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Compartmentalization and Selective Tagging for Disposal of Misfolded Glycoproteins
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Opinion
Compartmentalization and Selective Tagging for Disposal of Misfolded Glycoproteins Marina Shenkman1 and Gerardo Z. Lederkremer1,* The ability of mammalian cells to correctly identify and degrade misfolded secretory proteins, most of them bearing N-glycans, is crucial for their correct function and survival. An inefficient disposal mechanism results in the accumulation of misfolded proteins and consequent endoplasmic reticulum (ER) stress. N-glycan processing creates a code that reveals the folding status of each molecule, enabling continued folding attempts or targeting of the doomed glycoprotein for disposal. We review here the main steps involved in the accurate processing of unfolded glycoproteins. We highlight recent data suggesting that the processing is not stochastic, but that there is selective accelerated glycan trimming on misfolded glycoprotein molecules.
Highlights N-glycan processing in the early secretory pathway by glucosidases and mannosidases generates a code that tags glycoprotein molecules according to their folding status. The code is interpreted by specific lectins that route the glycoproteins towards productive maturation or towards disposal by ER-associated degradation. Compartmentalization of the mannosidases appears to slow down the trimming of mannose residues, allowing time for nascent unfolded molecules to fold.
The Glycan Code for Glycoprotein Quality Control Pioneering work by Ari Helenius in the early 1990s on the lectin (see Glossary) and endoplasmic reticulum (ER) chaperone calnexin (CNX) [1], coupled with the earlier discovery by Armando Parodi of a transient glucosylation of nascent glycoproteins [2], led to the realization that a glycan code exists in the cell that reflects glycoprotein folding status. The glycan code instructs the cell whether to continue to fold a glycoprotein molecule or to deem it terminally misfolded and target it for degradation [3–5]. This decision must be precise and finely tuned, leading otherwise to premature glycoprotein disposal or conversely to accumulation of misfolded molecules in the ER, causing ER stress. The modifications are on N-glycans, which are prevalent in the vast majority of membrane and secretory proteins. It was initially thought that the glycan code consists of three steps in mammalian cells: (i) trimming of the three terminal glucose residues from N-glycan precursors, (ii) a checkpoint where a folding-sensor glucosyltransferase re-adds one glucose, allowing rebinding of CNX, and (iii) removal of one mannose residue by ER mannosidase I (ERManI/MAN1B1), which somehow tags the glycoprotein molecule for degradation [6,7]. Recent work has unveiled a more complex and fascinating mechanism, with additional checkpoints, subcellular compartmentalization, and regulated extensive glycan trimming, that tags an unfit glycoprotein molecule for disposal (Figure 1, Key Figure).
Recent data indicates that misfolded glycoproteins are trimmed considerably faster than properly folded glycoproteins by folding-sensitive mannosidases associated with oxidoreductases and are consequently targeted for disposal.
The Initial Steps of a Nascent Glycoprotein Secreted or plasma membrane proteins, or those resident in secretory compartments, are initially translocated into the rough ER (RER) [8]. The presence of a signal sequence or internal anchor sequence makes them recognizable by the signal recognition particle (SRP) and allows binding to the SRP receptor on the RER membrane as well as to the translocation channel formed by the Sec61 complex [8]. Most secretory proteins are N-glycosylated on asparagine residues in the canonical sequons N–X–T/S, where X is any amino acid except proline [9,10]. In mammalian cells, N-linked glycans are usually cotranslationally added to proteins in the ER lumen as a presynthesized oligosaccharide precursor [10]. This 14-sugar oligosaccharide, Glc3Man9GlcNAc2 (G3M9) (Figure 2), is transferred from the carrier molecule dolichol pyrophosphate to asparagines by oligosaccharyltransferase (OST) [10,11]. This enzyme contains eight Trends in Biochemical Sciences, October 2019, Vol. 44, No. 10
1
School of Molecular Cell Biology and Biotechnology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
*Correspondence: [email protected] (G.Z. Lederkremer).
https://doi.org/10.1016/j.tibs.2019.04.012 © 2019 Elsevier Ltd. All rights reserved.
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subunits, of which only one, STT3, is catalytic [12]. STT3 comes in two variants, STT3A, which forms part of an OST complex that functions cotranslationally, and STT3B that is part of a complex that acts post-translationally [13]. OST cryo-electron microscopy (cryo-EM) structures, which have recently been determined at atomic resolution, reveal its association with the Sec61 translocon and with subunits recognizing the acceptor peptide during its appearance from the translocon [14–16]. Following N-glycosylation, the G3M9 oligosaccharide begins to undergo processing in which the nonreducing terminal Glc is first removed by glucosidase I (GI). The resulting G2M9 is bound specifically by malectin [17], which was shown to inhibit secretion of defective cargo glycoproteins [18]. Removal of an additional Glc by glucosidase II (GII) to yield G1M9 allows glycoprotein association with a unique chaperone system known as the calnexin/calreticulin cycle [19] (Figures 1 and 2). Association of glycoproteins with CNX, or with its soluble functional homolog calreticulin (CRT), involves binding and release cycles driven by opposing actions of two soluble ER enzymes, GII and UDP-Glc:glycoprotein glucosyltransferase 1 (UGGT1) [19]. GII removes the last remaining Glc to yield M9, causing dissociation of CNX, whereas UGGT1 reglucosylates Man9GlcNAc2 (M9), allowing reassociation with the lectin CNX. UGGT1 serves as a folding-sensor by recognizing the folding state of the glycoprotein [20]. If the glycoprotein is correctly folded, it is no longer a substrate of UGGT1 and continues its maturation through the secretory pathway. However, if it is still unfolded or misfolded, UGGT1 recognizes an exposed hydrophobic patch on the protein moiety and reattaches a Glc residue to M9, allowing reassociation with CNX for further folding attempts [21] (Figure 1). Therefore, proper folding allows glycoprotein release to the Golgi apparatus, where the N-glycans are further modified by trimming and extension to generate a diverse array of structures which are recognized for targeting by transport vesicles to different final destinations [22]. Terminally misfolded glycoproteins are at some point removed from the CNX cycle and are targeted to ER-associated degradation (ERAD) [23,24].
Mannosidases – The Mannose Trimming Code How is a misfolded glycoprotein removed from the CNX cycle and targeted to ERAD? As mentioned above, it was initially thought that ERManI was the only mannosidase acting on nascent glycoproteins in mammals. This mannosidase was believed to remove only one mannose residue from the middle B branch [Figure 2, mannose (b)] of the N-glycan precursor, producing M8B [25]. However, experiments with mutant mammalian cells that transfer to proteins a glycan precursor that lacks branches B and C showed that removal of the terminal mannose residue from branch A is necessary for ERAD [26]. Wild-type mammalian cells were also found to trim N-glycans of ERAD substrate glycoproteins to Man6GlcNAc2 and Man5GlcNAc2 (M6 and M5), removing most or all α1,2-linked mannose residues before routing to degradation [27,28]. Three aims are fulfilled by this extensive trimming: (i) because the terminal mannose (a) in Figure 2 is the glucose acceptor in the CNX cycle, removal of this residue precludes re-entry of the glycoprotein molecule in this folding cycle; (ii) the trimmed M5-6 structures bind with high affinity to the lectin OS-9 or to its functional homolog XTP-3B, which target the glycoprotein to ERAD, whereas these lectins cannot bind the untrimmed glycan; and (iii), the trimmed species do not bind to the lectins VIPL, VIP36, or ERGIC53, and are therefore selected against in ER–Golgi transport [29]. We now know that ERManI, although preferring mannose (b) in Figure 2, is capable of removing all four α1,2 mannose residues in vitro [30,31] and in cells in vivo [27,32]. Therefore, ERManI might act alone. However, it is puzzling that there are several other α1,2 mannosidases in mammalian cells, all part of the glycosylhydrolase family 47, which might have redundant activity, including the EDEMs 1–3 and the Golgi α1,2 mannosidases, Man IA (MAN1A1), IB (MAN1A2), and IC 828
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Glossary Calnexin/calreticulin cycle: calnexin (CNX) and calreticulin (CRT) are lectins that assist the folding of glycoproteins by interacting with folding intermediates in a cycle of binding and release driven by the addition and removal of a glucose residue. CNX or CRT binding provides a protective environment for folding, helps to inhibit aggregation, and prevents premature ER exit. Cargo glycoproteins: glycoproteins to be sorted to other organelles that are concentrated at the vesicle budding site in the donor organelle. EDEMs: ER degradation-enhancing αmannosidase-like proteins that belong to the glycosylhydrolase family 47 and have α-mannosidase-like domains with conserved catalytic residues. ER-associated degradation (ERAD): a process that includes recognition of misfolded domains of ER proteins, retrotranslocation from the ER to the cytosol, protein modifications (e.g., ubiquitination) necessary for targeting, recognition, and finally degradation by the cytosolic proteasome. ER-derived quality control compartment (ERQC): a pericentriolar compartment where ERAD substrate proteins concentrate in a microtubuledependent manner. ERAD machinery components are recruited to the ERQC, in a Herp-dependent process, making it a likely staging ground for ubiquitination and degradation. CNX and CRT are recruited to the ERQC, but not other ER proteins such as BiP or PDI. It is a distinct compartment that does not colocalize with the Golgi apparatus, the ERto-Golgi intermediate compartment (ERGIC), or other organelles. ER stress: a condition caused by a high load of unfolded proteins in the ER lumen, which results in disruption of folding and secretory capacity. ER stress triggers a defensive process, the unfolded protein response (UPR), that is aimed at re-establishing protein homeostasis. Lectins: sugar-recognition proteins that exhibit distinct sugar-binding specificities and affinities, and play an important role in glycan structure detection. N-glycans: oligosaccharides that are covalently attached by an N-glycosidic bond to the protein moiety in glycoproteins via asparagine residues within the N-X-T/S consensus sequence (sequon), where X is any amino acid except proline.
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(MAN1C1) [4,33] (although at least ManIA has recently been shown not to localize in the Golgi but instead is located in specialized vesicles, described later). ManIA has recently been implicated in targeting to ERAD [34]. Overexpression of ManIB or ManIC was also reported to accelerate ERAD [35]; however, their overexpression could increase their ER levels, and thus it remains to be shown if they are actually involved. The EDEMs were initially thought to be lectins and were only very recently shown to have bona fide mannosidase activity in vitro [31,36]. The extensive trimming creating the ERAD-targeting tag is probably a combination of the activities of these enzymes, which have slight differences in their preferred mannose residues in vitro (Box 1). ERManI would usually act first by preferentially removing mannose (b), and EDEM1 or EDEM2 would excise mannose residues (a) or (c) [31,37–39]. ManIA would then preferentially remove the remaining mannoses to yield M5-6 [34,40,41]. Although ERManI and the EDEMs can produce M5-6 in vitro, this is to a low extent [31]. Trimming to M5-6 allows binding of OS-9 or XTP3-B and targeting to ERAD [42–44] (Figure 2). M7A could also bind OS-9, but the absence of mannose (c) strongly reduces the sensitivity to GII [45], which would prevent the glycoprotein from being deglucosylated and leaving the CNX cycle. An alternative pathway where ManIA starts by excising its preferred mannose (a) [40,41] is less common. This would reduce exit to the Golgi (Box 1), and it would also prevent reglucosylation, thus immediately removing the glycoprotein from the CNX cycle. The specificity of EDEM3 in vitro has not yet been determined. There is also an alternative pathway by which glycoprotein molecules can escape the ER carrying a G1M9 glycan; without prior trimming of the last Glc residue, G1M9 glycans can be processed by an endomannosidase that cleaves between mannose residues (a) and (d) in Figure 2. After cleavage, these molecules can be retrieved and targeted to ERAD [46].
Subcellular Compartmentalization Explains the Slow Rate of Mannose Trimming As one can observe in Figures 1 and 2, the process of mannose trimming could rapidly lead to the detrimental tagging and targeting to ERAD of nascent, still unfolded, or partially folded molecules. How is this avoided? In our opinion, the secret lies in compartmentalization [4,47]. We have recently described a new type of vesicle, quality control vesicles (QCVs), where ERManI and ManIA are usually kept segregated from their substrate glycoproteins [34,48]. Only occasionally do the glycoproteins interact with the mannosidases at the QCVs or at their fusion sites with the ER. This would result in a slow mannose-trimming process, giving time for unfolded glycoproteins to fold (Figure 3). EDEM1 also resides mostly in vesicles [49,50], possibly in QCVs. The QCVs have an ER-like density, because they are probably derived from the ER but can be separated from compartments containing major ER components, such as CNX, by velocity sedimentation [34]. In addition to the segregation of the mannosidases, the ER is compartmentalized in a series of defined subcompartments [47,51]. Folded glycoproteins, released from the CNX cycle, are concentrated at ER exit sites before their departure from the ER to the ER-to-Golgi intermediate compartment (ERGIC), the Golgi, and beyond in the secretory pathway [52]. During their maturation, these glycoproteins have their α1,2 mannose residues removed, but their Golgi localization appears to preclude routing to ERAD [4]. On the other hand, misfolded or slow-folding glycoproteins are sent to a specialized quality control compartment, the ER-derived quality control compartment (ERQC) [53–56] (Figure 3). The ERQC is a proposed staging ground for misfolded protein dislocation to the cytosol and ERAD, recruiting luminal, membrane, and cytosolic ERAD components upon ERAD substrate accumulation [42,57,58]. The CNX cycle is suggested to involve physical movement of the glycoprotein between the RER and the ERQC [59]. During these cycles, the unfolded or partially folded glycoprotein is exposed to the slow process of mannose trimming. If folding is completed in a timely manner, the glycoprotein can escape to the ER exit sites. In the case of misfolding or if folding is too slow, extensive mannose trimming will remove the glycoprotein from the CNX folding cycle. It will then be trapped by OS-9 at the ERQC
Oxidoreductases: a class of enzymes that catalyze the transfer of electrons from one molecule (the reductant) to another molecule (the oxidant). There are ~20 sulfhydryl oxidoreductases in the ER, most with still uncharacterized function. The best-studied oxidoreductase, protein disulfide isomerase 1 (PDI), catalyzes a wide range of reactions including disulfide oxidation, reduction, and isomerization. Quality control vesicles (QCVs): vesicles with ER-like density that are dependent on microtubules and COP-II coat machinery, and contain mannosidases involved in glycoprotein quality control. Retrotranslocation: a conserved step in targeting to ERAD, when terminally misfolded secretory proteins are exported from the ER lumen or membrane to the cytosol for proteasomal degradation. The mechanism of retrotranslocation is still poorly understood, although evidence suggests that the ubiquitin ligase HRD1 is one of the facilitators. UDP-Glc:glycoprotein glucosyltransferase 1 (UGGT1): an enzyme that recognizes N-glycans without glucose residues, but only when present on partially folded glycoproteins. UGGT1 catalyzes reglucosylation by transfer of glucose from UDP-glucose, thus allowing rebinding of CNX or CRT.
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Key Figure
Creation of the Glycan Code and Its Decoding in a Mammalian Cell
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Figure 1. The N-linked oligosaccharide precursor on a nascent glycoprotein is processed by glucosidases GI and GII, and then enters the calnexin/calreticulin (CNX/CRT) folding cycle (1). The first folding-sensor checkpoint is encountered when UDP-Glc:glycoprotein glucosyltransferase 1 (UGGT1) must decide whether to reglucosylate the glycan or not (2). If the glycoprotein is properly folded, it is not reglucosylated by UGGT1 and it exits to the Golgi with the help of the indicated lectins (3). The second foldingsensor checkpoint (4) is in charge of mannosidases (green) that create the glycan code by trimming up to four outer mannose residues if the glycoprotein is misfolded (but not if properly folded), releasing it from the CNX cycle and allowing binding to the lectins OS-9 or XTP3-B and targeting to ER-associated degradation (ERAD) (5). Given the structural specificity of the lectins (pink), they constitute checkpoints that interpret the glycan code. Associated oxidoreductases are in grey. ERdj5 associates with EDEM1 during targeting to ERAD. For simplification, several machinery components such as GI, GII, and CRT are not shown. It is unknown whether ER mannosidase I (ERManI) and ManIA associate with oxidoreductases. ManIB and IC might also participate, although evidence is scarce at present.
and targeted to the ERAD complex for proteasomal degradation (Figure 3) [58]. Inhibition of mannose trimming by knockdown of ERManI or interference with the assembly of the ERAD complex by knockdown of Herp, an ER membrane-bound ERAD machinery-organizer protein, is enough to prevent accumulation of misfolded glycoproteins at the ERQC [27,58]. There may be additional factors that slow down the targeting of unfolded proteins to degradation, for example, the Slp1– Emp65 protein complex that was recently described in yeast [60]. It is possible that mammalian homologs of such factors (Suco and Tapt1) regulate the compartmentalization. Upon accumulation of unfolded proteins that causes ER stress, EDEM1 and ERManI accumulate at the juxtanuclear ERQC together with the substrate glycoproteins, which would accelerate mannose trimming and targeting to ERAD to reduce the ER load [39,48].
Differentially Faster Trimming of Misfolded Glycoproteins The mechanism described in the previous sections suggests nonselective slow mannose trimming for all glycoproteins in the ER, which is accelerated under ER stress. However, is there any discrimination in the targeting of misfolded glycoproteins? Indeed, several machinery components such as UGGT1 [20], EDEM1 and 2 [39,61], and OS-9 [43,62] associate with exposed hydrophobic domains of unfolded or misfolded glycoproteins. In the case of UGGT1, it reglucosylates its client glycoprotein only if it is not properly folded [20]. This constitutes the first folding checkpoint (Figure 1). For ERManI, EDEM1, and EDEM2, it has recently been shown that their in vitro mannosidase activity is considerably increased when acting on an unfolded glycoprotein compared with its activity on a well-folded glycoprotein [31]. In fact, EDEM1 and 830
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Box 1. Specificity of Glycosidases and Lectins The specificity of the glycosidases that modify the structure of the precursor glycan creates the code that is then interpreted by specific lectins. Binding to CNX or CRT requires removal of the outer two Glc residues by glucosidases I (GI) and GII, and these lectins will not recognize the glycan after the last remaining Glc is removed by GII (Figure I) [1,19]. As for the mannosidases (green), all those involved can trim to some extent any of the α1,2-linked mannose residues. However, as shown in the scheme, each residue is removed with higher probability by a specific enzyme. The scheme is based on the in vitro specificity of each enzyme [30,31,40,41]. Although EDEM3 functions as a mannosidase in vitro [36], its specificity has not been determined. The result of EDEM3 overexpression in cells suggests an ability to remove both mannoses (a) and (c) (Figure I) [78]. In vitro, EDEM2 removes mannoses (a) or (c), converting M8 to M7, as does EDEM1 [31]. EDEM2 might remove mannose (b), then proceeding to remove mannoses (a) or (c). In our opinion, this could explain a report based on the structures observed in cells after EDEM2 knockout, which concluded that it has a preference for mannose (b), as does ERManI [79]. The same report showed a low effect of ERManI knockout, and this is possibly due to compensatory mechanisms, such as upregulation of the other mannosidases, given the strong activity of ERManI in vitro [30,31], in cells [27,32], and in vivo [80]. The action of the mannosidases will determine whether the structure is recognized by the lectins or not. Removal of α1,2 mannose residues limits the exit of the glycoprotein molecules from the ER towards the Golgi because it abrogates binding to ERGIC53, VIP36, or VIPL [29]. Removal of mannose (a) is sufficient to block binding to VIP36 and VIPL. Conversely, removal of the α1,2 mannose residues is required for binding to OS-9 and targeting to ERAD, the excision of mannose (c) being essential [43].
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Figure I. Specificities for Sugar Residues of the Precursor G3M9. The residues are indicated as in Figure 2. Arrows point to sugar residues that are recognized by the indicated lectins (red) or to links that are cleaved by the glucosidases (blue) or mannosidases (green). The inhibition symbols denote residues that block the binding to the indicated lectins. Abbreviations: CNX, calnexin; GI/GII, glucosidases I/II; ERManI, ER mannosidase I.
EDEM2 are strictly dependent on the unfolded status of the glycoprotein, showing little activity on native glycoproteins or on free glycans [31]. This process constitutes a second folding checkpoint, allowing differentially faster targeting to ERAD of misfolded glycoproteins, while sparing well-folded molecules. Recent reports describe the association of some of the mannosidases with oxidoreductases, which aid their function [31,36]. This follows the finding that the Saccharomyces cerevisiae EDEM homolog Htm1 associates with protein disulphide isomerase 1 (PDI), and this is required for its mannosidase activity [63]. EDEM1 and EDEM2
Figure 2. The Main N-Glycan Structures Leading to Golgi Maturation or ER-Associated Degradation (ERAD). The structures are based on the in vitro specificities of the enzymes. The detailed sugar links are shown only for Glc3Man9GlcNAc2 (G3M9); branches A, B, C are also called D1, D2, D3. The mannosidase (s) acting with the highest probability at each step are shown in bold. Only the most usual pathway is shown; for example, mannose residues (a) or (c) could theoretically be removed first, but usually mannose (b) is the first to be removed because of the high activity of ER mannosidase I (ERManI). As explained in Box 1, EDEM2 could also remove mannose (b), whereas the specificity of EDEM3 has not been shown in vitro. Green rectangles highlight folded glycoproteins that can exit to the Golgi, whereas red rectangles highlight the species most likely to be targeted to ERAD. M7A could also be targeted to ERAD but is still engaged in the calnexin (CNX) cycle involving UDP-Glc:glycoprotein glucosyltransferase 1 (UGGT1) and glucosidase II (GII). CNX [or calreticulin (CRT)] binds to species with one Glc. Excision of mannose (a) removes the glycoprotein from the CNX cycle. OS-9 (or XTP3-B) associates with species in which mannose (c) has been removed. 832
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Figure 3. Compartmentalization of Glycoproteins and Machinery Components. Cotranslational translocation and N-glycosylation take place at the rough endoplasmic reticulum (RER), followed by removal of two Glc residues and binding of calnexin (CNX). This would initiate CNX cycles between the RER and the ERderived quality control compartment (ERQC), during which up to two mannose residues can be removed. Successful folding allows movement of the glycoprotein to endoplasmic reticulum (ER) exit sites and exit to the ER-to-Golgi intermediate compartment (ERGIC) and Golgi. Alternatively, failure to achieve proper folding causes further trimming to Man6GlcNAc2 or Man5GlcNAc2 (M6-M5), allowing association with OS-9 and delivery to the ER-associated degradation (ERAD) complex at the ERQC, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and proteasomal degradation. The mannosidases are compartmentalized in quality control vesicles (QCVs) and only seldom interact with their substrate glycoproteins, and trimming is therefore slow, allowing time for unfolded molecules to achieve proper folding. Mannose trimming is even slower if the mannosidases encounter properly folded molecules.
associate with mammalian PDI, and especially with TXNDC11 [31], a disulfide reductase that has recently been implicated in ERAD [64]. PDI or TXNDC11 stimulated the mannosidase activity in vitro of EDEM1 and 2 on a native glycoprotein, but not on free N-glycans or on the denatured glycoprotein. It was suggested that the targets were misfolded molecules in the sample of native glycoprotein, which would be reduced, increasing the accessibility of the EDEMs to the N-glycan, as well as to an exposed hydrophobic patch [31]. The misfolded molecules, under conformational strain, would be more susceptible to reduction. In the case of EDEM3, it was found to associate with another oxidoreductase, ERp46, which promoted its mannose-trimming activity in vitro [36]. ERp46 seems to act on the substrate glycoprotein, similarly to PDI and TXNDC11, because EDEM3 itself was not affected by the redox environment [36]. A free cysteine appears to be required, at least on some substrates (such as α1-antitrypsin mutants NHK and Z), to allow their association with EDEM1, 2, or 3 [65]. No direct disulfide-bonding with the EDEMs was observed, suggesting that the associated oxidoreductases might recognize and stabilize interactions with the glycoproteins. EDEM1 was also shown to associate with another reductase, ERdj5, that has a role in ERAD [66]. However, ERdj5 did not stimulate the mannosidase activity of EDEM1 [31]. It is intriguing that several components of the glycan ER quality control system associate and act in combination with oxidoreductases, including the described mannosidases, CNX, and CRT, which form complexes with ERp57, and UGGT1 with Sep15 (Figure 1). Sep15 has recently been Trends in Biochemical Sciences, October 2019, Vol. 44, No. 10
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shown to be required for preventing the secretion of misfolded glycoproteins [67]. The role of the oxidoreductases might be to temporarily ‘lock in place’ the glycoproteins with their associated lectins and enzymes after the initial low-affinity interaction of the carbohydrate-binding domains with the glycans.
Retrotranslocation, Deglycosylation, and ERAD After the described glycan-trimming events, misfolded glycoprotein molecules are targeted to ERAD. As explained above, the glycoprotein is removed from the CNX cycle and a structural determinant is created that allows binding to the lectins OS-9 and XTP3-B (Figure 1) [42,68]. This association takes place at the ERQC, where the terminally misfolded glycoproteins appear to be retained before their export to the cytosol (Figure 3) [58]. OS-9 delivers these glycoproteins to SEL1L, a type I membrane protein that recognizes misfolded polypeptides and associates with HRD1, establishing a functional connection with the cytoplasmic ubiquitin-proteasome system [69]. HRD1 is a conserved multispanning membrane-bound E3 ubiquitin ligase that ubiquitinates aberrant ER proteins for ERAD. The Derlin proteins 1–3 are integral membrane proteins that are recruited to the HRD1 complex and are crucial for ERAD [70]. The HRD1 complex facilitates protein transport to the cytosol in a process termed retrotranslocation because it reverses the initial protein translocation into the ER during synthesis [71]. Herp, attached by a hydrophobic hairpin to the cytosolic side of the ERQC membrane, organizes the HRD1 complex [72]. The whole process appears to be coupled and located at the ERQC, facilitating efficient misfolded protein degradation and preventing aggregation [58]. Although HRD1 is the major ubiquitin ligase for ERAD, and there is evidence for direct involvement of the S. cerevisiae HRD1 homolog in retrotranslocation [71,73], there are several other membrane-bound ubiquitin ligases that target other substrates in mammalian cells. These other ligases, or even the Sec61 complex, can be alternative routes for misfolded protein dislocation to the cytosol [74]. Following polyubiquitination, the cytosolic AAA-ATPase P97/VCP associates with the emerging substrate, where ATP hydrolysis provides a driving force for the retrotranslocation process [75]. P97 is linked to N-glycanase (Ngly-1), which deglycosylates the glycoprotein before proteasomal degradation [76]. Before deglycosylation, retrotranslocated glycoproteins encounter the lectins Fbs1 and 2, which are F-box proteins, components of cytosolic SCF E3 ubiquitin ligases [77]. It was observed that, although several luminal lectins and glycosidases can also recognize and target nonglycosylated proteins, cytosolic Fbs2 only targets glycoproteins [62].
Concluding Remarks The main elements of the code that allows selective cellular recognition and disposal of misfolded glycoproteins have been deciphered following the recent additions described above. The code is relatively simple, and is created by progressive and selective glycan-trimming events on a misfolded glycoprotein, including steps that determine its removal from folding cycles and from its ability to associate with ER–Golgi trafficking lectins, and steps that promote binding to ERAD-targeting lectins. The mechanism involves a complex series of compartmentalized enzymes and lectins and is driven by at least two decision checkpoints comprising foldingsensors. However, some important questions remain (see Outstanding Questions). Future studies should aim to decipher the degree of redundancy of the mannosidases, the exact function of the associated oxidoreductases, and the regulation of the system under ER stress. Acknowledgments We apologize to those authors we have been unable to cite owing to space limitations. We would like to thank Haddas Saad for critically reading the manuscript. Work related to this article was supported by a grant from the Israel Science Foundation (1593/16).
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Outstanding Questions What is the exact specificity of the mannosidases and the order of their intervention? Do ERManI and ManIA have associated oxidoreductases? Are additional oxidoreductases involved? Do the oxidoreductases have a role in substrate recognition or in stabilization of interactions? Do ManIB and ManIC participate in ER quality control? How do the mannosidases and their substrate glycoproteins traffic between the compartments? Most of the studied misfolded glycoproteins are substrates of HRD1. Are similar mechanisms involved in targeting to other E3 ligases? How is the mechanism regulated under ER stress?
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Opinion
Compartmentalization and Selective Tagging for Disposal of Misfolded Glycoproteins Marina Shenkman1 and Gerardo Z. Lederkremer1,* The ability of mammalian cells to correctly identify and degrade misfolded secretory proteins, most of them bearing N-glycans, is crucial for their correct function and survival. An inefficient disposal mechanism results in the accumulation of misfolded proteins and consequent endoplasmic reticulum (ER) stress. N-glycan processing creates a code that reveals the folding status of each molecule, enabling continued folding attempts or targeting of the doomed glycoprotein for disposal. We review here the main steps involved in the accurate processing of unfolded glycoproteins. We highlight recent data suggesting that the processing is not stochastic, but that there is selective accelerated glycan trimming on misfolded glycoprotein molecules.
Highlights N-glycan processing in the early secretory pathway by glucosidases and mannosidases generates a code that tags glycoprotein molecules according to their folding status. The code is interpreted by specific lectins that route the glycoproteins towards productive maturation or towards disposal by ER-associated degradation. Compartmentalization of the mannosidases appears to slow down the trimming of mannose residues, allowing time for nascent unfolded molecules to fold.
The Glycan Code for Glycoprotein Quality Control Pioneering work by Ari Helenius in the early 1990s on the lectin (see Glossary) and endoplasmic reticulum (ER) chaperone calnexin (CNX) [1], coupled with the earlier discovery by Armando Parodi of a transient glucosylation of nascent glycoproteins [2], led to the realization that a glycan code exists in the cell that reflects glycoprotein folding status. The glycan code instructs the cell whether to continue to fold a glycoprotein molecule or to deem it terminally misfolded and target it for degradation [3–5]. This decision must be precise and finely tuned, leading otherwise to premature glycoprotein disposal or conversely to accumulation of misfolded molecules in the ER, causing ER stress. The modifications are on N-glycans, which are prevalent in the vast majority of membrane and secretory proteins. It was initially thought that the glycan code consists of three steps in mammalian cells: (i) trimming of the three terminal glucose residues from N-glycan precursors, (ii) a checkpoint where a folding-sensor glucosyltransferase re-adds one glucose, allowing rebinding of CNX, and (iii) removal of one mannose residue by ER mannosidase I (ERManI/MAN1B1), which somehow tags the glycoprotein molecule for degradation [6,7]. Recent work has unveiled a more complex and fascinating mechanism, with additional checkpoints, subcellular compartmentalization, and regulated extensive glycan trimming, that tags an unfit glycoprotein molecule for disposal (Figure 1, Key Figure).
Recent data indicates that misfolded glycoproteins are trimmed considerably faster than properly folded glycoproteins by folding-sensitive mannosidases associated with oxidoreductases and are consequently targeted for disposal.
The Initial Steps of a Nascent Glycoprotein Secreted or plasma membrane proteins, or those resident in secretory compartments, are initially translocated into the rough ER (RER) [8]. The presence of a signal sequence or internal anchor sequence makes them recognizable by the signal recognition particle (SRP) and allows binding to the SRP receptor on the RER membrane as well as to the translocation channel formed by the Sec61 complex [8]. Most secretory proteins are N-glycosylated on asparagine residues in the canonical sequons N–X–T/S, where X is any amino acid except proline [9,10]. In mammalian cells, N-linked glycans are usually cotranslationally added to proteins in the ER lumen as a presynthesized oligosaccharide precursor [10]. This 14-sugar oligosaccharide, Glc3Man9GlcNAc2 (G3M9) (Figure 2), is transferred from the carrier molecule dolichol pyrophosphate to asparagines by oligosaccharyltransferase (OST) [10,11]. This enzyme contains eight Trends in Biochemical Sciences, October 2019, Vol. 44, No. 10
1
School of Molecular Cell Biology and Biotechnology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
*Correspondence: [email protected] (G.Z. Lederkremer).
https://doi.org/10.1016/j.tibs.2019.04.012 © 2019 Elsevier Ltd. All rights reserved.
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subunits, of which only one, STT3, is catalytic [12]. STT3 comes in two variants, STT3A, which forms part of an OST complex that functions cotranslationally, and STT3B that is part of a complex that acts post-translationally [13]. OST cryo-electron microscopy (cryo-EM) structures, which have recently been determined at atomic resolution, reveal its association with the Sec61 translocon and with subunits recognizing the acceptor peptide during its appearance from the translocon [14–16]. Following N-glycosylation, the G3M9 oligosaccharide begins to undergo processing in which the nonreducing terminal Glc is first removed by glucosidase I (GI). The resulting G2M9 is bound specifically by malectin [17], which was shown to inhibit secretion of defective cargo glycoproteins [18]. Removal of an additional Glc by glucosidase II (GII) to yield G1M9 allows glycoprotein association with a unique chaperone system known as the calnexin/calreticulin cycle [19] (Figures 1 and 2). Association of glycoproteins with CNX, or with its soluble functional homolog calreticulin (CRT), involves binding and release cycles driven by opposing actions of two soluble ER enzymes, GII and UDP-Glc:glycoprotein glucosyltransferase 1 (UGGT1) [19]. GII removes the last remaining Glc to yield M9, causing dissociation of CNX, whereas UGGT1 reglucosylates Man9GlcNAc2 (M9), allowing reassociation with the lectin CNX. UGGT1 serves as a folding-sensor by recognizing the folding state of the glycoprotein [20]. If the glycoprotein is correctly folded, it is no longer a substrate of UGGT1 and continues its maturation through the secretory pathway. However, if it is still unfolded or misfolded, UGGT1 recognizes an exposed hydrophobic patch on the protein moiety and reattaches a Glc residue to M9, allowing reassociation with CNX for further folding attempts [21] (Figure 1). Therefore, proper folding allows glycoprotein release to the Golgi apparatus, where the N-glycans are further modified by trimming and extension to generate a diverse array of structures which are recognized for targeting by transport vesicles to different final destinations [22]. Terminally misfolded glycoproteins are at some point removed from the CNX cycle and are targeted to ER-associated degradation (ERAD) [23,24].
Mannosidases – The Mannose Trimming Code How is a misfolded glycoprotein removed from the CNX cycle and targeted to ERAD? As mentioned above, it was initially thought that ERManI was the only mannosidase acting on nascent glycoproteins in mammals. This mannosidase was believed to remove only one mannose residue from the middle B branch [Figure 2, mannose (b)] of the N-glycan precursor, producing M8B [25]. However, experiments with mutant mammalian cells that transfer to proteins a glycan precursor that lacks branches B and C showed that removal of the terminal mannose residue from branch A is necessary for ERAD [26]. Wild-type mammalian cells were also found to trim N-glycans of ERAD substrate glycoproteins to Man6GlcNAc2 and Man5GlcNAc2 (M6 and M5), removing most or all α1,2-linked mannose residues before routing to degradation [27,28]. Three aims are fulfilled by this extensive trimming: (i) because the terminal mannose (a) in Figure 2 is the glucose acceptor in the CNX cycle, removal of this residue precludes re-entry of the glycoprotein molecule in this folding cycle; (ii) the trimmed M5-6 structures bind with high affinity to the lectin OS-9 or to its functional homolog XTP-3B, which target the glycoprotein to ERAD, whereas these lectins cannot bind the untrimmed glycan; and (iii), the trimmed species do not bind to the lectins VIPL, VIP36, or ERGIC53, and are therefore selected against in ER–Golgi transport [29]. We now know that ERManI, although preferring mannose (b) in Figure 2, is capable of removing all four α1,2 mannose residues in vitro [30,31] and in cells in vivo [27,32]. Therefore, ERManI might act alone. However, it is puzzling that there are several other α1,2 mannosidases in mammalian cells, all part of the glycosylhydrolase family 47, which might have redundant activity, including the EDEMs 1–3 and the Golgi α1,2 mannosidases, Man IA (MAN1A1), IB (MAN1A2), and IC 828
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Glossary Calnexin/calreticulin cycle: calnexin (CNX) and calreticulin (CRT) are lectins that assist the folding of glycoproteins by interacting with folding intermediates in a cycle of binding and release driven by the addition and removal of a glucose residue. CNX or CRT binding provides a protective environment for folding, helps to inhibit aggregation, and prevents premature ER exit. Cargo glycoproteins: glycoproteins to be sorted to other organelles that are concentrated at the vesicle budding site in the donor organelle. EDEMs: ER degradation-enhancing αmannosidase-like proteins that belong to the glycosylhydrolase family 47 and have α-mannosidase-like domains with conserved catalytic residues. ER-associated degradation (ERAD): a process that includes recognition of misfolded domains of ER proteins, retrotranslocation from the ER to the cytosol, protein modifications (e.g., ubiquitination) necessary for targeting, recognition, and finally degradation by the cytosolic proteasome. ER-derived quality control compartment (ERQC): a pericentriolar compartment where ERAD substrate proteins concentrate in a microtubuledependent manner. ERAD machinery components are recruited to the ERQC, in a Herp-dependent process, making it a likely staging ground for ubiquitination and degradation. CNX and CRT are recruited to the ERQC, but not other ER proteins such as BiP or PDI. It is a distinct compartment that does not colocalize with the Golgi apparatus, the ERto-Golgi intermediate compartment (ERGIC), or other organelles. ER stress: a condition caused by a high load of unfolded proteins in the ER lumen, which results in disruption of folding and secretory capacity. ER stress triggers a defensive process, the unfolded protein response (UPR), that is aimed at re-establishing protein homeostasis. Lectins: sugar-recognition proteins that exhibit distinct sugar-binding specificities and affinities, and play an important role in glycan structure detection. N-glycans: oligosaccharides that are covalently attached by an N-glycosidic bond to the protein moiety in glycoproteins via asparagine residues within the N-X-T/S consensus sequence (sequon), where X is any amino acid except proline.
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(MAN1C1) [4,33] (although at least ManIA has recently been shown not to localize in the Golgi but instead is located in specialized vesicles, described later). ManIA has recently been implicated in targeting to ERAD [34]. Overexpression of ManIB or ManIC was also reported to accelerate ERAD [35]; however, their overexpression could increase their ER levels, and thus it remains to be shown if they are actually involved. The EDEMs were initially thought to be lectins and were only very recently shown to have bona fide mannosidase activity in vitro [31,36]. The extensive trimming creating the ERAD-targeting tag is probably a combination of the activities of these enzymes, which have slight differences in their preferred mannose residues in vitro (Box 1). ERManI would usually act first by preferentially removing mannose (b), and EDEM1 or EDEM2 would excise mannose residues (a) or (c) [31,37–39]. ManIA would then preferentially remove the remaining mannoses to yield M5-6 [34,40,41]. Although ERManI and the EDEMs can produce M5-6 in vitro, this is to a low extent [31]. Trimming to M5-6 allows binding of OS-9 or XTP3-B and targeting to ERAD [42–44] (Figure 2). M7A could also bind OS-9, but the absence of mannose (c) strongly reduces the sensitivity to GII [45], which would prevent the glycoprotein from being deglucosylated and leaving the CNX cycle. An alternative pathway where ManIA starts by excising its preferred mannose (a) [40,41] is less common. This would reduce exit to the Golgi (Box 1), and it would also prevent reglucosylation, thus immediately removing the glycoprotein from the CNX cycle. The specificity of EDEM3 in vitro has not yet been determined. There is also an alternative pathway by which glycoprotein molecules can escape the ER carrying a G1M9 glycan; without prior trimming of the last Glc residue, G1M9 glycans can be processed by an endomannosidase that cleaves between mannose residues (a) and (d) in Figure 2. After cleavage, these molecules can be retrieved and targeted to ERAD [46].
Subcellular Compartmentalization Explains the Slow Rate of Mannose Trimming As one can observe in Figures 1 and 2, the process of mannose trimming could rapidly lead to the detrimental tagging and targeting to ERAD of nascent, still unfolded, or partially folded molecules. How is this avoided? In our opinion, the secret lies in compartmentalization [4,47]. We have recently described a new type of vesicle, quality control vesicles (QCVs), where ERManI and ManIA are usually kept segregated from their substrate glycoproteins [34,48]. Only occasionally do the glycoproteins interact with the mannosidases at the QCVs or at their fusion sites with the ER. This would result in a slow mannose-trimming process, giving time for unfolded glycoproteins to fold (Figure 3). EDEM1 also resides mostly in vesicles [49,50], possibly in QCVs. The QCVs have an ER-like density, because they are probably derived from the ER but can be separated from compartments containing major ER components, such as CNX, by velocity sedimentation [34]. In addition to the segregation of the mannosidases, the ER is compartmentalized in a series of defined subcompartments [47,51]. Folded glycoproteins, released from the CNX cycle, are concentrated at ER exit sites before their departure from the ER to the ER-to-Golgi intermediate compartment (ERGIC), the Golgi, and beyond in the secretory pathway [52]. During their maturation, these glycoproteins have their α1,2 mannose residues removed, but their Golgi localization appears to preclude routing to ERAD [4]. On the other hand, misfolded or slow-folding glycoproteins are sent to a specialized quality control compartment, the ER-derived quality control compartment (ERQC) [53–56] (Figure 3). The ERQC is a proposed staging ground for misfolded protein dislocation to the cytosol and ERAD, recruiting luminal, membrane, and cytosolic ERAD components upon ERAD substrate accumulation [42,57,58]. The CNX cycle is suggested to involve physical movement of the glycoprotein between the RER and the ERQC [59]. During these cycles, the unfolded or partially folded glycoprotein is exposed to the slow process of mannose trimming. If folding is completed in a timely manner, the glycoprotein can escape to the ER exit sites. In the case of misfolding or if folding is too slow, extensive mannose trimming will remove the glycoprotein from the CNX folding cycle. It will then be trapped by OS-9 at the ERQC
Oxidoreductases: a class of enzymes that catalyze the transfer of electrons from one molecule (the reductant) to another molecule (the oxidant). There are ~20 sulfhydryl oxidoreductases in the ER, most with still uncharacterized function. The best-studied oxidoreductase, protein disulfide isomerase 1 (PDI), catalyzes a wide range of reactions including disulfide oxidation, reduction, and isomerization. Quality control vesicles (QCVs): vesicles with ER-like density that are dependent on microtubules and COP-II coat machinery, and contain mannosidases involved in glycoprotein quality control. Retrotranslocation: a conserved step in targeting to ERAD, when terminally misfolded secretory proteins are exported from the ER lumen or membrane to the cytosol for proteasomal degradation. The mechanism of retrotranslocation is still poorly understood, although evidence suggests that the ubiquitin ligase HRD1 is one of the facilitators. UDP-Glc:glycoprotein glucosyltransferase 1 (UGGT1): an enzyme that recognizes N-glycans without glucose residues, but only when present on partially folded glycoproteins. UGGT1 catalyzes reglucosylation by transfer of glucose from UDP-glucose, thus allowing rebinding of CNX or CRT.
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Key Figure
Creation of the Glycan Code and Its Decoding in a Mammalian Cell
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Figure 1. The N-linked oligosaccharide precursor on a nascent glycoprotein is processed by glucosidases GI and GII, and then enters the calnexin/calreticulin (CNX/CRT) folding cycle (1). The first folding-sensor checkpoint is encountered when UDP-Glc:glycoprotein glucosyltransferase 1 (UGGT1) must decide whether to reglucosylate the glycan or not (2). If the glycoprotein is properly folded, it is not reglucosylated by UGGT1 and it exits to the Golgi with the help of the indicated lectins (3). The second foldingsensor checkpoint (4) is in charge of mannosidases (green) that create the glycan code by trimming up to four outer mannose residues if the glycoprotein is misfolded (but not if properly folded), releasing it from the CNX cycle and allowing binding to the lectins OS-9 or XTP3-B and targeting to ER-associated degradation (ERAD) (5). Given the structural specificity of the lectins (pink), they constitute checkpoints that interpret the glycan code. Associated oxidoreductases are in grey. ERdj5 associates with EDEM1 during targeting to ERAD. For simplification, several machinery components such as GI, GII, and CRT are not shown. It is unknown whether ER mannosidase I (ERManI) and ManIA associate with oxidoreductases. ManIB and IC might also participate, although evidence is scarce at present.
and targeted to the ERAD complex for proteasomal degradation (Figure 3) [58]. Inhibition of mannose trimming by knockdown of ERManI or interference with the assembly of the ERAD complex by knockdown of Herp, an ER membrane-bound ERAD machinery-organizer protein, is enough to prevent accumulation of misfolded glycoproteins at the ERQC [27,58]. There may be additional factors that slow down the targeting of unfolded proteins to degradation, for example, the Slp1– Emp65 protein complex that was recently described in yeast [60]. It is possible that mammalian homologs of such factors (Suco and Tapt1) regulate the compartmentalization. Upon accumulation of unfolded proteins that causes ER stress, EDEM1 and ERManI accumulate at the juxtanuclear ERQC together with the substrate glycoproteins, which would accelerate mannose trimming and targeting to ERAD to reduce the ER load [39,48].
Differentially Faster Trimming of Misfolded Glycoproteins The mechanism described in the previous sections suggests nonselective slow mannose trimming for all glycoproteins in the ER, which is accelerated under ER stress. However, is there any discrimination in the targeting of misfolded glycoproteins? Indeed, several machinery components such as UGGT1 [20], EDEM1 and 2 [39,61], and OS-9 [43,62] associate with exposed hydrophobic domains of unfolded or misfolded glycoproteins. In the case of UGGT1, it reglucosylates its client glycoprotein only if it is not properly folded [20]. This constitutes the first folding checkpoint (Figure 1). For ERManI, EDEM1, and EDEM2, it has recently been shown that their in vitro mannosidase activity is considerably increased when acting on an unfolded glycoprotein compared with its activity on a well-folded glycoprotein [31]. In fact, EDEM1 and 830
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(See figure legend at the bottom of the next page.)
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Box 1. Specificity of Glycosidases and Lectins The specificity of the glycosidases that modify the structure of the precursor glycan creates the code that is then interpreted by specific lectins. Binding to CNX or CRT requires removal of the outer two Glc residues by glucosidases I (GI) and GII, and these lectins will not recognize the glycan after the last remaining Glc is removed by GII (Figure I) [1,19]. As for the mannosidases (green), all those involved can trim to some extent any of the α1,2-linked mannose residues. However, as shown in the scheme, each residue is removed with higher probability by a specific enzyme. The scheme is based on the in vitro specificity of each enzyme [30,31,40,41]. Although EDEM3 functions as a mannosidase in vitro [36], its specificity has not been determined. The result of EDEM3 overexpression in cells suggests an ability to remove both mannoses (a) and (c) (Figure I) [78]. In vitro, EDEM2 removes mannoses (a) or (c), converting M8 to M7, as does EDEM1 [31]. EDEM2 might remove mannose (b), then proceeding to remove mannoses (a) or (c). In our opinion, this could explain a report based on the structures observed in cells after EDEM2 knockout, which concluded that it has a preference for mannose (b), as does ERManI [79]. The same report showed a low effect of ERManI knockout, and this is possibly due to compensatory mechanisms, such as upregulation of the other mannosidases, given the strong activity of ERManI in vitro [30,31], in cells [27,32], and in vivo [80]. The action of the mannosidases will determine whether the structure is recognized by the lectins or not. Removal of α1,2 mannose residues limits the exit of the glycoprotein molecules from the ER towards the Golgi because it abrogates binding to ERGIC53, VIP36, or VIPL [29]. Removal of mannose (a) is sufficient to block binding to VIP36 and VIPL. Conversely, removal of the α1,2 mannose residues is required for binding to OS-9 and targeting to ERAD, the excision of mannose (c) being essential [43].
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Figure I. Specificities for Sugar Residues of the Precursor G3M9. The residues are indicated as in Figure 2. Arrows point to sugar residues that are recognized by the indicated lectins (red) or to links that are cleaved by the glucosidases (blue) or mannosidases (green). The inhibition symbols denote residues that block the binding to the indicated lectins. Abbreviations: CNX, calnexin; GI/GII, glucosidases I/II; ERManI, ER mannosidase I.
EDEM2 are strictly dependent on the unfolded status of the glycoprotein, showing little activity on native glycoproteins or on free glycans [31]. This process constitutes a second folding checkpoint, allowing differentially faster targeting to ERAD of misfolded glycoproteins, while sparing well-folded molecules. Recent reports describe the association of some of the mannosidases with oxidoreductases, which aid their function [31,36]. This follows the finding that the Saccharomyces cerevisiae EDEM homolog Htm1 associates with protein disulphide isomerase 1 (PDI), and this is required for its mannosidase activity [63]. EDEM1 and EDEM2
Figure 2. The Main N-Glycan Structures Leading to Golgi Maturation or ER-Associated Degradation (ERAD). The structures are based on the in vitro specificities of the enzymes. The detailed sugar links are shown only for Glc3Man9GlcNAc2 (G3M9); branches A, B, C are also called D1, D2, D3. The mannosidase (s) acting with the highest probability at each step are shown in bold. Only the most usual pathway is shown; for example, mannose residues (a) or (c) could theoretically be removed first, but usually mannose (b) is the first to be removed because of the high activity of ER mannosidase I (ERManI). As explained in Box 1, EDEM2 could also remove mannose (b), whereas the specificity of EDEM3 has not been shown in vitro. Green rectangles highlight folded glycoproteins that can exit to the Golgi, whereas red rectangles highlight the species most likely to be targeted to ERAD. M7A could also be targeted to ERAD but is still engaged in the calnexin (CNX) cycle involving UDP-Glc:glycoprotein glucosyltransferase 1 (UGGT1) and glucosidase II (GII). CNX [or calreticulin (CRT)] binds to species with one Glc. Excision of mannose (a) removes the glycoprotein from the CNX cycle. OS-9 (or XTP3-B) associates with species in which mannose (c) has been removed. 832
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Figure 3. Compartmentalization of Glycoproteins and Machinery Components. Cotranslational translocation and N-glycosylation take place at the rough endoplasmic reticulum (RER), followed by removal of two Glc residues and binding of calnexin (CNX). This would initiate CNX cycles between the RER and the ERderived quality control compartment (ERQC), during which up to two mannose residues can be removed. Successful folding allows movement of the glycoprotein to endoplasmic reticulum (ER) exit sites and exit to the ER-to-Golgi intermediate compartment (ERGIC) and Golgi. Alternatively, failure to achieve proper folding causes further trimming to Man6GlcNAc2 or Man5GlcNAc2 (M6-M5), allowing association with OS-9 and delivery to the ER-associated degradation (ERAD) complex at the ERQC, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and proteasomal degradation. The mannosidases are compartmentalized in quality control vesicles (QCVs) and only seldom interact with their substrate glycoproteins, and trimming is therefore slow, allowing time for unfolded molecules to achieve proper folding. Mannose trimming is even slower if the mannosidases encounter properly folded molecules.
associate with mammalian PDI, and especially with TXNDC11 [31], a disulfide reductase that has recently been implicated in ERAD [64]. PDI or TXNDC11 stimulated the mannosidase activity in vitro of EDEM1 and 2 on a native glycoprotein, but not on free N-glycans or on the denatured glycoprotein. It was suggested that the targets were misfolded molecules in the sample of native glycoprotein, which would be reduced, increasing the accessibility of the EDEMs to the N-glycan, as well as to an exposed hydrophobic patch [31]. The misfolded molecules, under conformational strain, would be more susceptible to reduction. In the case of EDEM3, it was found to associate with another oxidoreductase, ERp46, which promoted its mannose-trimming activity in vitro [36]. ERp46 seems to act on the substrate glycoprotein, similarly to PDI and TXNDC11, because EDEM3 itself was not affected by the redox environment [36]. A free cysteine appears to be required, at least on some substrates (such as α1-antitrypsin mutants NHK and Z), to allow their association with EDEM1, 2, or 3 [65]. No direct disulfide-bonding with the EDEMs was observed, suggesting that the associated oxidoreductases might recognize and stabilize interactions with the glycoproteins. EDEM1 was also shown to associate with another reductase, ERdj5, that has a role in ERAD [66]. However, ERdj5 did not stimulate the mannosidase activity of EDEM1 [31]. It is intriguing that several components of the glycan ER quality control system associate and act in combination with oxidoreductases, including the described mannosidases, CNX, and CRT, which form complexes with ERp57, and UGGT1 with Sep15 (Figure 1). Sep15 has recently been Trends in Biochemical Sciences, October 2019, Vol. 44, No. 10
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shown to be required for preventing the secretion of misfolded glycoproteins [67]. The role of the oxidoreductases might be to temporarily ‘lock in place’ the glycoproteins with their associated lectins and enzymes after the initial low-affinity interaction of the carbohydrate-binding domains with the glycans.
Retrotranslocation, Deglycosylation, and ERAD After the described glycan-trimming events, misfolded glycoprotein molecules are targeted to ERAD. As explained above, the glycoprotein is removed from the CNX cycle and a structural determinant is created that allows binding to the lectins OS-9 and XTP3-B (Figure 1) [42,68]. This association takes place at the ERQC, where the terminally misfolded glycoproteins appear to be retained before their export to the cytosol (Figure 3) [58]. OS-9 delivers these glycoproteins to SEL1L, a type I membrane protein that recognizes misfolded polypeptides and associates with HRD1, establishing a functional connection with the cytoplasmic ubiquitin-proteasome system [69]. HRD1 is a conserved multispanning membrane-bound E3 ubiquitin ligase that ubiquitinates aberrant ER proteins for ERAD. The Derlin proteins 1–3 are integral membrane proteins that are recruited to the HRD1 complex and are crucial for ERAD [70]. The HRD1 complex facilitates protein transport to the cytosol in a process termed retrotranslocation because it reverses the initial protein translocation into the ER during synthesis [71]. Herp, attached by a hydrophobic hairpin to the cytosolic side of the ERQC membrane, organizes the HRD1 complex [72]. The whole process appears to be coupled and located at the ERQC, facilitating efficient misfolded protein degradation and preventing aggregation [58]. Although HRD1 is the major ubiquitin ligase for ERAD, and there is evidence for direct involvement of the S. cerevisiae HRD1 homolog in retrotranslocation [71,73], there are several other membrane-bound ubiquitin ligases that target other substrates in mammalian cells. These other ligases, or even the Sec61 complex, can be alternative routes for misfolded protein dislocation to the cytosol [74]. Following polyubiquitination, the cytosolic AAA-ATPase P97/VCP associates with the emerging substrate, where ATP hydrolysis provides a driving force for the retrotranslocation process [75]. P97 is linked to N-glycanase (Ngly-1), which deglycosylates the glycoprotein before proteasomal degradation [76]. Before deglycosylation, retrotranslocated glycoproteins encounter the lectins Fbs1 and 2, which are F-box proteins, components of cytosolic SCF E3 ubiquitin ligases [77]. It was observed that, although several luminal lectins and glycosidases can also recognize and target nonglycosylated proteins, cytosolic Fbs2 only targets glycoproteins [62].
Concluding Remarks The main elements of the code that allows selective cellular recognition and disposal of misfolded glycoproteins have been deciphered following the recent additions described above. The code is relatively simple, and is created by progressive and selective glycan-trimming events on a misfolded glycoprotein, including steps that determine its removal from folding cycles and from its ability to associate with ER–Golgi trafficking lectins, and steps that promote binding to ERAD-targeting lectins. The mechanism involves a complex series of compartmentalized enzymes and lectins and is driven by at least two decision checkpoints comprising foldingsensors. However, some important questions remain (see Outstanding Questions). Future studies should aim to decipher the degree of redundancy of the mannosidases, the exact function of the associated oxidoreductases, and the regulation of the system under ER stress. Acknowledgments We apologize to those authors we have been unable to cite owing to space limitations. We would like to thank Haddas Saad for critically reading the manuscript. Work related to this article was supported by a grant from the Israel Science Foundation (1593/16).
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Outstanding Questions What is the exact specificity of the mannosidases and the order of their intervention? Do ERManI and ManIA have associated oxidoreductases? Are additional oxidoreductases involved? Do the oxidoreductases have a role in substrate recognition or in stabilization of interactions? Do ManIB and ManIC participate in ER quality control? How do the mannosidases and their substrate glycoproteins traffic between the compartments? Most of the studied misfolded glycoproteins are substrates of HRD1. Are similar mechanisms involved in targeting to other E3 ligases? How is the mechanism regulated under ER stress?
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