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• W2147949504 abstract "Peptide:N-glycosidase (PNGase) F, the first PNGase identified in prokaryotic cells, catalyzes the removal of intact asparagine-linked oligosaccharide chains from glycoproteins and/or glycopeptides. Since its discovery in 1984, PNGase F has remained as the sole prokaryotic PNGase. Recently, a novel gene encoding a protein with a predicted PNGase domain was identified from a clinical isolate of Elizabethkingia meningoseptica. In this study, the candidate protein was expressed in vitro and was subjected to biochemical and structural analyses. The results revealed that it possesses PNGase activity and has substrate specificity different from that of PNGase F. The crystal structure of the protein was determined at 1.9 Å resolution. Structural comparison with PNGase F revealed a relatively larger glycan-binding groove in the catalytic domain and an additional bowl-like domain with unknown function at the N terminus of the candidate protein. These structural and functional analyses indicated that the candidate protein is a novel prokaryotic N-glycosidase. The protein has been named PNGase F-II. Peptide:N-glycosidase (PNGase) F, the first PNGase identified in prokaryotic cells, catalyzes the removal of intact asparagine-linked oligosaccharide chains from glycoproteins and/or glycopeptides. Since its discovery in 1984, PNGase F has remained as the sole prokaryotic PNGase. Recently, a novel gene encoding a protein with a predicted PNGase domain was identified from a clinical isolate of Elizabethkingia meningoseptica. In this study, the candidate protein was expressed in vitro and was subjected to biochemical and structural analyses. The results revealed that it possesses PNGase activity and has substrate specificity different from that of PNGase F. The crystal structure of the protein was determined at 1.9 Å resolution. Structural comparison with PNGase F revealed a relatively larger glycan-binding groove in the catalytic domain and an additional bowl-like domain with unknown function at the N terminus of the candidate protein. These structural and functional analyses indicated that the candidate protein is a novel prokaryotic N-glycosidase. The protein has been named PNGase F-II. Peptide:N-glycosidase (PNGase 5The abbreviations used are: PNGasepeptide:N-glycosidasePNGF domainPNGase F domainNBLN-terminal bowl-likeGlcNAcN-acetylglucosamineSe-Metselenomethioninecontiggroup of overlapping clones. ; EC 3.5.1.52), also known as peptide-N4-(N-acetyl-β-d-glucosaminyl) asparagine amidase, catalyzes the cleavage and release of N-linked glycan moieties from glycoproteins and/or glycopeptides. The first PNGase (PNGase A) was identified in almond emulsion in 1977 (1.Takahashi N. Demonstration of a new amidase acting on glycopeptides.Biochem. Biophys. Res. Commun. 1977; 76: 1194-1201Crossref PubMed Scopus (154) Google Scholar). Subsequently, similar enzymes with deglycosylation activities were identified in other plants and seeds (2.Berger S. Menudier A. Julien R. Karamanos Y. Do de-N-glycosylation enzymes have an important role in plant cells?.Biochimie. 1995; 77: 751-760Crossref PubMed Scopus (28) Google Scholar, 3.Plummer Jr., T.H. Phelan A.W. Tarentino A.L. Detection and quantification of peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidases.Eur. J. Biochem. 1987; 163: 167-173Crossref PubMed Scopus (63) Google Scholar4.Sugiyama K. Ishihara H. Tejima S. Takahashi N. Demonstration of a new glycopeptidase, from jack-bean meal, acting on aspartylglucosylamine linkages.Biochem. Biophys. Res. Commun. 1983; 112: 155-160Crossref PubMed Scopus (38) Google Scholar). A fungal cytoplasmic PNGase was identified in the budding yeast, Saccharomyces cerevisiae (5.Chiba Y. Suzuki M. Yoshida S. Yoshida A. Ikenaga H. Takeuchi M. Jigami Y. Ichishima E. Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae.J. Biol. Chem. 1998; 273: 26298-26304Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In 1991, it was observed that free oligosaccharides accumulate in mature fish eggs and during the early stages of embryogenesis of medaka fish (Oryzias latipes). This led to the discovery of PNGase in animal sources, and a possible role for PNGase-mediated deglycosylation in the embryogenesis of fish was suggested (6.Seko A. Kitajima K. Inoue Y. Inoue S. Peptide:N-glycosidase activity found in the early embryos of Oryzias latipes (Medaka fish): the first demonstration of the occurrence of peptide:N-glycosidase in animal cells and its implication for the presence of a de-N-glycosylation system in living organisms.J. Biol. Chem. 1991; 266: 22110-22114Abstract Full Text PDF PubMed Google Scholar). Studies on the tissue distribution and subcellular localization of PNGase in higher eukaryotes illustrated the basal level of PNGase expression in all of the tissues tested and relatively high levels of expression in the testis, liver, and brain (7.Kitajima K. Suzuki T. Kouchi Z. Inoue S. Inoue Y. Identification and distribution of peptide:N-glycanase (PNGase) in mouse organs.Arch. Biochem. Biophys. 1995; 319: 393-401Crossref PubMed Scopus (40) Google Scholar, 8.Suzuki T. Kwofie M.A. Lennarz W.J. Ngly1, a mouse gene encoding a deglycosylating enzyme implicated in proteasomal degradation: expression, genomic organization, and chromosomal mapping.Biochem. Biophys. Res. Commun. 2003; 304: 326-332Crossref PubMed Scopus (35) Google Scholar). Further studies suggested that PNGase is a non-lysosomal soluble protein that may be involved in the regulation of the thermal stability of glycosylated proteins. Chantret et al. (9.Chantret I. Fasseu M. Zaoui K. Le Bizec C. Yayé H.S. Dupré T. Moore S.E. Identification of roles for peptide:N-glycanase and endo-β-N-acetylglucosaminidase (Engase1p) during protein N-glycosylation in human HepG2 cells.PLoS One. 2010; 5: e11734Crossref PubMed Scopus (31) Google Scholar) reported that down-regulation of the PNGase gene led to 30% inhibition of total free oligosaccharides possessing the di-N-acetylchitobiose moiety in human HepG2 cells. Cytoplasmic studies also indicated that PNGase might be involved in the recycling of misfolded glycoproteins and endoplasmic reticulum-associated protein degradation-mediated events (10.Suzuki T. Cytoplasmic peptide:N-glycanase and catabolic pathway for free N-glycans in the cytosol.Semin. Cell Dev. Biol. 2007; 18: 762-769Crossref PubMed Scopus (55) Google Scholar, 11.Suzuki T. Seko A. Kitajima K. Inoue Y. Inoue S. Identification of peptide:N-glycanase activity in mammalian-derived cultured cells.Biochem. Biophys. Res. Commun. 1993; 194: 1124-1130Crossref PubMed Scopus (90) Google Scholar). peptide:N-glycosidase PNGase F domain N-terminal bowl-like N-acetylglucosamine selenomethionine group of overlapping clones. The first prokaryotic PNGase was isolated from Elizabethkingia meningoseptica (formerly known as Flavobacterium meningosepticum) in 1984 (12.Plummer Jr., T.H. Elder J.H. Alexander S. Phelan A.W. Tarentino A.L. Demonstration of peptide:N-glycosidase F activity in endo-β-N-acetylglucosaminidase F preparations.J. Biol. Chem. 1984; 259: 10700-10704Abstract Full Text PDF PubMed Google Scholar). It was named peptide:N-glycosidase F (PNGase F), after the organism in which it was first identified, and has been subjected to extensive structural and functional analyses (13.Plummer Jr., T.H. Tarentino A.L. Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum.Glycobiology. 1991; 1: 257-263Crossref PubMed Scopus (138) Google Scholar14.Kuhn P. Tarentino A.L. Plummer Jr., T.H. Van Roey P. Crystal structure of peptide-N4-(N-acetyl-β-d-glucosaminyl) asparagine amidase F at 2.2-Å resolution.Biochemistry. 1994; 33: 11699-11706Crossref PubMed Scopus (30) Google Scholar, 15.Tarentino A.L. Gómez C.M. Plummer Jr., T.H. Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F.Biochemistry. 1985; 24: 4665-4671Crossref PubMed Scopus (917) Google Scholar16.Norris G.E. Stillman T.J. Anderson B.F. Baker E.N. The three-dimensional structure of PNGase F, a glycosylasparaginase from Flavobacterium meningosepticum.Structure. 1994; 2: 1049-1059Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). PNGase F hydrolyzes a broad spectrum of asparagine-linked glycoproteins to generate carbohydrate-free peptides and detached full-length oligosaccharides. PNGase F has been used extensively in protein glycosylation/deglycosylation studies. Despite the fact that PNGase F remained the only proximal N-glycosidase identified in bacteria for many years, its biological significance has remained elusive. Recently, several genes encoding proteins with predicted PNGase structures, including candidate PNGase proteins from Deinococcus radiodurans (GenBankTM identifier (GI): 15807985) and Bacteroides fragilis (GI: 3288840), were identified in genome projects. However, to the best of our knowledge, these proteins have not been characterized so far. In 2012, our group isolated an E. meningoseptica strain (FMS-007) from a T-cell non-Hodgkin's lymphoma patient and obtained its complete genome sequence in one contig (GenBankTM accession number CP006576). 6G. Sun and L. Chen, submitted for publication. In addition to the known PNGase F gene, a putative gene encoding a protein with significant structural homology to PNGase F at its C-terminal end was identified by bioinformatics analysis in this E. meningoseptica strain. The protein was expressed, purified, and subjected to biochemical and structural analyses. The results suggest that the candidate protein is a new PNGase identified from Elizabethkingia meningoseptica with a novel domain structure and catalytic specificity. It has been named PNGase F-II or type 2 PNGase F. The DNA sequence of the putative PNGase gene was obtained from the whole genome sequence of the clinical isolate (FMS-007) of E. meningoseptica. Conserved domains in the putative PNGase were identified using the Basic Local Alignment Search Tool (BLASTp) from the National Center for Biotechnology Information (NCBI) Web site. Protein sequences for the other PNGase used in the multiple-sequence alignment were downloaded from GenBankTM. For proteins with known structures, the .pdb files were obtained from the Protein Data Bank. Multiple sequence alignments were performed using Align X®. A phylogenetic tree was constructed using the neighbor joining method in the Molecular Evolutionary Genetics Analysis (MEGA version 5.2.2) software. The position of the signal peptide on the putative PNGase was predicted using the online program, SignalP (version 4.1). The target gene was cloned into the pET28a vector (kanr; Novagen, Darmstadt, Germany) and expressed in Escherichia coli BL21(DE3) (Tiangen, Shanghai, China) as follows. The candidate gene without the predicted signal peptide was amplified by PCR using genomic DNA isolated from E. meningoseptica strain FMS-007, and a pair of designed oligonucleotides (5′-atccatggcccagacttatgaaattacttatc-3′ and 5′-acctcgagttcttgccctaagagaacg-3′). The amplified PCR product was ligated between the NcoI and XhoI cloning sites on the pET28a plasmid. The constructed expression vector was transformed into E. coli BL21(DE3), and the transformants were cultured at 37 °C for 12 h in Luria-Bertani medium containing 50 μg/ml kanamycin. Isopropyl 1-thio-β-d-galactopyranoside (1.0 mm) was used to induce the expression of the candidate protein at 28 °C for 12 h. The expressed protein was purified in sequential steps. The cells were harvested by centrifugation at 2683 × g (Beckman Coulter AllegraTM 25R centrifuge, TA-14-250 rotor) for 20 min, and the pellet was resuspended in a lysis buffer (20 mm Tris-HCl, 500 mm NaCl, 25 mm imidazole, pH 8.0). The suspended cells were treated with lysozyme (1 mg/ml) (Sigma-Aldrich) for 30 min and sonicated (program: 3 s of sonication followed by a pause (5 s) per cycle, 30% power, φ = 10) for 20 min on ice or disrupted using a high pressure homogenizer (JNBIO®, Guangzhou, China). The debris was removed by centrifugation (30 min, 15,455 × g using a TA-14-50 rotor) at 4 °C. The supernatant was purified using a B-PER His6 fusion protein purification kit (Thermo Scientific Pierce) or a HisTrapTM column (GE Healthcare). The loaded column was washed twice with a wash buffer (20 mm Tris, 500 mm NaCl, 25 mm imidazole, pH 8.0), and the candidate protein was eluted in an elution buffer (20 mm Tris, 500 mm NaCl, 300 mm imidazole, pH 8.0). The protein was further purified by gel filtration using a Superdex® 200 16/60 preparation grade column (GE Healthcare) in a gel filtration buffer (10 mm Tris-HCl, 100 mm NaCl, pH 8.0). For the in vitro biochemical assays, the elution buffer was replaced with 10 mm PBS (pH 7.4) by ultrafiltration through a filter with MWCO of 30,000 (Millipore, Bedford, MA) at 2173 × g (Beckman Coulter AllegtaTM 25R, TA-14-50 rotor) for 30 min at 4 °C. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce) or by measuring the absorption of UV light at A280. The purity of the candidate protein was verified by SDS-PAGE analysis. The amount of purified PNGase F-II obtained from different batches was 70–90 mg/liter of culture. PNGase F-II was stable at −80, −20, and 4 °C in PBS buffer for 1 month. Prior to crystallization, the purified protein was concentrated to 60 mg/ml using an Amicon® Ultra centrifugal device from Millipore. All other chemicals were purchased from Sigma unless indicated otherwise. PNGase F was used as the positive control for all subsequent assays. A similar procedure was adopted to clone, express, and purify PNGase F with a pair of PNGase F-specific oligonucleotides (5′-atccatggatgctccggctgataatacc-3′ and 5′-ccctcgaggtttgtaactatcggagcactaat-3′). To verify the N-glycosidase activity of the candidate protein, bovine pancreatic ribonuclease B (RNase B) with N-linked high-mannose oligosaccharide (Sigma-Aldrich) was chosen as the standard substrate glycoprotein and subjected to cleavage by PNGase F (New England Biolabs) and the candidate protein. The oligosaccharides cleaved by the two enzymes were analyzed by MALDI-TOF MS (17.Zhang W. Wang H. Tang H. Yang P. Endoglycosidase-mediated incorporation of 18O into glycans for relative glycan quantitation.Anal. Chem. 2011; 83: 4975-4981Crossref PubMed Scopus (55) Google Scholar, 18.Küster B. Wheeler S.F. Hunter A.P. Dwek R.A. Harvey D.J. Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography.Anal. Biochem. 1997; 250: 82-101Crossref PubMed Scopus (323) Google Scholar). RNase B was first dissolved in 50 mm ammonium bicarbonate buffer (pH 8.0) at a concentration of 10 mg/ml. For each reaction, 100 μg of RNase B in ammonium bicarbonate reaction buffer was denatured in a 95 °C water bath for 10 min and then mixed with 90 μl of PBS containing 5 μg of PNGase F and the purified candidate protein in separate vials. The digestion was carried out at 37 °C for 12 h, and the released glycans were collected by ultrafiltration using a filter with a molecular weight cut-off of 3000 (Millipore, Bedford, MA) at 2683 × g for 30 min at 4 °C. One microliter of the filtered solution was deposited on the MALDI plate with 1 μl of saturated 2,5-dihydroxybenzoic acid using the dried droplet method for MS analysis. Profiling of the glycans was performed in the positive ion reflectron mode in an AXIMA MALDI-quadrupole ion trap TOF mass spectrometer (Shimadzu Corp., Kyoto, Japan) using a nitrogen pulsed laser (337 nm) and an acceleration voltage of 20 kV (17.Zhang W. Wang H. Tang H. Yang P. Endoglycosidase-mediated incorporation of 18O into glycans for relative glycan quantitation.Anal. Chem. 2011; 83: 4975-4981Crossref PubMed Scopus (55) Google Scholar, 19.Cai Y. Zhang Y. Yang P. Lu H. Improved analysis of oligosaccharides for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using aminopyrazine as a derivatization reagent and a co-matrix.Analyst. 2013; 138: 6270-6276Crossref PubMed Scopus (25) Google Scholar). The enzymatic activity was also analyzed by SDS-PAGE. A panel of representative substrate N-glycoproteins, including RNase B with N-linked high-mannose oligosaccharides, ovalbumin with N-linked hybrid oligosaccharides, human IgG with N-linked complex oligosaccharides (provided by the Chinese Center for Disease Control and Prevention), and horseradish peroxidase (HRP) with α→1,3 core-fucosylated oligosaccharides (which is resistant to PNGase F digestion; Biodee Biotechnology, Beijing, China) were selected for use in this study. Glycoprotein (10 mg/ml) dissolved in ultrapure water (native substrate) or heat-denatured at 100 °C for 10 min (denatured substrate) was assayed. All reactions were conducted in 10 mm PBS buffer (pH 7.4) at 37 °C for 12 h. The treated samples were analyzed by SDS-PAGE. Periodic acid-Schiff staining was applied for positive and negative detection of the glycosylated substrate and deglycosylated product, respectively. Denatured HRP was treated with PNGase F and PNGase F-II separately in PBS buffer (pH 7.4) at 37 °C for 12 h and separated by electrophoresis. The gel was cut into two halves; one half was stained using Coomassie Blue for protein detection, and the other half was stained using periodic acid-Schiff reagent, according to the instructions provided in the glycoprotein staining kit manual (Thermo Scientific Pierce). Western blot using anti-HRP polyclonal antibodies (LifeSpan BioSciences) was performed to verify the HRP protein. The digestion of α→1,3 core-fucosylated asparagine oligosaccharide by PNGase F-II was verified by MALDI-TOF-MS. Selenomethionine (Se-Met)-substituted PNGase F-II was obtained by expressing the protein in M9 medium supplemented with 60 mg/liter Se-Met (Sigma-Aldrich). Se-Met PNGase F-II was purified as described above, except that 1 mm DTT was added to the gel filtration buffer. Se-Met PNGase F-II (15 mg/ml) was crystallized using the hanging-drop vapor diffusion method at 16 °C. The optimized crystallization condition contained 10% polyethylene glycol (PEG) 4000, 0.01 m MgCl2, 0.2 m KCl, and 0.05 m sodium cacodylate, pH 6.5. Native PNGase F-II crystals were obtained in a crystallization condition comprising 12% PEG 3350 and 0.1 m sodium malonate, pH 7.0. The crystals were flash-frozen in liquid nitrogen in the mother liquor supplemented with 20% glycerol as a cryoprotectant. X-ray diffraction data were collected at the BL17U beamline of the Shanghai Synchrotron Radiation Facility. All diffraction data were indexed, integrated, and scaled using HKL2000 (20.Otwinowski Z. Minor W. Processing of x-ray diffraction data collected in oscillation mode.Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38556) Google Scholar). The crystal structure of native PNGase F-II was determined by molecular replacement using the crystal structure of PNGBf (B. fragilis; Protein Data Bank code 3KS7) as the search model. The crystal structure for Se-Met PNGase F-II was determined by molecular replacement using the structure of native PNGase F-II as the search model. All of the structures were displayed using the PyMOL molecular graphics system (version 1.4.1). All structural refinement was carried out using the REFMAC5 program of the CCP4 suite (21.Murshudov G.N. Vagin A.A. Dodson E.J. Refinement of macromolecular structures by the maximum-likelihood method.Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13864) Google Scholar). Five percent of the data were randomly selected and set aside for free R-factor cross-validation calculations. 2Fo − Fc and Fo − Fc maps were used for manual rebuilding of the peptide chain as well as for the addition of solvent molecules using COOT (22.Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17196) Google Scholar). The refinement was continued until convergence of the free R-factor. The final refinement statistics are listed in Table 1.TABLE 1Crystallographic data collection, processing, structure refinement, and model quality statisticsSe-Met PNGase F-IINative PNGase F-IIData collectionWavelength (Å)1.00001.0000Space groupC2P21Unit cell parameters (Å)a161.44981.864bRwork = Σ‖Fo| − |Fc‖/Σ|Fo|, where Fo and Fc are the observed and calculated structure factors, respectively.55.43194.165cRfree = Σ‖Fo| − |Fc‖/Σ|Fo| for 5% of the data not used at any stage of structural refinement.71.187165.815α90.00090.000β102.23191.403γ90.00090.000Resolution range (Å)aValues in parentheses are for the highest resolution shell.30.00–1.90 (1.97–1.90)30.00–2.80 (2.90–2.80)No. of unique observations47,58555,844Completeness (%)97.9 (86.1)91.7 (82.0)Rsym (%)9.9 (42.4)13.6 (42.0)I/σI15.56 (3.02)9.87 (2.28)Redundancy4.0 (2.8)2.8 (2.0)RefinementResolution (Å)29.97–1.9029.50–2.81Rwork0.1650.199Rfree0.2030.257No. of reflections45,18752,973Model qualityEstimated coordinate error (Å)0.130.44Root mean square deviation bonds (Å)0.0100.009Root mean square deviation angles (degrees)1.3861.156a Values in parentheses are for the highest resolution shell.b Rwork = Σ‖Fo| − |Fc‖/Σ|Fo|, where Fo and Fc are the observed and calculated structure factors, respectively.c Rfree = Σ‖Fo| − |Fc‖/Σ|Fo| for 5% of the data not used at any stage of structural refinement. Open table in a new tab To further define the function of the catalytic domain of PNGase F-II, a similar procedure was applied for the cloning, expression, and purification of PNGase F-IIΔNBL (residues 184–537, without the NBL domain). The mutant gene was amplified by PCR using the following primer pair (5′-atccatgggaaagagcagatttatcactattcctg-3′ and 5′-acctcgagctattcttgccctaagagaacg-3′). The amplified product was inserted between the NcoI and XhoI cloning sites on the pET28a plasmid. The constructed expression vector was transformed into E. coli BL21(DE3), and the transformants were cultured at 37 °C for 12 h in Luria-Bertani medium containing 50 μg/ml kanamycin. The enzymatic activity of the mutant protein PNGase F-II-ΔNBL was subjected to the same assays applied for PNGase F-II. FMS-007 is a clinical strain of E. meningoseptica that was isolated from the sputum collected from a T-cell non-Hodgkin's lymphoma patient; its whole genome sequence (comprising 3,938,967 base pairs in one contig) was obtained in 2012. Bioinformatics analysis of the genome led to the identification of an ORF with a mature protein of 537 amino acids plus a signal peptide of 30 amino acids. The protein contained a domain with unknown function at the N terminus (residues 1–170) followed by a PNGase F domain (PNGF domain; residues 184–537). The protein was named PNGase F-II. The pngF-II gene (FMSX7GL000700) was localized in the Scaffold1:749867–751570 (−) of the genome sequence. Phylogenetic analysis of the PNGF domain with other PNGase (both published and predicted) in GenBankTM revealed that the proteins could be divided into two separate clades, with PNGase F in one clade and the newly predicted PNGase in the other (Fig. 1C). Clade-specific conserved amino acids were also identified from the alignment (Fig. 1B). These results indicated that the candidate protein might have PNGase activity distinct from that reported for well characterized PNGase F protein. The profile of the released glycans from RNase B (Fig. 2A) was compared with that obtained for the PNGase F control (Fig. 2B). The result generated by PNGase F was consistent with that reported in previous studies (23.van Hoek A.N. Wiener M.C. Verbavatz J.M. Brown D. Lipniunas P.H. Townsend R.R. Verkman A.S. Purification and structure-function analysis of native, PNGase F-treated, and endo-β-galactosidase-treated CHIP28 water channels.Biochemistry. 1995; 34: 2212-2219Crossref PubMed Scopus (71) Google Scholar). The glycans released by the candidate protein were identical to the ones produced by PNGase F (Fig. 2). This result indicated that the putative PNGase has activity similar to that of PNGase F; they both hydrolyzed the glycan moiety in the innermost N-acetylglucosamine (GlcNAc) from denatured RNase B. Therefore, the candidate protein was named PNGase F-II. To define the substrate specificity of PNGase F-II, three representative glycoproteins with N-linked glycans (RNase B (high mannose type), ovalbumin (hybrid type), and IgG (complex type)) were digested with PNGase F-II and PNGase F. Substrate digestion was verified by the downward shift of the substrate glycoprotein band observed on SDS-PAGE analysis (Fig. 3). The results revealed that PNGase F-II could release N-linked glycans from denatured but not native RNase B and ovalbumin (Fig. 3, A1 and A2), whereas PNGase F could cleave both denatured and native substrates (Fig. 3, B1 and B2). Both enzymes were more active toward native IgG than toward denatured IgG (Fig. 3, A3 and B3). To further distinguish the activities of PNGase F-II and PNGase F, plant N-glycoprotein HRP was used as the substrate in the deglycosylation assay. HRP contains α→1,3 core-fucosylated glycans and is known to be resistant to PNGase F digestion (24.Tretter V. Altmann F. März L. Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F cannot release glycans with fucose attached α1→3 to the asparagine-linked N-acetylglucosamine residue.Eur. J. Biochem. 1991; 199: 647-652Crossref PubMed Scopus (371) Google Scholar). When heat-denatured HRP was treated with PNGase F-II and PNGase F, a band down-shifted to an approximate size of 35 kDa was detected only for the PNGase F-II-treated sample (Fig. 4A). Western blot analysis indicated that anti-HRP antibodies recognized the down-shifted band. To confirm that the down-shifted band was a deglycosylated product of PNGase F-II digestion, the proteins on the gel were subjected to glycoprotein staining. The 35 kDa band was negative for glycan staining, whereas the intact HRP band stained positive (Fig. 4A). The glycans released from HRP on PNGase F-II digestion were analyzed by MALDI-TOF MS; the resultant profile was consistent with the reported N-glycan profile for HRP (25.Yang B.Y. Gray J.S. Montgomery R. The glycans of horseradish peroxidase.Carbohydr. Res. 1996; 287: 203-212Crossref PubMed Scopus (113) Google Scholar). Because PNGase F does not show activity toward proteins with α→1,3 core fucosylation, the digestion of HRP by PNGase F-II indicated that PNGase F-II might have a function distinct from that of PNGase F. PNGase F-II, crystallized in two different space groups, C2 (Fig. 5, A and B) and P21 (Fig. 5, C and D). Native PNGase F-II crystallized in the P21 space group with four molecules in the asymmetric unit; the crystals diffracted x-rays to a resolution of 2.8 Å. Se-Met PNGase F-II crystallized in the C2 space group with one molecule in the asymmetric unit; the crystals diffracted x-rays to a resolution of 1.9 Å. The crystal structures of both forms were solved by molecular replacement. Because the structures of both forms were identical, the Se-Met PNGase F-II structure was used as the reference structure for all structural analyses. The structure of PNGase F-II contained two spatially independent domains, an N-terminal bowl-like (NBL) domain with unknown function (residues 1–170; Fig. 5A) and a core catalytic domain. The bridge between the NBL domain and the PNGF domain was a “linker” α-helix. In the P21 space group (native PNGase F-II), there were four molecules in the asymmetric unit: chains A, B, C, and D (Fig. 5C). These four chains and the single chain in the C2 asymmetric unit (Se-Met PNGase F-II) shared a common structure with minor differences. Structural alignment of the five different chains revealed that the “linker” α-helix might function to provide flexibility (Fig. 5D) between the two domains. Chains B and C of the P21 space group had the same structure, whereas chains A, D, and Se-Met PNGase F-II showed minor variations. When the PNGF domains of chains A, D, and Se-Met PNGase-II were aligned with that of chain B/C as the reference molecule, the NBL domains of chains A, D, and Se-Met PNGase-II were shifted by 9.2, 11.9, and 17.6°, respectively, from the “linker” α helix. Thus, the “linker” α-helix may play a role in coordinating the location of the NBL domain with respect to the PNGF domain. The hexahistidine tag and the bound Zn2+ at the C terminus were derived from the pET28a expression vector. Like all of the other PNGases, the core catalytic domain of PNGase F-II consists of two eight-stranded antiparallel β sheets, PNGF-N (β1 and β4–10) and PNGF-C (β11–15 and β17–19) (Figs. 1B and 5A). These two eight-stranded antiparallel β sandwiches lie side by side with parallel principal axes. Unlike PNGase F, which contains three pairs of disulfide bonds, only one disulfide bond was detected in PNGase F-II (between Cys460 and Cys488), and these two residues were conserved in the PNGase family (Fig. 1B). The substrate-binding region of the core catalytic domain was identified by superimposing its crystal structure on the structure of PNGase F with or without a glycan product (Fig. 6, A and B; EmPNG (Protein Data Bank code 1PNF), crystal structure complexed with the product N,N′-diacetylchitobiose; EmPNG (Protein Data Bank code 1PNG), crystal structure without the product). As in PNGase F, an N-glycan-binding groove located between the two eight-stranded antiparallel β sandwiches with a negatively charged surface was identified (Fig. 6F). Previous studies indicated that the residues Asp60, Glu118, and Glu206 in EmPNG are essential for catalytic activity, with Asp60 as the primary catalytic residue (26.Kuhn P. Guan C. Cui T. Tarentino A.L. Plummer Jr., T.H. Van Roey P. Active site" @default.
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• W2147949504 title "Identification and Characterization of a Novel Prokaryotic Peptide" @default.
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