Glycosylation: mechanisms, biological functions and clinical implications

Abstract

Protein post-translational modification (PTM) is a covalent process that occurs in proteins during or after translation through the addition or removal of one or more functional groups, and has a profound effect on protein function. Glycosylation is one of the most common PTMs, in which polysaccharides are transferred to specific amino acid residues in proteins by glycosyltransferases. A growing body of evidence suggests that glycosylation is essential for the unfolding of various functional activities in organisms, such as playing a key role in the regulation of protein function, cell adhesion and immune escape. Aberrant glycosylation is also closely associated with the development of various diseases. Abnormal glycosylation patterns are closely linked to the emergence of various health conditions, including cancer, inflammation, autoimmune disorders, and several other diseases. However, the underlying composition and structure of the glycosylated residues have not been determined. It is imperative to fully understand the internal structure and differential expression of glycosylation, and to incorporate advanced detection technologies to keep the knowledge advancing. Investigations on the clinical applications of glycosylation focused on sensitive and promising biomarkers, development of more effective small molecule targeted drugs and emerging vaccines. These studies provide a new area for novel therapeutic strategies based on glycosylation.

Fig. 1

Timeline of the four major glycosylations development and further investigation. In the early 1980s, there was a preliminary understanding of glycosylation. As the research progressed, the corresponding initiating enzymes and chemical structures were gradually deciphered and recognized. For N-glycosylation, the determination of the Asn-X-Thr/Ser consensus sequence and the structure of the OST complex subunit and some accessory subunits around 2000 led to the in-depth study. Years later, N-linked intact glycopeptides in human serum were identified on a large scale using HILIC enrichment and spectral library searches, and the relationship between N-glycosylation levels and glucose metabolic stress, iron apoptosis, and so on were found. O-glycosylation is mainly classified into two types: O-GlcNAcylation and O-GalNAcylation. Since the 2010s, the close connection between O-glycosylation and physiological processes such as inflammatory response, immune escape, viral infection, cell adhesion, metastasis, apoptosis, etc. has been continuously discovered. Technological breakthroughs around 2020 have also led to the emergence of quantitative determination of maps of the O-glycosylated proteome and the development of gene editing libraries. The understanding of C-glycosylation began in the 1990s with the ongoing discovery of the structure of tryptophan residues in human Rnase linking mannose via C-C bonds. By the 2000s it was understood that C-glycosylation may be a necessary step for ER export in mucin biosynthesis. Recently it has been possible to construct C-glycosylation-modified glycopeptide drugs or glycan analogs in vitro, which greatly improve biological functions. GPI-anchoring has been well understood since the complete structural elucidation in 1988 and the discovery of the initiator enzyme, Transamidase, in 1992. In 2023, the phenomenon and molecular mechanism of feedback regulation of cellular maintenance of GPI-anchored proteins were first elucidated. DPY19L, dpy-19 like C-Man transferase; Gal, d-galactose; GalNAc, N-acetyl-d-galactosamine; GALNT, polypeptide GalNAc transferase; Glc, d-glucose; GlcNAc, N-acetyl-d-glucosamine; GPI, glycosylphosphatidylinositol; HILIC, hydrophilic interaction liquid chromatography; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase; OST, oligosaccharyltransferase; TSR, thrombospondin type 1 repeat

Fig. 2

Classification of major manifestations of glycosylation on proteins. Glycosylation of proteins is capable of occurring when a saccharide is covalently attached to the polypeptide backbone via N-linkage to Asn or O-linkage to Ser/Thr. N-glycans that have complex, hybrid, or high mannose forms, are linked to Asn via GlcNAc. GalNAc links O-glycans to Ser/Thr with a variety of core structures and extensions, most of which are sialylated and fucosylated. Single GlcNAc molecules are linked to the Ser/Thr residue of intracellular proteins in the cytoplasm, mitochondria, and nucleus through the process of O-GlcNAc glycosylation. Certain glycoproteins known as glycosylphosphatidylinositol (GPI)-anchored proteins are also present in the plasma membrane’s outer leaflet and are connected to a phosphatidylinositol. A significant portion of the cell plasma membrane’s outer leaflet is made up of glycosphingolipids. Terminal sialic acids can be used to further modify the various series of structures that make up these ceramide-linked glycans. GalNAc, N-acetyl-d-galactosamine; GlcNAc, N-acetyl-d-glucosamine; GPI, glycosylphosphatidylinositol

Fig. 3

Biological functions of glycosylation. Glycosylation has a variety of biological functions that can affect protein folding and stability, for example, O-GlcNAcylation accelerates protein degradation in cells and decreases protein stability, it also regulates activities such as protein aggregation and phase separation. Glycosylation modulates cell-matrix interactions and promotes integrin-dependent signaling, which regulates adhesion activity. An effective immune response depends on the successful activation and maturation of dendritic cells, whereas abnormally glycosylated protein antigens impair the function of dendritic cells, allowing the cells to evade the host’s immune response. O-GlcNAc modifications have been found to directly regulate a variety of important biological processes within cells, such as cellular metabolism. Glycosylation can regulate enzyme activities or interact with other proteins and participate in cell signaling processes. These include Notch signaling, JAK-STAT signaling, TGF-β signaling, Wnt/β-catenin signaling pathways. GlcNAc, N-acetyl-d-glucosamine; LLPS, liquid-liquid phase separation; PSD, postsynaptic density; Glc-6-P, Glucose-6-phosphate; JAK-STAT, Janus kinase (JAK)-signal transducer and activator of transcription; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; TGF, transforming growth factor

Fig. 4

Altered glycosylation in human disease. Glycosylation is widely present in organisms, and aberrant glycosylation is closely associated with the development of a variety of diseases. Altered glycosylation present in solid tumors, hematological malignancies, and autoimmune diseases are summarized in the figure