O-linked glycosylation occurs at a later stage during protein processing, probably in the Golgi apparatus. This is the addition of N-acetyl-galactosamine to serine or threonine residues by the enzyme UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (EC number 188.8.131.52), followed by other carbohydrates (such as galactose and sialic acid). This process is important for certain types of proteins such as proteoglycans, which involves the addition of glycosaminoglycan chains to an initially unglycosylated "proteoglycan core protein." These additions are usually serine O-linked glycoproteins, which seem to have one of two main functions. One function involves secretion to form components of the , adhering one cell to another by interactions between the large sugar complexes of proteoglycans. The other main function is to act as a component of mucosal secretions, and it is the high concentration of carbohydrates that tends to give mucus its "slimy" feel. GlcNAc-β-Ser/Thr, which are found in nuclear and cytoskeletal proteins, were the first reported example of glycosylated proteins found in a location other than secretory channels.
O-fucose is added between the second and third conserved cysteines of EGF-like repeats in the Notch protein, and other substrates by GDP-fucose protein O-fucosyltransferase 1, and to Thrombospondin repeats by GDP-fucose protein O-fucosyltransferase 2 (commonly referred to as POFUT2). In the case of EGF-like repeats, the O-fucose may be further elongated to a tetrasaccharide by sequential addition of N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and for Thrombospondin repeats, may be elongated to a disaccharide by the addition of glucose. Both of these fucosyltransferases have been localized to the endoplasmic reticulum, which is unusual for glycosyltransferases, most of which function in the Golgi apparatus. These enzymes are not restricted to mammalian systems and recently POFUT2 homologs were described in the apicomplexa parasites Plasmodium falciparum and Toxoplasma gondii.
O-glucose is added between the first and second conserved cysteines of EGF-like repeats in the Notch protein, and possibly other substrates by protein:O-glucosyltransferase (Poglut). This enzyme is known as Rumi in Drosophila, and is also localized to the ER like the O-fucosyltransferases. The O-glucose modification appears to be necessary for proper folding of the EGF-like repeats of the Notch protein, and increases secretion of this receptor.
O-GlcNAc is added to serines or threonines by O-GlcNAc transferase (OGT). O-GlcNAc appears to occur on most serines and threonines that would otherwise be phosphorylated by serine/threonine kinases. Thus, if phosphorylation occurs, O-GlcNAc does not, and vice versa. This apparently competitive modification of certain sites may have significant consequences for some research directions. Much cancer research is focused on phosphorylation, because of its important role in cell signalling pathways. As competitive or variable glycosylation occurs at the same sites, there is a risk that phosphorylation research has overlooked important roles that these modification sites play when glycosylated. O-GlcNAc addition and removal also appears to be a key regulator of the pathways that are disrupted in diabetes mellitus. The gene encoding the O-GlcNAcase (OGA) enzyme has been linked to non-insulin dependent diabetes mellitus. It is the terminal step in a nutrient-sensing hexosamine signaling pathway.
Recently, O-GlcNAc was reported to occur between the fifth and sixth conserved cysteines in some EGF-like repeats from the Notch protein. This modification is performed by a second O-N-acetyglucosaminyl-transferase named eOGT.
During O-mannosylation, a mannose residue is transferred from mannose-p-dolichol to a serine/threonine residue in secretory pathway proteins. O-mannosylation is common to both prokaryotes and eukaryotes.