc in autoimmunity, generating complex molecular fingerprints that can be used not only to indicate disease on-off, but even to phenotype patients according to subtypes with different therapeutic targets. Thus, measuring the levels of core-fucosylated versus afucosylated glycoforms of a given acute-phase protein, for instance, may be used to stratify inflammatory diseases into subtypes with differential responses to biologic treatment. In addition, glycan biomarkers may be used to dynamically track responses to therapy and to monitor for relapse, since glycans typically have a half-life in the range of hours as opposed to days. In particular, measurement of changes in levels of sialyl-Lewis antigens in circulation, has been shown to pre-date clinical or radiological evidence of metastatic tumor relapse, and thus may offer the opportunity to intervene early. In autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis, the levels of IgG Fc-glycosylation has also been shown to reflect disease activity and even precede clinical disease flare. Thus, by incorporating glycan signatures with additional metadata, there is the potential to use glycan dynamics to inform the intensity of personalized treatment.
From chemical glue to homeostasis regulation, glycans stand out as the most versatile language of the cell. Dynamic and responsive, glycans can completely change their alphabet within minutes, yet preserve evolutionary information across eons. Glycosylation, in contrast to linear protein synthesis, is not a sequential process: A given polypeptide chain can send out many, even competing, signals simply by donning different glycan arrays. Glycans serve not only as tags for protein folding and interaction, but also as docking codes that govern access to the immune system, and as dimmers that fine-tune biological signaling based on nutrient availability or physical forces. Glycosylation is also a dynamic, reversible process: Glycan modifications can be wiped out or restored, potentially giving the cell a rewritable memory buffer that shields organisms from environmental fluctuations. As a result, glycans provide a dynamic substrate where the bookkeeping of health and disease is written in real-time, and glycobiology is fast becoming a core field rather than a niche sub-discipline in life science. We are at a tipping point. The conceptual shift of glycans from simple sugar decorations to central players in biology has been achieved, but the translational shift to therapeutic applications, bio-based materials, and biofuels has been patchy at best. No longer is a lack of awareness the most pressing issue, but rather the lack of broadly accepted standards for glycan annotation, consistent analytical methods, and open-source platforms for federating datasets. Closing these gaps will help glycoscience move away from being a craft field with a few artisan labs around the world, and towards becoming an industrial-scale discipline that can be routinely implemented in clinics, biorefineries, and even undergraduate teaching.
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