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 asparagine-linked oligosaccharides from an adult female mouse submandibular gland mucin were released by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F or endo-beta-N-acetylglucosaminidase H. Endo-beta-N-acetylglucosaminidase H appeared to be more effective at releasing the asparagine-linked oligosaccharides from this mucin than was peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase F. After quantitative reductive labelling with the fluorophore, 8-aminonaphthalene-1,3,6-sulphonic acid, the oligosaccharides were separated by polyacrylamide gel electrophoresis and isolated. The individual oligosaccharides were sequenced by a battery of recombinant exoglycosidases. Approximately 50% of the oligosaccharides were of the high-mannose type. The five-mannose member of this family was the most prevalent. The second group of oligosaccharides were of the non-bisected hybrid type. No complex asparagine-linked oligosaccharides were detected. The hybrids exhibited both biantennary and triantennary branching patterns. The triantennary hybrid was the most common hybrid at > 30% of all oligosaccharides. With approximately 98% of the hybrid oligosaccharides sialylated and all lacking a bisecting N-acetylglucosamine, these oligosaccharides as a group have been only rarely observed in other glycoproteins. The fully sialylated triantennary hybrid may be unique.
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PMID:Characterization of asparagine-linked oligosaccharides on a mouse submandibular mucin. 856 46

The beta-subunit of the gastric H,K-ATPase is the most abundant glycoprotein in the tubulovesicular compartment of the acid-secreting parietal cells. The oligosaccharides of the beta-subunit have been shown to contain fucose, N-acetylglucosamine, mannose, galactose, and N-acetylgalactosamine. Previous studies have shown that the rabbit beta-subunit is devoid of N-acetylneuraminic acid. Here we report the structural features of the N-linked oligosaccharides of the beta-subunit from rabbit H,K-ATPase. We used glycosidase digestions and analysis by high-pH anion-exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ionization mass spectrometry to analyze the peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase (PNGase F)- and endo-beta-N-acetylglucosaminidase H (Endo H)-released oligosaccharides. The studies showed that the oligosaccharides of the beta-subunit are a mixture of both oligomannosidic and lactosamine-type structures. The high-mannose structures were identified as Man5Man8GlcNAc2 species. A striking finding was that all the branches of the lactosamine-type structures were terminated with Galalpha-->Galbeta-->GlcNAc extensions. All of the lactosamine-type structures were found to be core fucosylated and some of them contained one to three lactosamine repeats. We propose that a part of the adaptation of the gastric beta-subunit to the acidic environment of the stomach is through providing acid-stable terminal residues on the oligosaccharides.
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PMID:The Beta-subunit of the rabbit H,K-ATPase:a glycoprotein with all terminal lactosamine units capped with alpha-linked galactose residues. 860 59

The serum-mannan binding protein (S-MBP) is a calcium-dependent C-type lectin specific for mannose and N-acetylglucosamine. S-MBP is known as a host defense factor involved in innate immunity, where the target ligands for S-MBP should be on the surface of exogenous microorganisms. In this study, we tried to find endogenous ligands for this endogenous lectin. Among the cells tested, only the lymphocytes from thymus of BALB/c mice expressed ligands for S-MBP on their surface, those from bone marrow, spleen, mesenteric lymph nodes and peripheral blood all being negative. Interestingly, among the thymocytes, only the immature thymocytes with the CD4+CD8+CD3low phenotype expressed ligands for S-MBP, and ligands for S-MBP decreased on their maturation. A major cell surface glycoprotein bearing S-MBP ligands was isolated and identified as CD45RO, which is a transmembrane protein with tyrosine phosphatase activity. Deglycosylation experiments with N-glycanase and endoglycosidase H indicated that the S-MBP ligands on thymic CD45 are high mannose type or hybrid type N-linked oligosaccharides. This unique presentation of S-MBP ligands on this special CD45 isoform suggested the possibility that the oligosaccharide portion of CD45 on immature thymocytes is associated with the maturation, development or selection events of thymocytes.
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PMID:A unique CD45 glycoform recognized by the serum mannan-binding protein in immature thymocytes. 861 14

The glycosylation of Na+/H+ exchanger isoform NHE-3 was studied in brush border membrane (BBM) vesicles isolated from rabbit, dog, and rat kidney cortex. Western blot analyses were performed against BBM proteins by using polyclonal antibodies to an NHE-3 fusion protein. In rabbit kidney, NHE-3 antibody recognized a band with approximately 95 kd molecular mass. Treatment of rabbit cortical BBM with glycopeptidase F, at 16 U/ml, for 4 or 16 hours increased the apparent mobility of NHE-3 to 84 and 82 kd, respectively. Incubation of rabbit BBM proteins for 16 hours with endoglycosidase H, at 0.1 U/ml, did not alter the apparent mobility of NHE-3. Deglycosylation of NHE-3 with glycopeptidase F did not affect acid-stimulated, amiloride-sensitive sodium 22 influx in BBM vesicles as compared with that in controls (p > 0.05). Immunoblot analysis against BBM proteins from canine kidney cortex demonstrated the presence of an approximately 83 to 92 kd protein. Treatment of canine BBM with glycopeptidase F or endoglycosidase H or F for 16 hours did not alter the apparent mobility of NHE-3, suggesting that canine renal NHE-3 is not glycosylated. Treatment of canine kidney BBM with glycopeptidase F did not affect acid-stimulated 22Na+ influx as compared with that in controls (p > 0.05). Immunoblot analysis against BBM proteins from rat kidney cortex demonstrated the presence of a sharp band at 90 kd. Treatment of rat BBM with glycopeptidase F or endoglycosidase H or F for 16 hours did not alter the apparent mobility of NHE-3, suggesting that rat renal NHE-3 is not glycosylated. The above experiments suggest that NHE-3 glycosylation in mammalians is species specific and that glycosylation does not affect the exchanger activity.
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PMID:Glycosylation of the Na+/H+ exchanger isoform NHE-3 is species specific. 878 38

The transmembrane glycoprotein HEF and its acylation deficient mutant M1 were expressed in Sf9 insect cells infected with recombinant baculovirus and in CV1 mammalian cells using the vaccinia T7 system. In insect cells (Sf9), both wild type HEF and HEF(M1) are synthesized in their precursor form HEF0, which appears as a double band in SDS gels. Digestion with glycopeptidase F and endoglycosidase H reveals that the larger 84-kDa form is modified by the attachment of unprocessed carbohydrates of the high mannose type whereas the smaller 76-kDa form is non-glycosylated. As revealed by in vitro labeling experiments with palmitic acid another modification of HEF is the attachment of a long chain fatty acid to cysteine residue Cys-652 which is located at the internal border of the cytoplasmic membrane. After labeling with [3H]palmitic acid in both systems only HEF(WT) is acylated, whereas HEF(M1) is not. High performance liquid chromatography analysis of the fatty acids bound to HEF(WT) expressed in Sf9 insect cells reveals nearly 80% of palmitic acid. In contrast to this finding, the acylation pattern of HEF expressed in CV1 cells shows nearly the same amounts of stearic and palmitic acid (40%). Since the interconversion of the input [3H]palmitic acid to stearic acid is even lower in CV1 cells than in insect cells, it follows that only HEF expressed in mammalian, but not in insect cells selects for stearic acid during its biosynthetic acylation. We extended our study to acylation of endogenous proteins in Sf9 cells. In finding only palmitate linked to protein we present evidence that, in contrast to mammalian cells, insect cells (Sf9) cannot transfer stearic acid to polypeptide. This finding favors the hypothesis of enzymatic acylation over non-enzymatic mechanisms of acyl transfer to protein.
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PMID:Differential fatty acid selection during biosynthetic S-acylation of a transmembrane protein (HEF) and other proteins in insect cells (Sf9) and in mammalian cells (CV1). 879 73

Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.
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PMID:A source of glycosylated human T-cell lymphotropic virus type 1 envelope protein: expression of gp46 by the vaccinia virus/T7 polymerase system. 889 53

This study reports the identification and initial characterization of the precursors, modified forms, and oligomers of bovine herpesvirus 1 (BHV-1) gI and gE proteins with polyvalent rabbit serum specific for gI or gE. Our experiments used the Colorado strain of BHV-1 and mutant viruses with insertions of the Escherichia coli lacZ gene into the predicted gE and gI reading frames. We also translated the gE and gI open reading frames in vitro and expressed them in uninfected cells using eukaryotic expression vectors. Precursor-product relationships were established by pulse-chase analysis and endoglycosidase H and glycopeptidase F digestions. Like the homologous glycoproteins of herpes simplex virus type 1, pseudorabies virus, and varicella-zoster virus, BHV-1 gI and gE are modified by N-linked glycosylation and associate with each other soon after synthesis, forming a noncovalent complex in infected and transfected cells. An analysis of mutant and wild-type-virus-infected cells and transfected COS cells expressing gE or gI alone suggested that gE-gI complex formation is necessary for efficient processing of the gE precursor to its mature form. One new finding was that unlike the other alphaherpesvirus gI homologs, a fraction of pulse-labeled gI synthesized in BHV-1-infected cells apparently is cleaved into two relatively stable fragments 2 to 4 h after the pulse. Finally, we incubated BHV-1-infected cell extracts with nonimmune mouse, rabbit, horse, pig, and calf sera and found no evidence that gE or gI functioned as Fc receptors as reported for the herpes simplex virus type 1 and varicella-zoster virus homologs.
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PMID:Synthesis, processing, and oligomerization of bovine herpesvirus 1 gE and gI membrane proteins. 889 10

Point mutations in the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor have been shown to cause constitutive activation which results in precocious puberty in affected males. We introduced one of these mutations, Asp-556 --> Gly, into the rat LH/hCG receptor and demonstrated that the mutant receptor constitutively activated adenylate cyclase in transfected 293 T cells. The cell surface expression of the mutant receptor was lower than that of the wild type receptor. Pulse-chase studies showed that the 73-kDa precursor of both the mutant and wild type receptors was synthesized at comparable efficiencies. However, post-translational processing of the mutant receptor to the mature 92-kDa form, which has N-linked complex type oligosaccharide chains, was impaired. Sensitivity of the mutant receptor to peptide-N-glycanase F and endoglycosidase H, and insensitivity to sialidase indicated that the 73-kDa species represents the high mannose form that has not yet been trafficked through the medial and trans Golgi. Additionally, although the wild type receptor was palmitoylated, the mutant receptor was not. Although the high mannose 73-kDa species is capable of binding LH/hCG, our results show that post-translational processing in the Golgi is required for the mature 92-kDa receptor to reach the cell surface.
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PMID:Post-translational processing in the Golgi plays a critical role in the trafficking of the luteinizing hormone/human chorionic gonadotropin receptor to the cell surface. 903 11

The structures of N-linked oligosaccharides from various Leishmania life-cycle stages and species have been investigated in order to elucidate differences which may be correlated with virulence or tissue tropisms. The structure of gp63 glycans from L major log- and stationary-phase promastigotes were elucidated and compared with the total membrane associated oligosaccharides from five Leishmania spp. L. major gp63 glycans from promastigotes in either log or stationary phases of their growth cycle were shown to have two neutral oligosaccharides having Bio-Gel P4 hydrodynamic volumes of 10.5 and 9.6 glucose units (GU). Sequential exoglycosidase digestion, fragmentation by acetolysis and methylation analysis of hydrazine released glycans, revealed the structure of G9.6 to be a biantennary oligomannose type, having the composition Man6GlcNAc2. These data were confirmed by structural analysis of gp63 oligosaccharides released by digestion with endo-beta-N-acetylglucosaminidase H (Endo-H) and N-glycanase F. The larger glycan was found to be terminally glucosylated, having the composition GlcMan6GlcNAc2. These oligosaccharides were found to occupy only two of the three predicted N-linked glycosylation sites in the L. major gp63 molecule, at positions 300 and 407. On comparison with glycans from other Leishmania spp. and strains, these two oligosaccharides were consistently found to be the predominant promastigote structures. Following transformation to the amastigote stage, alterations in N-linked oligosaccharides appeared to be less consistent between species. L. m. mexicana amastigotes were found to display the same G10.5 and G9.6 glycans found on promastigotes while L. donovani LV9 amastigotes were found to be devoid of N-linked glycans.
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PMID:A unique, terminally glucosylated oligosaccharide is a common feature on Leishmania cell surfaces. 904 19

Prompted by previous observations that polymannose oligosaccharides are released from newly synthesized glycoproteins [Anumula and Spiro (1983) J. Biol. Chem. 258, 15274-15282], we examined rat liver endoplasmic reticulum (ER) for the presence of endoglycosidases that could be involved in an event presumed to be a function of the protein quality control machinery. Our investigations indicated that a peptide:N-glycanase (PNGase) is present in ER membranes that has the capacity to release from radiolabelled glycopeptides glucosylated as well as non-glucosylated polymannose oligosaccharides terminating at their reducing end in a di-N-acetylchitobiose sequence (OS-GlcNAc2). This enzyme, which was found to be luminal in orientation, was most active in the pH range 5.5-7.0 and although it had no exogenous bivalent-cation requirements it was inhibited by EDTA. Detailed studies with Man9GlcNAc2-peptides demonstrated that in addition to the free oligosaccharide (Man9GlcNAc2) an additional neutral product characterized as Man9GlcNAc2 linked to an as yet unidentified aglycone was released in a manner that suggests its role as an intermediate. Our observation that ER, in contrast with cytosol, had no endo-beta-N-acetylglucosaminidase activity would indicate that oligosaccharides terminating in a single GlcNAc residue (OS-GlcNAc1), which have been noted to appear in the extravesicular compartment shortly after N-glycosylation [Moore and Spiro (1994) J. Biol. Chem. 269, 12715-12721] are released from the protein as OS-GlcNAc2 and undergo an ER-to-cytosol translocation in that form before undergoing cleavage of their chitobiose core.
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PMID:Demonstration of a peptide:N-glycosidase in the endoplasmic reticulum of rat liver. 906 90


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