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

The significance of carbohydrate moieties containing the beta-adrenoceptor molecule in the rat brain was examined using radioligand binding assay methods. Thus, this experiment was designed to assess the effects of exoglycosidase (alpha-D-mannosidase and neuraminidase), endoglycosidase (endoglycosidase D and endoglycosidase H), and glycopeptidase A on the affinity of beta-adrenoceptor. The main reason why five kinds of enzymes were used in the present study is that they can hydrolyze different carbohydrate molecules from cell membranes. Rat brain was used and beta-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol (3H-DHA) as a ligand. 3H-DHA binding to beta-adrenoceptors was sensitive to very low concentration of endoglycosidase H and glycopeptidase A, thus indicating that the treatments with these enzymes of rat brain membrane appear to decrease the number of beta-receptor binding sites. On the other hand, the treatment with neuraminidase, endoglycosidase H, and glycopeptidase A of the membrane induced lower values of the dissociation constant (Kd) than those of the control. alpha-D-mannosidase and endoglycosidase D are without effect in spite of the removal of hexose contents and total carbohydrate contents with these treatments, respectively. These results imply that complex type N-linked acidic carbohydrate chains containing neuraminic acid and high mannose type N-linked carbohydrate chains, which are hydrolyzed with endoglycosidase H and glycopeptidase A, of the rat brain membrane containing beta-adrenoceptor molecules play a crucial role in the drug-receptor interaction.
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PMID:Binding characteristics of 3H-dihydroalprenolol to beta-adrenergic receptors of rat brain: influence of exo- and endo-glycosidases and glycopeptidase. 299 87

A sensitive and specific strategy has been developed for determining the sites of attachment of Asn-linked carbohydrates in glycoproteins, and defining the compositions and molecular heterogeneity of carbohydrates at each specific attachment site. In this carbohydrate 'fingerprinting' strategy, potential glycopeptides are identified by comparing the high pressure liquid chromatography (HPLC) chromatograms of proteolytic digests of a glycoprotein obtained before and after digestion with a glycosidase, usually peptide:N-glycosidase F (PNGase F). The glycopeptide-containing HPLC fractions are analyzed by fast atom bombardment mass spectrometry (FAB MS) prior to and after digestion with PNGase F to identify the former glycosylation site peptide and its sequence location (Carr and Roberts, (1986) Anal. Biochem. 157, 396-406). Carbohydrates are extracted from these fractions as the peracetates which are then permethylated and analyzed by FAB MS. The spectra exhibit molecular weight-related ions for each of the parent oligosaccharides present in the fraction which provide composition in terms of hexose, deoxyhexose, N-acetylhexosamine and sialic acid. The relative ratios of these peaks reflect the relative abundances of the various carbohydrate homologs present in the mixture. The derivatives formed are directly amenable to methylation analysis for determination of linkage. This strategy enables the structural classes of carbohydrates at specific attachment sites to be determined using only a few nmol of glycoprotein. The carbohydrate fingerprinting strategy has been applied to a number of glycoproteins including tissue plasminogen activator, the results for which are described herein.
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PMID:Structural fingerprinting of Asn-linked carbohydrates from specific attachment sites in glycoproteins by mass spectrometry: application to tissue plasminogen activator. 314 14

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.
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PMID:Structural study of the asparagine-linked oligosaccharides of lipophorin in locusts. 358 78

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

Because of the limited information available about the synthesis of N-linked glycoproteins in nerve cells, in regard to both processing steps and enzyme characterization, the biosynthetic processing of Thy-1 of the rat neuronal tumor cell line BN-1010-1 has been investigated using several inhibitors of biosynthesis and transport. (i) Tunicamycin completely inhibited mannose incorporation into Thy-1. Unglycosylated Thy-1 was not transported to the cell surface and was probably degraded rapidly following synthesis. (ii) Brefeldin A completely inhibited the transport of all [3H]mannose-labeled proteins releasable by phosphatidylinositol-specific phospholipase C, including Thy-1, to the surface of BN-1010-1 cells. Removal of the inhibitor led to rapid reversal of the inhibition. Pulse-chase experiments demonstrated that approximately 50% of Thy-1 was degraded after 4 h in the presence of brefeldin A. (iii) Castanospermine treatment slowed the appearance of Thy-1 on the cell surface. The surface Thy-1 contained mainly normal Man5, Man6, and Man7 oligosaccharides, suggesting that Golgi endo-alpha-D-mannosidase effected the removal of glucose. (iv) Treatment with deoxymannojirimycin resulted in the synthesis of Thy-1 that contained Man8 and Man9 oligosaccharides compared to Man5, Man6, and Man7 in the control. Neither the rate of appearance nor the level of surface expression was affected by the drug. (v) Swainsonine did not affect either the rate of appearance or the level of surface expression of Thy-1. The HPLC elution profile of neutral oligosaccharides resulting from Endo-H digestion of Thy-1 synthesized in the presence of swainsonine was indistinguishable from controls. The lack of an effect of swainsonine is explained by the unexpected absence of a complex type oligosaccharide in Thy-1 of BN-1010-1 cells, as shown in experiments with a variety of lectins as well as digestions with Endo-H or glycopeptidase F, or digestions with both enzymes in sequence. The fact that, after [3H]fucose-labeling, Endo-H digestion produced Thy-1 still labeled with fucose indicates that hybrid oligosaccharide is present in Thy-1.
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PMID:Biosynthetic and structural studies on Thy-1 in a rat neuronal tumor cell line. 809 81

Transforming growth factor-beta 1 (TGF-beta 1) stimulated growth and glucose uptake in Swiss mouse fibroblasts. DNA synthesis was increased 2-3-fold after 48 h incubation of growing 3T3 cells with TGF-beta 1 in calf serum-containing medium. Glucose transport activity in the cells was increased within 3 h after addition of TGF-beta 1 and this stimulation continued during incubation for 48 h. TGF-beta 1 also increased the levels of a brain type-glucose transporter (GLUT1) mRNA and the GLUT1 protein (55 kDa) in the membranes, consistent with the increase in glucose uptake. Furthermore, a longer exposure of TGF-beta 1 for 24-48 h induced a marked increase in the 65 kDa GLUT1 in 3T3 cell membranes. Other growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor-alpha, and insulin did not elevate glucose uptake and the levels of 55 and 65 kDa GLUT1 proteins. Adding tunicamycin or deoxymannojirimycin to the TGF-beta 1-treated and untreated cells caused these 55 and 65 kDa glucose transporters to migrate as one band at 40-43 kDa. In addition, treating membrane proteins with glycopeptidase F, which removes N-linked oligosaccharides, also generated a glucose transporter of 40 kDa, suggesting that the 55 and 65 kDa GLUT1 proteins have a similar or identical core polypeptide but with different N-linked oligosaccharides. These results indicate that TGF-beta 1 modulates the synthesis of GLUT1 protein as well as its glycosylation in Swiss 3T3 cells, and that these changes may contribute to the control of cell proliferation by TGF-beta 1.
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PMID:Modulation of the synthesis and glycosylation of the glucose transporter protein by transforming growth factor-beta 1 in Swiss 3T3 fibroblasts. 843 54

We have previously published a two-dimensional (2-D) mapping technique for N-linked oligosaccharides using pyridylaminated derivatives (PA-oligosaccharides) (N. Tomiya et al. Anal. Biochem. 171, 73-90, 1988). We now report an extension of this method to GalNAc-containing N-linked oligosaccharides. The new 2-D map was prepared from the elution data of 40 different GalNAc-containing oligosaccharides, 16 of which were obtained directly from human urinary kallidinogenase by digestion with glycopeptidase A. The other 24 oligosaccharides were derived by subsequent digestion of the 16 original oligosaccharides with beta-galactosidase or alpha-fucosidase. Each of the 40 oligosaccharide derivatives was separated by high-performance liquid chromatography using ODS-silica and amide-silica columns. The 2-D map constructed by plotting elution position of each oligosaccharide (expressed in terms of glucose units) can be useful as such in delineating the structure of an unknown oligosaccharide by direct placement of its elution positions in the 2-D map. Multiple regression analysis of the data as performed previously yielded parameters related to the contribution of each component monosaccharide unit to the elution profile. The best results were obtained when the GalNAc-containing PA-oligosaccharides were classified into an F-series (those containing Fuc alpha 6GlcNAc-PA) and a Z-series (all others), based on our previous classification method. These calculated values are useful in predicting oligosaccharide structure from known elution values as well as to predict elution volumn from a known structure. The structure of a minor GalNAc-containing oligosaccharide in human urinary kallidinogenase was elucidated using these newly calculated values.
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PMID:Two-dimensional elution map of GalNAc-containing N-linked oligosaccharides. 843 1

alpha-Dystroglycan is a heavily glycosylated protein, which is localized on the Schwann cell membrane as well as the sarcolemma, and links the transmembrane protein beta-dystroglycan to laminin in the extracellular matrix. We have shown previously that sialidase treatment, but not N-glycanase treatment, of bovine peripheral nerve alpha-dystroglycan greatly reduces its binding activity to laminin, suggesting that the sialic acid of O-glycosidically-linked oligosaccharides may be essential for this binding. In this report, we analyzed the structures of the sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan by two methods. O-Glycosidically-linked oligosaccharides were liberated by alkaline-borotritide treatment or by mild hydrazinolysis followed by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion exchange column chromatography that eluted at a position corresponding to monosialylated oligosaccharides were converted to neutral oligosaccharides by exhaustive sialidase digestion. The sialidases from Arthrobacter ureafaciens and from Newcastle disease virus resulted in the same degree of hydrolysis. The neutral oligosaccharide fraction, thus obtained, gave a major peak with a mobility of 3.8-3.9 glucose units upon gel filtration, and its reducing terminus was identified as a mannose derivative. Based on the results of sequential exoglycosidase digestion, lectin column chromatography, and reversed-phase high-performance liquid chromatography, we concluded that the major sialylated O-glycosidically-linked oligosaccharide of the alpha-dystroglycan was a novel O-mannosyl-type oligosaccharide, the structure of which was Siaalpha2-3Galbeta1-4GlcNAcbeta1-2Man-Ser/Thr (where Sia is sialic acid). This oligosaccharide constituted at least 66% of the sialylated O-linked sugar chains. Furthermore, a laminin binding inhibition study suggested that the sialyl N-acetyllactosamine moiety of this sugar chain was involved in the interaction of the alpha-dystroglycan with laminin.
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PMID:Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan. The role of a novel O-mannosyl-type oligosaccharide in the binding of alpha-dystroglycan with laminin. 899 17

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


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