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)

Horseradish peroxidase (E.C. 1.11.1.7) isozyme c (HRPc) is a glycoprotein found to contain 21.8% carbohydrate with the average composition: 2 mol GlcNAc, 2.6 mol Man, and 0.8 mol each of Fuc and Xyl. The oligosaccharides of HRPc were investigated by a combination of High pH Anion-Exchange Chromatography with Pulsed Amperometric Detection, methylation analysis and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. The structure of the major oligosaccharide released by digestion with glycopeptidase A, accounting for between 75 and 80% of the total, was confirmed to be [sequence: see text]. Most of the remaining oligosaccharides were found to belong to the (Xyl)xManm(Fuc)fGlcNAc2 (m = 2, 4, 5, 6; f = 0 or 1; x = 0 or 1) family. Less than 5% of the oligosaccharides were of the ManmGlcNAc2 (m = 4 to 7) type. Methylation analysis of holo- and apo-HRPc and its tryptic glycopeptides support the structures proposed for the oligosaccharides. Furthermore, methylation analysis of the tryptic glycopeptides provides evidence for the heterogeneity of the oligosaccharides occurring at each of the N-linked sites.
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PMID:The glycans of horseradish peroxidase. 876 7

Soybean hull peroxidase (SBP, E.C. 1.11.1.7), an anionic glycoprotein, was found to contain 18.2% carbohydrate with the average composition: 2 mol GlcNAc, 3.3 mol Man, 0.9 mol Fuc, and 0.7 mol Xyl. The oligosaccharides of SBP, after release with glycopeptidase A, were investigated by a combination of high pH anion exchange chromatography with pulsed amperometric detection, methylation analysis and matrix assisted laser desorption/ionization-time-of-flight mass spectrometry. The structure of the major oligosaccharide, accounting for 60 to 65% of the total, is Man alpha 1-->6(Man beta 1-->3)(Xyl beta 1-->2)Man beta 1-->4GlcNAc beta 1-->4(Fuc alpha 1-->3)GlcNAc. A further 20 to 25% of the released oligosaccharides belong to the (Xyl)xManm(Fuc)fGlcNAc2 (m = 2, 4, 5, 6; f = 0 or 1, x = 0 or 1) family. The rest of the oligosaccharides were of the high-mannose type. Investigation of the six tryptic fractions containing carbohydrate revealed considerable heterogeneity in the N-linked oligosaccharides present in each fraction. The major glycan (4, Table III) was present in each fraction. Two of the fractions contained the major part of the high-mannose type glycans, ManmGlcNAc2 (m = 5-9), the major species being Man7GlcNAc2. The other four fractions contained mainly members of the (Xyl)xManm(Fuc)fGlcNAc2 (m = 2, 4, 5, 6; f = 0 or 1; x = 0 or 1) family. Methylation analysis of the holo- and apo-SBP provide support for the structures proposed for the oligosaccharides as well as for the heterogeneity of the glycopeptide fractions.
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PMID:The glycans of soybean peroxidase. 899 5

Tryptic digestion of apo-soybean peroxidase (apo-SBP), with and without acetamidation, chromatographic separation of the tryptic fragments and MALDI-TOF analysis of the major components, both before and after digestion with glycopeptidase A, demonstrated the presence of six carbohydrate groups on five peptides. Five of the glycopeptides can be mapped with confidence to the peptides containing Asn16, Asn90, Asn104, Asn169, and Asn174. The sixth N-glycosylation site is not known and does not appear to be Asn145. It may be present on the N-terminus of SBP, which has not been sequenced.
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PMID:The N-glycosylation sites of soybean seed coat peroxidase. 925 49

The major cathepsin B-like proteinase of adult Schistosoma japonicum has been isolated for the first time. Affinity chromatography with the mammalian cathepsin B inhibitor glycyl-phenylalanyl-glycine-semicarbazone purified a protein that was identified by N-terminal sequencing as Sj31. Sensitivity of Sj31 to PNGase F demonstrated the presence of asparagine-linked N-glycan. Marked resistance to the action of Endo-beta-glycosidase H indicated that most of the N-glycan chains are of the complex type. Binding of horseradish peroxidase-conjugated lectins demonstrated the presence of N-mannose, N-acetylglucosamine, and N-acetyllactosamine type 2 in the N-glycan. Fucose was not detected, and the presence of sialic acid remained questionable. Sj31 degraded the fluorogenic substrates Z-Phe-Arg-NMec and Z-Arg-Arg-NMec with an optimum between pH 5.0 and 6.0. The specific activity was 18-21-fold higher with the Phe-Arg substrate compared with the Arg-Arg substrate, whereas this value was 4-6-fold for bovine spleen cathepsin B, thus suggesting differences in the S2 subsite between parasite and host proteinases. Quantitative purification of Sj31 led to the conclusion that cathepsin B-like activity predominates over cathepsin L-like activity in S. japonicum. Because Sj31 degraded hemoglobin in vitro and was localized in the parasite gut, the proteinase may degrade ingested proteins in vivo.
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PMID:Affinity isolation and characterization of the cathepsin B-like proteinase Sj31 from Schistosoma japonicum. 940 88

The cationic peanut peroxidase is a complex enzyme consisting of a heme group, two calcium ions and three complex carbohydrate chains at positions Asn60, 144 and 185. Details of the heme and calcium ligation, necessary for oxidation, have recently been revealed from the three-dimensional structure of the peroxidase. However, the three glycans that may be important for the stability of the enzyme as well as its activity were not resolved. In order to determine the configuration of one of these glycans, PNGase A was used to cleave the glycan from the enzyme at Asn-144. This glycan was studied by two dimensional 1H-NMR spectroscopy to identify the sugar linkages. The results indicated a glycan structure comprising a Man alpha1-6(Xyl beta1-2)Man beta1-4GlcNAc beta1-4(Fuc alpha1-3)GlcNAc beta core but with an additional Man alpha1-3 appendage linked to Man3. The glycan also appeared to be heterogeneous as was noted from a single terminating galactose being linked to approximately 20-25% glycan.
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PMID:Sequence specific analysis of the heterogeneous glycan chain from peanut peroxidase by 1H-NMR spectroscopy. 1065 21

A neutral peroxidase isozyme (TP) purified from turnip (Brassica napus L. var. purple top white globe) was partially deglycosylated, using chemical and enzymatic treatment. A 32% carbohydrate removal was achieved by exposing TP to a mixture of PNGase F, O-glycosidase, NANase, GALase III and HEXase I, while m-periodate treatment removed about 88% of TP carbohydrate moiety. The glycoprotein fraction of the TP contained a relatively high mannose and fucose content (37 and 31%, w/w, respectively), 16% (w/w) galactose, and 15% (w/w) GlcNAc. Thus, the carbohydrate moiety was classified as a hybrid type. Partially deglycosylated TP had reduced activity (by 50-85%), was more susceptible to proteolysis, and showed a slight decrease in thermostability compared to the native enzyme. Circular dichroism studies strongly suggested that although the carbohydrate moiety of TP did not influence the conformation of the polypeptide backbone, its presence considerably enhanced protein conformational stability toward heat. Removal of oligosaccharide chains from TP caused a decrease in K(m) and V(max) for hydrogen peroxide. Native and chemically deglycosylated TP were similarly immunodetected by rabbit polyclonal antibodies raised against TP. The results suggest that the carbohydrate moiety of TP is important for peroxidase activity and stability.
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PMID:Monosaccharide composition and properties of a deglycosylated turnip peroxidase isozyme. 1247 13

A peroxidase is present in the chorion of Aedes aegypti eggs and catalyzes chorion protein cross-linking during chorion hardening, which is critical for egg survival in the environment. The unique chorion peroxidase (CPO) is a glycoprotein. This study deals with the N-glycosylation site, structures, and profile of CPO-associated oligosaccharides using mass spectrometric techniques and enzymatic digestion. CPO was isolated from chorion by solubilization and several chromatographic methods. Mono-saccharide composition was analyzed by HPLC with fluorescent detection. Our data revealed that carbohydrate (D-mannose, N-acetyl D-glucosamine, D-arabinose, N-acetyl D-galactosamine, and L-fucose) accounted for 2.24% of the CPO molecular weight. A single N-glycosylation site (Asn328-Cys- Thr) was identified by tryptic peptide mapping and de novo sequencing of native and PNGase A-deglycosylated CPO using matrix-assisted laser/desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The Asn328 was proven to be a major fully glycosylated site. Potential tryptic glycopeptides and profile were first assessed by MALDI/TOF/MS and then by precursor ion scanning during LC/MS/MS. The structures of N-linked oligosaccharides were elucidated from the MS/MS spectra of glycopeptides and exoglycosidase sequencing of PNGase A-released oligosaccharides. These CPO-associated oligosaccharides had dominant Man3GlcNAc2 and Man3 (Fuc) GlcNAc2 and high mannose-type structures (Man(4-8)GlcNAc2). The truncated structures, Man2GlcNAc2 and Man2 (Fuc) GlcNAc2, were also identified. Comparison of CPO activity and Stokes radius between native and deglycosylated CPO suggests that the N-linked oligosaccharides influence the enzyme activity by stabilizing its folded state.
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PMID:Characterization of N-linked oligosaccharides in chorion peroxidase of Aedes aegypti mosquito. 1613 61

Aedes aegypti chorion peroxidase (CPO) plays a crucial role in chorion hardening by catalyzing chorion protein cross-linking through dityrosine formation. The enzyme is extremely resistant to denaturing conditions, which seem intimately related to its post-translational modifications, including disulfide bond formation and glycosylation. In this report, we have provided data that describe a new type of glycosylation in CPO, where a mannose is linked to the N-1 atom of the indole ring of Trp residue. Through liquid chromatography/electrospray ionization/tandem mass spectrometry and de novo sequencing of CPO tryptic peptides, we determined that three of the seven available Trp residues in mature CPO are partially (40-50%) or completely mannosylated. This conclusion is based on the following properties of the electrospray ionization/tandem mass spectrometry spectra and the enzymatic reaction of these peptides: 1) the presence of a 162-Da substituent in each Trp residue; 2) the presence of abundant fragments of m/z 163 ([Hex + H]) and [M + H - 162] (typical for N-glycosides); 3) the absence of a loss of 120 Da (this loss is typical for aromatic C-glycosides); and 4) the cleavage of the glycosidic linkage by PNGase A or F (typical for N-glycans). These results establish that a C-N bond is formed between the anomeric carbon of a mannose residue and the N-1 atom of the indole ring of Trp. This is the first report that provides definitive evidence for N-mannosylation of Trp residues in a protein. In addition, our data demonstrate that PNGase can hydrolyze Trp N-linked mannose in peptides, which is unusual because no typical beta-amide bond is present in the Trp-mannosyl moiety. Results of this study should stimulate research toward a comprehensive understanding of physiology and biochemistry of Trp N-mannosylation in proteins and the overall biochemical mechanisms of PNGase-catalyzed reactions.
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PMID:Novel glycosidic linkage in Aedes aegypti chorion peroxidase: N-mannosyl tryptophan. 1615 Jun 91

Hepatic asialoglycoprotein receptor, which may mediate the clearance of circulating thyroglobulin, is known to have a high affinity for GalNAc. Recently, the receptor has been reported to be present also in the thyroid, implicating interaction with thyroglobulin. Here, mammalian thyroglobulins were analyzed for GalNAc termini by Western blotting with GalNAc-recognizing lectins labeled with peroxidase or (125)I. Wistaria floribunda lectin was found to bind human thyroglobulin and, to some extent, bovine, but not porcine thyroglobulin. After desialylation, the lectin bound all of the thyroglobulins tested. The binding was inhibited by competitive inhibitor GalNAc. Peptide N-glycanase treatment of human desialylated thyroglobulin resulted in the complete loss of reactivity with W. floribunda lectin, indicating that the binding sites are exclusively on N-glycans. The binding sites on human desialylated thyroglobulin were partly sensitive to beta-galactosidase, and the remainder was essentially sensitive to beta-N-acetylhexosaminidase. On the other hand, the binding sites of bovine and porcine desialylated thyroglobulins were totally sensitive to beta-galactosidase. Thus the lectin binds beta-Gal termini, as well as beta-GalNAc. GalNAc-specific Dolichos biflorus lectin also bound human thyroglobulin weakly. In contrast to W. floribunda lectin, desialylation diminished binding, suggesting that these two lectins recognize different GalNAc-terminated structures. Again, the binding was inhibited by GalNAc and by treatment with peptide N-glycanase. These results strongly indicate the presence of distinct GalNAc termini of N-glycans on human thyroglobulin.
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PMID:Presence of beta-linked GalNAc residues on N-glycans of human thyroglobulin. 1709 89

High-throughput quantitative analytical method for plant N-glycan has been developed. All steps, including peptide N-glycosidase (PNGase) A treatment, glycan preparation, and exoglycosidase digestion, were optimized for high-throughput applications using 96-well format procedures and automatic analysis on a DNA sequencer. The glycans of horseradish peroxidase with plant-specific core alpha(1,3)-fucose can be distinguished by the comparison of the glycan profiles obtained via PNGase A and F treatments. The peaks of the glycans with (91%) and without (1.2%) alpha(1,3)-fucose could be readily quantified and shown to harbor bisecting beta(1,2)-xylose via simultaneous treatment with alpha(1,3)-mannosidase and beta(1,2)-xylosidase. This optimized method was successfully applied to analyze N-glycans of plant-expressed recombinant antibody, which was engineered to contain a minor amount of glycan harboring beta(1,2)-xylose. These results indicate that our DNA sequencer-based method provides quantitative information for plant-specific N-glycan analysis in a high-throughput manner, which has not previously been achieved by glycan profiling based on mass spectrometry.
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PMID:High-throughput quantitative analysis of plant N-glycan using a DNA sequencer. 1916 52


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