Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.52 (PNGase F)
1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The N-glycans have been removed by peptide-N-glycosidase F (PNGase F) from purified human non-secretory RNases derived from kidney, liver and spleen. The spleen RNase was purified by two procedures, one of which did not include the usual acid treatment step (0.25 M H2SO4, 45 min, 4 degrees C), to determine if acid treatment alters the carbohydrate moieties. The N-glycans of the RNases were fractionated by Bio-Gel P-4 chromatography and analysed by 600 MHz 1H-NMR spectroscopy and electrospray mass spectrometry. All four non-secretory RNase preparations contained the following structures: [formula: see text] The relative amounts of the trisaccharide, pentasaccharide and hexasaccharide appeared to vary slightly in the different tissue RNases. The overall results indicate: (i) that acid treatment during purification does not alter the N-glycans of non-secretory RNases; (ii) that the N-glycans from kidney, liver and spleen non-secretory RNases are very similar, if not identical, to one another, but different from the N-glycan structures reported for secretory RNase.
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PMID:Human non-secretory ribonucleases. II. Structural characterization of the N-glycans of the kidney, liver and spleen enzymes by NMR spectroscopy and electrospray mass spectrometry. 835 50

Calnexin is a membrane protein of the endoplasmic reticulum that associates transiently with newly synthesized N-linked glycoproteins in vivo. Using defined components, the binding of ribonuclease B (RNase B) Man7-Man9 glycoforms to the luminal domain of calnexin was observed in vitro only if RNase B was monoglucosylated. Binding was independent of the conformation of the glycoprotein. Calnexin protected monoglucosylated RNase B from the action of glucosidase II and PNGase F but not from that of Endo H, which completely released the protein from calnexin. These observations directly demonstrate that calnexin can act exclusively as a lectin.
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PMID:Conformation-independent binding of monoglucosylated ribonuclease B to calnexin. 901 2

A novel glycoprotein designated glycolactin, with a molecular weight of 64 kDa, a sequence hitherto unknown in the literature and capable of inhibiting the hemagglutinating activities of soybean lectin and Ricinus communis agglutinin 120, was isolated from bovine milk. Its lectin-inhibiting activity differed from that of lactoferrin, another milk protein. Like other milk proteins, glycolactin inhibited superoxide formation in vitro. Glycolactin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of about 31 nM. It exhibited ribonucleolytic (RNase) activity towards yeast transfer RNA with a pH optimum of 7.5, and specific RNase activity towards poly C. The purification protocol of glycolactin involved removal of globulin from the acid whey fraction of bovine milk by precipitation with 1.8 M (NH4)2SO4, and adsorption on the ion exchangers CM-Sepharose and Mono S. Deglycosylation of glycolactin using glycopeptidase F produced only a slight decrease of 4 kDa in the molecular weight of glycolactin.
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PMID:Purification and characterization of glycolactin, a novel glycoprotein from bovine milk. 1073 13

The characterization of high-mannose-type N-glycosylation by capillary electrophoresis-electrospray mass spectrometry (CE-ESI MS) was described. In addition to the use of a cationic noncovalent capillary coating, strong acidic buffer, and charge reversal to increase the glycoform resolving power, N-glycosidase F (PNGase F) combined with a basic protease and alpha-mannosidase combined with an acidic protease were used to analyze the high-mannose-type N-glycosylation in ribonuclease B (RNase B) and in a novel C-type lectin from the venom of Trimeresurus stejnegeri (TSL). The structures of oligosaccharide, glycosylation sites, and glycoform distributions were determined simultaneously, and the differential oxidation of Met residues in glycopeptides obtained from TSL protease digestion was also characterized successfully by CE-MS/MS. The results showed that the oligosaccharide attached to RNase B has a structure of GlcNAc2Man5 approximately 9, and that attached to TSL has a structure of GlcNAc2Min5 approximately 8. The glycoform distributions in these glycoproteins are quite different, with the GlcNAc2Man5 type predominant in RNase B, and the GlcNAc2Man8 type, in TSL This method may be useful not only for the characterization of glycosylation sites and glycan structures, but also for the determination of the relative abundance of individual glycoforms.
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PMID:Capillary electrophoresis-electrospray mass spectrometry for the characterization of high-mannose-type N-glycosylation and differential oxidation in glycoproteins by charge reversal and protease/glycosidase digestion. 1179 56

The research on glycoproteomes represents an interesting field in the functional proteomics research. Affinity chromatography and mass spectrometry are powerful techniques that are used for gaining valuable information on glycoproteomes because glycoproteins and their unusual forms resulting from protein glycosylation can be important indicators of several diseases. In this study, the concanavalin A (Con A) immobilized silica packing was prepared and used for the separation of glycoprotein and glycopeptides. A very low, non-specific adsorption on the Con A affinity column was demonstrated by mass recovery of bovine serum albumin at more than 98.5%. The effect of concentration of methyl-alpha-D-mannopyranoside (alpha-Me-D-Man) in the mobile phase and the effect of flow rate on the retention behavior of ribonuclease B (RNase B) were also investigated. The standard glycoprotein RNase B was separated under optimized conditions using 0.2 mol/L alpha-Me-D-Man in the mobile phase at a flow rate of 0.5 mL/min. Meanwhile, the oligosaccharides and glycopeptides were enriched using a Con A column after digestion of the purified RNase B with peptide-N-glycosidase F (PNGase F) and trypsin. The structure of N-linked glycan and the rate and the site of glycosylation of RNase B were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Glycoproteins and glycopeptides in human serum and digest solution could be separated by this method. The results showed that this method is rapid and sensitive for the purification and characterization of glycoproteins and glycopeptides.
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PMID:[Preparation of a concanavalin A immobilized affinity column and its application in the structural analysis of ribonuclease B]. 1716 31

Peptide:N-glycanase has been thought to be responsible for proteasome-dependent degradation of misfolded glycoproteins translocated from the endoplasmic reticulum (ER) to the cytosol. Therefore, the enzyme was supposed to be able to distinguish between native and non-native glycoproteins. In the present study, a recombinant, yeast peptide:N-glycanase, Png1p, was expressed in Escherichia coli as inclusion bodies and was purified, refolded and characterized. The results showed that the recombinant enzyme has a broad pH range adaptation, from pH 4.0 to pH 10.0, and has an optimum temperature of 30 degrees C. This enzyme is a zinc metalloenzyme. Its activity was abolished with the addition of EDTA and not restored by adding metal ions. Furthermore, the deglycosylation efficiency of recombinant Png1p from E. coli was investigated with respect to the substrate conformation in vitro. When ribonuclease B (RNase B) was denatured at 60-65 degrees C or by 40-60 mM dithiothreitol, indicated by its obvious structural change and sharpest activity change, its deglycosylation by Png1p was most prominent. The deglycosylation efficiency of RNase B by Png1p was found to be related to its structural conformation and enzymatic activity.
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PMID:Influence of substrate conformation on the deglycosylation of ribonuclease B by recombinant yeast peptide:N-glycanase. 1721 53

In glycoanalysis protocols, N-glycans from glycoproteins are most frequently released with peptide- N (4)-( N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F). As the enzyme is an amidase, it cleaves the NH-CO linkage between the Asn side chain and the Asn-bound GlcNAc residue. Usually, the enzyme has a low activity, or is not active at all, on native glycoproteins. A typical example is native bovine pancreatic ribonuclease B (RNase B) with oligomannose-type N-glycans at Asn-34. However, native RNase BS, generated by subtilisin digestion of native RNase B, which comprises amino acid residues 21-124 of RNase B, is sensitive to PNGase F digestion. The same holds for carboxymethylated RNase B (RNase B (cm)). In this study, NMR spectroscopy and molecular modeling have been used to explain the differences in PNGase F activity for native RNase B, native RNase BS, and RNase B (cm). NMR analysis combined with literature data clearly indicated that the N-glycan at Asn-34 is more mobile in RNase BS than in RNase B. MD simulations showed that the region around Asn-34 in RNase B is not very flexible, whereby the alpha-helix of the amino acid residues 1-20 has a stabilizing effect. In RNase BS, the alpha-helix formed by amino acid residues 23-32 is significantly more flexible. Using these data, the possibilities for complex formation of both RNase B and RNase BS with PNGase F were studied, and a model for the RNase BS-PNGase F complex is proposed.
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PMID:The structural basis of the difference in sensitivity for PNGase F in the de-N-glycosylation of the native bovine pancreatic ribonucleases B and BS. 1829 28

One pair of primers were designed and synthesized on the base of the cDNA sequence encoding Schizosaccharomyces pombe N-glycanase reported on the GenBank. The cDNA sequence encoding Peptide N-glycanase was cloned from the Schizosaccharomyces pombe by RT-PCR. And then the RT-PCR product was cloned into the expression vector pET-15b. The expression vector pET-15b(+)/Png1p was transformed into E. coli BL21(DE3). The results showed that the relative molecular weight of the enzyme was determined to be approximately 39 kD using SDS-PAGE. The expression products after induction and purification can catalyze the cleavage of N-linked oligosaccharides from glycoprotein coped with heat, but have no action on the native glycoprotein with the help of DTT. The percentage of deglycosylated RNase B treated with equate Png1p in different reaction temperature, pH, concentration of DTT and denatured temperature showed that the optimum temperature, the optimum pH is 30 degrees C; the optimum concentration of DTT is 10 mmol/L and the optimum denatured temperature is 100 degrees C.
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PMID:[Expression, purification and characterization of N-glycanase from Schizosaccharomyces pombe in Escherichia coli]. 1861 68

Peptide: N-glycosidase F (PNGase F) is an asparagine amidase produced by Flavobacterium meningosepticum that serves as a useful tool in the research on protein N-glycosylation. However, native PNGase F purified from F. meningosepticum and recombinant PNGase F expressed in Escherichia coli are obtained only at low levels, with the culture yield being no more than 15mg/L. Here, we report the efficient production of large amounts of recombinant PNGase F. First, a codon-optimized sequence encoding F. meningosepticum PNGase F was cloned into the pPICZaA vector, which was used to transform Pichia pastoris GS115. Clones were screened directly by dot blotting with an anti-6His-tag antibody, and then protein expression was induced in glass tubes to conduct validation assays. The clone expressing the highest level of PNGase F was selected for fermentation at a 5-L scale, and then the recombinant enzyme produced was purified in a single step using affinity chromatography, which yielded 800mg of the protein per liter of culture. The partly glycosylated recombinant PNGase F exhibited an identical specific activity as commercially available PNGase F when using RNase B or other N-glycoproteins as substrates. Thus, the method developed in this study can facilitate the large-scale production and use of PNGase F in the rapidly developing research field of N-glycomics.
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PMID:Highly efficient production of peptides: N-glycosidase F for N-glycomics analysis. 2458 22

Detailed characterization of glycoprotein structures requires determining both the sites of glycosylation as well as the glycan structures associated with each site. In this work, we developed an analytical strategy for characterization of intact N-glycopeptides in complex proteome samples. In the first step, tryptic glycopeptides were enriched using ZIC-HILIC. Secondly, a portion of the glycopeptides was treated with endoglycosidase H (Endo H) to remove high-mannose (Man) and hybrid N-linked glycans. Thirdly, a fraction of the Endo H-treated glycopeptides was further subjected to PNGase F treatment in ¹⁸O water to remove the remaining complex glycans. The intact glycopeptides and deglycosylated peptides were analyzed by nano-RPLC-MS/MS, and the glycan structures and the peptide sequences were identified by using the Byonic or pFind tools. Sequential digestion by endoglycosidase provided candidate glycosites information and indication of the glycoforms on each glycopeptide, thus helping to confine the database search space and improve the confidence regarding intact glycopeptide identification. We demonstrated the effectiveness of this approach using RNase B and IgG and applied this sequential digestion strategy for the identification of glycopeptides from the HepG2 cell line. We identified 4514 intact glycopeptides coming from 947 glycosites and 1011 unique peptide sequences from HepG2 cells. The intensity of different glycoforms at a specific glycosite was obtained to reach the occupancy ratios of site-specific glycoforms. These results indicate that our method can be used for characterizing site-specific protein glycosylation in complex samples. Graphical abstract Through integrating the information of intact glycopeptide, fragment ions filters and endoglycosidase digestion, the reliability of the identification could be significantly improved. We quantified the site-specific glycoforms occupancy ratios through the MS response signaling of each glycopeptide at the same time.
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PMID:Sequential fragment ion filtering and endoglycosidase-assisted identification of intact glycopeptides. 2825 64


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