Mechanism of class 1 (glycosylhydrolase family 47) {alpha}-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control

Quality control in the endoplasmic reticulum (ER) determines the fate of newly synthesized glycoproteins toward either correct folding or disposal by ER-associated degradation. Initiation of the disposal process involves selective trimming of N-glycans attached to misfolded glycoproteins by ER alpha-mannosidase I and subsequent recognition by the ER degradation-enhancing alpha-mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The unusual inverting hydrolytic mechanism catalyzed by members of this family is investigated here by a combination of kinetic and binding analyses of wild type and mutant forms of human ER alpha-mannosidase I as well as by structural analysis of a co-complex with an uncleaved thiodisaccharide substrate analog. These data reveal the roles of potential catalytic acid and base residues and the identification of a novel (3)S(1) sugar conformation for the bound substrate analog. The co-crystal structure described here, in combination with the (1)C(4) conformation of a previously identified co-complex with the glycone mimic, 1-deoxymannojirimycin, indicates that glycoside bond cleavage proceeds through a least motion conformational twist of a properly predisposed substrate in the -1 subsite. A novel (3)H(4) conformation is proposed as the exploded transition state.     

Structure of mouse Golgi alpha-mannosidase IA reveals the molecular basis for substrate specificity among class 1 (family 47 glycosylhydrolase) alpha1,2-mannosidases

Three subfamilies of mammalian Class 1 processing alpha1,2-mannosidases (family 47 glycosidases) play critical roles in the maturation of Asn-linked glycoproteins in the endoplasmic reticulum (ER) and Golgi complex as well as influencing the timing and recognition for disposal of terminally unfolded proteins by ER-associated degradation. In an effort to define the structural basis for substrate recognition among Class 1 mannosidases, we have crystallized murine Golgi mannosidase IA (space group P2(1)2(1)2(1)), and the structure was solved to 1.5-A resolution by molecular replacement. The enzyme assumes an (alphaalpha)(7) barrel structure with a Ca(2+) ion coordinated at the base of the barrel similar to other Class 1 mannosidases. Critical residues within the barrel structure that coordinate the Ca(2+) ion or presumably bind and catalyze the hydrolysis of the glycone are also highly conserved. A Man(6)GlcNAc(2) oligosaccharide attached to Asn(515) in the murine enzyme was found to extend into the active site of an adjoining protein unit in the crystal lattice in a presumed enzyme-product complex. In contrast to an analogous complex previously isolated for Saccharomyces cerevisiae ER mannosidase I, the oligosaccharide in the active site of the murine Golgi enzyme assumes a different conformation to present an alternate oligosaccharide branch into the active site pocket. A comparison of the observed protein-carbohydrate interactions for the murine Golgi enzyme with the binding cleft topologies of the other family 47 glycosidases provides a framework for understanding the structural basis for substrate recognition among this class of enzymes.     

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.     

Structural basis for catalysis and inhibition of N-glycan processing class I alpha 1,2-mannosidases

Endoplasmic reticulum (ER) class I alpha1,2-mannosidase (also known as ER alpha-mannosidase I) is a critical enzyme in the maturation of N-linked oligosaccharides and ER-associated degradation. Trimming of a single mannose residue acts as a signal to target misfolded glycoproteins for degradation by the proteasome. Crystal structures of the catalytic domain of human ER class I alpha1,2-mannosidase have been determined both in the presence and absence of the potent inhibitors kifunensine and 1-deoxymannojirimycin. Both inhibitors bind to the protein at the bottom of the active-site cavity, with the essential calcium ion coordinating the O-2' and O-3' hydroxyls and stabilizing the six-membered rings of both inhibitors in a (1)C(4) conformation. This is the first direct evidence of the role of the calcium ion. The lack of major conformational changes upon inhibitor binding and structural comparisons with the yeast alpha1, 2-mannosidase enzyme-product complex suggest that this class of inverting enzymes has a novel catalytic mechanism. The structures also provide insight into the specificity of this class of enzymes and provide a blueprint for the future design of novel inhibitors that prevent degradation of misfolded proteins in genetic diseases.     

Herp enhances ER-associated protein degradation by recruiting ubiquilins

ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3δ, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3δ was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates.     

Soluble tyrosinase is an endoplasmic reticulum (ER)-associated degradation substrate retained in the ER by calreticulin and BiP/GRP78 and not calnexin

Tyrosinase is a type I membrane protein regulating the pigmentation process in humans. Mutations of the human tyrosinase gene cause the tyrosinase negative type I oculocutaneous albinism (OCAI). Some OCAI mutations were shown to delete the transmembrane domain or to affect its hydrophobic properties, resulting in soluble tyrosinase mutants that are retained in the endoplasmic reticulum (ER). To understand the specific mechanisms involved in the ER retention of soluble tyrosinase, we have constructed a tyrosinase mutant truncated at its C-terminal end and investigated its maturation process. The mutant is retained in the ER, and it is degraded through the proteasomal pathway. We determined that the mannose trimming is required for an efficient degradation process. Moreover, this soluble ER-associated degradation substrate is stopped at the ER quality control checkpoint with no requirements for an ER-Golgi recycling pathway. Co-immmunoprecipitation experiments showed that soluble tyrosinase interacts with calreticulin and BiP/GRP78 (and not calnexin) during its ER transit. Expression of soluble tyrosinase in calreticulin-deficient cells resulted in the export of soluble tyrosinase of the ER, indicating the calreticulin role in ER retention. Taken together, these data show that OCAI soluble tyrosinase is an ER-associated degradation substrate that, unlike other albino tyrosinases, associates with calreticulin and BiP/GRP78. The lack of specificity for calnexin interaction reveals a novel role for calreticulin in OCAI albinism.     

Bypass of glycan-dependent glycoprotein delivery to ERAD by up-regulated EDEM1

Trimming of mannose residues from the N-linked oligosaccharide precursor is a stringent requirement for glycoprotein endoplasmic reticulum (ER)-associated degradation (ERAD). In this paper, we show that, surprisingly, overexpression of ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) or its up-regulation by IRE1, as occurs in the unfolded protein response, overrides this requirement and renders unnecessary the expression of ER mannosidase I. An EDEM1 deletion mutant lacking most of the carbohydrate-recognition domain also accelerated ERAD, delivering the substrate to XTP3-B and OS9. EDEM1 overexpression also accelerated the degradation of a mutant nonglycosylated substrate. Upon proteasomal inhibition, EDEM1 concentrated together with the ERAD substrate in the pericentriolar ER-derived quality control compartment (ERQC), where ER mannosidase I and ERAD machinery components are localized, including, as we show here, OS9. We suggest that a nascent glycoprotein can normally dissociate from EDEM1 and be rescued from ERAD by reentering calnexin-refolding cycles, a condition terminated by mannose trimming. At high EDEM1 levels, glycoprotein release is prevented and glycan interactions are no longer required, canceling the otherwise mandatory ERAD timing by mannose trimming and accelerating the targeting to degradation.     

The biological and chemical basis for tissue-selective amyloid disease

Factors controlling the onset and progression of extracellular amyloid diseases remain largely unknown. Central to disease etiology is the efficiency of the endoplasmic reticulum (ER) machinery that targets destabilized mutant proteins for degradation and the enhanced tendency of these variants to aggregate if secreted. We demonstrate that mammalian cells secrete numerous transthyretin (TTR) disease-associated variants with wild-type efficiency in spite of compromised folding energetics. Only the most highly destabilized TTR variants are subjected to ER-associated degradation (ERAD) and then only in certain tissues, providing insight into tissue selective amyloidosis. Rather than a "quality control" standard based on wild-type stability, we find that ER-assisted folding (ERAF), based on global protein energetics, determines the extent of export. We propose that ERAF (influenced by the energetics of the protein fold, chaperone enzyme distributions, and metabolite chaperones) in competition with ERAD defines the unique secretory aptitude of each tissue.     

The Png1-Rad23 complex regulates glycoprotein turnover

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1-Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.     

Human EDEM2, a novel homolog of family 47 glycosidases, is involved in ER-associated degradation of glycoproteins

In the endoplasmic reticulum (ER), misfolded proteins are retrotranslocated to the cytosol and degraded by the proteasome in a process known as ER-associated degradation (ERAD). Early in this pathway, a proposed lumenal ER lectin, EDEM, recognizes misfolded glycoproteins in the ER, disengages the nascent molecules from the folding pathway, and facilitates their targeting for disposal. In humans there are a total of three EDEM homologs. The amino acid sequences of these proteins are different from other lectins but are closely related to the Class I mannosidases (family 47 glycosidases). In this study, we characterize one of the EDEM homologs from Homo sapiens, which we have termed EDEM2 (C20orf31). Using recombinantly generated EDEM2, no alpha-1,2 mannosidase activity was observed. In HEK293 cells, recombinant EDEM2 is localized to the ER where it can associate with misfolded alpha1-antitrypsin. Overexpression of EDEM2 accelerates the degradation of misfolded alpha1-antitrypsin, indicating that the protein is involved in ERAD.     

Identification of critical residues of the mycobacterial dephosphocoenzyme a kinase by site-directed mutagenesis

Dephosphocoenzyme A kinase performs the transfer of the γ-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5-10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg(2+) for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.     

The N-glycans of jack bean alpha-mannosidase. Structure, topology and function.

The acid hydrolase alpha-mannosidase, which accumulates in plant vacuoles and probably is involved in the catabolism and turnover of N-linked glycoproteins, is itself a glycoprotein with at least one high-mannose-type and one complex-type N-glycan. The puzzling finding that alpha-mannosidase stably carries its own substrate suggests that the N-glycans have unique topologies, and important functions in protein folding, oligomerization or enzyme activity. As a first step towards the elucidation of this enigma, we purified the N-glycans of jack bean alpha-mannosidase and determined their structures by sugar composition analysis, mass spectrometry and 1H-NMR. The structures of two N-glycans were identified in an approximate ratio of one-to-one: a glucose-containing high-mannose-type glycan (Glc1Man9GlcNAc2) and a small xylose- and fucose-containing complex-type glycan (Xyl1Man1Fuc1GlcNAc2). Isolation and sequencing of glycopeptides strongly suggests that one high-mannose-type and one complex-type glycan are linked to specific glycosylation sites of the large alpha-mannosidase subunit. The high-mannose-type glycan, which is a good substrate of the endoglycosidase (endo-H), can only be removed from the enzyme after denaturation and cleavage of disulfide bonds by a reducing agent, suggesting that this glycan is buried within the folded polypeptide and, thus, protected from its hydrolytic activity. Denaturation and reduction of the native enzyme led to a marked decrease in alpha-mannosidase activity. However, the activity could largely be recovered by renaturation in an appropriate renaturation buffer. In contrast, recovery of alpha-mannosidase activity failed when the high-mannose-type glycan was removed by endo-H prior to renaturation, indicating that this glycan appears to be important for enzyme activity.     

Characterization of Schizosaccharomyces pombe ER alpha-mannosidase: a reevaluation of the role of the enzyme on ER-associated degradation

It has been postulated that creation of Man8GlcNAc2 isomer B (M8B) by endoplasmic reticulum (ER) alpha-mannosidase I constitutes a signal for driving irreparably misfolded glycoproteins to proteasomal degradation. Contrary to a previous report, we were able to detect in vivo (but not in vitro) an extremely feeble ER alpha-mannosidase activity in Schizosaccharomyces pombe. The enzyme yielded M8B on degradation of Man9GlcNAc2 and was inhibited by kifunensin. Live S. pombe cells showed an extremely limited capacity to demannosylate Man9GlcNAc2 present in misfolded glycoproteins even after a long residence in the ER. In addition, no preferential degradation of M8B-bearing species was detected. Nevertheless, disruption of the alpha-mannosidase encoding gene almost totally prevented degradation of a misfolded glycoprotein. This and other conflicting reports may be best explained by assuming that the role of ER mannosidase on glycoprotein degradation is independent of its enzymatic activity. The enzyme, behaving as a lectin binding polymannose glycans of varied structures, would belong together with its enzymatically inactive homologue Htm1p/Mnl1p/EDEM, to a transport chain responsible for delivering irreparably misfolded glycoproteins to proteasomes. Kifunensin and 1-deoxymannojirimycin, being mannose homologues, would behave as inhibitors of the ER mannosidase or/and Htm1p/Mnl1p/EDEM putative lectin properties.     

Role of Sec61p in the ER-associated degradation of short-lived transmembrane proteins

Misfolded proteins in the endoplasmic reticulum (ER) are identified and degraded by the ER-associated degradation pathway (ERAD), a component of ER quality control. In ERAD, misfolded proteins are removed from the ER by retrotranslocation into the cytosol where they are degraded by the ubiquitin-proteasome system. The identity of the specific protein components responsible for retrotranslocation remains controversial, with the potential candidates being Sec61p, Der1p, and Doa10. We show that the cytoplasmic N-terminal domain of a short-lived transmembrane ERAD substrate is exposed to the lumen of the ER during the degradation process. The addition of N-linked glycan to the N terminus of the substrate is prevented by mutation of a specific cysteine residue of Sec61p, as well as a specific cysteine residue of the substrate protein. We show that the substrate protein forms a disulfide-linked complex to Sec61p, suggesting that at least part of the retrotranslocation process involves Sec61p.     

Inhibition of p97-dependent protein degradation by Eeyarestatin I

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by ER-associated degradation involves substrate retrotranslocation from the ER lumen into the cytosol for degradation by the proteasome. For many substrates, retrotranslocation requires the action of ubiquitinating enzymes, which polyubiquitinate substrates emerging from the ER lumen, and of the p97-Ufd1-Npl4 ATPase complex, which hydrolyzes ATP to dislocate polyubiquitinated substrates into the cytosol. Polypeptides extracted by p97 are eventually transferred to the proteasome for destruction. In mammalian cells, ERAD can be blocked by a chemical inhibitor termed Eeyarestatin I, but the mechanism of EerI action is unclear. Here we report that EerI can associate with a p97 complex to inhibit ERAD. The interaction of EerI with the p97 complex appears to negatively influence a deubiquitinating process that is mediated by p97-associated deubiquitinating enzymes. We further show that ataxin-3, a p97-associated deubiquitinating enzyme previously implicated in ER-associated degradation, is among those affected. Interestingly, p97-associated deubiquitination is also involved in degradation of a soluble substrate. Our analyses establish a role for a novel deubiquitinating process in proteasome-dependent protein turnover.     

Lectin-deficient calnexin is capable of binding class I histocompatibility molecules in vivo and preventing their degradation

Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc(1)Man(9)GlcNAc(2), calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, we have generated six calnexin mutants defective in oligosaccharide binding using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc(1) Man(9)GlcNAc(2) oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean alpha-mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules K(b) and D(b) as model glycoproteins. In this system, lectin site-defective calnexin was able to replace wild type calnexin in forming a complex with K(b) and D(b) heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions.     

A complex of Pdi1p and the mannosidase Htm1p initiates clearance of unfolded glycoproteins from the endoplasmic reticulum

Endoplasmic reticulum (ER)-resident mannosidases generate asparagine-linked oligosaccharide signals that trigger ER-associated protein degradation (ERAD) of unfolded glycoproteins. In this study, we provide in vitro evidence that a complex of the yeast protein disulfide isomerase Pdi1p and the mannosidase Htm1p processes Man(8)GlcNAc(2) carbohydrates bound to unfolded proteins, yielding Man(7)GlcNAc(2). This glycan serves as a signal for HRD ligase-mediated glycoprotein disposal. We identified a point mutation in PDI1 that prevents complex formation of the oxidoreductase with Htm1p, diminishes mannosidase activity, and delays degradation of unfolded glycoproteins in vivo. Our results show that Pdi1p is engaged in both recognition and glycan signal processing of ERAD substrates and suggest that protein folding and breakdown are not separated but interconnected processes. We propose a stochastic model for how a given glycoprotein is partitioned into folding or degradation pathways and how the flux through these pathways is adjusted to stress conditions.     

Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast

Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD). To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast. We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover. Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.     

Single, context-specific glycans can target misfolded glycoproteins for ER-associated degradation

The endoplasmic reticulum (ER) maintains an environment essential for secretory protein folding. Consequently, the premature transport of polypeptides would be harmful to the cell. To avert this scenario, mechanisms collectively termed "ER quality control" prevent the transport of nascent polypeptides until they properly fold. Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway. To better understand the relationship between quality control and ERAD, we studied a new misfolded variant of carboxypeptidase Y (CPY). The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway. Systematic analysis revealed that a single, specific N-linked glycan of CPY was required for sorting into the pathway. The determinant is dependent on the putative lectin-like receptor Htm1/Mnl1p. The discovery of a similar signal in misfolded proteinase A supported the generality of the mechanism. These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold.     

Division of labor among oxidoreductases: TMX1 preferentially acts on transmembrane polypeptides

The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in eukaryotic cells. The lumen of the mammalian ER contains >20 members of the protein disulfide isomerase (PDI) superfamily, which ensure formation of the correct set of intramolecular and intermolecular disulfide bonds as crucial, rate-limiting reactions of the protein folding process. Components of the PDI superfamily may also facilitate dislocation of misfolded polypeptides across the ER membrane for ER-associated degradation (ERAD). The reasons for the high redundancy of PDI family members and the substrate features required for preferential engagement of one or the other are poorly understood. Here we show that TMX1, one of the few transmembrane members of the family, forms functional complexes with the ER lectin calnexin and preferentially intervenes during maturation of cysteine-containing, membrane-associated proteins while ignoring the same cysteine-containing ectodomains if not anchored at the ER membrane. As such, TMX1 is the first example of a topology-specific client protein redox catalyst in living cells.