Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human skin fibroblast lines of the infantile form of neuronal ceroid lipofuscinosis and control lines were cultured in the presence of [3H]glucosamine plus [3H]mannose and [35S]methionine. The labeled glycoconjugates were compared by quantitative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The infantile form of the disease showed a 75% decrease of four glycoprotein components of M(r) 120-140 kDa. These components appeared to be N-linked glycoproteins as peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase F) released 86-96% of the labeled carbohydrate from the labeled protein. These results suggest that the infantile form of this disease may be characterized by abnormalities in glycoconjugate metabolism leading to reduction of specific glycoproteins.
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PMID:Glycoprotein metabolism in neuronal ceroid lipofuscinosis fibroblasts. 141 45

Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F (PNGase F) and endo-beta-N-acetyl glucosaminidase F (Endo F) activities were monitored during cultivation of Flavobacterium meningosepticum using a new fluorescence-HPLC procedure based on a commercially available substrate. The PNGase F activity reached a maximum level at the end of the log phase and remained constant during the stationary phase, while Endo F continuously increased until late stationary phase. PNGase F obtained at the end of the log phase was less contaminated by other proteins compared with late stationary phase.
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PMID:Use of resorufin-labelled N-glycopeptide in a high-performance liquid chromatography assay to monitor endoglycosidase activities during cultivation of Flavobacterium meningosepticum. 142 35

The recent discovery of free oligosaccharides typical for the complex type of glycan chains terminating with a free di-N-acetylchitobiosyl structure in certain fish eggs and early embryos (Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Seko, A., Kitajima, K., Iwasaki, M., Inoue, S., and Inoue, Y. (1989) J. Biol. Chem. 264, 15922-15929; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J. Biol. Chem. 264, 18520-18526) led us to find an enzyme responsible for detachment of N-linked glycan chains from glycoproteins by hydrolyzing the beta-aspartyl-glucosylamine linkage in Oryzias latipes embryos. The enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase or peptide:N-glycosidase (PNGase), was partially (2090-fold) purified, and the reaction site at which this enzyme acts was specified by analysis and identification of the reaction products. This is the first demonstration showing PNGase in animal sources, although the presence of PNGases was reported in a variety of plant extracts and bacteria. Thus, the commonality of this type of enzyme is now demonstrated, and the possible physiological role of PNGase in de-N-glycosylation as a basic biologic process is proposed.
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PMID: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. 171 90

Four oligosaccharide chain-cleaving enzymes, including two new endoglycosidases distinct from endo-beta-acetylglucosaminidase (Endo) F1, have been identified and purified to homogeneity from cultural filtrates of Flavobacterium meningosepticum. FPLC-directed hydrophobic-interaction chromatography in conjunction with high-resolution ion-exchange chromatography provided a more simple, rapid method for the isolation of endoglycosidase F1, F2 and F3, and the amidase, peptide-N4-N-acetyl-beta-D-glucosaminyl)-asparagine amidase (PNGase F), in greater than 50% yield. The specificity of PNGase F and Endo F1 are well established. Endo F2 and Endo F3 represent new distinct endoglycosidases that prefer complex as compared to high-mannose asparagine-linked glycans. Endo F2 cleaved biantennary oligosaccharides, whereas Endo F3 cleaved both bi- and triantennary oligosaccharides.
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PMID:Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum. 179 38

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F(PNGase F) from Flavobacterium meningosepticum is a highly useful enzyme for the structural analysis of N (asparagine)-linked carbohydrate chains derived from glycoproteins. The enzyme was enriched using a published procedure [Tarentino AL, Gomez CM, Plummer TH, Jr (1984) Biochemistry 1985:4665-71; Tarentino AL, Plummer TH, Jr (1987) Methods Enzymol 138:770-78] and further purified by hydrophobic interaction HPLC on a weak hydrophobic TSK-Ether column from which it was eluted by a decreasing gradient of 1.7 M ammonium sulphate in 100 mM sodium phosphate, pH 7.0, containing 5 mM EDTA. To determine the optimal conditions for a complete deglycosylation of glycoproteins by PNGase F, experiments were performed with human alpha 1-acid glycoprotein, because the five complex type carbohydrate chains are quite resistant to enzymic hydrolysis. The influence of different detergents on the enzyme reaction was studied. Complete deglycosylation of human alpha 1-acid glycoprotein was achieved by the use of 60 mU/ml PNGase F in 0.25 M sodium phosphate buffer, pH 8.6, containing 0.2% (w/v) SDS, 20 mM mercaptoethanol and 0.5% Mega-10.
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PMID:Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase from Flavobacterium meningosepticum. 213 46

A polytropic recombinant retrovirus containing the envelope gene of Friend mink cell focus-inducing virus plus the remainder of the genome of an amphoropic murine leukemia virus was propagated on mouse embryo fibroblasts and mink lung cells. Virus particles, metabolically labeled with [2-3H]mannose, were harvested from the culture supernatants and lysed with detergents. The viral envelope glycoprotein was isolated from the lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Oligosaccharides were liberated by sequential treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F and fractionated by high-performance liquid chromatography. Individual glycans were characterized chromatographically, by methylation analyses and in part, by enzymic microsequencing. The results demonstrated that viral glycoproteins, synthesized in mouse embryo fibroblasts, carried as major constituents partially fucosylated diantennary, 2,4- and 2,6-branched triantennary and tetraantennary complex type N-glycans with 0-4 sialic acid residues and only small amounts of high-mannose type species with 5-9 mannose residues. As a characteristic feature, part of the complex type glycans contained additional Gal(alpha 1-3) substituents. Glycoprotein obtained from virions propagated on mink lung cells, contained partially fucosylated diantennary and 2,4-branched triantennary oligosaccharides with 1-3 sialic acid residues, in addition to trace amounts of high-mannose type species with 8 or 9 mannose residues. Thus, the results reveal that predominantly, the complex type N-glycans of the retroviral envelope glycoprotein display cell-specific variations including differences in oligosaccharide branching, sialylation and substitution by additional Gal(alpha 1-3) residues.
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PMID:Glycosylation of the envelope glycoprotein from a polytropic murine retrovirus in two different host cells. 217 68

A 3,000-base pair EcoRI fragment containing the Flavobacterium meningosepticum gene for peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was cloned into the Bluescript plasmid vector and expressed in Escherichia coli. The gene consists of an open reading frame of 1,062 base pairs coding for a 354-amino acid protein; the first 40 amino acids are presumed to be the natural secretory signal sequence, with the remaining 314 amino acids (34,779 Da) representing the catalytically active protein. The deduced amino acid sequence was verified independently by direct microsequencing of over 94% of the pure protein (Flavobacterium peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase) as tryptic and cyanogen bromide peptides. Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was not secreted by E. coli; molecular weight analysis of the partially purified recombinant enzyme suggested incomplete processing of the putative leader sequence.
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PMID:Molecular cloning and amino acid sequence of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from flavobacterium meningosepticum. 218 34

The peptide-N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) gene from Flavobacterium meningosepticum was cloned into a high copy number Escherichia coli plasmid. Levels of PNGase F activity produced in cultures of the recombinant strain were up to 100-fold higher than those obtained in cultures of F. meningosepticum. The complete PNGase F gene sequence was determined. Comparison of the predicted amino acid sequence of pre-PNGase F to the N-terminal sequence of the native mature enzyme indicates that the protein is synthesized with a 40-amino acid signal sequence that is removed during secretion in F. meningosepticum. The recombinant PNGase F produced in E. coli is a mixture of products comprised predominantly of two proteins with molecular masses of 36.3 and 36.6 kDa. These proteins have a higher apparent molecular mass than the 34.7-kDa native enzyme. N-terminal amino acid sequencing demonstrated that these higher molecular mass products result from cleavage of the pre-PNGase F in E. coli upstream of the native N terminus. The PNGase F gene was engineered to encode a preenzyme that was processed in E. coli to give an N terminus identical to that of the native enzyme. Purified preparations of this form of recombinant PNGase F were shown to be suitable for glycoprotein analyses since they possess no detectable endo-beta-N-acetylglucosaminidase F, exoglycosidase, or protease activity.
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PMID:Cloning and expression of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F in Escherichia coli. 218 35

As tools to study structural relationships of cobra venom factor (CVF) and human complement component C3, murine monoclonal antibodies to CVF were produced. In this paper we describe two of these monoclonal anti-CVF antibodies designated GV1.8 and GV1.10, both of which bind to carbohydrate epitopes. On immunoblotting, antibody GV1.8 binds to both the alpha- and beta-chains of CVF, whereas antibody GV1.10 binds only to the alpha-chain of CVF. After enzymatic deglycosylation of CVF with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase), both antibodies lose their ability to bind to the deglycosylated protein. Additionally, the free oligosaccharide chains of CVF are able to inhibit the binding of antibodies GV1.8 and GV1.10 to CVF on enzyme-linked immunosorbent assay, further demonstrating their carbohydrate specificity. Both monoclonal antibodies to CVF cross-react with human C3. Antibody GV1.8 binds to both chains of human C3 indicating that the shared antigenic epitope present on the two glycosylated chains of CVF is also present on the two chains of human C3. Antibody GV1.10 cross-reacts only with the beta-chain of human C3 which is the homologous chain to the alpha-chain of CVF. After enzymatic deglycosylation of human C3 by N-glycanase, both antibodies lose their ability to bind to the deglycosylated protein consistent with the carbohydrate nature of the recognized epitopes. These results indicate that CVF and human C3 share carbohydrate epitopes on their homologous and nonhomologous chains.
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PMID:Cobra venom factor and human C3 share carbohydrate antigenic determinants. 244 Sep 48

We have studied the differential susceptibility to N-glycanase (peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase) of oligosaccharides at the individual glycosylation sites of mouse TSH and free alpha-subunits. Mouse thyrotropic tumor tissue or hypothyroid pituitary tissue were incubated with D-[2-3H]mannose for 6 h. [3H]Mannose-labeled TSH or free alpha-subunits were obtained from homogenates using specific antisera and were digested with N-glycanase in their native state or after heat denaturation and reduction in the absence or presence of detergents. Tryptic fragments of the digestion products were then analyzed by reverse phase HPLC so that the effects of N-glycanase at the individual glycosylation sites could be determined. N-Glycanase treatment of native molecules did not cleave oligosaccharides efficiently at Asn56 of alpha-subunits and Asn23 of TSH beta, whereas oligosaccharides at Asn82 of alpha-subunits were more susceptible regardless of whether the alpha-subunits were combined with TSH beta. Heat denaturation, reduction, and the presence of detergents did not substantially increase the cleavage by N-glycanase of the protected oligosaccharides, suggesting that the primary structures of the TSH subunits influenced efficiency at specific sites. Pretreatment of free alpha-subunits with trypsin failed to enable N-glycanase to work fully, as oligosaccharides at Asn56 were cleaved less effectively than those at Asn82. Thus, the susceptibility to N-glycanase differs at the individual glycosylation sites of mouse TSH and free alpha-subunits, and these differences may result from effects of the primary structures of the TSH subunits.
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PMID:Differential susceptibility to N-glycanase at the individual glycosylation sites of mouse thyrotropin and free alpha-subunits. 245 9


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