Evolutional Conservation of the Glycan-mediated Quality Control System for Newly Synthesized Proteins in the Endoplasmic Reticulum (ER)

The endoplasmic reticulum (ER) is the site for biosynthesis of soluble/membrane proteins that go through the secretory pathway. A Eukaryotic cell has a mechanism called the "quality control system" in the ER that distinguishes the functional proteins from aberrant ones, so that only the former exit the compartment via vesicular transport to their respective final destinations. Recent studies have revealed that the asparagine(N)-linked oligosaccharides on proteins play pivotal roles in the folding and maturation of glycoproteins. For instance, the glycan can serve as a "tag" to represent the folding status of proteins, so they can be aided by molecular chaperones to acquire correct folding, or in other cases, they are targeted for degradation. The glycan-dependent quality control machinery involves (1) transfer of the oligosaccharide Glc3Man9GlcNAc2 from dolichylpyrophosphate to the nascent proteins; (2) rapid deglucosylation by a-glucosidase I/II; (3) reglucosylation of unfolded/misfolded proteins by UDP-Glucose: glycoprotein glucosyltransferase, a folding sensor to distinguish the folded proteins from the malfolded ones; (4) recognition of the monoglucosylated glycans by lectins called calnexin (CNX; membrane protein) and calreticulin (CRT; a soluble homologue of CNX) (Figure). CNX/CRT play a role as glycan-dependent molecular chaperones, avoiding the malfolded glycoproteins to aggregate and aiding their folding (though it is not clear at this moment if it is a direct or indirect effect as far as folding-promoting activity is concerned). The monoglucose recognized by these lectins is eventually removed by -glucosidase II, allowing UGGT to judge whether the proteins finally achieve correct folding. If not, UGGT again pronounces a sentence of guilty for being misfolded on the protein, and again reglucosylates it. This cycle of glucosylation/deglucosylation is often called the "calnexin cycle."

The above scheme holds true for most eukaryotes, but there are a few exceptions: (a) Trypanosomatides transfer unglucosylated oligosaccharides in protein N-glycosylation, namely Man6GlcNAc2, Man7GlcNAc2 or Man9GlcNAc2, depending on the species. Therefore, the sugars on proteins are ready for glucosylation by UGGT without deglucosylation. (b) A calnexin orthologue (Cne1p) does not appear to have lectin-activity nor are there functional orthologues for UGGT in budding yeast (baker`s yeast). Accordingly there is no "calnexin cycle" in this organism. The case of budding yeast seems to be a rare exception, however. For instance, fission yeast, another yeast widely used for life science studies, has all the components mentioned above.

If proteins consistently fail to achieve a correct folding state despite the help of CNX/CRT, not only are they not functional but they can also be toxic to cells as they form an aggregate inside the cell. Therefore, the cell has a mechanism to eliminate such deleterious proteins by retro-translocating them from the lumen of the ER into the cytosol, where the proteasome degrades them. This mechanism is called ER-associated degradation (ERAD). The EDEM protein (ER degradation enhancing -mannosidase-like protein) has been discovered as a putative lectin molecule that recognizes proteins bearing the Man8B (or smaller) oligosaccharides and targets them for proteasomal degradation. The detailed mechanism by which EDEM recognizes the substrate remains to be determined. This molecule is well conserved from budding yeast to human.

Figure Glycoprotein ERAD (GERAD)
Tetradecasaccharides, Glc3Man9GlcNAc2 synthesized on the dolichol pyrophosphate, are transferred en bloc onto the nascent proteins by oligosaccharyltransferase in the ER lumen. Soon after the transfer, glucoses at the non-reducing end are removed by -glucosidase I/II. The calnexin cycle (CNX/CRT cycle) is mediated by the interplay between UGGT and -glucosidase II, which are both inhibited by the action of ER -mannosidase I. The correctly folded proteins escape from the recognition by UGGT and therefore from the calnexin cycle, and if the folding occurs fast enough it will exit the ER without the prior action by ER -mannosidase I. On the other hand, glycoproteins bearing Man8B (and in some cases shorter ones in which more Man are trimmed) also exit the cycle and are recognized somehow by EDEM, which allow these proteins to be targeted for degradation. Once in the cytosol after the retrotranslocation, the SCF (Skp1-cullin-Roc1-Fbox protein) complex bearing sugar-binding Fbx2 (Fbs1) or other family members binds to the N-linked glycans on glycoproteins, and the proteins are polyubiquitinated by the action of this complex. The polyubiquitinated proteins are deglycosylated by PNGase prior to the action of the 26S proteasome.

In mammalian cells, once the misfolded glycoproteins are exported out of the ER, glycoprotein-specific ubiquitin ligase, Fbs1/2 (Fbx2 and their paralogues) binds to the carbohydrates on the proteins and modifies them with polyubiquitin, a recognition tag for proteasomal degradation. So far no apparent orthologues of this protein family in lower eukaryotes have been reported, and it remains to be revealed how widespread the glycoprotein-specific ubiquitination is. Finally, prior to the degradation by proteasome, the N-linked glycans are removed by the action of the cytoplasmic peptide:N-glycanase (PNGase). This deglycosylating enzyme is also distributed widely in eukaryotes. However, the metabolic pathway of free oligosaccharides formed by the PNGase-action seems quite distinct between human and yeast. The molecular details of this process have not been revealed in any system.

Tadashi Suzuki (Department of Biochemistry, Graduate School of Medicine, Osaka University)
References (1) Spiro RG: Role of N-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation. Cell Mol. Life Sci. 61, 1025-1041, 2004
(2) Helenius A, Aebi M: Role of N-linked glycans in the endoplasmic reticulum. Annu. Rev. Biochem. 73, 1019-1049, 2004
GP-B07Sweet ER Quality Control: The Role of N-glycosylation in Protein Quality Control (Sato Sachiko)
GP-B08Biological Roles of N-glycanase in Cell (Sadako Inoue)
GP-C04 N-linked Oligosaccharide as a Signal for Proteolysis (Tadashi Tai)
-Mannosidase-like Protein Involved in Quality Control in the Endoplasmic Reticulum(ER) (Nobuko Hosokawa, Kazuhiro Nagata)
Cytoplasmic Peptide:N-Glycanase (Tadashi Suzuki)


Dec. 28, 2004

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