Introduction to O-Glycans
O-glycans are glycosidically linked to the hydroxyl group of serine or threonine residues in proteins. They differ from N-glycans, which are attached to asparagine residues, and are typically more diverse in structure and function. O-glycans contribute to protein stability, folding, and functionality, and are involved in processes such as cellular communication, adhesion, and immune responses.
The biosynthesis of O-glycans is initiated by the attachment of a N-acetylgalactosamine (GalNAc) residue to the hydroxyl group of serine or threonine, forming the initial core structure. Subsequent elongation and modifications of this core structure lead to more complex O-glycans. Key enzymes responsible for O-glycan biosynthesis play essential roles in determining the diversity of glycan structures and their biological functions.
Core O-Glycan Structures
The initial and most common structures formed during O-glycan biosynthesis are Core 1 (T-antigen) and Core 2 O-glycans. These core structures serve as building blocks for more complex glycans, which can be further modified by the addition of sialic acid, fucose, sulfate groups, and other monosaccharides.
Core 1 O-Glycan (T-antigen)
Core 1 O-glycan, also known as the T-antigen, is one of the simplest O-glycan structures. It consists of a Galβ1-3GalNAc linkage, where GalNAc is attached to the hydroxyl group of serine or threonine, and Gal is added via a β1-3 linkage to form the core structure. Core 1 O-glycan is ubiquitous in various cell types and is involved in key physiological processes, including cell-cell adhesion and recognition, as well as modulating immune responses.
The Core 1 O-glycan structure can be further modified by sialylation or fucosylation, which impacts its functional properties and interaction with other cellular molecules.
Core 2 O-Glycan
Core 2 O-glycan is more complex than Core 1 and consists of a GlcNAcβ1-6GalNAc linkage attached to the serine or threonine residue of a protein. Core 2 structures are predominantly synthesized in tissues where high levels of O-glycosylation occur, such as the intestinal epithelium and immune cells. The addition of the GlcNAc residue in Core 2 O-glycan enhances its diversity, enabling further modifications that regulate immune function, protein folding, and the cell cycle.
Unlike Core 1, Core 2 O-glycans play a significant role in immune modulation, particularly in the context of immune cell recognition and response. Core 2 O-glycans are often found on mucins and glycoproteins involved in immune cell signaling.
Other Core O-Glycans
Besides Core 1 and Core 2, there are additional minor core structures, such as Core 3 and Core 4 O-glycans, which are formed through different enzymatic pathways. Core 3 O-glycans consist of GlcNAcβ1-3GalNAc and are typically found in specialized glycoproteins and mucins. Core 4 O-glycans are similar to Core 3, with a β1-4 linkage between GlcNAc and GalNAc. These core structures contribute to the diversity of O-glycans and have distinct roles in cellular processes.
O-Glycan structures Core 1-8. Core 1 and 2 are the most abundant out of the eight identified mammalian Core structures (Watson et al., 2015).
Key Enzymes in O-Glycan Biosynthesis
The biosynthesis of O-glycans is a highly coordinated process that involves a variety of glycosyltransferases and glycosidases. These enzymes control the addition of monosaccharides to growing glycan chains and the modification of existing glycans. Below is an overview of the most important enzymes involved in the synthesis of core 1 and core 2 O-glycans.
Core 1 β3-Galactosyltransferase (C1GalT1)
The C1GalT1 enzyme is responsible for transferring galactose (Gal) from UDP-Gal to GalNAc, forming the T-antigen (Core 1 O-glycan). This reaction is a key step in the formation of the simplest O-glycan structure and is essential for the proper functioning of many glycoproteins. C1GalT1 is expressed in a variety of tissues, with its activity regulated by both developmental and pathological signals.
Core 2 β6-N-acetylglucosaminyltransferase (C2GnT1)
The C2GnT1 enzyme catalyzes the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to the GalNAc residue of Core 1, thereby converting it into Core 2. This modification is critical for expanding the diversity of O-glycans and is required for the formation of complex glycans involved in immune cell signaling, including glycosylation patterns on mucins and cell adhesion molecules.
C2GnT1 is expressed predominantly in the gastrointestinal tract and immune cells, where its role in glycosylation is particularly vital for mucosal immunity and cellular interactions.
Other Key Transferases
In addition to C1GalT1 and C2GnT1, other glycosyltransferases contribute to the synthesis of alternative core structures and modifications of O-glycans. These include:
- Core 3 β1,3-N-acetylglucosaminyltransferase (C3GnT1): Responsible for the synthesis of Core 3 O-glycans, which are involved in mucosal protection and immune modulation.
- Core 4 β1,3-N-acetylglucosaminyltransferase (C4GnT1): Catalyzes the formation of Core 4 O-glycans, which play roles in cellular adhesion and migration.
Other enzymes involved in O-glycan biosynthesis include sialyltransferases and sulfotransferases, which add sialic acid and sulfate groups, respectively, to the growing glycan chains, thereby modifying the functional properties of the O-glycans.
Pathways of O-glycan biosynthesis (Freire-de-Lima et al., 2014).
UDP-Sugar Transporters
Transporters responsible for the delivery of nucleotide sugars to the glycosyltransferases are equally important. UDP-GalNAc and UDP-Gal transporters ensure the availability of the nucleotide sugar precursors needed for the formation of GalNAc and Gal-containing O-glycans.
O-Glycan Biosynthesis Pathways
O-glycan biosynthesis occurs through a series of enzymatic reactions that add monosaccharides sequentially to a protein-bound GalNAc residue. The two primary biosynthesis pathways that lead to Core 1 and Core 2 O-glycans are outlined below.
Core 1 O-Glycan Biosynthesis Pathway
The biosynthesis of Core 1 O-glycans begins with the attachment of GalNAc to serine or threonine residues in proteins by UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases. Following this initial modification, C1GalT1 adds a Gal residue to form the T-antigen (Core 1). The simple structure of Core 1 serves as the foundation for more complex O-glycans, which can be further modified by the addition of sialic acid or fucose.
Core 2 O-Glycan Biosynthesis Pathway
Core 2 O-glycans are synthesized by the action of C2GnT1, which adds GlcNAc to the GalNAc residue on Core 1, creating a branch structure. The Core 2 pathway plays a critical role in the development of complex glycoproteins, particularly those involved in immune regulation, cell signaling, and tissue morphogenesis. The final structure can undergo additional modifications, including sialylation and fucosylation, contributing to its biological function.
Regulation of O-Glycan Biosynthesis
The biosynthesis of O-glycans is tightly regulated by both genetic and environmental factors. Tissue-specific expression of glycosyltransferases and other biosynthetic enzymes ensures that O-glycans are produced in the appropriate cell types and at the correct stages of development. For instance, C2GnT1 is upregulated in the immune system to facilitate immune cell interactions, while C1GalT1 is highly expressed in epithelial cells.
Moreover, post-translational modifications of glycosyltransferases, such as phosphorylation, can modulate their activity, further fine-tuning the production of specific O-glycan structures.
Pathophysiological Roles of O-Glycans
O-Glycans in Cancer
O-glycans, especially the Core 1 (T-antigen) and Core 2 structures, undergo significant alterations in cancer cells. These changes often facilitate tumor progression, metastasis, and immune evasion. Tumors typically exhibit altered glycosylation profiles, characterized by overexpression or silencing of specific glycosyltransferases that are crucial for O-glycan synthesis. For example, C1GalT1, which catalyzes the formation of Core 1 O-glycans, is often upregulated in cancerous tissues, leading to an increase in T-antigen expression. This altered glycosylation pattern can enhance tumor cell adhesion to the extracellular matrix, promoting invasion and metastasis.
The Core 2 O-glycan pathway, mediated by C2GnT1, is another critical axis in cancer biology. Increased activity of C2GnT1 and the subsequent formation of Core 2 glycans on cancer cell surfaces can promote immune evasion. The presence of sialylated Core 2 structures, for instance, can shield cancer cells from immune recognition by preventing the binding of immune receptors. Moreover, such O-glycans can influence the interaction between tumor cells and immune cells, such as T cells and macrophages, by altering cell surface molecules involved in immune synapse formation.
O-glycan alterations also affect the expression of mucins, which are glycoproteins involved in tumor cell protection and resistance to chemotherapy. For example, MUC1, a heavily glycosylated transmembrane protein, is frequently aberrantly glycosylated in cancer, with a shift towards truncated O-glycan structures. These modified mucins can contribute to immune suppression and promote tumor growth by creating a protective barrier around the tumor cells.
In addition to structural changes in glycosylation, enzymes involved in O-glycan biosynthesis, such as C1GalT1 and C2GnT1, can be directly regulated by oncogenic signaling pathways, including those driven by PI3K/Akt or Wnt/β-catenin. Such regulation of glycosylation enzymes contributes to the altered glycan structures found in tumors, further promoting cancer progression.
O-Glycans in Inflammatory Diseases
In inflammatory diseases, changes in O-glycan biosynthesis contribute to abnormal immune responses and the persistence of inflammation. Alterations in the expression and activity of O-glycan biosynthetic enzymes can influence the recruitment and activation of immune cells at sites of inflammation. Core 1 and Core 2 O-glycans are involved in immune cell signaling and the regulation of cell adhesion molecules, such as selectins and integrins. These interactions are critical in controlling the migration of immune cells into inflamed tissues.
In diseases like rheumatoid arthritis (RA), Crohn's disease, and ulcerative colitis, altered glycosylation of adhesion molecules, including E-selectin and P-selectin, results in enhanced leukocyte trafficking to sites of inflammation. For example, E-selectin, which normally binds to sialylated Core 2 O-glycans on leukocytes, can exhibit changes in its glycosylation pattern, affecting its binding efficiency and leading to excessive immune cell infiltration.
Furthermore, inflammatory cytokines can upregulate the expression of specific glycosyltransferases that modify O-glycans on immune cells. In particular, the upregulation of C2GnT1 in inflammatory environments can lead to the production of Core 2 O-glycans that alter immune cell adhesion and signaling. The increased presence of these glycans on the surface of leukocytes can enhance the interaction with endothelial cells and contribute to tissue infiltration, exacerbating inflammation.
In autoimmune diseases, such as systemic lupus erythematosus (SLE), changes in glycosylation patterns on immunoglobulins or other immune proteins may alter their function, contributing to the development of autoimmune responses. For instance, O-glycans on IgG antibodies have been shown to modulate their effector functions, influencing processes like antigen recognition and immune complex clearance.
O-Glycans in Aging
Aging is associated with a gradual accumulation of alterations in O-glycosylation, affecting various cellular processes, including immune function, tissue regeneration, and protein stability. One of the key changes observed in aging is a decrease in the diversity of O-glycan structures on glycoproteins, particularly in immune cells. This loss of glycan complexity has been linked to immune senescence, where the immune system becomes less effective at responding to new infections or eliminating dysfunctional cells.
The decrease in Core 2 O-glycans, which are often involved in immune cell signaling, is particularly notable in aging. For instance, as people age, the reduced synthesis of Core 2 O-glycans on T cell receptors can impair T cell function, diminishing the ability of the immune system to mount an effective response to pathogens or tumors. Additionally, the accumulation of truncated O-glycan structures on proteins like MUC1 or MUC16 in aging tissues may contribute to the development of chronic inflammation, a hallmark of aging.
Moreover, altered glycosylation patterns in the extracellular matrix (ECM) contribute to age-related changes in tissue stiffness and regenerative capacity. Core 1 O-glycans, found in key ECM components, are involved in regulating the interaction between cells and the matrix. As O-glycan biosynthesis becomes dysregulated with age, ECM remodeling is compromised, resulting in diminished tissue repair and increased susceptibility to age-related diseases, such as osteoarthritis and fibrosis.
References
- Watson, Michel E., et al. "Glycosylation-related diagnostic and therapeutic drug target markers in hepatocellular carcinoma." J Gastrointestin Liver Dis 24.3 (2015): 349-57. http://dx.doi.org/10.15403/jgld.2014.1121.243.mew
- Freire-de-Lima, Leonardo. "Sweet and sour: the impact of differential glycosylation in cancer cells undergoing epithelial–mesenchymal transition." Frontiers in oncology 4 (2014): 59. https://doi.org/10.3389/fonc.2014.00059
