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)

Saccharomyces cerevisiae contains an amphiphilic cAMP-binding glycoprotein at the outer face of the plasma membrane (M(r) = 54,000). It is converted to a hydrophilic form by treatment with glycosyl-phosphatidylinositol-specific phospholipases C and D (GPI-PLC/D), suggesting membrane anchorage by a covalently bound glycolipid. Determination of the constituents of the purified anchor by gas-liquid chromatography and amino acid analysis reveals the presence of glycerol, myo-inositol, glucosamine, galactose, mannose, ethanolamine, and asparagine (as the carboxyl-terminal amino acid of the Pronase-digested protein to which the anchor is attached). Complementary results are obtained by metabolic labeling, indicating that fatty acids and phosphorus are additional anchor constituents. The phosphorus is resistant to alkaline phosphatase, whereas approximately half is lost from the protein after treatment with GPI-PLD or nitrous acid, and all is removed by aqueous HF indicating the presence of two phosphodiester bonds. Inhibition of N-glycosylation by tunicamycin or removal of protein-bound glycan chains by N-glycanase or Pronase does not abolish radiolabeling of the anchor structure by any of the above compounds. Analysis of the products obtained after sequential enzymic and chemical degradation of the anchor agrees with the arrangement of constituents in GPIs from higher eucaryotes. Evidence for anchorage of the yeast cAMP-binding protein by a GPI anchor is strengthened additionally by the reactivity of the GPI-PLC-cleaved anchor with antibodies directed against the cross-reacting determinant of trypanosomal variant surface glycoproteins.
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PMID:The cAMP-binding ectoprotein from Saccharomyces cerevisiae is membrane-anchored by glycosyl-phosphatidylinositol. 133 92

The contribution of N-linked carbohydrate to the complement-inhibitory function of the human erythrocyte membrane glycoprotein, CD59, was investigated. Amino acid sequence analysis of tryptic peptides labeled with [3H]borohydride revealed an N-linked carbohydrate moiety at the Asn18 residue. No O-linked carbohydrate was detected, as judged by the failure of asialo-CD59 to bind peanut agglutinin and by its resistance to digestion by O-glycanase. The apparent molecular mass of CD59 was reduced from 18-20 to 14 kDa upon complete digestion with N-glycanase, with no detectable proteolysis. N-glycanase digestion of CD59 was associated with an 88 +/- 4% loss of the complement-inhibitory activity of the protein, as assessed by its capacity to protect chicken erythrocytes from lysis by the human C5b-9 proteins. By contrast, no change in function was observed after digestion of CD59 with neuraminidase, under conditions that removed greater than 60% of [3H]sialic acid residues. Despite loss of functional activity after N-glycanase digestion, we detected no change in the capacity of the deglycosylated CD59 to incorporate into erythrocyte membranes or to bind specifically and with species selectivity to the C8 and C9 components of the membrane attack complex. In order to alter the branched-chain structure of the N-linked carbohydrate of CD59 without enzymatic digestion, Chinese hamster ovary (CHO) cells transfected with cDNA for human CD59 were grown in the alpha-mannosidase inhibitor, 1-deoxymannojirimycin, resulting in conversion of approximately 70% of the membrane glycoprotein to a high mannose. When grown in the presence of 1-deoxymannojirimycin, the C5b-9-inhibitory activity of CD59 expressed on the surface of the transfected CHO cells was reduced by an amount comparable to that observed for the N-glycanase digested protein. Taken together, these data suggest that normal glycosylation of Asn18 in CD59 is required for the normal expression of its complement-inhibitory activity on membrane surfaces, although these N-linked sugar residues do not contribute to CD59's affinity for the C8 and C9 components of the C5b-9 complex.
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PMID:Contribution of the N-linked carbohydrate of erythrocyte antigen CD59 to its complement-inhibitory activity. 137 27

A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum. After in-vivo radiolabelling of rats with L-[6-3H]fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride. Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains. Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-[3H]fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine.
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PMID:Occurrence of alpha 1-2-fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver. Aberrant type of fucosylation in a malignant tissue. 139 74

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

A rapid quantitative analysis of the sialylated N-linked oligosaccharides of recombinant erythropoietin (EPO) expressed in Chinese hamster ovary (CHO) cells has been developed. The procedure utilizes a glycoamidase (glycopeptidase F) to release all of the N-linked oligosaccharides from the native glycoprotein, followed by direct chromatographic analysis using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. The eight sialyloligosaccharides isolated from HPAEC were characterized by derivatizing with 2-aminopyridine followed by two-dimensional HPLC mapping of the pyridylaminated asialooligosaccharides (Tomiya et al., 1988, Anal. Biochem. 171, 73-90). Seven kinds of complex-type asialooligosaccharides were identified ranging from a biantennary structure to N-acetyllactosamine-extended tetraantennary structure. Approximately 3% of the terminal galactose residues of the oligosaccharides released from EPO were not sialylated whereas 97% contained an alpha(2-->3)-linked sialic acid. Quantitative oligosaccharide mapping of four different lots of EPO from CHO cells was performed to quantify the molar balance and distribution of the N-linked oligosaccharides. The sialyloligosaccharides were distributed with approximately 5% disialylated (single type), 20% trisialylated (six types), and 75% tetrasialylated (four types) oligosaccharides with an average molar recovery of 85% starting from 750 pmol of EPO.
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PMID:Quantitative mapping of the N-linked sialyloligosaccharides of recombinant erythropoietin: combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization. 144 98

Chromatographically purified endopolygalacturonase (PG) from Aspergillus niger was deglycosylated with N-glycosidase F (PNGase F) and characterized by means of sodium dodecyl sulfate (SDS)-electrophoresis, polyacrylamide gel electrophoresis (PAGE) without denaturing agents, isoelectric focusing (IEF) and lectin affino-blotting. The results show that PG, which is apparently homogeneous in SDS-PAGE but heterogeneous in IEF and PAGE, consists of at least two polypeptide chains with different glycosylation patterns. The component with the higher electrophoretic mobility is deglycosylated with PNGase F and reacts with concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA), indicating a "high mannose" or "hybrid"-type of glycoprotein (GP). The other component may contain O-glycosidically linked mannose, N-acetylglucosamine or glucose.
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PMID:Characterization of endopolygalacturonase (EC 3.2.1.15) from Aspergillus niger as glycoprotein by electrophoretic methods and lectin affino-blotting. 145 18

Electrospray and tandem mass spectrometry are used to characterize underivatized oligosaccharides that have been digested from asparagine side chains of glycoproteins. Oligosaccharides that contain sialic acids were detected with the best sensitivity in the negative-ion detection mode whereas those that do not contain sialic acid were detected with the best sensitivity in the positive-ion detection mode. The positive-ion abundances of oligosaccharides were greatly enhanced in electrospray mass spectra by adding 10 mM sodium acetate or ammonium acetate to the sample solvent. Tandem mass spectrometry was used to determine primary structural features of the oligosaccharides. Methodology that has been developed on branched high-mannose, hybrid, and complex carbohydrate standards was applied to a mixture of oligosaccharides that were digested with N-glycanase from the glycoprotein, ovalbumin. The composition and relative abundances of individual oligosaccharides obtained from the electrospray mass spectrum compare favorably to those obtained by anion-exchange chromatography/pulsed amperometric detection and by gel permeation chromatography of the oligosaccharides after radiolabelling the reducing end of the carbohydrates. The oligosaccharide content of ovalbumin was independently determined from the heterogeneity observed in the electrospray mass spectrum of the intact 44-kDa glycoprotein. Comparison of the oligosaccharide compositions determined before and after enzymatic digestion shows a selective digestion of high-mannose and low molecular weight oligosaccharides by N-glycanase.
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PMID:Characterization of N-linked oligosaccharides by electrospray and tandem mass spectrometry. 150 19

Variant surface glycoproteins (VSGs) of Trypanosoma brucei contain two distinct glycosylation sites: (1) N-linked glycans within the protein portion of the molecules, and (2) the glycosyl-phosphatidylinositol (GPI) membrane anchor. Since galactose residues show uncommon alpha-glycosidic linkages in the GPI membrane anchor, we were prompted to investigate galactosylation of the GPI anchor. On comparing a trypanosome clone galactosylated exclusively in N-glycans (clone MITat 1.5) with clones galactosylated predominantly in the glypiated membrane anchor (clones MITat 1.4, MITat 1.6 and AnTat 1.8), clone MITat 1.5 showed a 10-fold increased enzyme activity when using a protocol including Triton X-100 to assay UDPgalactose:N-acetylglucosaminyl glycopeptide beta 1,4-galactosyltransferase (EC 2.4.1.38). Only the VSG of clone MITat 1.5 could be radiochemically labelled with UDP[14C]galactose, and galactosylation of N-glycans was confirmed by digestion with peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase F). However, in a modified enzyme assay without detergent, galactosyltransferase activity was increased considerably (15-fold) in clone MITat 1.4. VSG galactosylation of clones MITat 1.4, MITat 1.6 and AnTat 1.8 was readily detected by fluorography of the respective SDS/polyacrylamide gels, suggesting that galactosyltransferase activity modifies the VSG membrane anchor in these clones. In this case, [14C]galactose labelling of immunoprecipitated VSG (clone MITat 1.4) was resistant to the release of N-glycans by PNGase F treatment, and thus revealed galactosylation in vitro of a VSG membrane anchor. Exoglycosidase digestions of VSG MITat 1.4 confirmed the presence of alpha-linked galactose residues. We suggest that these specific alpha-galactosyltransferases are inhibited by the action of detergent, but can be activated in a detergent-free buffer system.
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PMID:Identification of two distinct galactosyltransferase activities acting on the variant surface glycoprotein of Trypanosoma brucei. 153 12

Human transferrin receptor was isolated from placenta and from the hepatocarcinoma cell line Hep G2. Asparagine-linked oligosaccharides were released by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols were fractionated by anion-exchange high-performance liquid chromatography and by high-pH anion-exchange chromatography. Glycans from placental transferrin receptor were further characterized, after desialylation, by methylation analysis and, in part, by liquid secondary-ion mass spectrometry. Sialylation of placental transferrin receptor was examined by lectin affinity blotting with Sambucus nigra agglutinin and Maackia amurensis agglutinin. In order to trace possible inter-individual differences in N-glycosylation of the receptor, two preparations of placental transferrin receptor purified from two donors were compared. The results demonstrate that human transferrin receptor from placenta predominantly carries diantennary and triantennary N-acetyllactosaminic glycans as well as hybrid-type species, the galactose residues of which being almost completely substituted with (alpha 2-3)-linked sialic acid residues. Distinct differences were noted in the glycosylation pattern of the receptor from different individuals. Transferrin receptor from donor A carried predominantly diantennary and triantennary complex-type glycans, in part fucosylated at the innermost N-acetylglucosamine residue, in addition to small amounts of bisected and of incomplete diantennary species. Placental transferrin receptor from donor B predominantly carried triantennary N-acetyllactosaminic glycans without fucose and hybrid-type oligosaccharides with four or five mannose residues. Distinct from placental transferrin receptor, the receptor from Hep G2 cells contained larger amounts of oligomannosidic glycans with six to nine mannose residues and tetrasialylated complex-type oligosaccharides apart from mono-, di- and trisialylated species.
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PMID:Structure of the N-linked oligosaccharides of the human transferrin receptor. 155 86

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.
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PMID:alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins. 158 69


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