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 ability of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N-glycopeptides and N-glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase-C glycoprotein, which contain xylose linked beta 1----2 to beta-mannose and fucose linked alpha 1----3 to the innermost N-acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100-fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of alpha 1----3-linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that alpha 1----3 fucosylation of the inner N-acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.
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PMID:Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached alpha 1----3 to the asparagine-linked N-acetylglucosamine residue. 186 49

The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five oligosaccharides obtained are a series of compounds with xylose-containing common structural core, Xyl beta 1----2 (Man alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures containing monoantennary complex-type were characterized. (formula; see text)
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PMID:Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin. 233 5

The glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen was treated with pepsin and glycopeptidase A to release asparagine-linked oligosaccharides. The reducing ends of the oligosaccharides were aminated with the fluorescent reagent 2-aminopyridine. The oligosaccharide derivatives were purified by gel permeation chromatography and reversed-phase HPLC. Their structures were determined by sequential exoglycosidase digestion and 500 MHz 1H-NMR spectroscopy. Four oligosaccharide structures, A, B, C, and D, were identified as the xylose-containing complex-type. They were present at a molar ratio of 8:1:6:1. By amino acid sequence analyses of the tryptic peptides, Asn-170 and Asn-333 of Cry j I were found to carry asparagine-linked oligosaccharides. [formula: see text]
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PMID:Carbohydrate structures of the glycoprotein allergen Cry j I from Japanese cedar (Cryptomeria japonica) pollen. 760 14

We report a carbohydrate-dependent supramolecular architecture in the extracellular giant hemoglobin (Hb) from the marine worm Perinereis aibuhitensis; we call this architectural mechanism carbohydrate gluing. This study is an extension of our accidental discovery of deterioration in the form of the Hb caused by a high concentration of glucose. The giant Hbs of annelids are natural supramolecules consisting of about 200 polypeptide chains that associate to form a double-layered hexagonal structure. This Hb has 0.5% (wt) carbohydrates, including mannose, xylose, fucose, galactose, glucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). Using carbohydrate-staining assays, in conjunction with two-dimensional polyacrylamide gel electrophoresis, we found that two types of linker chains (L1 and L2; the nomenclature of the Hb subunits followed that for another marine worm, Tylorrhynchus heterochaetus) contained carbohydrates with both GlcNAc and GalNAc. Furthermore, two types of globins (a and A) have only GlcNAc-containing carbohydrates, whereas the other types of globins (b and B) had no carbohydrates. Monosaccharides including mannose, fucose, glucose, galactose, GlcNAc, and GalNAc reversibly dissociated the intact form of the Hb, but the removal of carbohydrate with N-glycanase resulted in irreversible dissociation. These results show that carbohydrate acts noncovalently to glue together the components to yield the complete quaternary supramolecular structure of the giant Hb. We suggest that this carbohydrate gluing may be mediated through lectin-like carbohydrate-binding by the associated structural chains ("linkers").
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PMID:Carbohydrate gluing, an architectural mechanism in the supramolecular structure of an annelid giant hemoglobin. 763 98

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A (PNGase A) was purified from almonds (Prunus amygdalus var. dulcis). Contrary to previous results in the literature, the enzyme appeared to be a heterodimer with subunits of 55 and 27 kDa when analysed by SDS/PAGE and two-dimensional electrophoresis. Peaks corresponding to molecular masses of 54.2, 21.2 and 75.5 kDa were observed with matrix-assisted laser-desorption/ionization mass spectrometry. The N-terminal sequences of the larger and the smaller chain were determined to be LASGYHSWAD and EPTPLHDFPP, respectively. Both polypeptides reacted with concanavalin A, indicating their glycoprotein nature. Upon digestion of PNGase with pepsin, the N-linked oligosaccharides were released with active PNGase and analysed as their 2-aminopyridine derivatives by two-dimensional HPLC and by matrix-assisted laser-desorption mass spectrometry. The most abundant N-glycan of the four species found exhibited the well known vacuole type structure, i.e. the pentasaccharide core with xylose and alpha1,3-linked fucose. The other structures either had an additional mannose residue and/or lacked the fucose. PNGase A was largely but not absolutely resistant to self-deglycosylation. However, only at an extremely high enzyme/substrate ratio, N-glycans released from PNGase A itself caused a detectable contamination of a PNGase digest of a glycopeptide.
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PMID:Characterisation of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A and its N-glycans. 952 20

We report here the isolation and characterization of a peptide-N4-(acetyl-beta-glucosaminyl) asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds. The enzyme was purified to homogeneity with 6.5% yield from defatted soybean meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite chromatography, and hydrophobic chromatography. The purified enzyme, designated PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90 kDa by gel filtration, indicating this PNGase is a monomeric protein. The enzyme showed maximal activity at pH 4.5-5.0. PNGase-GM was capable of hydrolyzing the beta-aspartylglycosylamine linkage (GlcNAc beta 1-->Asn) of various glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing plant complex type N-glycan units, while this amidase was far less active on the glycopeptides bearing sialylated animal complex-type glycans.
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PMID:A new peptide-N4-(acetyl-beta-glucosaminyl)asparagine amidase from soybean (Glycine max) seeds: purification and substrate specificity. 953 7

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose, and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanvalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host.
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PMID:Cell wall antigens of Pneumocystis carinii trophozoites and cysts: purification and carbohydrate analysis of these glycoproteins. 962 93

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

Mucuna pruriens seeds are used in some countries as a human prophylactic oral anti-snake remedy. Aqueous extracts of M. pruriens seeds possess in vivo activity against cobra and viper venoms, and protect mice against Echis carinatus venom. It was recently demonstrated that the seed immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc), and the immunogenic properties of gpMuc seemed to mainly reside in its glycan chains. In the present study, gpMuc was found to contain only N-glycans. Part of the N-glycans could be released with peptide-(N (4)-(N-acetyl-beta -glucosaminyl)asparagine amidase F (PNGase F-sensitive N-glycans); the PNGase F-resistant N-glycans were PNGase A-sensitive. The oligosaccharides released were analyzed by a combination of MALDI-TOF mass spectrometry, HPLC profiling of 2-aminobenzamide-labelled derivatives and (1)H NMR spectroscopy. The PNGase F-sensitive N-glycans comprised a mixture of oligomannose-type structures ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), and two xylosylated structures, Xyl(1)Man(3)GlcNAc(2) and Xyl(1)Man(4)GlcNAc(2). The PNGase A-sensitive N-glycans, containing (alpha 1-3)-linked fucose, were identified as Fuc(1)Xyl(1)Man(2)GlcNAc(2) and Fuc(1)Xyl(1)Man(3)GlcNAc(2). In view of the determined N-glycan ensemble, the immunoreactivity of gpMuc was ascribed to the presence of core (beta 1-2)-linked xylose- and core alpha (1-3)-linked fucose-modified N-glycan chains.
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PMID:Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds. 1700 51

A novel dopachrome conversion enzyme (DCE) is present in insects and involved in their melanization pathway. DCE shares no sequence homology with any noninsect species from bacteria to humans. Several DCE sequences have been available, but enzyme structure and catalytic mechanism are unclear. This study concerns DCE PTMs, especially glycosylation. A mosquito DCE was purified and its monosaccharide composition, N-glycosylation site, and oligosaccharide structures were determined. Results showed that N-acetyl D-glucosamine and D-mannose are the major monosaccharides and L-fucose, D-xylose, and D-arabinose are the minor ones in mosquito DCE. Glycosylation site and oligosaccharide structures were elucidated from MS and MS/MS spectra of trypsin-digested DCE glycopeptides. A single N-glycosylation site (Asn285 -Glu-Thr) was identified in DCE and was proven to be fully glycosylated. Man3GlcNAc2, Man3(Fuc)1-2GlcNAc2, and their truncated structures were the dominant oligosaccharides. In addition, high mannose-type structures (Man4-7(Fuc)GlcNAc2) were also identified. Removal of DCE N-oligosaccharides with peptide N-glycosidase (PNGase F) decreased its activity and thermal stability. However, partial DCE deglycosylation with alpha-mannosidase or alpha-fucosidase somewhat stimulated its activity and improved its thermal stability. During mass spectrometric analysis of DCE glycopeptides, their CID patterns were highly intriguing, in that some glycopeptides underwent both C-terminal rearrangement and formation of dimeric structures during CID. Results of this study provide an interesting example in terms of potential complexity of the glycopeptide CID fragmentation pattern.
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PMID:Proteomic analysis of N-glycosylation in mosquito dopachrome conversion enzyme. 1762 77


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