Glycans function in protein folding, trafficking, and stability, and can directly or indirectly affect protein function. First, glycans serve as molecular quality control signals to promote protein folding, including serving as a holdase to prevent aggregation and targeting misfolded proteins for degradation. Glycosylation can also promote thermodynamic stability by sterically shielding against proteases and by affecting conformational entropy. In its functions, glycosylation has the ability to regulate specificity and affinity of cell signaling. This is accomplished by regulating ligand-receptor binding, as well as clustering of the receptors into membrane microdomains. Glycosylation defects have been linked to cancer, and human diseases, like Alzheimer's disease, where altered glycosylation can influence protein stability and function.
Glycosylation is a post-translational modification that results in the covalent attachment of glycans (oligosaccharides) to proteins. It has evolved as a biological strategy to extend the information encoded by the genome. Within the secretory pathway glycosylation takes place where N-linked glycosylation starts in the endoplasmic reticulum (ER) by attaching a preformed oligosaccharide to a new protein and O-linked glycosylation initiates in the Golgi apparatus through sequential monosaccharide addition to the protein. Glycosylation utilizes the energy of nucleotide sugars to drive the transfer of monosaccharides to acceptor proteins by glycosyltransferases. It is frequently critical to protein function, as suggested by the participation of glycoproteins in such varied biological processes as immune recognition, adhesion, and signaling. Glycans can modify the protein folding landscape and serve as a recognition element for cellular quality control.
Fig.1 Central dogma of molecular biology and different forms of post-translational modifications.1,5
Proteins are heteropolymers whose function is strongly linked to their structure on multiple scales. The order of amino acids in a protein establishes its secondary structure by determining the local conformation of polypeptide segments and shapes its tertiary structure through long-range amino acid interactions that form a three-dimensional structure. The tertiary structure of proteins is responsible for most of the functional diversity of proteins, as it leads to 3D domains which can perform specific catalytic, recognition and scaffolding functions. The quaternary structure, the way in which polypeptides come together to form oligomeric assemblies, provides additional functional diversity. Variation in structure is thus strongly linked to functional variation. The reason for this is that small changes in the conformation of a protein can result in large changes in its function. Proteins are also often subject to post-translational modifications (PTMs), chemical modifications that can also affect their structure and properties. Glycosylation, one type of PTM, can influence a protein's solubility, net charge, and hydrodynamic volume. This mechanism enables proteins to shield hydrophobic patches or prevent aggregation-prone regions from forming while simultaneously revealing new binding surfaces for different purposes.
Glycosylation can increase protein stability through several cooperative mechanisms. N-linked glycans are folding scaffolds that recruit chaperones such as the calnexin/calreticulin cycle that both prevent aggregation and promote correct disulfide bond formation. Glycans can sterically block proteases to increase the half-lives of glycoproteins. They can also protect against denaturation by forcing the hydration shell of the protein into a more ordered state, giving the protein additional stability. This protection has been demonstrated to be especially crucial for secreted glycoproteins, as they are more susceptible to changes in their environment. Glycosylation can also serve as a degradation tag in quality control mechanisms in which if the protein is not folded properly the glycan can target it for ER-associated degradation. The glycan attached to a particular residue can also restrict conformational entropy of the region, such as a flexible loop. In certain circumstances, glycosylation can be destabilizing if the glycan distorts the protein conformation by disrupting long-range native contacts.
Glycosylation has a great effect on folding pathways. Glycosylation in the ER is a folding sensor, which determines the fates of the folded or degraded proteins. When a polypeptide enters the ER lumen, the N-linked glycans on the polypeptide are recognition signals for the calnexin/calreticulin chaperone system. The glycoprotein will be cycled through a series of refolding, reglucosylation, and so on, until it attains its native folded structure. Glycosylation can also be used to connect the thermodynamics of protein folding to protein homeostasis. Glycoproteins that are terminally misfolded are segregated away from the folding machinery and instead targeted for degradation by the ERAD system. Glycosylation also plays a role in the kinetics of protein folding. It has been reported that the bulky glycans sterically protect aggregation-prone hydrophobic residues from interacting with each other. Glycosylation also has a role in chaperoning folding enzymes that assist the folding process by catalyzing disulfide formation and proline isomerization. A dysregulation of the glycosylation machinery is known to lead to misfolded protein aggregation that is seen in a number of protein folding diseases.
Carbohydrates are known to play an important role in protein folding by recruiting ER-resident chaperones to the folding glycoprotein. The addition of N-linked oligosaccharides is co-translational and the preassembled oligosaccharide, after some processing, is trimmed to yield a monoglucosylated glycan that serves as the docking signal for the lectin chaperones calnexin and calreticulin. The carbohydrate recognition domains of these proteins bind the glycoprotein substrates while the P-domains are directly involved in the recruitment of proline isomerases and disulfide-forming enzymes. These P-domains, which are rich in proline, are thus able to target the various enzymatic activities involved in disulfide bond formation and peptidyl-prolyl isomerization to specific protein substrates. Calnexin and calreticulin also prevent aggregation of the substrate by masking hydrophobic regions of the polypeptide while they are bound. UDP-glucose glycoprotein glucosyltransferase (UGGT) binds to glycoproteins with incorrectly formed disulfide bonds and reglucosylates the glycan, targeting it for another round of binding to calnexin. Calnexin therefore also acts as a folding sensor. As soon as the folding process is complete and correct, the glycoprotein is no longer recognized by calnexin and its function is served. The glycan thus has two roles. It acts as a retention signal which prevents glycoproteins from progressing forward in the secretory pathway and it is also a marker for quality control, allowing only properly folded proteins to be secreted.
Glycoproteins that repeatedly fail to fold adopt folding-enhancing glycosylation that is replaced by degradation-enhancing glycosylation. Terminal mannose residues are removed from the core glycan by slowly acting mannosidases. The resulting glycan is recognized by lectins in the ER that target terminally misfolded substrates for ubiquitination and subsequent degradation by the ER-associated degradation pathway. The misfolded glycoproteins are not allowed to accumulate. Failure of ER-associated degradation is one cause of conformational diseases. Proteins that have a propensity to aggregate (amyloidogenic proteins) are glycosylated in neurodegenerative diseases, which then hinders quality control. This results in ER stress and loss of proteostasis. Viruses rely on host glycosylation for folding of viral glycoproteins. Misfolded viral proteins can initiate an immune response, as well as act as a driving force of viral adaptation. ER-to-lysosome-associated degradation pathway and ER-phagy serve as an alternative clearance for aggregated glycoproteins which are above proteasomal capacity, and calnexin shuttles such recalcitrant glycoproteins towards lysosomal compartments. For this reason, defects in glycosylation are also a feature of diseases associated with protein misfolding, where anomalous glycan processing both signifies and contributes to proteostatic collapse.
Post-translational glycosylation can alter a protein's physicochemical features in order to influence its activity. For example, the addition of oligosaccharide residues can directly affect a protein's catalytic function, substrate recognition or interactions with other proteins. The added volume and hydrophilicity of glycans can shift surface charge and steric hindrance, factors that may alter the binding of a substrate and its subsequent conversion to a product. For secreted proteins, the sialic acid termini of glycans can also directly alter the surface charge of the protein. Secreted glycoproteins can have their half-life in circulation affected by the terminal sialic acid on the attached glycan, since these molecules can confer both resistance to proteolytic enzymes and prevent renal filtration. The positioning of glycans in extracellular proteins (e.g. on the luminal surface of transmembrane receptors) can also affect the protein's function. In this context, attached glycans may act as antennae to bind to or sterically shield ligands. Glycosylation may also shift a protein's conformational equilibrium to favor states that restrict or enable particular protein motions that can have an indirect effect on a protein's catalytic activity by altering the geometry of the active site. A protein's glycosylation profile can even control its subcellular routing to ensure it is trafficked to the right cellular compartment. This is significant for secreted proteins such as proteases, receptors, and hormones in regards to their activity. For example, cathepsin D is a lysosomal enzyme and an aspartyl protease that is only active in the acidic lysosome. Glycosylation allows for precise control of protein function while glycosylation profiles show variation between different cell types and developmental stages.
Glycosylation is a specific kind of post-translational modification that can have significant allosteric consequences. Due to their steric demands, carbohydrate moieties can serve as potent allosteric effectors that can communicate changes in conformation over large distances. If a glycan is added to an outward facing loop or domain interface that is distant from the active site, the size of the glycan and its relative immobility can communicate that change in conformation across the protein structure, slightly shifting the positioning of amino acids in the active site. This can affect the volume and charge complementarity of the substrate binding pocket and thereby tune the Michaelis-Menten kinetics of an enzyme without directly binding to the substrate. The presence of charged sialic acids or neutral hexoses can also change the local electrostatic environment and pKa of surrounding catalytic residues, which may affect proton transfer dynamics during the reaction. Glycosylation can also act to stabilize an enzyme in a particular conformational substate by limiting domain motions and reducing the entropic cost of substrate binding. In some cases, a dynamic interplay of glycan addition and removal can provide a mechanism for rapid enzyme regulation in metabolic processes in response to changes in nutrient levels.
Glycosylation frequently serves to compartmentalize receptor activity both at the level of the ligand-receptor interaction and in subsequent signalling cascades. The glycan structures on receptor ectodomains manage the accessible space to ligands which controls the frequency of binding interactions between ligands and receptors along with the signaling thresholds. Glycosylation also can partition receptors into certain membrane domains, concentrating signaling elements. In the case of growth factor receptors, glycosylation can determine the specific orientation of receptor dimers, which affects the pattern of phosphotyrosines that are produced in the activated receptor, which in turn affects the recruitment of specific adaptor proteins. Both the receptors and ligands involved may themselves be glycosylated, raising the possibility of combinatorial glycan matching, with the positive and negative binding outcomes resulting from compatible and incompatible receptor and ligand glycans respectively. Glycosylation of intracellular receptor domains is also possible. O-GlcNAc modification of receptor proteins may directly influence receptor phosphorylation, by directly competing with kinases for serine and threonine residues. Glycosylation of G-protein-coupled receptors is also known to be a mode of regulation, where it may influence coupling to specific G-proteins as well as arrestin binding and receptor desensitization.
By the presence of glycans the interaction network can be modified. Glycosylation often provides steric hindrance that can be utilized to prevent or promote interactions. Glycosylation can also make new binding sites accessible. The glycan itself may serve as a binding site. The modification can also affect the interaction between the glycoprotein and its binding partner in terms of binding kinetics and affinities. This is often related to glycosylation sites that create a new protein surface structure that sterically hinders access to a binding site. Glycans attached to membrane proteins extend into the extracellular milieu and are important in controlling the way in which receptors interact with each other and with other proteins. Glycans can play a role in regulating transient or stable interactions by constraining the conformational dynamics of the glycoprotein such that certain domain motions are restricted while others are allowed or favored. Examples can be found in the endoplasmic reticulum quality control network and at the plasma membrane. Changes in the glycosylation status can modify the protein's interactome: in development or disease, where glycosylation is dynamically changed, some interactions can be lost and others formed, thus, affecting the protein's function.
Glycosylation can be understood as a systems-level regulator of protein interaction networks that is defined by the topology of its interactions rather than by changes in discrete interactions. As glycans concurrently modify multiple members of a protein complex, they define context-specific subnetworks whose interaction topologies are dynamically remodeled in response to cellular metabolic conditions. Network-wide changes in signaling flow can thus be effected by changes in glycan composition of receptors, adaptors, and scaffolds that modulate complex stoichiometry and stability, partitioning the glycan-dependent subnetwork into co-enhanced and co-suppressed modules. Glycosylation thus partitions the proteome into glycan-defined, functional modules that can be differentially activated to rapidly reprogram cellular responses to environmental changes, and such rewiring can occur without the need for new protein synthesis. Glycosylation also acts to define spatially restricted membrane microdomains within which interaction partners are concentrated, in effect creating specialized cellular interaction hubs with context-dependent connectivity rules. Dysregulation of glycan processing can cause such network remodeling, leading to the cancer metastasis and neurodegeneration seen when misglycosylated glycoproteins ectopically form signaling complexes.
Glycosylation provides a means of complex recognition patterns by which specificity is created by carbohydrate identity tags on the surface of proteins. Glycans serve as readable information for the recognition events, and can be deciphered by specific lectin domains of binding partners for very specific recognition that is crucial to the immune system and cell-cell recognition in general. The great variation possible from combinations of monosaccharides, their linkages and branching provides combinatorial variation enough to label thousands of different proteins. Glycan labels can thus be used for recognition of self and non-self as a means of identification and/or targeting, without triggering an autoimmune response. Recognition in a receptor-ligand interaction is by two binding partners, both of which can be glycosylated, and productive recognition necessitates that their glycans, which also need to be compatible, are oriented in a complementary fashion in three-dimensional space so as to support the interface. Binding on or off can thus be turned by a specific terminal sugar, that can be sensitive to developmental signals. Binding kinetics can be tuned by glycosylation by varying the association and dissociation rates. Glycosylation thus provides an additional layer of information, to the protein-protein interactions, in the form of carbohydrates.
Disorders of glycosylation are involved in a large number of diseases, ranging from single enzyme deficiencies to acquired conditions, in which changes in glycosylation patterns are found to be instrumental in the onset and/or progression of the disorder. In this sense, mutations, metabolic alterations, and environmental factors may interact to cause pathological conditions by altering protein glycosylation on a global level. Congenital disorders of glycosylation for example, where glycan processing is defective, have a detrimental effect on the folding and trafficking of a multitude of glycoproteins, causing multi-system effects that include neurological, hepatic and immunological symptoms. In cancer and neurodegeneration, aberrant glycosyltransferase expression can modify cell surface glycans, altering adhesion properties and deregulating adhesion controls. This in turn can lead to a number of pathological processes such as metastasis and synaptic degeneration. The glycan changes can also become characteristic of the disease, acting as a marker of the biochemical changes. In addition to this, they can have active roles in the progression of the disease. These multi-system effects highlight how a single post-translational modification can have a role in pathology, and how alteration in glycosylation could be used to correct these effects.
Fig. 2 Altered glycosylation in human disease.2,5
Congenital Disorders of Glycosylation (CDG) are a group of genetic diseases, each caused by mutations in an enzyme involved in the synthesis, modification, or transport of glycans. The fact that such mutations can cause profound effects in humans shows how critical the glycome is for normal function. In each case, hundreds of glycoproteins are no longer properly glycosylated as a result of only a partial loss in the activity of a single enzyme. Mutations are most commonly in elements of the core biosynthetic machinery, but recent reports also involve chaperone or Golgi-trafficking machinery that simultaneously impacts on more than one glycosylation pathway. In these cases, the effects on glycosylation again impact on multiple systems giving rise to broad symptoms of developmental delay, bleeding disorders, and immune deficiency as different tissues/cell types are affected by the aberrant glycosylation of different sets of glycoproteins. On a molecular level, the resulting defects in function can be traced back to a failure of the quality control machinery to properly assess the status of aberrantly folded glycoproteins leading either to retention/degradation in the ER or release of misfolded glycoproteins to the cell surface. These two mechanisms underlie the pleiotropic nature of the symptoms of CDG as each cell type will have a different susceptibility based on the glycoproteins it expresses. Investigation of these rare diseases can be very informative in that they can teach us which glycans are non-essential and which are absolutely critical for particular physiological roles.
Aberrant glycosylation on cellular proteins plays an important role in cancer progression and escape from immune detection. These changes in glycosylation patterns can create an immunosuppressive tumor microenvironment that shields the tumor cells from immune attack. For example, hyper-sialylation and aberrant fucosylation can protect tumor cells from immune clearance by engaging lectin receptors on immune cells with immunosuppressive effects. In addition to creating an immune-suppressive environment, tumor-associated glycans can promote metastasis through interactions with selectins and altering extracellular matrix (ECM) interactions. Similar to immune cells, abnormal glycosylation of tau proteins in neurons can increase the rate of aggregation in tauopathies. Aggregation is influenced by the O-GlcNAc modifications on tau, which act in competition with phosphorylation. O-GlcNAcylation of tau is negatively correlated with its hyperphosphorylation and aggregate formation. Microglia cell surface receptors, like TREM2, also depend on N-glycosylation to maintain normal surface expression and signaling capacity. These receptors are important for the clearance of apoptotic cells and amyloid-beta (A-beta), and disruption of glycan processing in microglia may contribute to neuroinflammation and neuronal damage. The link between metabolic dysfunction and aberrant glycosylation also contributes to the accumulation of A-beta and tau in AD. In response to stress, neurons undergo metabolic changes that decrease the available substrate for glycosylation, which may further contribute to proteostatic failure.
Glycosylation is an essential post-translational modification that crucially regulates protein stability and activity through mechanisms including structural support, allosteric effects, and metabolic integration. Glycosylation connects gene sequence with protein function by adding a metabolically regulated and dynamic layer of carbohydrates that can reflect the state of the cell, including its metabolic status, developmental stage, and response to stress. Stability is influenced by mechanisms such as chaperone-mediated folding in the ER, steric protection from proteases, and regulation of clearance receptors, while activity can be modulated by glycan-mediated constraints on the active site, control of receptor dimerisation, and competition with phosphorylation. In the context of disease, the importance of these regulatory mechanisms is underscored: defects in the glycosylation machinery lead to congenital disorders with often fatal outcomes, while altered glycosylation patterns in diseases such as cancer and neurodegeneration contribute to disease progression. Therapeutic strategies to modulate glycosylation are emerging, including targeting glycosyltransferases to restore normal glycan structures on tumor cells or increasing O-GlcNAcylation to prevent neurofibrillary tangle formation. The integration of glycomic analysis into clinical practice is an area of active research, with the potential to provide predictive biomarkers and real-time monitoring of therapeutic response. In conclusion, glycosylation’s influence on protein stability and activity underscores its potential as a therapeutic target. By modulating these post-translational modifications, it may be possible to reprogram disease pathways with a precision that is unachievable by directly targeting proteins themselves. This paradigm positions carbohydrates not as mere structural adornments but as active determinants of protein fate, whose regulation is vital for health.
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