Endoplasmic Reticulum Glucosidase Research Articles

Reproductive and developmental toxicology studies of iminosugar UV-4

UV-4 (N‐(9‐methoxynonyl)‐1‐deoxynojirimycin) is a host-targeted antiviral agent, which targets mammalian proteins (endoplasmic reticulum glucosidases) rather than virally encoded proteins. This mechanism confers both broad-spectrum activity and low potential for generation of viral drug resistance mutations. Reproductive and developmental studies of UV-4 evaluated effects on fertility and early embryonic development in rats, embryo-fetal development in rats and rabbits, and pre- and postnatal development including maternal function in rats. All reproductive and developmental studies conducted achieved dose levels where parental toxicity (generally decreased body weight, decreased food consumption and adverse clinical signs) were observed. Toxicokinetic evaluations confirmed UV-4 crossed the placenta exposing fetal rats and rabbits in utero. Adverse findings in reproductive and developmental studies included decreases in sperm motility with histopathology correlates, visceral and skeletal malformations, changes in eye opening, air drop reflex, vaginal opening and preputial separation. The combined results of the fertility and early embryonic developmental study and pre- and postnatal study suggested that there may be an increased risk for male fertility. These effects are similar to those reported in pre-clinical studies of the structurally related drug Miglustat (N-butyl-1-deoxynojirimycin), therefore UV-4 may have risk of developmental or reproductive adverse outcomes in humans similar to existing approved agents in this drug class.

A role for N-glycosylation in active adenosine deaminase 2 production

BackgroundAdenosine deaminase 2 (ADA2) regulates extracellular levels of adenosine and the optimal expression of ADA2 is essential for modulating the immune system. However, the mechanisms regulating the production of active ADA2 enzyme are not fully understood. In this study, we examined the role of N-glycosylation in the formation of functional structures and the secretory pathway of ADA2. MethodsWe investigated the roles of N-glycosylation in the activity, homodimerization, and secretion of ADA2 via site-directed mutagenesis and the application of N-glycosylation inhibitors. Subcellular localization of ADA2 along with the endoplasmic reticulum (ER) glucosidase inhibitor was observed under confocal fluorescence microscope. ResultsInhibiting the initial N-glycosylation of ADA2 in the ER via site-directed mutagenesis or treatment with N-glycosylation inhibitors reduced the intracellular ADA2 activity and secretion. At this time, decreases in the ADA2 homodimers and ADA2 aggregation were observed in the cells. Treating the cells with castanospermine, an inhibitor of N-glycan editing in the ER, resulted in a reduction of the localization rate to the Golgi and markedly suppressed the ADA2 secretion. ConclusionsThese data suggest that the initial N-glycosylation and N-glycan editing in the ER are essential for the production of an active ADA2 enzyme and proper trafficking to the extracellular space. General significanceWith sufficient N-glycosylation in the ER, ADA2 exerts its function and is secreted extracellularly.

Review] Development of Fluorogenic Substrates and Inhibitors of Human Intracellular Glycosidases: Focusing on the Application of Endoplasmic Reticulum Glucosidase Inhibitors to Antiviral Agents

Review] Development of Fluorogenic Substrates and Inhibitors of Human Intracellular Glycosidases: Focusing on the Application of Endoplasmic Reticulum Glucosidase Inhibitors to Antiviral Agents

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2 precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo-acting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation.

Antiviral Activity and Mechanism of Action of Endoplasmic Reticulum Glucosidase Inhibitors: A Mini Review

Antivirals are used to treat viral infections, and antibiotics are used to treat bacterial infections. However, the mechanisms of action and number of commercially available antivirals are very limited compared to those for antibiotics. Accordingly, our group is engaged in ongoing research to develop host-targeting antivirals for the virus infections. This work is primarily focused on the endoplasmic reticulum (ER) glucosidases involved in N-glycan synthesis as the host-dependent factors of viral infection. It is widely accepted that a key mechanism by which those inhibitors act as antivirals is their ability to disrupt virus glycoprotein folding via their inhibition of ER glucosidases. Importantly, very few virus strains are resistant to ER glucosidase inhibitors because ER glucosidase enzymes are not encoded on virus genomes. This avoids problems arising from resistance mutations occurring in the viral target. A number of ER glucosidase inhibitors with antiviral activity have been reported in the past, and several clinical trials of these have been performed. In this paper, we examine the factors preventing the development of these inhibitors as antivirals and our attempts to overcome them.

Antiviral Activity and Mechanism of Action of Endoplasmic Reticulum Glucosidase Inhibitors: A Mini Review

Antiviral Activity and Mechanism of Action of Endoplasmic Reticulum Glucosidase Inhibitors: A Mini Review

Inhibition of endoplasmic reticulum glucosidases is required for in vitro and in vivo dengue antiviral activity by the iminosugar UV-4

The antiviral activity of UV-4 was previously demonstrated against dengue virus serotype 2 (DENV2) in multiple mouse models. Herein, step-wise minimal effective dose and therapeutic window of efficacy studies of UV-4B (UV-4 hydrochloride salt) were conducted in an antibody-dependent enhancement (ADE) mouse model of severe DENV2 infection in AG129 mice lacking types I and II interferon receptors. Significant survival benefit was demonstrated with 10–20 mg/kg of UV-4B administered thrice daily (TID) for seven days with initiation of treatment up to 48 h after infection. UV-4B also reduced infectious virus production in in vitro antiviral activity assays against all four DENV serotypes, including clinical isolates. A set of purified enzyme, in vitro, and in vivo studies demonstrated that inhibition of endoplasmic reticulum (ER) α-glucosidases and not the glycosphingolipid pathway appears to be responsible for the antiviral activity of UV-4B against DENV. Along with a comprehensive safety package, these and previously published data provided support for an Investigational New Drug (IND) filing and Phases 1 and 2 clinical trials for UV-4B with an indication of acute dengue disease.

Structural basis for substrate recognition mechanism of ER glucosidase II

The endoplasmic reticulum (ER) possesses a sophisticated quality control system to proofread newly synthesized proteins. A series of N-linked oligosaccharide intermediates attached on the nascent proteins serves as specific tags for the quality control system. In this system, glucosidase II is involved in trimming of non-reducing terminal glucose residue of N-glycan intermediates. Glucosidase II consists of approximately 110 kDa catalytic α subunit (GIIα) and 60 kDa non-catalytic regulatory β subunit (GIIβ). It has been shown that GIIα alone can hydrolyze a small α-glycosidase model substrate (pNP-glucose), while it cannot catalyze deglucosylation of the N-linked oligosaccharide substrates unless it makes a complex with GIIβ. In this study, we determined the first crystal structure of GIIα in the absence and presence of its inhibitor 1-deoxynojirimycin at 1.6-Å resolution. The crystal structure revealed that GIIα has a characteristic segment at the N-terminus as compared with the cognate glycoside hydrolases (GH31). Interestingly, the N-terminal segment was accommodated on the substrate-binding pocket. Based on these results, we suggest that the N-terminal segment of GIIα undergoes structural rearrangement through interaction with GIIβ, thereby promoting the substrate-binding capacity for the N-linked oligosaccharide substrates.

Viral resistance of MOGS-CDG patients implies a broad-spectrum strategy against acute virus infections.

Sadat et al. reported in the 24 April 2014 issue of the New England Journal of Medicine that patients genetically deficient in the gene encoding mannosyl-oligosaccharide glucosidase (MOGS), also known as endoplasmic reticulum (ER) glucosidase I, manifested a severe hypogammaglobulinaemia without clinical evidence of an infectious diathesis. This paradox phenomenon is, at least in part, because the impaired N-linked glycan processing of the patients compromises their ability to support efficient replication and cellular entry of viruses. This finding unambiguously validates ER glucosidases as valuable targets for antiviral agents against a broad-spectrum of enveloped viruses.

Deciphering the Roles of Glycan Processing in Glycoprotein Quality Control through Organic Synthesis

Protein quality control (QC) in the endoplasmic reticulum (ER) comprises many aspects, including folding and transport of nascent proteins and degradation of misfolded proteins. Recent studies have revealed that high-mannose-type glycans play pivotal roles in the QC process. To gain knowledge of the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives. A major part of our study focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER glucosidase II, which play crucial roles in glycoprotein QC, to clarify their specificities. In addition, we established an in vitro assay system mimicking the in vivo condition, which is highly crowded due to the presence of various macromolecules.

Isolation and functional characterization of Sporothrix schenckii ROT2, the encoding gene for the endoplasmic reticulum glucosidase II

The N-linked glycosylation is a ubiquitous protein modification in eukaryotic cells. During the N-linked glycan synthesis, the precursor Glc3Man9GlcNAc2 is processed by endoplasmic reticulum (ER) glucosidases I, II and α1,2-mannosidase, before transporting to the Golgi complex for further structure modifications. In fungi of medical relevance, as Candida albicans and Aspergillus, it is well known that ER glycosidases are important for cell fitness, cell wall organization, virulence, and interaction with the immune system. Despite this, little is known about these enzymes in Sporothrix schenckii, the causative agent of human sporotrichosis. This limited knowledge is due in part to the lack of a genome sequence of this organism. In this work we used degenerate primers and inverse PCR approaches to isolate the open reading frame of S. schenckii ROT2, the encoding gene for α subunit of ER glucosidase II. This S. schenckii gene complemented a Saccharomyces cerevisiae rot2Δ mutant; however, when expressed in a C. albicans rot2Δ mutant, S. schenckii Rot2 partially increased the levels of α-glucosidase activity, but failed to restore the N-linked glycosylation defect associated to the mutation. To our knowledge, this is the first report where a gene involved in protein N-linked glycosylation is isolated from S. schenckii.

An MBoC Favorite: Malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation

N-linked glycans are originally transferred as triglucosylated species in the endoplasmic reticulum (ER) and then subsequently have their glucoses trimmed by ER glucosidases. The lectin molecular chaperones calnexin and calreticulin bind to monoglucosylated glycan side chains. No role had been ascribed to the diglucosylated species until the Schallus et al. (2008) article filled this void. In this paper, a comprehensive study of a newly identified ER carbohydrate-binding protein called malectin was performed. Carbohydrate microarray analysis was used to demonstrate that malectin specifically binds diglucosylated glycans, and nuclear magnetic resonance structural studies exposed the carbohydrate-binding site of malectin. In light of its ER localization, malectin appears to be positioned to play an important role in assisting the maturation and quality control of glycoproteins in the early secretory pathway.

Analysis of glycoprotein processing in the endoplasmic reticulum using synthetic oligosaccharides

Protein quality control (QC) in the endoplasmic reticulum (ER) comprises many steps, including folding and transport of nascent proteins as well as degradation of misfolded proteins. Recent studies have revealed that high-mannose-type glycans play a pivotal role in the QC process. To gain knowledge about the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives. We focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER Glucosidase II, which play crucial roles in glycoprotein QC; however, their specificities remain unclear. In addition, we established an in vitro assay system mimicking the in vivo condition which is highly crowded because of the presence of various biomacromolecules.

Demonstration that endoplasmic reticulum-associated degradation of glycoproteins can occur downstream of processing by endomannosidase

During quality control in the ER (endoplasmic reticulum), nascent glycoproteins are deglucosylated by ER glucosidases I and II. In the post-ER compartments, glycoprotein endo-α-mannosidase provides an alternative route for deglucosylation. Previous evidence suggests that endomannosidase non-selectively deglucosylates glycoproteins that escape quality control in the ER, facilitating secretion of aberrantly folded as well as normal glycoproteins. In the present study, we employed FOS (free oligosaccharides) released from degrading glycoproteins as biomarkers of ERAD (ER-associated degradation), allowing us to gain a global rather than single protein-centred view of ERAD. Glucosidase inhibition was used to discriminate between glucosidase- and endomannosidase-mediated ERAD pathways. Endomannosidase expression was manipulated in CHO (Chinese-hamster ovary)-K1 cells, naturally lacking a functional version of the enzyme, and HEK (human embryonic kidney)-293T cells. Endomannosidase was shown to decrease the levels of total FOS, suggesting decreased rates of ERAD. However, following pharmacological inhibition of ER glucosidases I and II, endomannosidase expression resulted in a partial switch between glucosylated FOS, released from ER-confined glycoproteins, to deglucosylated FOS, released from endomannosidase-processed glycoproteins transported from the Golgi/ERGIC (ER/Golgi intermediate compartment) to the ER. Using this approach, we have identified a previously unknown pathway of glycoprotein flow, undetectable by the commonly employed methods, in which secretory cargo is targeted back to the ER after being processed by endomannosidase.

N-Glycans of Phaeodactylum tricornutum Diatom and Functional Characterization of Its N-Acetylglucosaminyltransferase I Enzyme

N-glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc(3)Man(9)GlcNAc(2)-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.

Promiscuous activity of ER glucosidase II discovered through donor specificity analysis of UGGT

In glycoprotein quality control system in the endoplasmic reticulum (ER), UGGT (UDP-glucose:glycoprotein glucosyltransferase) and glucosidase II (G-II) play key roles. UGGT serves as a glycoprotein folding sensor by virtue of its unique specificity to glucosylate glycoproteins at incompletely folded stage. By using various UDP-Glc analogues, we first analyzed donor specificity of UGGT, which was proven to be rather narrow. However, marginal activity was observed with UDP-galactose and UDP-glucuronic acid as well as with 3-, 4- and 6-deoxy glucose analogues to give corresponding transfer products. Intriguingly, G-II smoothly converted all of them back to Man 9GlcNAc 2, providing an indication that G-II has a promiscuous activity as a broad specificity hexosidase.

Uncoupling of sustained MAMP receptor signaling from early outputs in an Arabidopsis endoplasmic reticulum glucosidase II allele

Recognition of microbe-associated molecular patterns (MAMPs), conserved structures typical of a microbial class, triggers immune responses in eukaryotes. This is accompanied by a diverse set of physiological responses that are thought to enhance defense activity in plants. However, the extent and mechanisms by which MAMP-induced events contribute to host immunity are poorly understood. Here we reveal Arabidopsis priority in sweet life4 (psl4) and psl5 mutants that are insensitive to the bacterial elongation factor (EF)-Tu epitope elf18 but responsive to flagellin epitope flg22. PSL4 and PSL5, respectively, identify beta- and alpha-subunits of endoplasmic reticulum-resident glucosidase II, which is essential for stable accumulation and quality control of the elf18 receptor EFR but not the flg22 receptor FLS2. We notice that EFR signaling is partially and differentially impaired without a significant decrease of the receptor steady-state levels in 2 weakly dysfunctional gIIalpha alleles, designated psl5-1 and rsw3. Remarkably, rsw3 plants exhibit marked supersusceptibility against a virulent bacterial phytopathogen despite nearly intact coactivation of MAPKs, reactive oxygen species, ethylene biosynthesis, and callose deposition in response to elf18, demonstrating that these signaling outputs alone are insufficient to mount effective immunity. However, rsw3 plants fail to maintain high transcript levels of defense-promoting WRKY, PR1, and PR2 genes at late time points (4 to 24 h) after elf18 elicitation. This points to an unexpected separation between initial and sustained activation of EFR-mediated signaling in the absence of proper glucosidase II-mediated endoplasmic reticulum quality control. Our findings strongly suggest the importance of sustained MAMP receptor signaling as a key step in the establishment of robust immunity.

Synthesis and Biological Characterisation of Novel N‐Alkyl‐Deoxynojirimycin α‐Glucosidase Inhibitors

The N-alkylated deoxynojirimycin compound, N-(6'-(4''-azido-2''-nitrophenylamino)hexyl)-1-deoxynojirimycin (6) was synthesised as a potential photoaffinity probe for endoplasmic reticulum (ER) alpha-glucosidases I and II. Surprisingly this compound was a highly potent inhibitor of alpha-glucosidase I (IC(50), 17 nM) in an in vitro assay and proved equally effective at inhibiting cellular ER glucosidases, as determined by a free oligosaccharide (FOS) analysis. A modest library of compounds was synthesised to obtain structure-activity information by variation of the N-alkyl chain length and modifications to the azido-nitrophenyl group. All of these compounds failed to improve on the efficacy of compound 6, but most showed greater enzyme inhibitory potency than N-butyl-deoxynojirimycin (NB-DNJ), a pharmacological agent that has been evaluated for the treatment of several viruses for which infectivity is dependent on host cell glycosylation.

Endoplasmic Reticulum Glucosidase II Is Inhibited by Its End Products

The calnexin/calreticulin cycle is a quality control system responsible for promoting the folding of newly synthesized glycoproteins entering the endoplasmic reticulum (ER). The association of calnexin and calreticulin with the glycoproteins is regulated by ER glucosidase II, which hydrolyzes Glc 2Man X GlcNAc 2 glycans to Glc 1Man X GlcNAc 2 and further to Glc 0Man X GlcNAc 2 ( X represents any number between 5 and 9). To gain new insights into the reaction mechanism of glucosidase II, we developed a kinetic model that describes the interactions between glucosidase II, calnexin/calreticulin, and the glycans. Our model accurately reconstructed the hydrolysis of glycans with nine mannose residues and glycans with seven mannose residues, as measured by Totani et al. [Totani, K., Ihara, Y., Matsuo, I., and Ito, Y. (2006) J. Biol. Chem. 281, 31502-31508]. Intriguingly, our model predicted that glucosidase II was inhibited by its nonglucosylated end products, where the inhibitory effect of Glc 0Man 7GlcNAc 2 was much stronger than that of Glc 0Man 9GlcNAc 2. These predictions were confirmed experimentally. Moreover, our model suggested that glycans with a different number of mannose residues can be equivalent substrates of glucosidase II, in contrast to what had been previously thought. We discuss the possibility that nonglucosylated glycans, existing in the ER, might regulate the entry of newly synthesized glycoproteins into the calnexin/calreticulin cycle. Our model also shows that glucosidase II does not interact with monoglucosylated glycans while they are bound to calnexin or calreticulin.