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Chapter 5 Protein Synthesis, Processing, and Trafficking 53

ER
N-acetylglucosamine
UGT1
Mannose
GI GII Lumen
Glucose MNSI
GII
NH 2
Sialic acid
Galactose Donor
Fucose
Glycan transfer Trimming and processing Cytosol

GOLGI

Lumen

Trimming Terminal glycosylation Cytosol
Fig. 5.5 N-GLYCOSYLATION OF PROTEINS. In the lumen of the endoplasmic reticulum (ER), a core
oligosaccharide, Glc 3Man 9GlcNac 2, is transferred from a lipid-linked precursor (dolichol donor) to the aspara-
gine residue in an N-X-S/T motif in the nascent polypeptide chain. The terminal glucose residues are removed
by GI and GII and cycles of reglucosylation by UGT1 (UGGT1) can occur (curved arrows). When the protein
is folded, one mannose is trimmed by ER-mannosidase I and the protein is transported to the Golgi. Core
oligosaccharides are further trimmed by mannosidases to produce the Man 5GlcNac 2 unit. Further elaboration
is catalyzed by glycosyltransferases that add various sugars and create branches. Bi, tri and tetra antennary
chains are generated. In the figure, only one pathway of terminal glycosylation is shown. (Modified from Helenius
A, Aebi M: Intracellular function of N-linked glycans. Science 291:2364, 2001.)

ERO1, a membrane associated oxidoreductase. ERO1 returns to the complex type of N-glycan chains for its biologic function to stimulate
oxidized state by transfer of electrons to molecular oxygen via its erythropoiesis.
cofactor flavin adenine dinucleotide (FAD). In contrast to the highly In the recent years several studies have revealed the importance of
reducing environment of the cytosol where disulfide bonds do not protein N-glycosylation in promoting folding. The addition of glycan
typically form, the lumen of the ER is very oxidizing so that disulfide chains may prevent aggregation or provide steric influences that affect
bonds formation is favored. polypeptide folding and disulfide bond formation and also mediate
interaction with specific chaperones. In mammalian cells, N-linked
oligosaccharides are also used as signal for monitoring protein folding
Protein Modifications in the ER and trafficking. They are ligands for a complex chaperone system
composed of the lectin chaperones calnexin (CNX) and calreticulin
Most proteins that enter the secretory pathway are modified by (CRT), Erp57 (an oxidoreductase), two α-glucosidases (GI and
N-glycosylation (Fig. 5.5). This process starts with the transfer GII) and one folding sensor (UGGT1) endowed with reglucosyl-
of a core oligosaccharide from a lipid-linked dolichol donor to ation activity (UDP-glucose:glycoprotein glucosyltransferase). GI
an asparagine residue within the consensus sequence N-X-S/T removes the most terminal glucose and diglucosylated polypeptides
of a nascent polypeptide (X can be any amino acid except for associated with malectin, an ER resident lectin. Then, GII removes
proline). The N-linked oligosaccharide is composed of a glucose 3 - the second terminal glucose residue to form a monoglucosylated
mannose 9 -N-acetylglucosamine 2 unit (Glc 3 Man 9 GlcNac 2 ). The N-linked chain (see Fig. 5.5) that is a ligand for CNX and CRT.
oligosaccharide is transferred to the asparagine residue by an ER CNX and CRT associate with the thiol-disulfide oxidoreductase
oligosaccharyltransferase (OST) that is composed of a catalytic ERP57 which promotes proper rearrangement of disulfide bonds.
subunit (STT3A or STT3B) and a set of accessory subunits. Further Then the remaining glucose residue is removed by GII. UGGT1
processing of the terminal sugars occurs in the ER and after the recognizes and reglucosylates N-linked oligosaccharides on proteins
polypeptide transits the Golgi compartment (see Fig. 5.5). Many that have not completed the folding process. The addition of
blood proteins, for example immunoglobulins, antiproteases, glucose residues allows reassociation with the CNX/CRT chaperone
coagulation factors, and many membrane proteins of the cell are system for another attempt for the polypeptide to attain its proper
glycosylated. conformation. 5
Although glycan chains are often not required for the enzy- Besides N-core glycosylation and oxidative folding, the ER is also
matic activity of glycoproteins, they are important for the physical site of other protein modifications. A remarkable one is γ-carboxylation
properties they confer and for many physiologic functions. Glycans of glutamic acid residues. Although this is a rather rare modification,
protect proteins from protease digestion and heat denaturation, it is crucial for the functionality of specific proteins, such as the
confer hydrophilicity and adhesive properties to the proteins, and coagulation factors VII, X, IX and prothrombin, and is essential for
mediate interaction with other proteins or receptors. A remarkable life as described earlier (see Box 5.1). Another modification is the
example is the hormone erythropoietin that requires a particular attachment of a GPI to the C-terminal end of protein with