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.     

Influence of Substrate Conformation on the Deglycosylation of Ribonuclease B by Recombinant Yeast Peptide：N-glycanase

Peptide:N-glycanase has been thought to be responsible for proteasome-dependent degradationof misfolded glycoproteins translocated from the endoplasmic reticulum (ER) to the cytosol.Therefore,theenzyme was supposed to be able to distinguish between native and non-native glycoproteins.In the presentstudy,a recombinant,yeast peptide:N-glycanase,Png lp, was expressed in Escherichia coli as inclusionbodies and was purified,refolded and characterized.The results showed that the recombinant enzymehas a broad pH range adaptation,from pH 4.0 to pH 10.0,and has an optimum temperature of 30 ℃.This enzyme is a zinc metalloenzyme.Its activity was abolished with the addition of EDTA and notrestored by adding metal ions.Furthermore,the deglycosylation efficiency of recombinant Pnglpfrom E.coli was investigated with respect to the substrate conformation in vitro.When ribonuclease B(RNase B) was denatured at 60-65 ℃ or by 40-60 mM dithiothreitol, indicated by its obvious structuralchange and sharpest activity change,its deglycosylation by Pnglp was most prominent.The deglycosylationefficiency of RNase B by Pnglp was found to be related to its structural conformation and enzymaticactivity.     

Malectin Forms a Complex with Ribophorin I for Enhanced Association with Misfolded Glycoproteins

Malectin is an endoplasmic reticulum-resident lectin, which recognizes di-glucosylated Glc₂Man₉GlcNAc₂ (G2M9) N-glycans on newly synthesized glycoproteins. We previously demonstrated that malectin preferentially associates with misfolded glycoproteins and inhibits their secretion (Chen, Y., Hu, D., Yabe, R., Tateno, H., Qin, S. Y., Matsumoto, N., Hirabayashi, J., and Yamamoto, K. (2011) Mol. Biol. Cell 22, 3559–3570). The sugar binding activity of malectin is required for this process. However, because G2M9 N-glycans are generated at the very early stage of processing and are typically found on both misfolded glycoproteins and glycoproteins undergoing folding, other mechanisms must underlie the preference of malectin for misfolded glycoproteins. Here, we searched for proteins that were co-immunoprecipitated with malectin, and we found that malectin formed a stable complex with an endoplasmic reticulum-resident transmembrane protein, ribophorin I. Co-expression of malectin and ribophorin I significantly enhanced the association between malectin and a folding-defective α1-antitrypsin variant (null Hong Kong) and reduced its secretion; however, secretion of wild-type α1-antitrypsin was not affected. The enhanced association and reduced secretion were counteracted by siRNA-mediated down-regulation of ribophorin I. Also, a reporter assay revealed that ribophorin I preferentially interacted with misfolded ribonuclease A but not with the native form, suggesting that ribophorin I may function as a chaperone that recognizes misfolded proteins inside cells. These results provide the first evidence of the mechanism by which malectin preferentially associates with misfolded glycoproteins.     

Structural Analysis of Free N-Glycans in α-Glucosidase Mutants of Saccharomyces cerevisiae: Lack of the Evidence for the Occurrence of Catabolic α-Glucosidase Acting on the N-Glycans

Saccharomyces cerevisiae produces two different α-glucosidases, Glucosidase 1 (Gls1) and Glucosidase 2 (Gls2), which are responsible for the removal of the glucose molecules from N-glycans (Glc₃Man₉GlcNAc₂) of glycoproteins in the endoplasmic reticulum. Whether any additional α-glucosidases playing a role in catabolizing the glucosylated N-glycans are produced by this yeast, however, remains unknown. We report herein on a search for additional α-glucosidases in S. cerevisiae. To this end, the precise structures of cytosolic free N-glycans (FNGs), mainly derived from the peptide:N-glycanase (Png1) mediated deglycosylation of N-glycoproteins were analyzed in the endoplasmic reticulum α-glucosidase-deficient mutants. 12 new glucosylated FNG structures were successfully identified through 2-dimentional HPLC analysis. On the other hand, non-glucosylated FNGs were not detected at all under any culture conditions. It can therefore be safely concluded that no catabolic α-glucosidases acting on N-glycans are produced by this yeast.     

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.     

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.     

Arabidopsis Class I α-Mannosidases MNS4 and MNS5 Are Involved in Endoplasmic Reticulum–Associated Degradation of Misfolded Glycoproteins

To ensure that aberrantly folded proteins are cleared from the endoplasmic reticulum (ER), all eukaryotic cells possess a mechanism known as endoplasmic reticulum–associated degradation (ERAD). Many secretory proteins are N-glycosylated, and despite some recent progress, little is known about the mechanism that selects misfolded glycoproteins for degradation in plants. Here, we investigated the role of Arabidopsis thaliana class I α-mannosidases (MNS1 to MNS5) in glycan-dependent ERAD. Our genetic and biochemical data show that the two ER-resident proteins MNS4 and MNS5 are involved in the degradation of misfolded variants of the heavily glycosylated brassinosteroid receptor, BRASSINOSTEROID INSENSITIVE1, while MNS1 to MNS3 appear dispensable for this ERAD process. By contrast, N-glycan analysis of different mns mutant combinations revealed that MNS4 and MNS5 are not involved in regular N-glycan processing of properly folded secretory glycoproteins. Overexpression of MNS4 or MNS5 together with ER-retained glycoproteins indicates further that both enzymes can convert Glc_0-1Man_8-9GlcNAc₂ into N-glycans with a terminal α1,6-linked Man residue in the C-branch. Thus, MNS4 and MNS5 function in the formation of unique N-glycan structures that are specifically recognized by other components of the ERAD machinery, which ultimately results in the disposal of misfolded glycoproteins.     

Wild Type RTA and Less Toxic Variants Have Distinct Requirements for Png1 for Their Depurination Activity and Toxicity in Saccharomyces cerevisiae

Ricin A chain (RTA) undergoes retrograde trafficking and is postulated to use components of the endoplasmic reticulum (ER) associated degradation (ERAD) pathway to enter the cytosol to depurinate ribosomes. However, it is not known how RTA evades degradation by the proteasome after entry into the cytosol. We observed two distinct trafficking patterns among the precursor forms of wild type RTA and nontoxic variants tagged with enhanced green fluorescent protein (EGFP) at their C-termini in yeast. One group, which included wild type RTA, underwent ER-to-vacuole transport, while another group, which included the G83D variant, formed aggregates in the ER and was not transported to the vacuole. Peptide: N-glycanase (Png1), which catalyzes degradation of unfolded glycoproteins in the ERAD pathway affected depurination activity and toxicity of wild type RTA and G83D variant differently. PreG83D variant was deglycosylated by Png1 on the ER membrane, which reduced its depurination activity and toxicity by promoting its degradation. In contrast, wild type preRTA was deglycosylated by the free pool of Png1 in the cytosol, which increased its depurination activity, possibly by preventing its degradation. These results indicate that wild type RTA has a distinct requirement for Png1 compared to the G83D variant and is deglycosylated by Png1 in the cytosol as a possible strategy to avoid degradation by the ERAD pathway to reach the ribosome.     

The PUB domain: a putative protein-protein interaction domain implicated in the ubiquitin-proteasome pathway

Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al. J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism. Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified. Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway. We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions.     

Lack of the evidence for the enzymatic catabolism of Man1GlcNAc2 in Saccharomyces cerevisiae

In the cytosol of Saccharomyces cerevisiae, most of the free N-glycans (FNGs) are generated from misfolded glycoproteins by the action of the cytoplasmic peptide: N-glycanase (Png1). A cytosol/vacuole α-mannosidase, Ams1, then trims the FNGs to eventually form a trisaccharide composed of Manβ1,4GlcNAc β1,4GlcNAc (Man₁GlcNAc₂). Whether or not the resulting Man₁GlcNAc₂ is enzymatically degraded further, however, is currently unknown. The objective of this study was to unveil the fate of Man₁GlcNAc₂ in S. cerevisiae. Quantitative analyses of the FNGs revealed a steady increase in the amount of Man₁GlcNAc₂ produced in the post-diauxic and stationary phases, suggesting that this trisaccharide is not catabolized during this period. Inoculation of the stationary phase cells into fresh medium resulted in a reduction in the levels of Man₁GlcNAc₂. However, this reduction was caused by its dilution due to cell division in the fresh medium. Our results thus indicate that Man₁GlcNAc₂ is not enzymatically catabolized in S. cerevisiae.     

Fluorescently labeled inhibitor for profiling cytoplasmic peptide:N-glycanase

Cytoplasmic peptide:N-glycanase (PNGase) is an enzyme that removes N-glycans from misfolded glycoproteins. The function of cytoplasmic PNGase plays a significant role in the degradation of misfolded glycoproteins, which is critical for cell viability. Recently, we reported that haloacetoamidyl derivatives of high-mannose-type oligosaccharides selectively modify the catalytic cysteine of cytoplasmic PNGase and serve as its specific inhibitor. Interestingly, a drastically simplified chloroacetamidyl chitobiose derivative [(GlcNAc)(2)-ClAc] was also reactive to PNGase. In our work, it was conjugated to a hydrophobic fluorophore in order to render (GlcNAc)(2)-ClAc cells permeable. We demonstrated that this compound [BODIPY-(GlcNAc)(2)-ClAc] specifically binds to cytoplasmic PNGase from budding yeast (Png1). To date, only Z-VAD-fmk is known as an inhibitor of PNGase. BODIPY-(GlcNAc)(2)-ClAc and Z-VAD-fmk share the same binding site on Png1, while BODIPY-(GlcNAc)(2)-ClAc has markedly stronger inhibitory activity. The functional analysis of PNGase using Z-VAD-fmk should be carefully interpreted because of its intrinsic property as a caspase inhibitor. In sharp contrast, chloroacetamidyl chitobiose was not reactive to caspase. In addition, BODIPY-(GlcNAc)(2)-ClAc did not bind either chitobiose-binding lectins or PNGase from other sources. Moreover, fluorescent microscopy clearly showed that BODIPY-(GlcNAc)(2)-ClAc was efficiently introduced into cells. These results suggest that this compound could be an in vivo inhibitor of cytoplasmic PNGase.     

A remodeling system for the oligosaccharide chains on glycoproteins with microbial endo-β-N-acetylglucosaminidases

Endo-M, endo-β-N-acetylglucosaminidase from Mucor hiemalis, transferred the complex type oligosaccharide of sialoglycopeptide to partially deglycosylated proteins (N-acetylglucosamine-attached proteins), which were prepared by excluding high-mannose type oligosaccharides from glycoproteins with Endo-H, endo-β-N-acetylglucosaminidase from Streptomyces plicatus. This finding indicated that the high-mannose type oligosaccharides on glycoproteins can be changed to complex type ones by the transglycosylation activity of Endo-M. This is the first report of the establishment of a remodeling system for the different types of oligosaccharides on glycoproteins with microbial endo-β-N-acetylglucosaminidases having different substrate specificities. Endo-M is a powerful tool for the in vitro synthesis of glycoproteins containing complex type oligosaccharides from glycoproteins produced by yeast.     

Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75–85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.     

Non-lysosomal degradation pathway for N-linked glycans and dolichol-linked oligosaccharides

There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins. During the N-glycosylation of proteins, significant amounts of free oligosaccharides (fOSs) and phosphorylated oligosaccharides (POSs) are generated at the endoplasmic reticulum (ER) membrane by unclarified mechanisms. fOSs are also formed in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins destined for proteasomal degradation. This article summarizes the current knowledge of the molecular and regulatory mechanisms underlying the formation of fOSs and POSs in mammalian cells and Saccharomyces cerevisiae.     

The Neurospora Peptide:N-Glycanase Ortholog PNG1 Is Essential for Cell Polarity despite Its Lack of Enzymatic Activity

Secretory proteins are subjected to a stringent endoplasmic reticulum-based quality control system that distinguishes aberrant from correctly folded proteins. The cytoplasmic peptide:N-glycanase cleaves oligosaccharides from misfolded glycoproteins and prepares them for degradation by the 26 S proteasome. In contrast to abundant in vitro data on its enzymatic function, the in vivo relevance of peptide:N-glycanase activity remains unclear. Here we show that the PNG1 ortholog from the filamentous ascomycete Neurospora crassa is an essential protein, and its deletion results in strong polarity defects. PNG1 and its predicted binding partner RAD23 have distinct functions in N. crassa and are involved in cell wall integrity and DNA repair, respectively. Moreover, wild type PNG1 has substitutions in essential catalytic amino acids, and its deglycosylation activity is lost. These substitutions are conserved in many PNG1 orthologs of the fungal kingdom, implying a so far unrecognized enzyme-independent function of PNG1 that may only become apparent in highly polar cells such as fungal hyphae.     

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.     

PNG1, a yeast gene encoding a highly conserved peptide:N-glycanase

It has been proposed that cytoplasmic peptide:N-glycanase (PNGase) may be involved in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not correctly fold in the ER. However, a lack of information about the structure of the enzyme has limited our ability to obtain insight into its precise biological function. A PNGase-defective mutant (png1-1) was identified by screening a collection of mutagenized strains for the absence of PNGase activity in cell extracts. The PNG1 gene was mapped to the left arm of chromosome XVI by genetic approaches and its open reading frame was identified. PNG1 encodes a soluble protein that, when expressed in Escherichia coli, exhibited PNGase activity. PNG1 may be required for efficient proteasome-mediated degradation of a misfolded glycoprotein. Subcellular localization studies indicate that Png1p is present in the nucleus as well as the cytosol. Sequencing of expressed sequence tag clones revealed that Png1p is highly conserved in a wide variety of eukaryotes including mammals, suggesting that the enzyme has an important function.     

Changes in N-Linked Oligosaccharides during Seed Development of Ginkgo biloba

Structural changes in N-linked oligosaccharides of glycoproteins during seed development of Ginkgo biloba have been explored to discover possible endogenous substrate(s) for the Ginko endo-β-N-acetylglucosaminidase (endo-GB; Kimura, Y., et al. (1998) Biosci. Biotechnol. Biochem., 62, 253-261), which should be involved in the production of high-mannose type free N-glycans.

The structural analysis of the pyridylaminated oligosaccharides with a 2D sugar chain map, by ESI-MS/MS spectroscopy, showed that all N-glycans expressed on glycoproteins through the developmental stage of the Ginkgo seeds have the xylose-containing type (GlcNAc_2~0Man₃Xyl₁Fuc_1~0GlcNAc₂) but no high-mannose type structure. Man3Xyl1Fuc1GlcNAc2, a typical plant complex type structure especially found in vacuolar glycoproteins, was a dominant structure through the seed development, while the amount of expression of GlcNAc₂Man₃Xyl₁Fuc₁GlcNAc₂ and GlcNAc₁Man₃Xyl₁Fuc₁GlcNAc₂ decreased as the seeds developed. The dominantly occurrence of xylose-containing type structures and the absence of the high-mannose type structures on Ginkgo glycoproteins were also shown by lectin-blotting and immunoblotting of SDS-soluble glycoproteins extracted from the developing seeds at various developmental stages.

Concerning the endogenous substrates for plant endo-β-N-acetylglucosaminidase, these results suggested that the endogenous substrates might be the dolicol-oligosaccharide intermediates or some glycopeptides with the high-mannose type N-glycan(s) derived from misfolded glycoproteins in the quality control system for newly synthesized 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.