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 azurophil granules of neutrophil granulocytes contain neutral proteases such as leukocyte elastase and cathepsin G. These are synthesized as inactive precursors, but following proteolytic processing, they are stored in granules as active enzymes. We describe the establishment of a transgenic cellular model for expression of the human myeloid serine protease cathepsin G. The cDNA for preprocathepsin G was stably expressed in the rat basophilic/mast cell line RBL-1 and the translation product was characterized by use of biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Conversion into complex form of an asparagine-linked carbohydrate unit of approximately 3.5 kDa was shown, as judged by the products obtained upon treatment with endoglycosidase H and N-glycanase. Proteolytic processing of 32.5-kDa procathepsin G into a 31-kDa form, within 1-2 h after synthesis, was demonstrated by pulse-chase experiments. Further processing into a 30-kDa form also occurred to a minor extent. The processed forms were enzymatically active, as judged by affinity for the serine protease inhibitors diisopropylfluorophosphate and aprotinin. Translocation of processed forms of cathepsin G to high density fractions, indicating targeting of the protease to granules, was demonstrated by subcellular fractionation. The weak base NH4Cl was shown to delay the processing and enzymatic activation of cathepsin G, whereas the monovalent ionophore monensin completely inhibited both events. Our data demonstrate that human cathepsin G transfected to rat RBL-1 cells, is proteolytically processed into enzymatically active forms and that subcellular transfer to granular organelles occurs. As the processing of transgenic human cathepsin G corresponds to that of endogenous protease of myeloid cells, the model should provide new unique possibilities to further characterize the activation and granular targeting of myeloid serine proteases.
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PMID:Processing of human cathepsin G after transfection to the rat basophilic/mast cell tumor line RBL. 792 11

The voltage-gated Cl- channel from Torpedo electroplax was purified in functional form by an immunoaffinity procedure. Channel activity was assayed by 36Cl- uptake into reconstituted liposomes and by direct recording after insertion into planar lipid bilayers. The purified channel displays the same "double-barreled" gating kinetics observed with native membranes, as well as the correct single-channel permeation characteristics. Preparations of active channels consist of a 90-kDa polypeptide, as expected from the known cDNA sequence. No associated subunits are present in the purified material. Direct protein sequencing confirms the absence of a cleavable signal sequence and demonstrates an N-terminus at Ser-2 of the cDNA-derived sequence. This "ClC-0" protein is lightly glycosylated, losing only approximately 2 kDa of sugar upon treatment with endoglycosidase H or N-glycanase. Most if not all of this glycosylation is found on Asn-365. This result necessitates revision of current transmembrane topology proposals, which have placed this residue on the cytoplasmic side of the membrane. Sedimentation in sucrose density gradients under activity-preserving conditions suggests the ClC-0 channel is slightly larger than the Na/K-ATPase alpha/beta-protomer (approximately equal to 150 kDa) and substantially smaller than the reduced form of the nicotinic acetylcholine receptor (approximately equal to 300 kDa). The detergent-solubilized ClC-0 channel, which invariably displays two Cl- diffusion pores in the active complex, is therefore built most likely as a homodimer of the 90-kDa protein purified here.
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PMID:Purification, reconstitution, and subunit composition of a voltage-gated chloride channel from Torpedo electroplax. 794 26

Blastocrithidia culicis is a trypanosomatid protozoon that transfers Man6GlcNAc2 in protein N-glycosylation. Compounds containing mannosyl, xylosyl, and rhamnosyl residues were found among the endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides of whole cell glycoproteins of this parasite. The compositions of some of them were as follows: Man5GlcNAc2, Man6GlcNAc2, Rha1Man5GlcNAc2, Rha2Man6GlcNAc2, Xyl1Rha2Man6-GlcNAc2, Xyl1Rha3Man6GlcNAc2, and Xyl2Rha3Man6-GlcNAc2. On the other hand, oligosaccharides containing mannosyl, xylosyl, rhamnosyl, and ribosyl units were liberated from endo-beta-N-acetylglucosaminidase-resistant glycopeptides upon treatment with N-glycanase. This is the first report on the presence of ribosyl units in eukaryote glycoconjugates, of rhamnosyl residues in asparagine-linked oligosaccharides, and of xylosyl units in high mannose-type compounds.
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PMID:Novel (rhamnosyl and ribosyl) and uncommon (xylosyl) monosaccharide residues are present in asparagine-linked oligosaccharides of the trypanosomatid Blastocrithidia culicis. 805 Nov 23

The biosynthesis and processing of a lysosomal cysteine proteinase, cathepsin C (dipeptidylaminopeptidase I), was investigated by pulse-chase experiments in cultured rat macrophages. Cathepsin C is first synthesized as procathepsin C with a molecular mass of 55 kDa. Procathepsin C is then cleaved and modified within 1 h into mature cathepsin C with two chains of 25 and 7.8 kDa. A combination of pulse-chase experiments and the subcellular fractionation analysis showed that procathepsin C and cathepsin C are located in low-buoyant-density organelles and lysosomes, respectively. The reactivity of endoglycosidase H and N-glycanase and analysis of phosphorylation indicated that both precursor and mature cathepsin C are phosphorylated and N-glycosylated to give a high-mannose-type. The addition of 300-kDa mannose 6-phosphate receptor antiserum to the chase medium caused extensive release of procathepsin C into the medium, whereas the addition of control serum did not. The membrane association of procathepsin C was tested by successive extraction of cells pulse labeled for 75 min with hypotonic buffer, alkaline solution, and Triton X-100. Procathepsin C was totally extracted by hypotonic solution, whereas procathepsin D was a membrane-associated form requiring Triton X-100 for its extraction. Gel-filtration chromatography analysis of the pulse-labeled products revealed that the precursor product exists as an oligomeric form. It is suggested that the oligomerization of cathepsin C occurs before its entry into lysosomes.
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PMID:Processing and transport of the precursor of cathepsin C during its transfer into lysosomes. 821 89

Swainsonine, a known inhibitor of the alpha-mannosidase II involved in processing of asparagine-linked glycoproteins, causes accumulation of hybrid-type oligosaccharide-containing glycoproteins in mammalian cells. Swainsonine augments lymphokine-activated, killer-cell induction at suboptimal doses of interleukin-2; the amount needed to increase LAK activity is 100-1000 fold higher than required to completely inhibit mannosidase II. Human mononuclear lymphocytes, when treated with these relatively high (58 microM) concentrations of swainsonine showed a 3-4 fold increase in D-[3H]mannose incorporation into the glycan as compared to glycans of untreated cells. Analysis indicated accumulation of high-mannose type, free oligosaccharides in the soluble fractions of the cell. Chromatographic analysis of glycan obtained by D-[2-3H]mannose labeling of human mononuclear lymphocytes showed synthesis of a new oligosaccharide, at 58 microM of swainsonine, that contained 36% of the total radioactivity incorporated into the glycan (oligosaccharide pool). This oligosaccharide fraction was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, O and N-glycanase, but was susceptible to cleavage by Jack bean alpha-mannosidase and was bound > 90% to concanavalin A-Sepharose. A similar chromatographic elution profile was obtained from glycans labeled with D-[2-3H]mannose from mouse B16F10 melanoma and baby hamster kidney cells subsequent to swainsonine treatment. Methylation analysis of free oligosaccharides obtained from MNL revealed the presence of a pentamannose. These results indicate the accumulation of a free high-mannose oligosaccharide rather than expected hybrid-type structure on treatment of cells with relatively high concentrations of swainsonine.
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PMID:Accumulation of pentamannose oligosaccharides in human mononuclear leukocytes by action of swainsonine, an inhibitor of glycoprotein processing. 825 42

The protein encoded by the envelope gene of Friend spleen focus-forming virus is responsible for the acute leukaemogenicity of this virus. In order to correlate glycosylation and intracellular processing of this protein with viral pathogenicity, envelope gene products of pathogenic and apathogenic glycosylation mutants were expressed in Rat-1 cells and metabolically labelled with [6-3H]glucosamine. Following immunoprecipitation, primary and secondary gene products (gp55, gp65) were separated by preparative polyacrylamide gel electrophoresis. Oligosaccharides were released from tryptic glycopeptides by treatment with endo-beta-N-acetylglucosaminidase H (gp55), peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (gp65) or by reductive beta-elimination. Resulting glycans were characterized by co-chromatography with authentic oligosaccharide standards using different HPLC systems and digestion with exoglycosidases. The results revealed that the primary envelope gene products of pathogenic glycosylation mutants were, in part, further processed in Rat-1 cells similar to wild-type glycoprotein, resulting in polypeptides carrying complex-type N-glycans as well as partially sialylated O-linked oligosaccharides. In contrast, corresponding glycoproteins encoded by apathogenic mutants were found to remain at the level of the primary translation product exclusively comprising high-mannose-type N-glycans. Hence, intracellular maturation of the envelope gene products in this model cell line seems to correlate with the in vivo pathogenicity of the glycosylation mutants studied.
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PMID:Glycosylation pattern and processing of envelope gene products encoded by glycosylation mutants of Friend spleen focus-forming virus. 828 59

Recombinant human uterine tissue plasminogen activator (tPA) glycosylation mutants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain due to the replacement of either Tyr67 by Asn (YN-tPA) or Gly60 by Ser (GS-tPA) were expressed in mouse epithelial cells (C127) in the presence of [6-3H]glucosamine. Glycopeptides comprising individual glycosylation sites were isolated and oligosaccharides attached were liberated by treatment with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols obtained after reduction were either directly characterized by high-pH anion-exchange chromatography (high-mannose and hybrid-type glycans) or preparatively subfractionated after enzymic desialylation and separation from sulphated asialooligosaccharides (complex-type sugar chains). Individual (sub)fractions of glucans were studied by methylation analysis, liquid secondary-ion mass spectrometry and, in part, by exoglycosidase digestion, whereas corresponding deglycosylated peptides were identified by amino acid analysis and N-terminal amino acid sequencing. The results revealed that Asn117 of YN-tPA carried exclusively high-mannose-type glycans with five to nine mannose residues similar to wild-type tPA expressed in this cell line [Pfeiffer, G., Schmidt, M., Strube, K.-H. & Geyer, R. (1989) Eur. J. Biochem. 186, 273-286]. In contrast, Asn117 of GS-tPA carried only small amounts (about 25%) of high-mannose and hybrid-type species and predominantly complex-type sugar chains (about 75%) which were partially incomplete and mostly devoid of fucose. Newly introduced N-glycosylation sites at Asn67 (YN-tPA) or Asn58 (GS-tPA) as well as those at Asn184 and Asn448 were solely substituted by complex-type glycans. Each carbohydrate attachment site displayed a peculiar oligosaccharide pattern with regard to branching and substitution by Gal alpha 3-residues, sulphate groups, intersecting GlcNAc and lactosamine repeats. Our study clearly demonstrates that creation of a new glycosylation site at Asn58 influenced the oligosaccharide processing and, hence, the glycosylation pattern at Asn117, whereas introduction of a new site at Asn67 did not. The relative amounts of complex-type glycans at Asn117 of GS-tPA correlated with the degree of carbohydrate substitution of Asn58. Therefore, it can be concluded that the presence of a sugar chain at the position and not the Gly to Ser mutation itself is responsible for the observed alteration of GS-tPA glycosylation.
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PMID:Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain. 830

In an approach to examine the lectin-hypothesis in the pathogenesis of coeliac disease, the presence of lectin-like components in three wheat gluten preparations known to induce coeliac disease, gliadin, Frazer fraction III and an acetic acid/ethanol extract of gluten, was investigated. Lectin-like components in these wheat gluten preparations were traced in binding studies employing a variety of model glycoproteins glycosylated with the different types of N-linked oligosaccharides, i.e., those of the high mannose-, complex- and hybrid-type. Binding affinity of wheat proteins to these glycoproteins was analyzed by affinity dotting and blotting techniques and was compared to that of the well characterized lectins Galanthus nivalis agglutinin, Concanavalin A and wheat germ agglutinin. Though the three wheat gluten preparations exhibited binding reactivity for distinct model glycoproteins, no correlation was found between the type of N-glycosylation of the model glycoproteins and their binding capability to the different wheat gluten preparations. Moreover, binding of the three gluten preparations to the model glycoproteins could not be inhibited by competitive saccharides (methyl-alpha-D-mannopyranoside, N-acetyl-D-glucosamine, mannan). Enzymatic deglycosylation of the ligand glycoproteins with endo-beta-N-acetylglucosaminidase H (Endo H, EC 3.2.1.96) or peptide N-glycosidase F (PNGase F, EC 3.5.1.52) abolished their binding reactivity for the plant lectins, but did not affect binding of the wheat gluten preparations. These results give no evidence for the presence of lectin-like components in wheat gluten preparations and do question the 'lectin hypothesis' of coeliac disease.
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PMID:Studies on the aetiology of coeliac disease: no evidence for lectin-like components in wheat gluten. 831 50

This study was undertaken to investigate the nature and microheterogeneity of the carbohydrate moiety of the Fc epsilon receptors of RBL-CA10 and RBL-CA10.7 cells. Treatment using the glycosylation processing inhibitors, castanospermine (CN), 1-deoxymannojirimycin (DMJ), and swainsonine (SW) resulted in a decrease of the relative molecular mass (M(r)) of both the alpha-chain of the high affinity receptor for IgE, Fc epsilon RI(alpha), and the low affinity receptor for IgE, Fc epsilon RL. Exposure to DMJ had the greatest effect on the M(r), while CN seemed to lead to a decreased cell surface expression of Fc epsilon RI. Both receptors are largely resistant to endoglycosidase H as their M(r) decreased only by approximately 2 kDa. These results suggest that both receptors are composed primarily of complex oligosaccharides with a single high mannose, N-glycosylated site. Both Fc epsilon receptors become endoglycosidase H sensitive if first exposed to DMJ which indicates that the carbohydrate composition is indeed altered by this processing inhibitor presumably by blocking the formation of complex structures. When the Fc epsilon receptors were reduced and hydrolyzed by N-glycanase, the M(r) values for Fc epsilon RI(alpha) and Fc epsilon RL decreased to approximately 28 and 34-38 kDa respectively. In the case of Fc epsilon RI(alpha), this implies the presence of only a small amount of O-linked oligosaccharides.
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PMID:The N-linked oligosaccharides of the Fc epsilon receptors of rat basophilic leukemia cells. 843 10

The H and M antigens of Histoplasma capsulatum are glycoproteins, and both possess epitopes found on the C antigen, a cross-reactive galactomannan shared by the major genera of systemic dimorphic fungi. We modified the H and M glycoproteins by chemical and enzymatic digestion to determine the relative contributions of the carbohydrate and protein moieties to the immunological reactivities and the apparent molecular weights of these antigens. Endoglycosidases with known action patterns were used to determine the nature of the glycopeptide bonds in the H and M antigens. The effects of these treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lectin binding, and enzyme-linked immunoelectrotransfer blots probed with polyclonal and monoclonal antibodies (MAbs). Oxidation with 100 mM periodate destroyed the common fungal epitope recognized by MAb CA1-CB4 and nearly all of the concanavalin A-binding sites on both the H and M antigens; it also caused the molecular mass of the M antigen to shift from 94 to 88 kDa. Treatment of samples with O-glycanase had little, if any, effect on the H and M glycoproteins. On the other hand, treatments with endo-beta-N-acetylglucosaminidase H, and particularly peptide N-glycoproteins F (PNGase F), produced pronounced shifts in the M(r) but did not completely eliminate concanavalin A- or MAb CA1-CB4-binding sites. PNGase F treatment caused the molecular mass of the H antigen to shift from 116 to 94 kDa and that of the M antigen to shift from 94 to 74 kDa. The susceptibilities of the H and M glycoproteins to endo-N-acetyl-beta-D-glucosaminidases suggest that their glycosidic moieties are N linked.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Immunochemical analysis of the H and M glycoproteins from Histoplasma capsulatum. 855 2


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