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 human choriocarcinoma cell line, BeWo, synthesizes the glycoprotein hormone, human chorionic gonadotropin (hCG). We have undertaken this study to compare the synthesized and secreted forms of hCG and their alpha- and beta-subunits in cell cultures of BeWo cells to those forms of normal placental cells by immunobinding techniques. BeWo cells appeared to synthesize and secrete one species of the respective hCG subunit. The immature alpha- and beta-subunits, synthesized in BeWo cells as well as those of placental cells, were digested by endoglycosidase H indicating N-linked sugar chain(s) to be the high-mannose type. The mature alpha- and beta-subunits, secreted by BeWo cells as well as subunits of urinary hCG which are usually used as a standard hCG secreted by normal placental cells, were sensitive to neuraminidase treatment indicating that these subunits have terminal sialic acid(s). Contrary to placental cells, mature subunits of BeWo hCG could not be found in any subcellular fraction indicating a rapid secretion rate or supporting the hypothesis that BeWo cells secrete hCG subunits without the formation of secretory granules. The alpha-subunit synthesized in BeWo cells had a slightly lower molecular weight than that of placental cells; however, the alpha-subunit secreted by BeWo cells had a slightly higher molecular weight than the alpha-subunit of urinary hCG. The beta-subunits synthesized and secreted by BeWo cells had slightly higher molecular weights than beta-subunits of both placental cells and urinary hCG. Even after digestion by N-glycanase as well as endoglycosidase H, molecular weights were still different between the respective subunits of BeWo and placental cells indicating that the apoprotein structures of BeWo hCG subunits may differ from those of placental cells. Moreover, urinary beta-subunit was sensitive to endo-alpha-N-acetylgalactosaminidase treatment but the beta-subunit secreted by BeWo cells was not, indicating that the structure of O-linked sugar chain(s), if present, may be unusual. Analysis of assembled and free forms of subunits of BeWo cell cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by immunobinding methods revealed that subunits are associated intracellularly and then secreted to the media as hCG. Moreover, only free beta-subunits, but not alpha-subunits, of BeWo hCG were found intra- and extracellularly.
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PMID:Synthesis and secretion of human chorionic gonadotropin and its subunits in choriocarcinoma cells: a comparative study with normal placental cells. 169 20

The characteristics of glycosylation of a brain-specific glycoprotein, 1D4 antigen, and the epitope recognized by its monoclonal antibody were studied. Removal of high-mannose and hybrid types of N-linked oligosaccharides by treatment with endoglycosidase H converted the molecular mass of the 1D4 antigen from 89 kDa to 78 kDa, but did not affect its reactivity with the 1D4 monoclonal antibody. Removal of all types of N-linked oligosaccharides by treatment with glycopeptidase F or removal of both N- and O-linked oligosaccharides by chemical treatment caused both reduction of the molecular mass of the antigen to 63 kDa and loss of its reactivity with the monoclonal antibody. These results suggest that the 1D4 monoclonal antibody recognizes a complex-type oligosaccharide-related epitope specific for the 1D4 antigen. Results also showed that N-linked glycosylation was not responsible for the charge heterogeneity of the 1D4 antigen. The oligosaccharide chain-related epitope was detected in rat brain but not in mouse, rabbit, or bovine brain, but the 1D4 antigen was recognized in rat and mouse brains with antiserum (polyclonal antibodies). These findings indicate that the oligosaccharide-related epitope is species specific. Furthermore, results with neuraminidase-treated 1D4 antigen indicated that sialic acids were not involved in the oligosaccharide-related epitope. These findings suggest that the 1D4 antigen may have the oligosaccharide structure specific for rat brain and itself.
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PMID:Oligosaccharide-related epitope specific for a brain-specific glycoprotein, 1D4 antigen. 169 90

A polyclonal antiserum was raised to a gel purified preparation of the major water-soluble surface glycoprotein (gp29) of adult Brugia malayi, and used to define the stage specificity of expression, localisation (by immunoelectron microscopy) and the dynamics of biosynthesis and turnover via pulse-chase experiments. Gp29 was not detected in surface-labelled preparations of either pre- or post-parasitic third stage larvae (L3), but was present in fourth stage larvae (L4), where its mass was estimated to be 30 kDa by SDS-PAGE. In both L4 and adult worms, the protein resolved as 3 distinct species in 2-dimensional electrophoresis, with pIs from 6.5 to 7.5. Pulse-chase studies via metabolic labelling of adult worms with [35S]methionine in vitro indicated that gp29 was processed from a 32-kDa precursor to the mature molecule within 45 min and that it was secreted into culture medium within 5 h of synthesis. On extended culture, gp29 was converted to a 56-kDa product, presumably either by complex formation or covalent linkage with another secreted molecule. This higher molecular weight component had a more acidic pI of 4.5 and was insensitive to digestion with N-glycanase. Immunoelectron microscopy showed that gp29 was distributed throughout the cuticle and hypodermal cell layer of adult worms, suggesting that the protein was synthesised in the hypodermis, and that turnover into culture medium occurred through the cuticle. The protein appeared to concentrate at the distal cell membrane of the hypodermis, particularly at the stacked invaginations. Additional immunostaining was found on the basement membrane of the basal lamina of the intestine.
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PMID:Cuticular localisation and turnover of the major surface glycoprotein (gp29) of adult Brugia malayi. 170 Feb 98

The conformational epitopes reactive with neutralizing monoclonal antibodies (MAbs) appear to be clustered at the middle third of the glycoprotein (G) of the New Jersey serotype of vesicular stomatitis virus (VSV-NJ) and are flanked by two N-linked carbohydrate chains (W. Keil and R.R. Wagner, Virology 170, 392-407, 1989). We report here studies on the effect of glycosylation on the reactivity of VSV-NJ G protein derived from released virions or immunoprecipitated from pulse-labeled cells was not significantly affected in its reactivity with MAbs directed to epitope IV mapped toward the amino-terminus, nor to the centrally located conformational epitopes VI, VIII, and IX. However, there was a 5- to 15-fold decrease in the reactivity with MAb of epitopes VI, VIII, and IX on unglycosylated G protein either isolated from a ribosome-enriched membrane fraction or immunoprecipitated from whole VSV-infected cells labeled for 15 hr in the presence of tunicamycin. In sharp contrast, epitope V and to a somewhat lesser extent epitope VII exhibited decreased reactivity with their respective MAbs when unglycosylated G protein was isolated from released viral particles or from pulse-labeled cells infected with VSV-NJ in the presence of tunicamycin. Enzymatic removal of preformed carbohydrate chains with N-glycanase had little or no effect on the MAb-reactivity of epitopes V and VII, indicating that the carbohydrate chains per se do not influence the antigenic specificity of VSV-NJ G protein. These data suggest that the formation of N-linked carbohydrate chains influences the structure of the VSV-NJ G protein in such a way that epitopes V and VII are shielded from reactivity with their specific MAbs from an early stage of G-protein processing and to a much lesser extent epitopes VI, VIII, and IX at late stages of intracellular processing. These results are compatible with, but do not prove, the hypothesis that N-linked glycosylation plays a key role in promoting the formation and the stability of the disulfide bonds that determine the epitope-specific conformational integrity of the VSV-NJ glycoprotein.
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PMID:Effect of glycosylation on the conformational epitopes of the glycoprotein of vesicular stomatitis virus (New Jersey serotype). 170 43

The glycoprotein allergen Art v II, from the pollen of mugwort (Artemisia vulgaris L.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides. The oligosaccharides were isolated by gel permeation chromatography and their structures determined by 500-MHz 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, and high-pH anion-exchange chromatography. The high-mannose oligosaccharides Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 were present in the ratios 2:49:19:24:6 and accounted for all the asparagine-linked oligosaccharides released from Art v II by PNGase F. The NH2-terminal amino acid sequences of Art v II and of four peptides generated by cyanogen bromide (CNBr) cleavage of deglycosylated Art v II were determined. The first 30 amino acid residues of Art v II did not contain any potential N-glycosylation sites. One potential N-glycosylation site was identified in one of the CNBr fragments. The native protein conformation was shown by enzyme-linked immunosorbent assay inhibition assays to be essential for the binding of rabbit IgG to Art v II and for the binding of human IgE to the major IgE-binding epitope(s) in this allergen. At least one minor IgE-binding epitope still bound IgE after denaturation of the allergen. Removal of the high-mannose chains from denatured Art v II had no significant effect on the binding of human IgE to the minor IgE-binding epitope(s).
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PMID:Structural analysis of the glycoprotein allergen Art v II from the pollen of mugwort (Artemisia vulgaris L.). 170 33

Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase expressed at high levels in rat kidney, liver and lung. We established eight monoclonal antibodies against partially purified DPP IV from rat liver plasma membranes. By means of a competitive dot blot assay with purified DPP IV, these antibodies were shown to recognize four different epitopes of the glycoprotein, designated A - D. The epitopes are located on the extracellular domain of DPP IV, as shown by papain digestion of liver plasma membranes. Treatment of DPP IV with neuraminidase and glycopeptide N-glycosidase F, as well as incubation of hepatocytes with the alpha-mannosidase I inhibitor deoxymannojirimycin, revealed that epitope A may be formed by a mannose-rich sugar chain and epitope D might represent a complex carbohydrate structure in the mature glycoprotein, while the epitopes B and C are formed by the protein moiety. Concanavalin A reduced the binding of monoclonal antibody to epitope A by 78%. Binding to epitope D was blocked by 73% with wheat germ lectin, and by more than 99% with sialic acid; epitopes B and C were unaffected by any of the lectins or sugars tested. The immunological cross-reactivity with DPP IV from Morris hepatoma 7777 was demonstrated with monoclonal antibodies against epitopes A-C. Epitope D was not recognized on hepatoma DPP IV. However, in addition to DPP IV, four hepatoma plasma membrane glycoproteins were precipitated by the monoclonal antibody against the epitope D, indicating that this epitope is not uniquely restricted to DPP IV.
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PMID:Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes. 170 62

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.
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PMID:Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line. 170 83

The synthesis and secretion of mucin-like high-molecular glycoprotein was studied in 2 human colon cancer cell lines that spontaneously differentiate in culture (Caco-2 and T84) and in 2 cell lines that do not spontaneously differentiate (LS174T and HT29). Mucin, quantitated by 3H-glucosamine labelling and chromatography on Sepharose CL-4B was found to be produced by all 4 cell lines. The mucinous nature of the labelled high-molecular glycoprotein was verified by enzymatic degradation treatments (heparinase, hyaluronidase, chondroitinase ABC, and N-glycanase), alkaline-borohydride treatment, inhibition of labelling by the glycosylation inhibitor benzyl-alpha-GalNAc, and by CsCl-density-gradient centrifugation. In all 4 cell lines, an inverse correlation of mucin synthesis with cell density was demonstrated. In Caco-2 cells, the spontaneous post-confluent enterocytic differentiation with increased brush-border enzyme expression was associated with a decrease in mucin synthesis and in the activities of polypeptidyl GalNAc transferase and beta 1,3-galactosyltransferase activity. Using cDNA probes for 2 distinct human intestinal mucins (MUC2 and MUC3), we found that all 4 colon cancer cell lines expressed mucin message, but the types of mucin mRNA expressed differed. These data indicate that mucin-like glycoproteins can be synthesized by cell lines derived from non-mucinous colon cancer, whether or not they undergo spontaneous differentiation in culture. These cell lines may serve as in vitro models for studying apomucin heterogeneity and control of mucin gene expression.
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PMID:Mucin synthesis and secretion in relation to spontaneous differentiation of colon cancer cells in vitro. 172 5

The structure of the N-linked oligosaccharides of Semliki Forest viral glycoproteins produced in infected mosquito cells (C6/36) was investigated by biosynthetic labeling, enzymic deglycosylation using endo-beta-N-acetylglucosaminidases H, D, F/glycopeptidase F, exoglycosidase and analysis of the sugars on Concanavalin A-Sepharose columns and by gel filtration chromatography. The results demonstrated that the glycoproteins decorating the virus shed from infected cells have N-linked glycans with a trimannosyl core similar to the core glycans produced by vertebrate and yeast cells. However, the E1 glycoprotein produced by infected C6/36 cells exhibited both a trimannosyl core and a modified trimannosyl core most probably with terminal N-acetylglucosamine. The carbohydrate side chains of Semliki Forest envelope proteins displayed two types of structural heterogeneities existing either at different N-glycosylation sites as in the case of E2, or at the same N-glycosylation site as in the case of E1. In the presence of 1-deoxymannojirimycin, no structural heterogeneities in the glycan chains were found. This strongly suggests that the glycosylation events that lead to the observed sugar heterogeneities occur in the Golgi membranes.
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PMID:Asparagine-linked oligosaccharides of Semliki Forest virus grown in mosquito cells. 172 85

A glycoprotein reactive with antibodies against corneal keratan sulfate proteoglycan (KSPG) was purified 300-fold from extracts of bovine aorta using DEAE ion-exchange, gel-filtration, hydrophobic interaction, and reverse-phase chromatographic separations. The intact glycoprotein was 70-80 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Deglycosylation with endo-beta-galactosidase and N-glycanase reduced the size to 48 and 37 kDa, respectively, similar to the large isoforms of corneal KSPG. N-terminal amino acid sequence of the arterial KSPG was identical with lumican, the 37B isoform of corneal KSPG, and the arterial KSPG reacted with an antibody to synthetic peptide duplicating this sequence. Arterial KSPG and corneal lumican displayed identical tryptic maps. Arterial lumican contains fucose and mannose in amounts similar to corneal KSPG, but galactose, glucosamine, and sulfate were reduced compared to KSPG from cornea. Treatment of arterial lumican with endo-beta-galactosidase released 8-9 mol of glucosamine and galactose per mol of protein as oligosaccharides. These eluted as neutral, nonsulfated oligosaccharides on high pH anion-exchange chromatography. The size of arterial lumican was not altered by glycosidases having specificity for sulfated keratan sulfate, nor was the charge of the lumican molecule altered by digestion with endo-beta-galactosidase. These data show arterial lumican to be a glycoprotein containing unsulfated lactosaminoglycan chains. Abundance of low sulfate lumican in many tissues indicates that this protein occurs predominantly as a glycoprotein rather than as the more widely studied, highly sulfated proteoglycan present in the cornea.
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PMID:Arterial lumican. Properties of a corneal-type keratan sulfate proteoglycan from bovine aorta. 176 72


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