N-Terminal Deletion of Peptide:N-Glycanase Results in Enhanced Deglycosylation Activity

Peptide:N-glycanase catalyzes the detachment of N-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing the β-aspartylglucosaminyl bond. Peptide:N-glycanase in yeast binds to Rad23p through its N-terminus. In this study, the complex formed between Peptide:N-glycanase and Rad23p was found to exhibit enhanced deglycosylation activity, which suggests an important role for this enzyme in the misfolded glycoprotein degradation pathway in vivo. To investigate the role of this enzyme in this pathway, we made stepwise deletions of the N-terminal helices of peptide:N-glycanase. Enzymatic analysis of the deletion mutants showed that deletion of the N-terminal H1 helix (Png1p-ΔH1) enhanced the deglycosylation activity of N-glycanase towards denatured glycoproteins. In addition, this mutant exhibited high deglycosylation activity towards native glycoproteins. Dynamic simulations of the wild type and N-terminal H1 deletion mutant implied that Png1p-ΔH1 is more flexible than wild type Png1p. The efficient deglycosylation of Png1p-ΔH1 towards native and non-native glycoproteins offers a potential biotechnological application.     

Identification of an Htm1 (EDEM)-dependent,Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) Pathway in Saccharomyces cerevisiae: APPLICATION OF A NOVEL ASSAY FOR GLYCOPROTEIN ERAD*

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related α-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.     

A role for N-glycanase in the cytosolic turnover of glycoproteins

Successful maturation determines the intracellular fate of secretory and membrane proteins in the endoplasmic reticulum (ER). Failure of proteins to fold or assemble properly can lead to their retention in the ER and redirects them to the cytosol for degradation by the proteasome. Proteasome inhibitors can yield deglycosylated cytoplasmic intermediates that are the result of an N-glycanase activity, believed to act prior to destruction of these substrates by the proteasome. A gene encoding a yeast peptide:N-glycanase, PNG1, has been cloned, but this N-glycanase and its mammalian homolog were reported to be incapable of deglycosylating full-length glycoproteins. We show that both the yeast PNG1 enzyme and its mammalian homolog display N-glycanase activity towards intact glycoproteins. As substrates, cytosolic PNGase activity prefers proteins containing high-mannose over those bearing complex type oligosaccharides. Importantly, PNG1 discriminates between non-native and folded glycoproteins, consistent with a role for N-glycanase in cytoplasmic turnover of glycoproteins.     

Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A

Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins. We hereby report the discovery and characterization of a novel bacterial N-glycanase from Terriglobus roseus with an extremely low pH optimum of 2.6, and annotated it therefore as PNGase H⁺. The gene of PNGase H⁺ was cloned and the recombinant protein was successfully expressed in Escherichia coli. The recombinant PNGase H⁺ could liberate high mannose-, hybrid- and complex-type N-glycans including core α1,3-fucosylated oligosaccharides from both glycoproteins and glycopeptides. In addition, PNGase H⁺ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A. The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H⁺ a versatile tool in N-glycan analysis.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.     

Yeast N-glycanase distinguishes between native and non-native glycoproteins

N-glycanase from Saccharomyces cerevisiae (Png1) preferentially removes N-glycans from misfolded proteins. The ability of Png1 to distinguish between folded and misfolded glycoproteins is reminiscent of substrate recognition by UDP-glucose glycoprotein glucosyl transferase, an enzyme that possesses this trait. The only known in vivo substrates of Png1 are aberrant glycoproteins that originate in the endoplasmic reticulum, and arrive in the cytoplasm for proteasomal degradation. The substrate specificity of Png1 is admirably suited for this task.     

Calcineurin Subunits A and B Interact to Regulate Growth and Asexual and Sexual Development in Neurospora crassa

Calcineurin is a calcium/calmodulin dependent protein phosphatase in eukaryotes that consists of a catalytic subunit A and a regulatory subunit B. Previous studies in the filamentous fungus Neurospora crassa had suggested that the catalytic subunit of calcineurin might be an essential protein. We generated N. crassa strains expressing the A (cna-1) and B (cnb-1) subunit genes under the regulation of P_tcu-1, a copper-responsive promoter. In these strains, addition of bathocuproinedisulfonic acid (BCS), a copper chelator, results in induction of cna-1 and cnb-1, while excess Cu²⁺ represses gene expression. Through analysis of these strains under repressing and inducing conditions, we found that the calcineurin is required for normal growth, asexual development and female fertility in N. crassa. Moreover, we isolated and analyzed cnb-1 mutant alleles generated by repeat-induced point mutation (RIP), with the results further supporting roles for calcineurin in growth and fertility in N. crassa. We demonstrated a direct interaction between the CNA-1 and CNB-1 proteins using an assay system developed to study protein-protein interactions in N. crassa.     

Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans

Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS–PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and six secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and nine secreted proteins. Most of the C. albicans cell wall proteins are found in the cell walls of both yeast and hyphae cells, but some cell type-specific cell wall proteins were observed. The analyses showed that the pattern of cell wall proteins present in N. crassa vegetative hyphae and conidia (asexual spores) are quite different. Almost all of the cell wall proteins identified in N. crassa have close homologs in the sequenced fungal genomes, suggesting that these proteins have important conserved functions within the cell wall.     

Lectin-like ERAD players in ER and cytosol

Protein quality control in the endoplasmic reticulum (ER) is an elaborate process conserved from yeast to mammals, ensuring that only newly synthesized proteins with correct conformations in the ER are sorted further into the secretory pathway. It is well known that high-mannose type N-glycans are involved in protein-folding events. In the quality control process, proteins that fail to achieve proper folding or proper assembly are degraded in a process known as ER-associated degradation (ERAD). The ERAD pathway comprises multiple steps including substrate recognition and targeting to the retro-translocation machinery, retrotranslocation from the ER into the cytosol, and proteasomal degradation through ubiquitination. Recent studies have documented the important roles of sugar-recognition (lectin-type) molecules for trimmed high-mannose type N-glycans and glycosidases in the ERAD pathways in both ER and cytosol. In this review, we discuss a fundamental system that monitors glycoprotein folding in the ER and the unique roles of the sugar-recognizing ubiquitin ligase and peptide:N-glycanase (PNGase) in the cytosolic ERAD pathway.     

A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death

Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G₁/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25–100 μm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function. In vivo, pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. The tryptophan at the N terminus of pep5 is essential for its cell death activity, and N terminus acetylation reduced the potency of pep5-cpp. WELVVL is the minimal active sequence of pep5, whereas Leu-Ala substitutions totally abolished pep5 cell death activity. Findings from the initial characterization of the cell death/signaling mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation, activation of p38α and -γ, and inhibition of proteasome activity. Further pharmacological analyses suggest that pep5 can trigger cell death by distinctive pathways, which can be blocked by IM-54 or a combination of necrostatin-1 and q-VD-OPh. These data further support the biological and pharmacological potential of intracellular peptides.     

Immunological cross-reactivity and inhibitor sensitivities of the plasma membrane H+-ATPase from plants and fungi

Properties of the plasma membrane proton pump (H⁺-ATPase) isolated from several species of higher plants were compared to those isolated from the mycelial fungus, Neurospora crassa. Under identical experimental conditions, differences were observed in the vanadate concentrations required for half-maximal inhibition (1 μM and 10 μM, respectively, for fungal and plant enzymes) and in the stability towards treatment with detergents at 30°C. Similarities were noted in the reactivity to N,N′-dicyclohexylcarbodiimide, an irreversible inhibitor that reacts with an essential amino acid in the putative proton-transport site (Sussman, M.R. and Slayman, C.W. (1983) J. Biol. Chem. 258, 1839–1843). A structural comparison was performed using immunoblot analysis with specific polyclonal antibodies directed towards the M_r = 100 000 polypeptide of the enzyme isolated from hyphal cells of N. crassa and from root cells of oat. Weak cross-reactivity was observed between the fungal and plant enzymes. Strong cross-reactivity was observed between the M_r = 100 000 H⁺-ATPases of oat and tomato or potato roots, providing evidence for structural homology between the enzymes isolated from phylogenetically diverse species of higher plant.     

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.     

Endo-β-N-acetylglucosamidases (ENGases) in the fungus Trichoderma atroviride: Possible involvement of the filamentous fungi-specific cytosolic ENGase in the ERAD process

N-Glycosylation is an important post-translational modification of proteins, which mainly occurs in the endoplasmic reticulum (ER). Glycoproteins that are unable to fold properly are exported to the cytosol for degradation by a cellular system called ER-associated degradation (ERAD). Once misfolded glycoproteins are exported to the cytosol, they are subjected to deglycosylation by peptide:N-glycanase (PNGase) to facilitate the efficient degradation of misfolded proteins by the proteasome. Interestingly, the ortholog of PNGase in some filamentous fungi was found to be an inactive deglycosylating enzyme. On the other hand, it has been shown that in filamentous fungi genomes, usually two different fungi-specific endo-β-N-acetylglucosamidases (ENGases) can be found; one is predicted to be localized in the cytosol and the other to have a signal sequence, while the functional importance of these enzymes remains to be clarified. In this study the ENGases of the filamentous fungus Trichoderma atroviride was characterized. By heterologous expression of the ENGases Eng18A and Eng18B in Saccharomyces cerevisiae, it was found that both ENGases are active deglycosylating enzymes. Interestingly, only Eng18B was able to enhance the efficient degradation of the RTL protein, a PNGase-dependent ERAD substrate, implying the involvement of this enzyme in the ERAD process. These results indicate that T. atroviride Eng18B may deglycosylate misfolded glycoproteins, substituting the function of the cytoplasmic PNGase in the ERAD process.     

Nonself Recognition Through Intermolecular Disulfide Bond Formation of Ribonucleotide Reductase in Neurospora

Type I ribonucleotide reductases (RNRs) are conserved across diverse taxa and are essential for the conversion of RNA into DNA precursors. In Neurospora crassa, the large subunit of RNR (UN-24) is unusual in that it also has a nonself recognition function, whereby coexpression of Oak Ridge (OR) and Panama (PA) alleles of un-24 in the same cell leads to growth inhibition and cell death. We show that coexpressing these incompatible alleles of un-24 in N. crassa results in a high molecular weight UN-24 protein complex. A 63-amino-acid portion of the C terminus was sufficient for un-24^PA incompatibility activity. Redox active cysteines that are conserved in type I RNRs and essential for their catalytic function were found to be required for incompatibility activity of both UN-24^OR and UN-24^PA. Our results suggest a plausible model of un-24 incompatibility activity in which the formation of a complex between the incompatible RNR proteins is potentiated by intermolecular disulfide bond formation.     

A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated degradation in yeast

Background

The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous biochemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can function as not only PNGase but also transglutaminase, while its in vivo function remained unclarified.

Methods

AtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD pathway was examined.

Results

AtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant of AtPNG1 (AtPNG1(C251A)) was found to significantly impair the ERAD process. This result was found to be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA? (ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA? was confirmed by co-immunoprecipitation analysis.

Conclusion

The plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates.

General significance

Our studies underscore the functional importance of a plant PNGase orthologue as a deglycosylating enzyme involved in the ERAD.     

The common ancestral core of vertebrate and fungal telomerase RNAs

Telomerase is a ribonucleoprotein with an intrinsic telomerase RNA (TER) component. Within yeasts, TER is remarkably large and presents little similarity in secondary structure to vertebrate or ciliate TERs. To better understand the evolution of fungal telomerase, we identified 74 TERs from Pezizomycotina and Taphrinomycotina subphyla, sister clades to budding yeasts. We initially identified TER from Neurospora crassa using a novel deep-sequencing–based approach, and homologous TER sequences from available fungal genome databases by computational searches. Remarkably, TERs from these non-yeast fungi have many attributes in common with vertebrate TERs. Comparative phylogenetic analysis of highly conserved regions within Pezizomycotina TERs revealed two core domains nearly identical in secondary structure to the pseudoknot and CR4/5 within vertebrate TERs. We then analyzed N. crassa and Schizosaccharomyces pombe telomerase reconstituted in vitro, and showed that the two RNA core domains in both systems can reconstitute activity in trans as two separate RNA fragments. Furthermore, the primer-extension pulse-chase analysis affirmed that the reconstituted N. crassa telomerase synthesizes TTAGGG repeats with high processivity, a common attribute of vertebrate telomerase. Overall, this study reveals the common ancestral cores of vertebrate and fungal TERs, and provides insights into the molecular evolution of fungal TER structure and function.     

Molecular identification and characterization of peptide: N-glycanase from Schizosaccharomyces pombe

Peptide:N-glycanase (PNGase) is an enzyme responsible for deglycosylation of misfolded glycoproteins in so-called endoplasmic reticulum-associated degradation (ERAD) system. In this study, we reported the molecular identification and characterization of SpPNGase (Schizosaccharomyces pombe PNGase). Enzymatic analysis revealed that SpPNGase deglycosylated the misfolded glycoproteins and distinguished native and denatured high-mannose glycoproteins in vitro. The deglycosylation activity was lost with the addition of chelating agent EDTA and was not restored by re-addition of metal ions. By construction of deletion mutant, we confirmed that N-terminal α-helix of SpPNGase was responsible for the protein-protein interaction. Combining the results from ternary structure prediction and dendrogram analysis, we suggested that the N-terminal α-helices of PNGase are derived from evolutionary motif/peptide fusion.     

Endoplasmic Reticulum Glucosidases and Protein Quality Control Factors Cooperate to Establish Biotrophy in Ustilago maydis

Secreted fungal effectors mediate plant–fungus pathogenic interactions. These proteins are typically N-glycosylated, a common posttranslational modification affecting their location and function. N-glycosylation consists of the addition, and subsequent maturation, of an oligosaccharide core in the endoplasmic reticulum (ER) and Golgi apparatus. In this article, we show that two enzymes catalyzing specific stages of this pathway in maize smut (Ustilago maydis), glucosidase I (Gls1) and glucosidase II β-subunit (Gas2), are essential for its pathogenic interaction with maize (Zea mays). Gls1 is required for the initial stages of infection following appressorium penetration, and Gas2 is required for efficient fungal spreading inside infected tissues. While U. maydis Δgls1 cells induce strong plant defense responses, Δgas2 hyphae are able to repress them, showing that slight differences in the N-glycoprotein processing can determine the extent of plant–fungus interactions. Interestingly, the calnexin protein, a central element of the ER quality control system for N-glycoproteins in eukaryotic cells, is essential for avoiding plant defense responses in cells with defective N-glycoproteins processing. Thus, N-glycoprotein maturation and this conserved checkpoint appear to play an important role in the establishment of an initial biotrophic state with the plant, which allows subsequent colonization.     

Systematic synthesis and inhibitory activity of haloacetamidyl oligosaccharide derivatives toward cytoplasmic peptide:<Emphasis Type="Italic">N</Emphasis>-glycanase

A series of glycosyl haloacetamides were synthesized as potential inhibitors of cytoplasmic peptide:N-glycanase (PNGase), an enzyme that removes N-glycans from misfolded glycoproteins. Chloro-, bromo-, and iodoacetamidyl chitobiose and chitotetraose derivatives exhibited a significant inhibitory activity. No inhibitory activity was observed with of fluoroacetamididyl derivatives. Moreover, N-acetylglucosamine derivatives, β-chloropropionamidyl chitobiose, and chloroacetamidyl cellooligosaccharide derivatives did not show any activity. These results underscore the importance of the N-acetyl groups of chitobiose for PNGase recognition. In addition, reactivity and position of the leaving group at the reducing end are also important factors.