The oligosaccharides attached to glycoproteins serve various functions. For example, some proteins require N-linked oligo saccharides in order to fold properly in the ER. This function has been demonstrated in studies with the antibiotic tunicamycin, which blocks the first step in the formation of the dolichol linked oligosaccharide precursor and therefore inhibits synthesis of all N-linked oligosaccharides in cells (see Figure 1, top left). For example, in the presence of tunicamycin, the influenza virus hemagglutinin precursor polypeptide (HA0) is synthesized, but it cannot fold properly and form a normal trimer; in this case, the protein remains, misfolded, in the rough ER. Moreover, mutation of a particular asparagine in the hemagglutinin sequence to a glutamine residue prevents addition of an N-linked oligosaccharide to that site and causes the protein to accumulate in the ER in an unfolded state.
Fig1. Biosynthesis of the oligosaccharide precursor. Dolichol phosphate is a strongly hydrophobic lipid, containing 75–95 carbon atoms, that is embedded in the ER membrane. Two N- acetylglucosamine (GlcNAc) and five mannose residues are added one at a time to a dolichol phosphate on the cytosolic face of the ER membrane (steps 1–3). The nucleotide-sugar donors in these and later reactions are synthesized in the cytosol. Note that the first sugar residue is attached to dolichol by a high-energy pyrophosphate linkage. Tunicamycin, which blocks the first enzyme in this pathway, inhibits the synthesis of all N-linked oligosaccharides in cells. After the seven-residue dolichol pyrophosphoryl intermediate is flipped to the luminal face (step 4), the remaining four mannose residues and all three glucose residues are added one at a time (steps 5–6). In the later reactions, the sugar to be added is first transferred from a nucleotide sugar to a carrier dolichol phosphate on the cytosolic face of the ER; the carrier is then flipped to the luminal face, where the sugar is transferred to the growing oligosaccharide, after which the “empty” carrier is flipped back to the cytosolic face. See C. Abeijon and C. B. Hirschberg, 1992, Trends Biochem. Sci. 17:32.
In addition to promoting proper folding, N-linked oligo saccharides confer stability on many secreted glycoproteins. Many secretory proteins fold properly and are transported to their final destination even if the addition of all N-linked oligosaccharides is blocked, for example, by treatment with tunicamycin. However, such nonglycosylated proteins have been shown to be less stable than their glycosylated forms. For instance, glycosylated fibronectin, a normal component of the extracellular matrix, is degraded much more slowly by tissue proteases than is nonglycosylated fibronectin.
Oligosaccharides on certain cell-surface glycoproteins also play a role in cell-cell adhesion. For example, the plasma mem brane of white blood cells (leukocytes) contains cell-adhesion molecules (CAMs) that are extensively glycosylated. The oligosaccharides in these molecules interact with a sugar-binding domain in certain other CAMs found on endothelial cells lining blood vessels. This interaction tethers the leukocytes to the endothelium and assists in their movement into tissues during an inflammatory response to infection. Other cell-surface glycoproteins possess oligosaccharide side chains that can induce an immune response. A common example is the ABO blood group antigens, which are O-linked oligosaccharides attached to glycoproteins and glycolipids on the surfaces of erythrocytes and other cell types. In both cases, oligosaccharides are added to the luminal face of these membrane proteins, in a manner similar to what is shown in Figure 2 for soluble proteins. The luminal face of these membrane proteins is topologically equivalent to the exterior face of the plasma membrane, where these proteins eventually end up.
Fig2. Addition and initial processing of N-linked oligosaccharides. In the rough ER of vertebrate cells, the Glc3Man9(GlcNAc)2 precursor is transferred from the dolichol carrier to a susceptible asparagine residue on a nascent protein as soon as the asparagine crosses to the luminal side of the ER (step 1). In three separate reactions, first one glucose residue (step 2), then two glucose residues (step 3), and finally one mannose residue (step 4) are removed. Re-addition of one glucose residue (step 3a) plays a role in the correct folding of many proteins in the ER, as discussed later. The process of N-linked glycosylation of a soluble secretory protein is shown here, but the luminal portions of an integral membrane protein can be modified on asparagine residues by the same mechanism. See R. Kornfeld and S. Kornfeld, 1985, Annu. Rev. Biochem. 45:631, and M. Sousa and A. J. Parodi, 1995, EMBO J. 14:4196.
