Udp A

Dolichol phosphate

ER lumen

Dolichol phosphate

ER lumen

▲ FIGURE 16-17 Biosynthesis of the dolichol pyrophosphoryl oligosaccharide precursor of W-linked oligosaccharides.

Dolichol phosphate is a strongly hydrophobic lipid, containing 75-95 carbon atoms, that is embedded in the ER membrane. Two W-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 hi - 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 W-linked oligosaccharides in cells. After the seven-residue dolichol pyrophosphoryl intermediate is flipped to the luminal face (step 14), the remaining four mannose 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. [After C. Abeijon and C. B. Hirschberg, 1992, Trends Biochem. Sci. 17:32.]

(Glc)3(Man)9(GlcNAc)2

(Man)8(GlcNAc)2

(Man)8(GlcNAc)2

To cis-

Golgi

ER lumen

Dol = Dolichol ■ = N-Acetylglucosamine

▲ FIGURE 16-18 Addition and initial processing of W-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 11). In three separate reactions, first one glucose residue

(step 12), 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. [See R. Kornfeld and S. Kornfeld, 1985, Ann. Rev. Biochem. 45:631, and M. Sousa and A. J. Parodi, 1995, EMBO J. 14:4196.]

coproteins. 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 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 membrane 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 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 (see Figure 6-30). Other cell-surface glycoproteins possess oligosaccharide side chains that can induce an immune response. A common example is the A, B, O blood-group antigens, which are O-linked oligosaccharides attached to glycoproteins and glycolipids on the surface of erythrocytes and other cell types (see Figure 5-16).

Disulfide Bonds Are Formed and Rearranged by Proteins in the ER Lumen

In Chapter 3 we learned that both intramolecular and intermolecular disulfide bonds (-S-S-) help stabilize the tertiary and quaternary structure of many proteins. These covalent bonds form by the oxidative linkage of sulfhydryl groups (-SH), also known as thiol groups, on two cysteine residues in the same or different polypeptide chains. This reaction can proceed spontaneously only when a suitable oxidant is present. In eukaryotic cells, disulfide bonds are formed only in the lumen of the rough ER; in bacterial cells, disulfide bonds are formed in the periplasmic space between the inner and outer membranes. Thus disulfide bonds are found only in secretory proteins and in the exoplasmic domains of membrane proteins. Cytosolic proteins and organelle proteins synthesized on free ribosomes lack disulfide bonds and depend on other interactions to stabilize their structures.

The efficient formation of disulfide bonds in the lumen of the ER depends on the enzyme protein disulfide isomerase (PDI), which is present in all eukaryotic cells. This enzyme is especially abundant in the ER of secretory cells in such organs as the liver and pancreas, where large quantities of proteins that contain disulfide bonds are produced. As shown in Figure 16-19a, the disulfide bond in the active site of PDI can be readily transferred to a protein by two sequential thiol-disulfide transfer reactions. The reduced PDI generated by this reaction is returned to an oxidized form by the action of an ER-resident protein, called Ero1, which carries a disulfide bond that can be transferred to PDI. It is not yet understood how Ero1 itself becomes oxidized. Figure 16-20 depicts the organization of the pathway for protein disulfide-bond formation in the ER lumen and the analogous pathway in bacteria.

In proteins that contain more than one disulfide bond, the proper pairing of cysteine residues is essential for normal structure and activity. Disulfide bonds commonly are

(a) Formation of a disulfide bond

SH SH

Reduced substrate protein

Oxidized substrate protein

(b) Rearrangement of disulfide bonds

(b) Rearrangement of disulfide bonds

Protein with incorrect disulfide bonds

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