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)

Conversion of factor X to factor Xa results in release of a heavily glycosylated activation peptide. Analysis of protease-digested glycopeptides derived from the activation peptides of bovine and human blood coagulation factor X allowed the identification of sites of the O-linked oligosaccharide chains in these peptides. Glycopeptides were prepared from the activation peptides by digestion with chymotrypsin or Staphylococcus aureus V8 protease. By combined analysis of amino acid sequence and sialic acid content, we found that bovine factor X had an O-linked oligosaccharide chain linked to Thr26, and human factor X had four carbohydrate-attachment sites, namely, O-glycosidic linkages to Thr17 and Thr29, respectively, and N-glycosidic linkages to Asn39 and Asn49, respectively, in their activation peptides. The O-linked carbohydrate-attachment sites were identified since the yields of phenylthiohydantoin derivatives of amino acids that corresponded to their residues were increased during amino acid sequencing after deglycosylation of the glycopeptides with sialidase and O-glycanase. The effect of deglycosylation of bovine factor X1 was investigated with factor-X-activating enzyme from Russell's viper venom or extrinsic Xase (factor VIIa/tissue factor/phospholipid) by examining the activation rates of derivatives of factor X prepared using O-glycanase, sialidase, and/or N-glycanase. The removal of O-linked carbohydrate resulted in a decrease in the rate of activation. It appears that carbohydrate residues in factor X play an important role in the activation of the zymogen.
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PMID:Identification of O-linked oligosaccharide chains in the activation peptides of blood coagulation factor X. The role of the carbohydrate moieties in the activation of factor X. 824 61

The acceptor for Clostridium botulinum type B neurotoxin was solubilized from rat brain synaptic membrane with nonionic detergent, nonanoyl-N-methylglucamide (MEGA-9). The solubilized acceptor was assayed for the binding activity by precipitating the acceptor with acetone in the presence of phosphatidylcholine. 125Ilabeled neurotoxin specifically bound to the lipid vesicles having incorporated the acceptor together with gangliosides. The lipid vesicles having incorporated either the acceptor or gangliosides alone showed extremely low binding activity. The treatment of the solubilized acceptor with lysyl endopeptidase and glycopeptidase F but not with sialidase resulted in decreased toxin binding, indicating that the putative acceptor is a glycoprotein accompanying an N-linked carbohydrate moiety. The observations suggest also that a protein acceptor/ganglioside complex may be required to form the functional toxin receptor.
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PMID:Solubilization and characterization of the acceptor for Clostridium botulinum type B neurotoxin from rat brain synaptic membranes. 825 34

When a rat liver Golgi apparatus-enriched subcellular fraction is incubated with UDP-[3H]Gal, CMP-[3H] Neu5Ac, or [acetyl-3H]acetyl (Ac)-CoA, label is efficiently transferred to endogenous acceptors, which are resistant to added proteases, unless detergent is added at a sufficiently high concentration. Thus, the acceptors are within the lumen of intact compartments of correct topological orientation, which are likely to be similar to those of the Golgi apparatus in the intact cell. In each case, approximately 90% of the macromolecular radioactivity is specifically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase digestion, as labeled N-linked oligosaccharides. Label from UDP-[3H]Gal is transferred to several distinct N-linked oligosaccharides, and many of these carry sialic acid (Sia) residues. This amount increases if the transfer reaction is chased with CMP-Neu5Ac. A major fraction of the [3H]Gal is directly "covered" with Sia residues, indicating that at least a portion of the beta-galactosyltransferase(s) are co-localized with one or more sialyltransferases. The majority of the [3H]Gal is found in a beta 1,3-linkage, rather than the more common beta 1,4-linkage. The N-linked oligosaccharides labeled by CMP-[3H] Neu5Ac carry labeled Sia residues in either alpha 2,3 or alpha 2,6 linkage, and showed a range of charge distribution. The transferred [3H]Neu5Ac is not O-acetylated even when Ac-CoA is added at saturating concentrations, implying that the sialyltransferases and the O-acetyltransferase(s) are not functionally co-localized. However, approximately 20% of label released from N-linked oligosaccharides by sialidase does not co-migrate with authentic Neu5Ac in high performance liquid chromatography analysis, indicating that transferred [3H] Neu5Ac is modified by unknown enzymes in the Golgi. Most of the [3H]acetate transferred from [acetyl-3H] Ac-CoA to N-linked oligosaccharides is on Sia residues that are exclusively alpha 2,6-linked, and is enriched on tri- and tetra-antennary chains that do not appear to carry any 2,3-linked Sia residues. These data indicate a restricted substrate preference of the O-acetyltransferase(s). About one-quarter of the [3H]acetate transferred is sialidase-resistant, indicating either transfer to monosaccharides other than sialic acid, or to sialidase-resistant sialic acids. While most of these sialidase-resistant oligosaccharides remain negatively charged, about 10% are neutralized by sialidase, confirming transfer of [3H]acetate to monosaccharides other than sialic acid.
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PMID:Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked glycans labeled by UDP-[6-3H]galactose, CMP-[9-3H]N-acetylneuraminic acid, and [acetyl-3H]acetyl-coenzyme A. 834

uK2t-PA is a hybrid plasminogen activator in which the epidermal growth factor-like domain of the urokinase-type plasminogen activator precedes the kringle 2 and catalytic domains of tissue-type plasminogen activator. The molecules are expressed in Chinese hamster ovary cells in two variant forms, a type II form in which only the protease domain is glycosylated, and a type I form in which both the kringle 2 and the protease domains carry N-acetyllactosamine type glycans. The two forms differed slightly in their affinity for fibrin and fibrinogen, which allowed their separation, but the stimulation of plasminogen activation of the type II form by fibrin was up to eight-fold lower than that of the type II form. The sensitivity to fibrin could be restored by treatment of the type I form with N-glycanase or sialidase. Enzymatic activity vs low molecular weight substrates was not influenced by the glycosylation of kringle 2.
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PMID:Functional effects of kringle 2 glycosylation in a hybrid plasminogen activator. 838 98

The serine protease, prostate-specific antigen (PSA), its protein substrates, semenogelin (Sg) I and II, and protein C inhibitor (PCI) have been described as components of human seminal plasma. PCI was found to inhibit the PSA-catalyzed degradation of insoluble coagula Sg I + II by forming a PSA-PCI complex. Digestion of seminal coagula with PSA released PCI and PSA-PCI complex from the coagula into a soluble phase, suggesting the presence of active PCI binding to the coagula. To investigate the molecular interaction of Sg with PSA and PCI, we purified Sg II from seminal coagula as a soluble form and found that Sg II is glycosylated with heterogeneous carbohydrate moieties. Sg II bound to the solid-phase complex of diisopropylfluorophosphate (iPr2FP) and PSA with an apparent dissociation constant (kd) of 41 nM and to PCI with a Kd of 28 nM. The binding of Sg II to iPr2P-PSA was not affected by PCI and that of Sg II to PCI was not affected by iPr2P-PSA, suggesting that Sg II forms a ternary complex with PSA and PCI. The bindings of Sg II to both iPr2P-PSA and PCI were influenced by pH, ionic strength, heparin, dextran sulfate, and divalent cations, particularly by Zn2+. Treatment of Sg II with heparinase, heparitinase, N-glycanase, or with O-glycanase following sialidase did not affect the binding of Sg II to iPr2P-PSA and PCI. These findings suggested that PCI bound to Sg in seminal vesicles regulates the PSA-catalyzed degradation of Sg in seminal plasma, and that the binding of PCI and PSA to Sg is modulated by several factors such as pH, ionic strength, divalent cations, and heparin-like substances in seminal plasma.
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PMID:Characterization of semenogelin II and its molecular interaction with prostate-specific antigen and protein C inhibitor. 866 56

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

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

Lectin blot analysis of bovine, goat, human, rabbit and mouse serum immunoglobulin G (IgG) samples revealed that Wisteria floribunda agglutinin (WFA) binds to the heavy chains of bovine, goat and human serum IgG proteins but not those of the rabbit and mouse proteins. WFA-positive light chain bands were also detected in bovine, goat and human serum IgG samples only after the filters were treated with Arthrobacter ureafaciens sialidase. The WFA-binding to these IgG proteins was abolished by treatment of the filter with sialidase and then beta-N-acetylhexosaminidase or N-glycanase prior to incubation with the lectin. WFA-agarose column chromatography of the oligosaccharides released by hydrazinolysis from the IgG samples followed by reduction with NaB3H4 revealed that 0.15, 0.09 and 0.07% of the total oligosaccharides from bovine, goat and human serum IgG samples bind to the column, respectively. Partial characterization of WFA-positive bovine IgG oligosaccharides by Bio-Gel P-4 column chromatography suggested that the major oligosaccharide is of non-fucosylated biantennary complex-type. These results indicate that beta-N-acetylgalactosaminylation occurs to N-linked sugar chains of heavy and light chains of IgG proteins in a species-specific manner.
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PMID:Species-specific beta-N-acetylgalactosaminylation of serum IgG proteins. 910 15

Using a CD4-binding assay to assess the conformation of the human immunodeficiency virus envelope glycoprotein (CHO+ Env), we studied the effect of treatment with various glycosidases on the stability of Env in denaturing environments and in biological media: cleavage from Env of either high-mannose-type glycans (HMT- Env) by endoglycosidase H or sialic acid residues (Sial- Env) by sialidase did not alter Env stability whereas its complete deglycosylation (CHO- Env) by N-glycanase had a large effect. The influence of glycan removal on Env sensitivity to proteases was also studied. Thrombin cleavage within V3 was affected by N-glycanase treatment; both HMT- Env and CHO- Env displayed an increased sensitivity to other endoproteases. Thus, partial deglycosylation increases Env sensitivity to proteases but only its total deglycosylation alters its stability.
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PMID:Effect of various glycosidase treatments on the resistance of the HIV-1 envelope to degradation. 910 16

The effect of ammonium chloride was determined on a culture of CHO cells transfected with the human erythropoietin (EPO) gene. Cell growth was inhibited above a culture concentration of 5 mM NH(4)Cl with an IC-50 determined to be 33 mM. The specific production of EPO increased with the addition of NH(4)Cl above 5 mM. At 10 mM NH(4)Cl, the final cell density after 4 days in culture was significantly lower but the final yield of EPO was significantly higher. This appeared to be due to continued protein production after cell growth had ceased. The metabolic effects of added NH(4)Cl included higher specific consumption rates of glucose and glutamine and an increased rate of production of alanine, glycine, and glutamate. The EPO analyzed from control cultures had a molecular weight range of 33-39 kDa and an isoelectric point range of 4.06-4.67. Seven distinct isoforms of the molecule were identified by two-dimensional electrophoresis. This molecular heterogeneity was ascribed to variable glycosylation. Complete enzymatic de-glycosylation resulted in a single molecular form with a molecular mass of 18 kDa. Addition of NH(4)Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased molecular weight and pI range. Enzymatic de-sialylation of the EPO from the ammonia-treated and control cultures resulted in identical electrophoretic patterns. This indicated that the effect of ammonia was in the reduction of terminal sialylation of the glycan structures which accounted for the increased pI. Selective removal of the N-glycan structures by PNGase F resulted in two bands identified as the O-glycan linked structure (19 kDa) and the completely de-glycosylated structure (18 kDa). The proportion of the O-linked glycan structure was reduced, and its pI increased in cultures to which ammonia was added. Thus, the glycosylation pattern altered by the presence of ammonia included a reduction in terminal sialylation of all the glycans and a reduction in the content of the O-linked glycan. The addition of a sialidase inhibitor to the cultures had no effect on the ammonia-induced increase in EPO heterogeneity. Also, the effect of ammonia on glycosylation could not be mimicked using the weak base chloroquine in our system.
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PMID:Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. 1074 5


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