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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)

Molecular ions and fragment ions of underivatized and permethylated oligosaccharides derived from human urinary kallikrein and some model glycoproteins gave information about the sugar composition and arrangement of sugars. These ions were conveniently created by FAB-MS in a JEOL JMS-HX110 high field high resolution mass spectrometer. Glycoprotein samples were digested with N-glycanase and the asparagine(Asn)-linked oligosaccharides were separated from polypeptides by Sephadex G-50 chromatography. The mixture of oligosaccharides was converted to the reduced p-aminobenzoic ethyl ester (ABEE) derivative and the components separated by HPLC on an ion-exchange (AX-10) column. Individual components were analyzed by negative FAB-MS using glycerol as a matrix. Permethylated oligosaccharides were also analyzed by positive FAB-MS.
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PMID:Fast atom bombardment mass spectrometry (FAB-MS): analysis of complex carbohydrate chains of tissue kallikreins. 260 19

Transfer of truncated oligosaccharides to protein in vivo and the structure of Man2GlcNAc2 synthesized by intact yeast (Saccharomyces cerevisiae) were investigated in the alg2 mutant. At the nonpermissive temperature the alg2 mutant accumulates lipid-linked oligosaccharides that migrate on Bio-Gel P4 in the range expected for Man2GlcNAc2 and Man1GlcNAc2 (T.C. Huffaker and P.W. Robbins (1983) Proc. Natl. Acad. Sci. USA 80, 7466-7470). We characterized the oligosaccharides, derived from protein and lipid, by comigration with standards on HPLC and by Smith degradation followed by HPLC. Man2GlcNAc2 and Man1GlcNAc2 are found on protein in alg2, since their release from a protein-containing precipitate of alg2 cells is N-glycanase (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) dependent. Transfer also occurred in alg2/pAC3 cells, which carry ALG2 on a multicopy plasmid that confers partial correction of the oligosaccharide phenotype. The alg2/pAC3 cells are viable at 36 degrees C. Two isomers of Man2GlcNAc2, Man1----3ManGlcNAc2 and Man1----6ManGlcNAc2, were present on lipid and protein. The transfer of Man2GlcNAc2 and Man1GlcNAc2 to protein by intact cells supports topological models that postulate access by early intermediates to the lumen of the endoplasmic reticulum.
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PMID:Synthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Man2GlcNAc2 and Man1GlcNAc2 are transferred from dolichol to protein in vivo. 266 Jul 43

To examine the function of the carbohydrate chains of cobra venom factor (CVF), the molecule was enzymatically deglycosylated under non-denaturing conditions with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase). The deglycosylation of CVF chains seems to proceed independently of each other, leading to partially deglycosylated intermediates. Complete deglycosylation of CVF was found to abolish the activity of CVF. The deglycosylated molecule is unable to activate the alternative pathway of complement. Deglycosylated CVF no longer consumes the serum complement activity, it does not induce C3 activation in serum, nor does it induce complement-mediated hemolysis. These results indicate that the carbohydrate moieties of CVF are essential for its role in complement activation.
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PMID:The oligosaccharide chains of cobra venom factor are required for complement activation. 277 Jul 49

Factor B is a glycoprotein which plays an essential role in the alternative pathway of complement activation. It carries the proteolytic activity of the convertases, and its physiological breakdown products Ba and Bb have some effects on the cells of the immune system. Human factor B exhibits a microheterogeneity and five isoforms are present in serum. The nature and origin of the microheterogeneity was investigated by using electrophoretic techniques. Treatments of factor B with neuraminidase and glycopeptidase F show that this microheterogeneity is mainly due to differences in its sialic acid content, varying from seven to eleven residues per molecule, and resulting in different oligosaccharide structures. However, deglycosylated factor B reveals a residual, nonallotypic variation in the Bb region of the polypeptide backbone. We confirm the presence of four asparagine-linked oligosaccharide chains of the complex type in native factor B, two of which are located in the Ba fragment, and the two others in the Bb fragment. The prevalent isoform of the native protein carries two sialic acid residues per oligosaccharide chain. Biosynthesis experiments show that the microheterogeneity of secreted factor B from HepG2 cells is acquired during the processing of its glycans. However, in vitro-secreted factor B is more heterogeneous than the serum protein. We propose a structural model for the microheterogeneity of the native protein and its physiological fragments. We discuss as well the feasibility of electrophoretic techniques to deal with microheterogeneity analysis.
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PMID:Heterogeneous nature of human complement factor B: an electrophoretic approach for the analysis of its oligosaccharide chain structure and its physiological breakdown products. 277 35

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 ligand-binding subunit of the porcine striatal dopamine D2 receptor was identified by photoaffinity labeling with [125I]N-azidophenethylspiperone ([125I]NAPS). Upon photolysis, [125I]NAPS covalently incorporated into a broad band of apparent Mr congruent 140,000 with an appropriate pharmacological profile for D2 receptors as assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Smaller subunits of apparent Mr congruent 94,000 and 34,000 were specifically labeled by [125I]NAPS with an appropriate D2 receptor profile and were similar to the major ligand-binding subunits of photoaffinity-labeled canine striatal D2 receptors. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of the Mr congruent to 140,000/94,000 subunits upon denaturing electrophoresis in either the absence or presence of thiol-reducing/alkylating reagents. In order to investigate the possible basis for the existence of these high molecular weight forms of the D2 receptor, we assessed the carbohydrate nature of photolabeled D2 ligand-binding subunits by the use of lectin affinity chromatography and specific exo- and endoglycosidase treatments. Both photoaffinity-labeled D2 receptor proteins from porcine striatum (Mr congruent to 140,000 and 94,000) were glycoproteins as indexed by their absorption and specific elution from wheat germ agglutinin lectin resins. The exoglycosidase neuraminidase altered the electrophoretic mobility of both the Mr congruent to 140,000 and 94,000 labeled subunits to a single band of apparent Mr congruent to 51,000. Prior removal of sialic acid residues did not alter the reversible binding characteristics of [3H]spiperone to D2 receptors. Complete removal of receptor-associated N-linked carbohydrate by the endoglycosidase glycopeptidase F (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) produced a further increase in the mobility of the Mr congruent to 51,000 subunit to apparent Mr congruent to 44,000. The porcine Mr congruent to 34,000 photolabeled peptide is an N-linked glycoprotein as assessed by lectin affinity chromatography and susceptibility to digestion by glycopeptidase F to a peptide of apparent Mr congruent to 23,000.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Dopamine D2 receptor binding subunits of Mr congruent to 140,000 and 94,000 in brain: deglycosylation yields a common unit of Mr congruent to 44,000. 297 May 86

Recombinant transforming growth factor-beta 1 (TGF-beta 1) precursor produced and secreted by a clone of Chinese hamster ovary cells was found to be glycosylated and phosphorylated. Treatment of 32P-labeled precursor protein with N-glycanase indicated that phosphate was incorporated into asparagine-linked complex carbohydrate moieties. Fractionation of 32P-labeled glycopeptides followed by amino acid sequence analysis indicated that greater than 95% of the label was incorporated into two out of three glycosylation sites at Asn-82 and Asn-136 of the TGF-beta 1 precursor. Two-dimensional electrophoretic analysis of acid hydrolyzed precursor protein and precursor protein-derived glycopeptides indicated that 32P was incorporated as mannose 6-phosphate. Binding studies with the purified receptor for mannose 6-phosphate indicated that the TGF-beta 1 precursor could bind to this receptor and the binding was specifically inhibited with mannose 6-phosphate.
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PMID:Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-beta 1 precursor. 297 54

A commercially available endoglycosidase (N-glycanase, Genzyme, Boston, Mass.) purified from Flavobacterium meningosepticum with a specificity for cleaving asparagine-linked carbohydrate moieties in glycoproteins was tested on several pituitary and chorionic gonadotropins as substrates. All intact hormones tested were resistant to the action of the enzyme as were all beta subunits from the respective gonadotropins. All alpha subunits, however, were susceptible to the enzyme as evidenced by a decrease in molecular size when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Preparative experiments with ovine luteinizing hormone subunit (oLH alpha) indicated that only 35-40% of the carbohydrate was removed after N-glycanase treatment, suggesting that perhaps only one of the two carbohydrate moieties was cleavable under the conditions employed. The enzyme-modified subunit (DG-oLH alpha) was able to recombine with untreated oLH beta. An in vitro steroidogenic bioassay (rat Leydig cell) showed that the recombinant (DG-oLH alpha-oLH beta) was about 22% as potent as the native oLH, but in a testicular membrane binding assay for LH, it was equal in potency to the native hormone in competing with the radioligand.
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PMID:Deglycosylation of gonadotropins with an endoglycosidase. 308 Jul 56

We have elucidated the structures of the anionic asparagine-linked oligosaccharides present on the glycoprotein hormones lutropin (luteinizing hormone), follitropin (follicle-stimulating hormone), and thyrotropin (thyroid-stimulating hormone). Purified hormones, isolated from bovine, ovine, and human pituitaries, were digested with N-glycanase, and the released oligosaccharides were reduced with NaB[3H]4. The 3H-labeled oligosaccharides from each hormone were then fractionated by anion-exchange high performance liquid chromatography (HPLC) into populations differing in the number of sulfate and/or sialic acid moieties. The anionic oligosaccharides were further purified as well as structurally characterized using a variety of preparative and analytical techniques, including HPLC, endo- and exoglycosidase digestions, and lectin affinity chromatography. The sulfated, sialylated, and sulfated/sialylated structures, which together comprised 67-90% of the asparagine-linked oligosaccharides on the pituitary glycoprotein hormones, were highly heterogeneous and displayed hormone- as well as animal species-specific features. The sulfated oligosaccharides consisted of hybrid and complex type oligosaccharides with one or two branches terminating in SO4-4GalNAc beta 1,4. In contrast, the sialylated oligosaccharides consisted of a wide array of differing structures containing two or three peripheral branches as well as one, two, or three sialic acid moieties. A previously uncharacterized dibranched oligosaccharide, bearing one residue each of sulfate and sialic acid, was found on all of the hormones except bovine lutropin. In this study, we describe the purification and detailed structural characterizations of the sulfated, sialylated, and sulfated/sialylated oligosaccharides found on lutropin, follitropin, and thyrotropin from several animal species. In the accompanying paper (Green, E.D., and Baenziger, J.U.(1987) J. Biol. Chem. 262, 36-44) we demonstrate the marked quantitative differences among the pituitary glycoprotein hormones in terms of sulfation, sialylation, and underlying oligosaccharide structures, as well as provide evidence for site-specific synthesis of oligosaccharides on individual hormones.
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PMID:Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. I. Structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. 312 9

The structures of the N-linked oligosaccharides of the urinary erythropoietin (u-EPO) purified from urine of aplastic anemic patients were analyzed and compared with those for recombinant erythropoietin (r-EPO) prepared with baby hamster kidney (BHK) cells. Asparagine-linked neutral oligosaccharides were released from each EPO protein by N-oligosaccharide 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 8 and 13 kinds of oligosaccharide fractions for u-EPO and r-EPO (BHK), respectively, were completely separated by the one-step HPLC procedure. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amide-silica column. Furthermore, high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy and methylation analyses were carried out in the case of r-EPO (BHK).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins. 317 69


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