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)

Freshly isolated rat islets and cultured hamster insulinoma cells (HIT T15) were incubated with a membrane-permeable octanoyl tripeptide (N-octanoyl-ASN-TYR-THR-NH2), which contains an acceptor sequence for ASN-linked glycosylation. Labeled octanoyltripeptide (125[I]TYR) was glycosylated by both islets and HIT cells. The carbohydrate moiety of this glycotripeptide was removed by N-glycanase indicating that glycotripeptide was formed in the lumen of endoplasmic reticulum and, subsequently was secreted via the route for secretory protein. Secretion of glycotripeptide began more rapidly than that of insulin newly synthesized from 3[H]leucine. At 30 min glycotripeptide secretion was already significant but, over a 3-h period, it never represented more than 21% of glycotripeptide produced. Glycotripeptide secretion was not affected by compounds shown to regulate insulin secretion (glucose, forskolin, EGTA and streptozotocin). Thus in beta cells, it appears that glycotripeptide secretion is unregulated and that its cellular secretory pathway is different from that for insulin.
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PMID:Unregulated secretion of an exogenous glycotripeptide by rat islets and HIT cells. 284 81

After pepsin digestion, all of the carbohydrates in ovalbumin were recovered in two glycopeptides, Glu-Glu-Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val and Glu-Gln-Lys-Tyr-Asn(CHO)-Leu-Thr-Ser-Val. Almond glycopeptidase released quantitatively oligosaccharides from the glycopeptides. The products from both glycopeptides contained both the high-mannose-type oligosaccharides and the hybrid-type oligosaccharides in the same ratio. Thus, either the high-mannose-type or the hybrid-type oligosaccharide is attached to the unique asparagine residue in the ovalbumin molecule.
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PMID:Either high-mannose-type or hybrid-type oligosaccharide is linked to the same asparagine residue in ovalbumin. 728 36

Crystallographic analysis and site-directed mutagenesis have been used to identify the catalytic and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F), an amidohydrolase that removes intact asparagine-linked oligosaccharide chains from glycoproteins and glycopeptides. Mutagenesis has shown that three acidic residues, Asp-60, Glu-206, and Glu-118, that are located in a cleft at the interface between the two domains of the protein are essential for activity. The D60N mutant has no detectable activity, while E206Q and E118Q have less than 0.01 and 0.1% of the wild-type activity, respectively. Crystallographic analysis, at 2.0-A resolution, of the complex of the wild-type enzyme with the product, N,N'-diacetylchitobiose, shows that Asp-60 is in direct contact with the substrate at the cleavage site, while Glu-206 makes contact through a bridging water molecule. This indicates that Asp-60 is the primary catalytic residue, while Glu-206 probably is important for stabilization of reaction intermediates. Glu-118 forms a hydrogen bond with O6 of the second N-acetylglucosamine residue of the substrate and the low activity of the E118Q mutant results from its reduced ability to bind the oligosaccharide. This analysis also suggests that the mechanism of action of PNGase F differs from those of L-asparaginase and glycosylasparaginase, which involve a threonine residue as the nucleophile.
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PMID:Active site and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F. 749 89

We previously reported that activated platelets stimulated neutrophils and monocytes to produce superoxide anion (O2-) through the interaction between P-selectin and its carbohydrate ligand, sialyl Lewis X (sLeX) (Nagata, K., Tsuji, T., Todoroki, N., Katagiri, Y., Tanoue, K., Yamazaki, H., Hanai N., and Irimura, T. (1993) J. Immunol. 151, 3267-3273). In the present study, we investigated the role of cell surface carbohydrate chains of leukocytes in this process. Glycoconjugate-specific hydrolytic enzymes and inhibitors of glycosylation processing were applied. Granulocyte-like differentiated HL-60 (gHL-60) cells released an increased amount of O2- in response to activated platelets in a P-selectin-dependent manner. When HL-60 cells were differentiated in the presence of benzyl-alpha-N-acetylgalactosaminide (Bzl-alpha-GalNAc), an inhibitor of chain elongation of O-linked carbohydrates, the enhanced generation of O2- was abrogated in parallel with decrease in the expression of sLex structure and in the adhesion capacity to activated platelets. In contrast, treatment with swainsonine or 1-deoxymannojirimycin, inhibitors of processing of N-linked carbohydrate chains, did not show such effects. O-Sialoglycoprotease treatment of gHL-60 cells decreased the activated platelet-induced O2- production with concomitant reduction of cell surface sLex expression. Treatment of these cells with N-glycanase did not affect the O2- production. These results strongly suggested that serine/threonine-linked carbohydrate chains containing sLex played an essential role in the P-selectin-mediated leukocyte activation. By Western blotting analysis of gHL-60 cell lysates, we identified two glycoproteins which carried sLex structures and were sensitive to Bzl-alpha-GalNAc treatment.
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PMID:Role of O-linked carbohydrate chains on leukocyte cell membranes in platelet-induced leukocyte activation. 752 76

Monosaccharide analysis by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) was used to identify the glycopeptides in the reversed-phase (RP-HPLC) separation of a bovine fetuin tryptic digest (1.6 nmol). This method, requiring no sample derivatization, identified four asparagine-linked (N-linked) glycopeptides and at least seven serine/threonine-linked (O-linked) glycopeptides. Glycopeptide identification was confirmed by Edman sequencing. Monosaccharide quantification of each glycopeptide suggested that all of the N-linked glycopeptides were the complex type and all the O-linked glycopeptides were sialylated. We determined that glycopeptides could be prepared by acidic reversed-phase chromatography with less than 3% loss of N-acetylneuraminic acid (Neu5Ac). The N-linked glycopeptides of bovine fetuin were prepared, digested with N-glycosidase F (PNGase F), and their oligosaccharides analyzed by HPAEC/PAD. These oligosaccharide profiles revealed that the Asn-138 oligosaccharide attachment site contained the majority of the disialylated and monosialylated oligosaccharides. The Asn-158 oligosaccharide attachment site contained the majority of the tetrasialylated oligosaccharides.
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PMID:Identification, quantification, and characterization of glycopeptides in reversed-phase HPLC separations of glycoprotein proteolytic digests. 769 Jan 96

The L-glutamate transporter GLAST-1 belongs to the newly discovered family of Na(+)-dependent, high-affinity glutamate transporters, which are involved in the regulation of synaptic excitatory neurotransmitter concentration in mammalian brain. The members of this family have a similar topological organisation with at least six transmembrane helices (TMHs) and two putative N-glycosylation sites located in the extracellular loop connecting TMH 3 and TMH 4. Besides these two conserved N-glycosylation motifs at Asn206 and Asn216, GLAST-1 possesses an additional one at Asn35. The putative N-glycosylation consensus motifs (Asn-Xaa-Ser/Thr) were deleted by replacement of Asn206 and/or Asn216 by Thr using site-directed mutagenesis (mutants N206T, N216T and N206,216T). The cDNAs encoding wild-type GLAST-1 and the three glycosylation-defective transport proteins were expressed in the Xenopus laevis oocyte system. Immunoprecipitation of the [35S]methionine-labeled and glycopeptidase-F-treated transporter molecules indicates that GLAST-1 is glycosylated at Asn206 and Asn216, whereas Asn35 remains unglycosylated. To assess a possible functional role of the two glycosylation sites wild-type and glycosylation-deficient GLAST-1 were expressed in Xenopus oocytes and characterized functionally by using the whole-cell voltage-clamp technique. The results prove that N-glycosylation has no impact on the transport activity of GLAST-1.
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PMID:Localization of N-glycosylation sites and functional role of the carbohydrate units of GLAST-1, a cloned rat brain L-glutamate/L-aspartate transporter. 775 63

Addition of N-acetylgalactosamine to threonine and serine is the first step in the synthesis of O-glycosidically linked oligosaccharides. A UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (EC 2.4.1.41) from porcine submaxillary glands was recently purified to electrophoretic homogeneity, and polyclonal antibodies against the purified transferase were raised. Immunoblots of porcine, bovine, and ovine submaxillary gland extracts with the anti-transferase antibodies gave a single band and the antibodies reacted equally well with the purified glycosylated and N-glycanase-treated transferase. Immunoelectron microscopic localization of the transferase was achieved in Lowicryl K4M thin sections and frozen-thawed thin sections of porcine and bovine submaxillary gland by using the protein A-gold technique. Specific gold particle labeling was observed in the cis Golgi apparatus and smooth-membraned vesicular structures in close topological relation with it. Labeling was undetectable in the rough endoplasmic reticulum, its transitional elements, and smooth-membraned structures close to them, the trans Golgi apparatus, mucin droplets, and the plasma membrane. The onset of labeling for peptide-bound GalNAc as detected with Vicia villosa isolectin G4 mirrored the transferase immunolocalization as directly shown by double labeling and extended into the trans Golgi apparatus and mucous droplets. Apomucin immunolabeling was found throughout the endoplasmic reticulum and the intermediate compartment and partially overlapped the region of transferase labeling in the Golgi apparatus as demonstrated by double immunolabeling. Thus, the initial step of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation in porcine and bovine submaxillary gland cells occurs in the cis Golgi apparatus. The possible involvement of the intermediate compartment remains to be clarified.
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PMID:Subcellular localization of the UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-mediated O-glycosylation reaction in the submaxillary gland. 809 Jul 48

Muscarinic acetylcholine receptors (mAChR, human m2 subtype) expressed in Sf9 (Spodoptera frugiperda) cells using the baculovirus system were purified and subjected to phosphorylation by a mAChR kinase, which was partially purified from porcine cerebrum. Two bands with apparent molecular masses of 59 kDa and 39 kDa as determined by SDS/PAGE were found to be phosphorylated in an agonist-dependent manner. Both bands were labeled by the irreversible muscarinic ligand [3H]propylbenzilylcholine mustard. Molecular masses of the [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled bands decreased following treatment with N-glycanase. The 59-kDa and 39-kDa bands were converted to 52-kDa and 32-kDa bands, respectively, indicating that both the 59-kDa and 39-kDa bands contain the amino-terminal region where glycosylation sites are present. The ratio of incorporated [32P]phosphate and bound [3H]propylbenzilylcholine mustard was essentially the same for the 59-kDa and 39-kDa bands, indicating that all the phosphorylation sites reside in the sequence of 39 kDa from the amino-terminal region. The amounts of incorporated [32P]phosphate were estimated to be 10-11/receptor, with 7-8 serine and 3-4 threonine, but no phosphorylated tyrosine residues. Further treatment of [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled receptors with V8 protease indicated that the phosphorylation sites were not present in 30-kDa amino-terminal segment. These results indicate that the phosphorylation sites are localized in the range 30-39 kDa from the amino terminus, which consists of primarily the central part of the third intracellular loop. Consistent with this conclusion, a fusion protein containing glutathione S-transferase linked to a peptide corresponding to residues 227-324 of the central part of the third intracellular loop was found to be phosphorylated by the mAChR kinase in a heparin-sensitive manner.
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PMID:Location of agonist-dependent-phosphorylation sites in the third intracellular loop of muscarinic acetylcholine receptors (m2 subtype). 811 96

The electrophoretic analysis of purified Ole e I, the major allergen from Olea europaea pollen, reveals the presence of two main variants, glycosylated (20.0 kDa) and non-glycosylated (18.5 kDa) components. The glycosylated variant has been identified as a concanavalin A-binding glycoprotein. Its carbohydrate moiety has a molecular mass of about 1.3 kDa (5% weight of the glycosylated allergen), based on mass spectrometry analysis. Enzymatic treatment of native Ole e I with the specific glycosidase PNGase F accounts for an oligosaccharide N-linked to the polypeptide chain. This treatment does not sensibly modify the secondary structure of the protein but diminishes the affinity of the allergen for specific IgE antibodies. Tryptic digestion of Ole e I reveals the presence of a single carbohydrate-containing peptide. This peptide was recognized by the sera of hypersensitive individuals. The amino acid sequence of this peptide is Phe-Lys-Leu-Asn-Thr-Val-Asn-Gly-Thr-Thr-Arg, asparagine at the seventh being the carbohydrate attaching site. The obtained data are discussed in terms of the potential role of the sugar moiety in the allergenic activity of Ole e I.
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PMID:Glycosylation site of the major allergen from olive tree pollen. Allergenic implications of the carbohydrate moiety. 830 97

The amino acid sequence for the envelope protein(s) predicted from the nucleotide sequence of the E and E2/NS1 regions of the hepatitis C virus (HCV) genome is enriched with an N-linked glycosylation site motif, Asn-X-Thr/Ser, suggesting oligosaccharide moieties are present on the virion surface. We attempted to characterize the sugar moiety on the surface of HCV virions recovered from sera of infected humans to assess the natural properties of the virus. Six kinds of lectins were used to bind HCV virions in affinity column chromatographies: RCAI, WGA, Con A, AAL, LCA, and PNA. Lectin-bound virions were identified by detecting HCV RNA in eluted chromatography fractions with a polymerase chain reaction (PCR) method. Our results showed that HCV was similar to hepatitis B virus (HBV) in characteristics of binding to lectins: HCV showed a strong binding to RCAI and WGA, weak binding to Con A, and no detectable binding to AAL, LCA, or PNA. Treatment of the HCV virion preparation with an enzyme, glycopeptidase A, or a detergent, NP-40, resulted in a significant decrease in the ability to bind these lectins. Our results suggest that asparagine-linked sugar chains are present on the surface of native virions of HCV, very similar to those for HBV.
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PMID:Demonstration of sugar moiety on the surface of hepatitis C virions recovered from the circulation of infected humans. 839 23


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