Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.25.1 (proteasome)
 28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Structural basis for catalysis and inhibition of N-glycan processing class I alpha 1,2-mannosidases. 1099 65

The endoplasmic reticulum (ER) has a mechanism to block the exit of misfolded or unassembled proteins from the ER for the downstream organelles in the secretory pathway. Misfolded proteins retained in the ER are subjected to proteasome-dependent degradation in the cytosol when they cannot achieve correct folding and/or assembly within an appropriate time window. Although specific mannose trimming of the protein-bound oligosaccharide is essential for the degradation of misfolded glycoproteins, the precise mechanism for this recognition remains obscure. Here we report a new alpha-mannosidase-like protein, Mnl1p (mannosidase-like protein), in the yeast ER. Mnl1p is unlikely to exhibit alpha1,2-mannosidase activity, because it lacks cysteine residues that are essential for alpha1,2-mannosidase. However deletion of the MNL1 gene causes retardation of the degradation of misfolded carboxypeptidase Y, but not of the unglycosylated mutant form of the yeast alpha-mating pheromone. Possible roles of Mnl1p in the degradation and in the ER-retention of misfolded glycoproteins are discussed.
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PMID:Mnl1p, an alpha -mannosidase-like protein in yeast Saccharomyces cerevisiae, is required for endoplasmic reticulum-associated degradation of glycoproteins. 1125 55

A soluble form of ribophorin I (RI(332)) is rapidly degraded in Hela and Chinese hamster ovary (CHO) cells by a cytosolic proteasomal pathway, and the N-linked glycan present on the protein may play an important role in this process. Specifically, it has been suggested that endoplasmic reticulum (ER) mannosidase I could trigger the targeting of improperly folded glycoproteins to degradation. We used a CHO-derived glycosylation-defective cell line, MadIA214, for investigating the role of mannosidase(s) as a signal for glycoprotein degradation. Glycoproteins in MadIA214 cells carry truncated Glc(1)Man(5)GlcNAc(2) N-glycans. This oligomannoside structure interferes with protein maturation and folding, leading to an alteration of the ER morphology and the detection of high levels of soluble oligomannoside species caused by glycoprotein degradation. An HA-epitope-tagged soluble variant of ribophorin I (RI(332)-3HA) expressed in MadIA214 cells was rapidly degraded, comparable to control cells with the complete Glc(3)Man(9)GlcNAc(2) N-glycan. ER-associated degradation (ERAD) of RI(332)-3HA was also proteasome-mediated in MadIA214 cells, as demonstrated by inhibition of RI(332)-3HA degradation with agents specifically blocking proteasomal activities. Two inhibitors of alpha1,2-mannosidase activity also stabilized RI(332)-3HA in the glycosylation-defective cell line. This is striking, because the major mannosidase activity in the ER is the one of mannosidase I, specific for a mannose alpha1,2-linkage that is absent from the truncated Man(5) structure. Interestingly, though the Man(5) derivative was present in large amounts in the total protein pool, the two major species linked to RI(332)-3HA shortly after synthesis consisted of Glc(1)Man(5 )and Man(4), being replaced by Man(4 )and Man(3) when proteasomal degradation was inhibited. In contrast, the untrimmed intermediate of RI(332)-3HA was detected in mutant cells treated with mannosidase inhibitors. Our results unambiguously demonstrate that an alpha1,2-mannosidase that is not ER mannosidase I is involved in ERAD of RI(332-)3HA in the glycosylation-defective cell line, MadIA214.
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PMID:N-glycan structure of a short-lived variant of ribophorin I expressed in the MadIA214 glycosylation-defective cell line reveals the role of a mannosidase that is not ER mannosidase I in the process of glycoprotein degradation. 1144 36

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI(332), which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive alpha1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI(332) in the ERAD pathway. In a first step, the Man(8) isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI(332) intermediates. Some other trimmed N-glycan species, in particular Glc(1)Man(7)GlcNAc(2), were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI(332) accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.
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PMID:Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I. 1295 21

Metachromatic leukodystrophy is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). Biosynthesis studies of ASA with various structure-sensitive monoclonal antibodies reveal that some epitopes of the enzyme form within the first minutes of biosynthesis whereas other epitopes form later, between 10 and 25 min. When we investigated 12 various ASAs, with amino acid substitutions according to the missense mutations found in metachromatic leukodystrophy patients, immunoprecipitation with monoclonal antibodies revealed folding deficits in all 12 mutant ASA enzymes. Eleven of the 12 mutants show partial expression of the early epitopes, but only six of these show, in addition, incomplete expression of late epitopes. In none of the mutant enzymes were the late forming epitopes found in the absence of early epitopes. Thus, data from the wild-type and mutant enzymes indicate that the enzyme folds in a sequential manner and that the folding of early forming epitopes is a prerequisite for maturation of the late epitopes. All mutant enzymes in which the amino acid substitution prevents the expression of the late forming epitopes are retained in the endoplasmic reticulum (ER). In contrast, all mutants in which a single late epitope is at least partially expressed can leave the ER. Thus, irrespective of the missense mutation, the expression of epitopes forming late in biosynthesis correlates with the ability of the enzyme to leave the ER. The degradation of ER-retained enzymes can be reduced by inhibitors of the proteasome and ER alpha1,2-mannosidase I, indicating that all enzymes are degraded via the proteasome. Inhibition of degradation did not lead to an enhanced delivery from the ER for any of the mutant enzymes.
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PMID:Missense mutations as a cause of metachromatic leukodystrophy. Degradation of arylsulfatase A in the endoplasmic reticulum. 1572 Mar 92

Alpha-mannosidases in eukaryotic cells are involved in both glycan biosynthetic reactions and glycan catabolism. Two broad families of enzymes have been identified that cleave terminal mannose linkages from Asn-linked oligosaccharides (Moremen, 2000), including the Class 1 mannosidases (CAZy GH family 47 (Henrissat and Bairoch, 1996)) of the early secretory pathway involved in the processing of N-glycans and quality control and the Class 2 mannosidases (CAZy family GH38 [Henrissat and Bairoch, 1996]) involved in glycoprotein biosynthesis or catabolism. Within the Class 1 family of alpha-mannosidases, three subfamilies of enzymes have been identified (Moremen, 2000). The endoplasmic reticulum (ER) alpha1,2-mannosidase I (ERManI) subfamily acts to cleave a single residue from Asn-linked glycans in the ER. The Golgi alpha-mannosidase I (GolgiManI) subfamily has at least three members in mammalian systems (Herscovics et al., 1994; Lal et al., 1994; Tremblay and Herscovics, 2000) involved in glycan maturation in the Golgi complex to form the Man(5)GlcNAc(2) processing intermediate. The third subfamily of GH47 proteins comprises the ER degradation, enhancing alpha-mannosidase-like proteins (EDEM proteins) (Helenius and Aebi, 2004; Hirao et al., 2006; Mast et al., 2005). These proteins have been proposed to accelerate the degradation of misfolded proteins in the lumen of the ER by a lectin function that leads to retrotranslocation to the cytosol and proteasomal degradation. Recent studies have also indicated that ERManI acts as a timer for initiation of glycoprotein degradation via the ubiquitin-proteasome pathway (Hosokawa et al., 2003; Wu et al., 2003). This article discusses methods for analysis of the GH47 alpha-mannosidases, including expression, purification, activity assays, generation of point mutants, and binding studies by surface plasmon resonance.
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PMID:Family 47 alpha-mannosidases in N-glycan processing. 1711 66

Terminally misfolded or unassembled proteins are degraded by the cytoplasmic ubiquitin-proteasome pathway in a process known as ERAD (endoplasmic reticulum-associated protein degradation). Overexpression of ER alpha1,2-mannosidase I and EDEMs target misfolded glycoproteins for ERAD, most likely due to trimming of N-glycans. Here we demonstrate that overexpression of Golgi alpha1,2-mannosidase IA, IB, and IC also accelerates ERAD of terminally misfolded human alpha1-antitrypsin variant null (Hong Kong) (NHK), and mannose trimming from the N-glycans on NHK in 293 cells. Although transfected NHK is primarily localized in the ER, some NHK also co-localizes with Golgi markers, suggesting that mannose trimming by Golgi alpha1,2-mannosidases can also contribute to NHK degradation.
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PMID:Stimulation of ERAD of misfolded null Hong Kong alpha1-antitrypsin by Golgi alpha1,2-mannosidases. 1772 18