About 150 years ago, biologists E. Hoppe-Seyler and E. Eichwal first discovered that O-glycans isolated from human secretions mainly exist on mucins. This process was later referred to as mucin-type O-glycosylation or O-N-acetylgalactosamine (O-GalNAc) glycosylation. One of the most abundant and unique post-translational modifications, O-GalNAc glycosylation is the process of sequential addition of monosaccharides to oligosaccharide chains leading to glycan elongation. This process involves cell adhesion, communication, signal transduction, immune surveillance, epithelial cell protection, and host-pathogen interactions. There are many kinds of O-GalNAc glycans with four main core structures: Galβ1-3GalNAcαSer/Thr, GlcNAcβ1-6(Galβ1-3)GalNAcαSer/Thr, GlcNAcβ1-3GalNAcαSer/Thr, GlcNAcβ1-6(GlcNAcβ1-3)GalNAcαSer/Thr. The O-GalNAc core structure can be extended by various sugar residues to form complex O-GalNAc glycans, such as polysialic acid and linear i antigen.
The biosynthetic pathway of O-GalNAc glycans in healthy cells is restricted to the Golgi apparatus and can include the following steps: initiation, core elongation, elongation, capping, and sulfation. O-GalNAc glycan Synthesis begins with polypeptide N-acetylgalactosaminyltransferase (GALNT)-catalyzed transfer of GalNAc monosaccharides from UDP-GalNAc to threonine or serine (Ser/Thr) residues in newly synthesized proteins. Subsequent addition of one or two neutral sugars to the GalNAc group under the action of different enzymes resulted in four O-GalNAc core structures. Core 1 is produced by core 1 β1,3-galactosyltransferase 1 (C1GALT1). β1,6 N-Acetylglucosaminyltransferases 1, 3 and 4 (GCNT1/3/4) are responsible for adding β1-6 N-acetylglucosamine (GlcNAc) branches to the core 1 GalNAc group to form core 2 O-GalNAc glycans. Synthesis of core 3 O-GalNAc glycans is associated with β1-3 N-acetylglucosaminyltransferase 6 (B3GNT6). The enzyme is responsible for adding GlcNAc to the GalNAc group of the starting protein. GCNT1/3/4 is responsible for adding β1-6GlcNAc branches to the core 3 GalNAc group to form core 4 O-GalNAc glycans. The elongation of O-GalNAc glycans is catalyzed by β1-3 GlcNAc transferase, β1-3 galactose (Gal) transferase, or β1-4 Gal Transferase to form complex type 1 and type 2 poly-N-acetyllactosamines.
Fig.1 Structure and biosynthesis of O-GalNAc glycan. (Bagdonaite, et al., 2021)
O-GalNAc glycans have a variety of different biological functions, which are related to the structure, position, and density of glycans. Clustered mucin-like O-glycans are functionally different from O-GalNAc of individual glycosylation sites. Clustered mucin-like O-glycans provide structural modifications of the protein backbone to support larger mucin domains. In addition, clustered mucin-like O-glycans also contribute to hydration, protect healthy host-pathogen interactions, and protect against proteolysis. The O-GalNAc function of individual glycosylation sites differs from that of unregulated clustered O-GalNAc. O-GalNAc glycosylation at one or several sites in some proteins regulates protein cleavage by proprotein transferase and the cleavage and shedding of extracellular domains by ADAM (a disintegrin and metalloproteases). O-GalNAc glycans at individual glycosylation sites also affect ligand binding and cell-cell and cell-matrix interactions, thereby affecting tissue formation and differentiation. Either O-GalNAc glycans with a single glycosylation site or clustered mucin-like O-glycans can be used as carriers for terminal ligands.
Fig.2 Biological functions of O-GalNAc glycan. (Bagdonaite, et al., 2021)
Disorders of Protein O-Glycosylation lead to specific glycosylation disorders. GALNT is required to initiate mucin O-GalNAc Glycosylation. Genetic deletion of this enzyme family triggers corresponding biological changes. GALNT1 has been reported to affect cardiac development, hemostasis, and immune cell homing in mice. The researchers found that genetic deletion of GALNT2 in humans is associated with abnormal metabolism of Lipids, thereby affecting HDL cholesterol and triglyceride levels. Defects of the GALNT3 gene in O-GalNAc synthesis develop familial tumor calcinosis. This is a severe autosomal-recessive metabolic disorder characterized clinically by phosphateemia and massive calcium deposits in the skin and subcutaneous tissue.
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