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O-Linked Glycosylation Process

What is O-Linked Glycosylation?

O-linked glycosylation refers to the covalent attachment of sugar molecules, known as glycans, to the hydroxyl group of serine (Ser) or threonine (Thr) amino acids present in proteins. It is one of the two major types of glycosylation, the other being N-linked glycosylation, which involves the attachment of glycans to the nitrogen atom of asparagine (Asn) residues.

Common O-GalNAc core structures

O-Linked Glycosylation Process


The process begins with the addition of a monosaccharide, often N-acetyl-galactosamine (GalNAc), to the hydroxyl group of a Ser or Thr residue. This initiation step is catalyzed by a family of enzymes known as UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). These enzymes recognize specific amino acid motifs and transfer the GalNAc residue from the donor substrate UDP-GalNAc to the acceptor amino acid residue.


Following the initial attachment of GalNAc, the glycan chain can undergo further elongation through the addition of additional sugar residues. This process involves the activity of various glycosyltransferases that sequentially add different monosaccharides, such as glucose, galactose, N-acetylglucosamine, and fucose, to the growing glycan chain. The specific glycosyltransferases involved in O-glycan elongation depend on the cell type and tissue context.


In certain cases, the elongated O-glycans can undergo branching, where additional sugar residues are added to create a branched structure. This branching pattern is important for determining the functional properties of glycoproteins and is regulated by specific branching enzymes.

Protein folding and processing:

During and after O-glycosylation, the attached glycans can influence protein folding and processing. The presence of O-glycans can affect the conformation, stability, and solubility of the protein, as well as its trafficking and localization within the cell.

How Does O-linked Glycosylation Occur?

O-linked glycosylation occurs within the endoplasmic reticulum (ER) and Golgi apparatus of eukaryotic cells, where the necessary enzymes and substrates for glycosylation are localized.

The process of O-linked glycosylation begins with the activation of the sugar donor molecule, such as UDP-GalNAc, by specific nucleotide sugar transporters present in the ER and Golgi membranes. The activated sugar is then transferred to the acceptor Ser or Thr residues in the protein backbone by the corresponding glycosyltransferases.

The glycosylation process is highly regulated and influenced by various factors, including the availability of sugar donors, the expression and activity of glycosyltransferases, and the presence of specific recognition motifs in the protein sequence. Different cell types and tissues may exhibit distinct patterns of O-glycosylation due to the differential expression of glycosyltransferases and other enzymes involved in the process.

N-linked and O-linked protein glycosylation occurs in the ER and Golgi apparatus (Wang et al., 2014).

Regulation of O-Linked Glycosylation

The regulation of O-linked glycosylation, like other cellular processes, involves a complex interplay of factors that influence the enzymes, substrates, and conditions required for the attachment of glycosyl groups to serine (Ser) and threonine (Thr) residues in proteins. The following will describe what effect the regulation of O-linked glycosylation:

Gene expression and transcriptional regulation

The expression of glycosyltransferases and other enzymes involved in O-linked glycosylation is tightly regulated at the transcriptional level. Transcription factors and regulatory elements in the promoter regions of these genes can affect their expression levels in a tissue-specific or developmentally regulated manner. Changes in glycosyltransferase expression can affect the type and pattern of O-linked glycosylation in cells and tissues.

Golgi localization and enzyme activity

Many of the enzymes responsible for O-linked glycosylation are localized within the Golgi, where the final steps of sugar extension and processing occur. The precise localization of these enzymes within the Golgi helps in the correct glycosylation of proteins. Regulatory mechanisms that control the trafficking and localization of glycosyltransferases, such as signal sequences, targeting patterns, and protein-protein interactions, ensure proper enzymatic activity and substrate recognition.

Post-translational modifications

Post-translational modifications of glycosyltransferases and other proteins involved in O-linked glycosylation can regulate their activity and stability. For example, phosphorylation of glycosyltransferases can enhance or inhibit their catalytic activity and affect the efficiency and specificity of O-glycosylation reactions.

Substrate availability and competition

The availability of sugar donors, such as UDP-GalNAc, UDP-GlcNAc, and other nucleotide sugars, is crucial for O-linked glycosylation. The intracellular levels of these sugar nucleotides are regulated by metabolic pathways and the availability of precursors. Changes in the levels of sugar donors can impact the extent and types of O-glycosylation. Additionally, competition for sugar donors between different glycosylation pathways, such as O-linked and N-linked glycosylation, can influence the glycosylation patterns of proteins.

Protein sequence and substrate recognition

The sequence context surrounding potential O-glycosylation sites can influence the efficiency and specificity of glycosylation. Certain amino acid motifs, such as Proline-Serine/Threonine (Pro-Ser/Thr) motifs, are preferentially recognized by specific glycosyltransferases. The presence of adjacent amino acids and the overall protein structure can also influence the accessibility and recognition of potential O-glycosylation sites.

Cellular stress and signaling pathways

Cellular stress conditions, such as hypoxia, nutrient deprivation, or endoplasmic reticulum (ER) stress, can impact O-linked glycosylation processes. These stress conditions can induce changes in gene expression, alter the activity of glycosyltransferases, and affect the availability of sugar donors. Signaling pathways, such as the unfolded protein response (UPR) or the mammalian target of rapamycin (mTOR) pathway, can also modulate O-glycosylation by regulating the expression and activity of glycosyltransferases.

What is the Function of O-linked Glycosylation?

Protein function and stability: The presence of glycans enhances protein stability, prevents protein hydrolysis, and regulates protein-protein interactions.

Cellular signaling: Attached glycans can act as recognition sites for lectins and other carbohydrate-binding proteins and facilitate intercellular interactions. They can affect receptor activation, ligand binding and downstream signaling pathways. For example, O-glycosylation of cell surface receptors and adhesion molecules plays a key role in cell adhesion, migration and immune cell recognition.

Immune response: Glycans present on cell surface or secreted glycoproteins can act as antigens, triggering immune responses and influencing host-pathogen interactions. O-glycans are also involved in the recognition and adhesion of immune cells to target cells, facilitating immune surveillance and immune cell recruitment to sites of inflammation.

Development and disease: During embryogenesis, regulated glycosylation of specific proteins is essential for proper tissue development and organ formation. alterations in O-linked glycosylation patterns are associated with many diseases, including cancer, cardiovascular disease, genetic disorders, and inflammation. Changes in glycosylation affect protein function, cell signaling and immune responses, thereby contributing to disease progression and pathogenesis.

Learn more about what is glycosylation?


  1. Kamerling, Johannis P., and Geert-Jan Boons, eds. Cell Glycobiology and Development: Health and Disease in Glycomedicine. Elsevier, 2007.
  2. Wang, Yu-Chieh, Suzanne E. Peterson, and Jeanne F. Loring. "Protein post-translational modifications and regulation of pluripotency in human stem cells." Cell research 24.2 (2014): 143-160.
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