1. Dolichol-linked oligosaccharide synthesis: As with the O-linked glycoproteins, the protein is synthesized on the RER and enters its lumen. However, it does not become glycosylated with individual sugars. Instead, a lipid-linked oligosaccharide is first constructed. This consists of dolichol (an RER membrane lipid made from an intermediate of cholesterol synthesis; see p. 221) attached through a pyrophosphate linkage to an oligosaccharide containing GlcNAc, mannose, and glucose. The sugars to be added sequentially to the dolichol by membrane-bound glycosyltransferases are first GlcNAc, followed by mannose and glucose (see Fig. 14.16). The entire 14-sugar oligosaccharide is then transferred from dolichol to the amide nitrogen of an asparagine residue in the protein to be glycosylated by a protein–oligosaccharide transferase present in the RER. [Note: Tunicamycin inhibits N-linked glycosylation.]
Congenital disorders of glycosylation (CDG) are syndromes caused primarily by defects in the N-linked glycosylation of proteins, either oligosaccharide assembly (type I) or processing (type II).
2.
N-Linked oligosaccharide processing: After addition to the protein, the N-linked oligosaccharide is processed by the removal of specific mannosyl and glucosyl residues as the glycoprotein moves through the RER. Finally, the oligosaccharide chains are completed in the Golgi by addition of a variety of sugars (for example, GlcNAc, GalNAc, and additional mannoses and then fucose or NANA as terminal groups) to produce a complex glycoprotein. Alternatively, they are not processed further, leaving branched, mannose-containing chains in a high-mannose glycoprotein (see Fig. 14.16). The ultimate fate of N-linked glycoproteins is the same as that of the O-linked glycoproteins (for example, they can be released by the cell or become part of a cell membrane). In addition, N-linked glycoproteins can be targeted to the lysosomes. [Note: Nonenzymatic glycosylation of proteins is known as glycation (see p. 33).] 3.
Lysosomal enzymes: N-Linked glycoproteins being processed in the Golgi can be phosphorylated on carbon 6 of one or more mannosyl residues. UDP-GlcNAc provides the phosphate in a reaction catalyzed by a phosphotransferase. Receptors, located in the Golgi membrane, bind the mannose 6-phosphate residues of these proteins, which are then packaged into vesicles and sent to the lysosomes (Fig. 14.17). I-Cell disease is a rare lysosomal storage disease in which the phosphotransferase is deficient. This causes the proteins to be secreted, rather than being targeted to lysosomes. Consequently, the acid hydrolases normally found in the lysosomal matrix are absent, resulting in an accumulation of the substrates for these missing enzymes. [Note: I-Cell disease is so named because of the large inclusion bodies seen in cells of patients with this disease.] In addition, high amounts of lysosomal enzymes are found in the patient’s plasma and urine, indicating that the targeting process to lysosomes (rather than the synthetic pathway of these enzymes) is deficient. I-Cell disease is characterized by skeletal abnormalities, restricted joint movement, coarse (dysmorphic) facial features, and severe psychomotor impairment. [Note: