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Intracellular Functions of N-Linked Glycans Ari Helenius and and Markus Aebi Science 291, 2364 (2001); DOI

: 10.1126/science.291.5512.2364

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N-linked glycans are added to proteins in the ER as “core oligosaccharides” that have the structure shown. Bertozzi. Glycobiology 10. J. W. J. The trimming and processing that the glycans undergo when the glycoprotein is still in the ER introduce only limited additional diversity. J. K. C. 89. L. Lugemwa. the glycans acquire more complex structures and a new set of functions. P. E. C. K. L. 83. R. Science 287. K. 79. Saxon. 87. W. 170 (1996). Lemieux. 123.. Eds. among glycoproteins. Science 276. A. W. New York). 75. on April 24. T. Biophys. Bertozzi. Chem. D. 50. Biol. Chem. 1293 (1998). where the repertoire of oligosaccharide structures is still rather small. Am. Jacobs. J. L. R. K. Fan et al. Butcher. E. G. 266. Soc. Pozsgay. 669 (2000). Universita ¨ tstrasse 16. R. D. J. S. E. Ichikawa. 1. L. Collins. The authors thank J. L. Chem. REVIEW Intracellular Functions of N-Linked Glycans Ari Helenius1* and Markus Aebi2 N-linked oligosaccharides arise when blocks of 14 sugars are added cotranslationally to newly synthesized polypeptides in the endoplasmic reticulum (ER). K. 1125 (1997). 3323 (1995). J. Gordon.. 4278 (1999). R. 437 (1998). Beck. Bertozzi. Science 245. R. F. 16934 (1992). Strong. E. 2). C. Keppler et al. J. 76. Wieser. E. “Metabolic substrate engineering as a tool for glycobiology. Bioorg. Bertozzi. F. Biochem. J. Med. Schnaar. Synthesis and Applications. H. C. J. R. Pathol. The switch from structural uniformity in 1 the glycans have a common role in promoting protein folding.S. Goon. J. Rosen.. Chem. Golgi enzymes prepare them for the spectrum of novel functions that the sugars display in the mature proteins (3). C. The sugar composition and the number and size of branches in the sugar tree varies among glycans bound to a protein. 91. E. I. L. Biol.. Sanders. Natl. Keppler et al. the glycans play a pivotal role in protein folding. D. and the NIH and NSF for research support. Jutila. J. Am. T. 84. the spectrum of glycoforms remains rather uniform until the glycoproteins reach the medial stacks of the Golgi apparatus. 78. J. R. Int. C. Kiessling. CH-8092 Zu ¨ rich. J. Chem. a 14-saccharide Institute of Biochemistry. it is the terminal glycosylation in the Golgi complex that gives rise to the tremendous diversity seen in glycoconjugates that reach the cell surface. T. Rodriguez. Fig. Downloaded from www. L. S. E. Exp. J. C. P. Chem. the glycans do not display such heterogeneity. R.sciencemag. 97. Bertozzi. Biol. R. Biol. we mainly address events in the early secretory pathway. 274.. quality control. J. N-Linked Glycan Synthesis and Modification During the synthesis of N-linked glycans in mammalian cells (Fig. V. Switzerland. 395. tissues. Yarema. W. Itonori. Angew. 121. In the early secretory pathway. 267.A. *To whom correspondence should be addressed. J. Chem. 138 (1998). Am. E. P. Y. These are bound to the polypeptide chain through an N-glycosidic bond with the side chain of an asparagine that is part of the Asn-X-Ser/Thr consensus sequence. Gestwicki. U. Acad. 273. Esko. 2). Gordon. 80. E. T. Reutter. Baker. B. sorting. A. A. We focus on observations that are starting to unmask the logic of the various early trimming and modification events. when initially added in the ER to growing nascent polypeptides. P. In the ER and in the early secretory pathway. These glycans are then subjected to extensive modification as the glycoproteins mature and move through the ER via the Golgi complex to their final destinations inside and outside the cell. J. 90. A. 2663 (2000). In mature glycoproteins. 4076 (1975). Res. and certain sorting events. 37. R. 122. The symbols for the different sugars are used in the following figures. R. 39. C. in press.. 2364 23 MARCH 2001 VOL 291 SCIENCE www. Sanders. Chem. because the alterations are shared by all glycoproteins. Strong. Proc. 92. The division of synthesis and processing between the ER and the Golgi complex represents an evolutionary adaptation that allows efficient exploitation of the potential of oligosaccharides. Chem. L. Later. C. Am. Kishimoto. L. Soc. D. 274. Kitov et al. T. D. Terminal glucose and mannose residues are removed in the ER by glucosidases and mannosidases. Particularly in vertebrate and plant cells. Sci. 2007 (2000). Eidgeno ¨ ssische Technische Hochschule Zu ¨ rich. J. A. N-linked glycan moieties are structurally diverse. 21878 (1999). 270. J. and K. Here. Fritz. H. 11 (2000). H. Kiessling. Heisner. 2012 . where structural diversification is introduced through a series of nonuniform modifications. Sarkar. J. J. J. 6. S. Kiessling. Soc. Chem. and among cell types. U. quality control. J.CARBOHYDRATES AND GLYCOBIOLOGY 74. 98. 1). Bertozzi. and species (1. 88. 92. Taylor. 1013 (1999). K. E. 96. However. A. O. E. J. 1242 (2001). 99. Yarema. 81. M. Ed. Danishefsky. 2Institute of Microbiology. Oesch. Am. Thus. The N-linked core oligosaccharide. Gordon. 31168 (1998). L. S. C. Nature 403. Chem. Hang. 1308 (1995). F. Lett. Goebel. Nature 392. O. J. 85. 77. Esko. Fralich. K. Yarema. Biol. 5271 (1999). The “core glycans” are homogeneous and relatively simple (Fig. Angew. Alon. T. 95. Allen. Soc. Int. L.sciencemag. Med. Berg. 837 (2000). 521 (1929). R. Bertozzi. 144. Lemieux. FEBS Lett. L. Chem. L. Kayser et al. H. Yarema for their comments on the manuscript. Bundle. Bruehl. 1238 (1989). Lee et al. 94. Chem. J. We also discuss glycan structure and function in light of fundamental differences between the two biosynthetic organelles. R. 86. Biol. N. 82. oligomerization. 6674 (1991). the ER and the Golgi complex. Mahal. They are used as universal “tags” that allow specific lectins and modifying enzymes to establish order among the diversity of maturing glycoproteins. Mahal. J. O. and transport. R. Avery. Wang. K. Ed. In the Golgi complex. Commun. 93. Stehling. 97. J. (Dekker. W. C. the ER to diversification in the Golgi complex coincides with a marked change in glycan function.” in Glycochemistry: Principles. G. 30 (1998).

most of the biosynthetic machinery faces the cytosol and can directly use precursors such as sugar nucleotides and amino acids provided by cytosolic enzymes. www. the ER and the Golgi complex are fundamentally different with respect to operational principle. the glycan chains undergo further trimming of mannoses. During subsequent terminal glycosylation there is addition of new terminal sugars including GlcNAc. The addition of a GlcNAc residue is followed by trimming of two additional mannoses. it is first useful to discuss the division of labor between the ER and the Golgi complex. and is coupled through an N-glycosidic bond to the side chain of an asparagine residue. 5). 10). and terminal mannoses by one or more different ER mannosidases. In the ER. The oligosaccharyltransferase responsible for this transfer is a complex enzyme with its active site in the ER lumen (6 ). The completed core oligosaccharide is transferred from the dolichylpyrophosphate carrier to a growing. Synthesis starts on the cytosolic surface of the ER membrane by the addition of sugars. 8). SCIENCE VOL 291 23 MARCH 2001 2365 Downloaded from www. Whereas the glycoforms in the ER are homogeneous. the Golgi-generated forms are highly diverse and differ widely between species. When the glycoprotein has folded (gray oval) and reached the Golgi complex. Immediately after coupling to the polypeptide chain. as are the number and identity of sugars added. new sugars are added during terminal glycosylation to produce complex N-linked glycans. Why does the cell assemble a large oligosaccharide.or extracellular locations. Biosynthesis of the N-linked core oligosaccharide. 2012 . so that molecules originating in the ER pass through the Golgi complex on their way to their final intra. Mature glycoproteins carry a mixture of complex glycans as well as some high-mannose glycans that have escaped terminal glycosylation. the oligosaccharyltransferase enzyme complex catalyzes the transfer of the core oligosaccharide to the asparagine residues of nascent. 4. nascent polypeptide chain emerges from the translocon (7. one by one. with glucosidase II. nascent polypeptide chain. and then trim it down in order to rebuild it again in a different form? Why is the synthesis and modification shared between two separate biosynthetic organelles? Why does the ER contain enzymes both to remove and to restore glucose residues? Operating Principles of the ER and the Golgi Complex To better understand the switch that occurs in N-linked glycan biosynthesis midway through the maturation of a glycoprotein. The two organelles follow each other in series. the oligosaccharide is flipped to the lumenal side of the membrane. Only one of many possible terminal glycosylation pathways is shown. it has been estimated that 90% are glycosylated (9). sialic acid. and seven further sugars are added from lipid precursors. Although they are partners in the same pathway. After the last of the three glucoses have been added.CARBOHYDRATES AND GLYCOBIOLOGY “core” unit is assembled as a membranebound dolichylpyrophosphate precursor by enzymes located on both sides of the ER membrane (1. 2. a deglucosylation-reglucosylation cycle. When the glycoprotein moves to the Golgi complex. This Fig. The three glucoses are trimmed away by glucosidase I and II. just five sugars remain. Of all sequons. terminal glucose and mannose residues are removed by ER glucosidases and mannosidases (1. and probably evolutionary on April 24. galactose. and fucose. It recognizes a specific conformation of the glycosylation sequon (the sequence Asn-X-Ser/Thr) transiently formed when the growing.sciencemag. the number of branches generated is variable. the products are translocated across the membrane into the lumen. further mannose trimming occurs. growing polypeptide chains. The ER also contains a glucosyltransferase that can regluco- sylate glucose-free chains and thus establish. This biosynthesis pathway is puzzling in several respects. Of the original core glycan. Sometimes there is variability in the efficiency by which individual sequons are recognized. In many cases. giving rise to heterogeneously glycosylated products. After synthesis. When two N-acetylglucosamines and five mannoses have been added. overall architecture.sciencemag. One of many possible terminal glycosylation pathways is shown in Fig. to dolichylphosphate.

whereas many display no dependence at all. Another major difference is that synthesis in the Golgi complex makes use of soluble precursors (nucleotide sugars and other nucleotide precursors). Even severe glycosylation defects or deficiencies are. This eukaryotic adaptation of glycan function allows cells to produce and secrete larger and more complex proteins at higher levels. proteolytic enzymes. This implies that oligosaccharide appendices have local effects on protein folding. Like protein translocation. In vitro refolding studies using glycosylated and nonglycosylated versions of the same protein confirm that oligosaccharide moieties have a positive effect on the folding process (27. In those few cases where precursors are transported to the lumenal side. for example. This is dictated by the fact that the proteins or protein domains to be modified have already been translocated to the lumenal side in the ER. 25).e. Substrate proteins constitute a minority population in this large organelle (12). The Calnexin-Calreticulin Cycle The most important indirect effect of glycans on folding involves a unique chaper- 2366 23 MARCH 2001 VOL 291 SCIENCE www. it alters the conformational preferences close to the glycosylation site. 28). The sequences of Golgi glycosyltransferases suggest homology with cytosolic enzymes responsible for the use of sugar nucleotides (22. it is important to point out that glycans are not usually essential for maintaining the overall folded structure once a glycoprotein has folded (25. However. as nucleotide sugars and soluble precursors would diffuse away if transported out of the cell. the Golgi complex assembles its synthetic products on the lumenal side. ER-like exit criteria on individual molecules is one of the reasons why the Golgi complex is divided into on April 24. resulting in more compact conformations (28). 28)—the effect is usually rather small. 27. In contrast. One-third of N-linked glycans in glycoproteins occur in such locations (29. N-Linked Glycans Help Proteins Fold Whereas N-glycosidic links serve a structural role in the archaeal cell wall (19. It also applies. as is the origin of the Golgi complex itself. plasma membrane (18. although lacking ER-like quality control and degradation systems. where Ac is acetyl and Glc is glucose)] attached to an asparagine tends. The ER contains a high concentration of soluble molecular chaperones and folding enzymes. glycans can. 30). or cisternal progression (17 )— ensures a high degree of completeness in the biosynthetic processes without imposing direct quality control. 25. Before discussing the indirect effects of glycans on the folding process. lectins. the oligosaccharides seem to have both “local” and “global” effects on the folding process. A truncated N-linked glycan [the disaccharide chitobiose (GlcNAc␤1-4GlcNAc. folding may be compromised even though none of the glycans are needed individually. Thus. as well as to the synthesis of phospholipids. 31) and can keep the proteins in solution. These are present in considerably higher concentrations than in the ER (13). This. as a rule. For example. the Golgi complex does not have a rigorous system for controlling the fidelity of its biosynthetic processes. The importance of the added glycans varies between proteins and depends on the physiological context. 2012 . In addition.and dolichylpyrophosphatelinked oligosaccharides. and. 15). some become temperature-sensitive for folding. resident proteins (glycosidases. permeases. or when specific glycosyltransferases are mutated. a variety of other functions dominate in eukaryotes. aggregated proteins that fail to reach a functional state (2. the most commonly observed effect is the generation of misfolded. most likely archaeal. 33). be removed from a folded glycoprotein without major effects on protein conformation. Some are partially dependent. Synthetic events thus occur on the inside surface of the membrane.CARBOHYDRATES AND GLYCOBIOLOGY topological arrangement applies to the polypeptide synthesis that occurs on membrane-bound ribosomes. In the Golgi complex. makes it possible for the Golgi to produce such a wide diversity of structures. to induce a compact ␤ turn.sciencemag. as a rule.sciencemag. it is often found that some are more important than others. soluble sugar nucleotides would diffuse away. 21). 2). It is tempting to speculate that the need to ensure full processing without imposing stringent. when cells are treated with glycosidase inhibitors. Lipid-linked intermediates. The evolutionary origin of the glycosylation machinery in the Golgi complex is unclear. before the folding process has begun). glycosyltransferases. The programmed cis-to-trans movement—whether by vesicular transport. all archaeal genomes sequenced so far contain one or more homologs of the eukaryotic oligosaccharyltransferase subunit STT3 (20. The presence of a large polar saccharide unit is also likely to affect the folding process locally by orienting the polypeptide segment toward the surface of protein domains. and transport receptors) are membrane-bound. When glycosylation is inhibited. with the lumen being occupied by substrate molecules and other cargo. in turn. are not used. tolerated by the Golgi without retention or degradation. 23). 26 ). In archaea. Consequently. Although the presence of oligosaccharides does influence the properties of the polypeptide moiety—for example. Analysis at the genomic level confirms the presence of a biosynthetic pathway for dolichylphosphate-linked oligosaccharides in Archaeoglobus fulgidus (5). The single most important one is the promotion of proper folding of newly synthesized polypeptides in the ER (2. These are imported from the cytosol through the Golgi membrane by specific transporters (11). As in eukaryotes. This implies that the enzyme system in the Golgi and the machinery in the ER may have different evolutionary origins. transport occurs almost exclusively through lipid-linked intermediates (such as dolichylphosphomannose or dolichylphosphoglucose) rather than as soluble precursors. to the synthesis of dolichol precursors for N-linked glycans (Fig. the oligosaccharides are transferred to asparagine side chains in the same Asn-X-Ser/Thr sequence motif that is used by the ER oligosaccharyltransferase in eukaryotic cells (19). In archaea. Substrate molecules are progressively modified as they move from the entry side (cis) to the exit side (trans) (16 ). percolation. The explanation lies in the multicompartmental architecture of the Golgi complex. the Golgi complex does not export unfinished products. by increasing stability (27. glycoproteins carrying defective glycan moieties are readily exported (14. This makes sense because the coupling of protein and carbohydrate is likely to occur extracellularly. Studies performed using peptides indicate that although an N-linked glycan does not induce permanent secondary structure. synthesis makes use of dolichylphosphate. Glycoproteins must traverse the whole stack before they can be exported. if exported. It would also seem reasonable to assume that the machinery now present in the Golgi complex evolved after an intracellular membrane system was already in place. Most glycoproteins end up spending about the same length of time (usually 5 to 15 min) in the Golgi exposed to the full panel of modifying enzymes. But analysis of the secreted products of normal cells shows that they are appropriately modified. Thus. N-linked glycosylation clearly belongs to the functions that the ER has inherited from the prokaryotic. Unlike the ER. It is well known that glycans can have a direct effect on the folding process (27. When the importance of individual glycosylation sites is evaluated. and some are glycan-dependent in one cell type but not in another. It also explains why the addition of N-linked glycans must occur cotranslocationally in the ER (i. N-linked glycans occur in the S-layer of the cell wall. thus mimicking the role played by many molecular chaperones (32).org Downloaded from www. 24 ). Some proteins are completely dependent on glycosylation. 19).. such as those in the ER. in part. when multiple sites are modified.

a glycoprotein is no longer recognized by the glucosyltransferase. 3). Shortlived disulfide bonds occur between ERp57 and cysteines in calnexin.sciencemag. Calnexin (membrane-bound) and calreticulin (soluble) are homologous ER lectins that bind transiently to virtually all newly synthesized glycoproteins (37. 2012 . If the glycoproteins have cysteines. Association of glycoproteins with calnexin and calreticulin involves a bindingand-release cycle driven by the opposing actions of two soluble ER enzymes. blocks premature oligomerization. 43). 49). The calnexin-calreticulin cycle promotes correct folding. inhibits aggregation of folding intermediates. The protein is thereby exposed to another folding factor. hydrophilic peptide arm formed by the P-domain (39 – 41). and the latter removes it (Fig. ERGIC- 53. It can now leave the ER. Transgenic mice devoid of calreticulin die on embryonic day 18 (55). If there is a glycosylation site within the first 50 amino acids of a nascent glycoprotein. calreticulin. Once correctly folded. or features such as excessive dynamic mobility. The calnexin-calreticulin cycle seems to be essential in vivo. In this cycle. GT serves as the folding sensor. the formation of disulfide bonds is catalyzed through the formation of transient mixed disulfides with ERp57. the lectins retain misfolded conformers in the ER and thus mediate the quality control process that regulates ER-to-Golgi transport.and calreticulinbound substrate glycoproteins (46 ). the complexes dissociate. The calnexin-calreticulin cycle. GT forces incompletely folded conformers to remain in the calnexin-calreticulin cycle. In vivo and in vitro studies have shown that this remarkable enzyme only reglucosylates incompletely folded glycoproteins. glucose addition depends on the folding status of the protein moiety (36. There are also reports that calnexin and calreticulin can interact with polypeptide chains devoid of oligosaccharides (48. which binds to mannose residues. and possibly the ER degradation process as well (see below). depending on the rate of folding. www. Calnexin and calreticulin form a complex with ERp57. When the remaining third glucose residue is trimmed by glucosidase II. Substrate recognition by GT is triggered not by specific signal sequences but by general. it is unlikely to form the lectin binding site. a thiol oxidoreductase that binds to both calnexin and calreticulin. the oligosaccharides are reglucosylated by an ER glucosyltransferase. where Man is mannose) (38. the interaction with calnexin. and provides quality control by preventing incompletely folded glycoproteins from exiting to the Golgi complex. 42. 50). 52). the nascent or newly synthesized glycoproteins bind to calnexin (CNX) and/or calreticulin (CRT). and because it is no longer reglucosylated. By recognizing the folding status.sciencemag. that is. 3). The cycle is repeated until the protein is either folded or degraded. For many glycoproteins. are not glucosylated (54 ). and ERp57 slows down the rate of folding but increases efficiency. it will not bind back to calnexin and/or calreticulin. and the protein reassociates with the lectins.CARBOHYDRATES AND GLYCOBIOLOGY one system found in the ER of nearly all eukaryotes. but it is unclear whether this is important for protein maturation in cells. binding to the lectins continues for a period ranging from a few minutes to several hours. SCIENCE VOL 291 23 MARCH 2001 2367 Downloaded from www. 38). whereas folded conformers are allowed to proceed further in the secretory pathway. The essential nature of the pathway is also Fig. 3. interaction with calnexin and calreticulin begins cotranslationally (44 ). Even though not absolutely necessary for the folding of a protein. They are highly asymmetric molecules with a long. 51. or it may form a region of the chaperone that protects the bound substrate molecule during folding. These may include exposed hydrophobic patches. the so-called calnexin-calreticulin cycle (34 –36 ) (Fig. Calnexin and calreticulin interact with the glycan moieties of substrate glycoproteins after they have been trimmed by glucosidases I and II to the monoglucosylated form (Glc1Man9-6 GlcNAc2. 38. or in domains that have a random coil conformation. biophysical properties common to partially folded glycoproteins. two homologous ER lectins specific for monoglucosylated core oligosaccharides. regulates ER degradation. a thiol oxidoreductase homolog of protein disulfide isomerase (45). Such bonds serve as intermediates in oxidation and isomerization reactions and lead to the formation of correctly paired disulfide bonds (47 ). uridine diphosphate (UDP)–glucose:glycoprotein glucosyltransferase (GT) and glucosidase II (36. When two of the glucoses in the N-linked core glycans have been trimmed away by glucosidases I and II. Exit of certain glycoproteins from the ER to the Golgi complex is assisted by another membrane-bound lectin. The former adds a glucose residue to high-mannose glycans. If the glycoprotein is not folded at this time. After chain on April 24. It is apparent that GT specifically reglucosylates glycans in glycoprotein domains that have a partially folded structure (53). Glycans in nearby folded domains. This domain may interact with the cochaperone ERp57. curved. Glycans serve as an “admission ticket” to this cycle.

the Golgi complex. suggesting a hepatic secretion defect. In this on April 24. endosomes. the late endosome ( pH 5). with mannose trimming as one of the most conspicuous sorting criteria. It has been proposed that ERGIC-53 serves as a cargo capture and transport receptor for ER-to-Golgi traffic of glycoproteins (Fig. however. multifunctional insulin-like growth factor II/cation-independent M-6-P receptor (IGF-II/CI-MPR). by orphan subunits of oligomers. That these receptors serve a cargo capture. Given that the mannosidase is relatively slow-acting.sciencemag. resulting in a Glc0-3Man8GlcNAc2 structure (B isoform) (Fig. On the whole. and transport function is beyond doubt. the most abundant of which are the homologs LAMP-1 (lgp110) and LAMP-2 (lgp120) (80 – 82). additional lysines direct the enzyme to one of multiple Nlinked glycans. able to endocytose lysosomal enzymes from the cell surface. it relies on conformation-dependent substrate recognition. This cannot be the only pathway. It is a homo-oligomeric membrane protein that shuttles among the ER. the remaining mannose-6-phosphate group is recognized by mannose-6-phosphate receptors (M-6-P receptors) in the TGN. patients with nonfunctional ERGIC-53 have been found to suffer from an autosomal recessive bleeding disorder with combined deficiency of coagulation factors V and VIII (F5F8D) (75). degradation of glycoproteins essentially stops (62– 64 ). In this well-characterized process.CARBOHYDRATES AND GLYCOBIOLOGY illustrated by the lethal outcome of an inherited glucosidase I deficiency involving a neonate born with severe generalized hypotonia and dysmorphic features (56 ). VIP36 may play a similar function. ERGIC-53 (also called p58) is the better characterized of the two (69. targeting. however. 67 ). Their role is to sequester lysosomal enzymes in the TGN by associating with one or more of the oligosaccharides. With the mannose gone from the glycans. Like GT. It selects the lysosomal enzymes among all cargo coming from the ER. They are both membrane proteins with a lumenal domain homologous to leguminous plant lectins. multisubunit enzyme in the cis-Golgi membrane. LAMP-2 was recently dem- 2368 23 MARCH 2001 VOL 291 SCIENCE www. ERAD has a central clearance function in the cell. Recognition requires critically spaced lysine residues in the surface of the protein and involves. flattened ␤ barrel with the mannose-6-phosphate binding site in a cavity between two loops (79). because mannosidase-dependent degradation of some glycoproteins occurs without involvement of either calnexin or GT (66. Moreover. How the system works is not clear. The available data suggest multiple. Because misfolded side products are common even under unstressed conditions.2–linked mannose residue from the ␣-1. it has been suggested that it provides a timer function that awards protection against premature degradation to the most recently synthesized glycoproteins (25. and are thus clearly different from the other intracellular lectins described above. The limiting membrane contains a collection of highly glycosylated membrane proteins.5). and by some heterologously expressed proteins. a wide surface of contact (78). the TGN ( pH 6. Lysosomes and related organelles constitute an exception. early in the pathway they carry mainly high-mannose glycans. and pH dependence of ligand binding. oligomerization state. These include the dimeric cation-dependent M-6-P receptor (CD-MPR) and the much larger. 65). and the Golgi complex. This mannosidase removes a single ␣-1. Although it is not essential for the secretion or maturation of any known glycoprotein. the phosphotransferase.3 branch of the core oligosaccharide. Recent studies with mutant ␣1-antitrypsin have led to a model in which calnexin plays a central role by sequestering Glc1Man8GlcNAc2-containing glycoproteins (64 ). the glycoprotein can be degraded. Together they constitute the P-type family of animal lectins. That glycan modifications in the Golgi complex could still occur normally without glucose removal in the ER is explained by the presence of an endomannosidase enzyme in the Golgi complex (57 ). avidin-like. but differs in that it only modifies folded forms of the appropriate substrate proteins. The M-6-P receptors are homologous type I membrane proteins that differ in size. where they are ubiquitinated and degraded by 26S proteasomes (59 – 61). 3) (69). 2012 . effective mannosidase I– dependent ERAD system (65) but lacks a calnexin cycle and GT activity (68). Instead of using a lumenal degradation system that might endanger protease-sensitive folding intermediates. This fate is shared by misfolded and mutant proteins. because most proteins that have folded normally are mannose-trimmed before leaving the ER. carbohydrate-covered coat over the lumenal surface of the lysosomal membrane (81). and return empty to the TGN. Proteins that fail to reach their native conformation in the ER are selectively eliminated by ER-associated degradation (ERAD) (58. In some of the substrates.3 to 6. Removal of the mannose is not sufficient. the intermediate compartment. Oligosaccharides as Signals in ER-Associated Protein Degradation Trimming of the N-linked glycans also plays a role in the sorting process leading to glycoprotein degradation in the ER. IGF-II/CIMPR also binds mannose-6-phosphate at neutral pH and is therefore. 64. 1). The crystal structure of the lumenal domain of CDMPR shows that the protein is a homodimer with each monomer composed of a ninestranded. and lysosomes are themselves often glycoproteins. a large. These have up to 20 N-linked glycans each. parallel pathways leading to degradation. Their sugar composition reflects their location. The glycans have been shown to protect the LAMPs against degradation by lysosomal proteases (83). in addition. escort them via clathrin-coated vesicles to endosomes. Role in Transport and Targeting ERGIC-53 and VIP36 are homologous. The key event occurs in the cis-Golgi compartment where a resident enzyme (UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase) specifically modifies N-linked glycan moieties in lysosomal hydrolases by adding GlcNAc-phosphate to terminal or subterminal mannoses. and it has been estimated that they occur in sufficient concentrations to form a nearly continuous. Moreover. unlike CDMPR. 74 ). The pH dependence of the receptors is critical because net transport depends on the higher affinity for the ligand at the pH prevailing in the loading compartment. The plasma levels of these two highly glycosylated coagulation factors are reduced to 5 to 30% of normal. ERGIC-53 has been shown to associate with human cathepsin Z and accelerate its transport to the Golgi in a glycan-dependent fashion (69). 59). It is apparent that the resulting Man8 structures serve as part of the signal needed for ERAD. mannose-specific lectins in the Golgi complex and the early secretory pathway (69 –73). than in the release Downloaded from www. Saccharomyces cerevisiae has an Mannose-6-Phosphate and Targeting of Lysosomal Hydrolases The selective targeting of lysosomal hydrolases from the trans-Golgi network ( TGN) to endosomes and lysosomes also makes use of lectins (76 ).sciencemag. Other Intracellular Functions of N-Linked Glycans Resident proteins of the ER. GT would be the folding sensor for degradation. whereas in the Golgi complex or in post-Golgi organelles they contain complex sugars. they are more sparingly glycosylated than glycoproteins in the plasma membrane and extracellular space. provided that it is incompletely folded. When trimming by ER–mannosidase I is prevented by inhibitors or genetic manipulation. serves as the primary sensor (77 ). The defects were likely caused by a shutdown of the calnexin-calreticulin cycle. cells transport proteins that need to be destroyed to the cytosol. After removal of the GlcNAc residue.

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