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)

We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.
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PMID:Purification and characterization of a novel growth factor from human breast cancer cells. 132 10

Membrane polypeptides (relative mass (Mr) 48,000--55,000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42,000--47,000) during sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS-polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41,000--48,000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by greater than 95% had little, if any, effect on either the affinity (Kd values, 0.1-0.2 nM) or abundance (Bmax values, 200,000--220,000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 degrees C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.
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PMID:Effects of inhibition of N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of L1210 leukemia cells. 235 Apr 87

Acid carboxypeptidase from Aspergillus saitoi is a glycoprotein that contains both N- and O-linked sugar chains. The N-glycanase released high-mannose type oligosaccharides that were separated into eight components on HPLC. One, which had a unique structure of Man11GlcNAc2, was characterized. Mild alkali treatment of the carboxypeptidase, under conditions that effect beta-elimination, yielded D-mannose. Deglycosylation of the carboxypeptidase with endo-beta-N-acetylglucosaminidase and alpha-mannosidase effected the reduction of the molecular mass from 72 kDa to 60 kDa. Partial changes of CD spectra of the native and the deglycosylated enzymes indicate that some conformational changes on the peptide of the enzyme occurred after deglycosylation. Other enzymatic properties, such as catalytic activity, pH, and thermal stability and resistivity to protease digestion, did not appear to change. Tunicamycin halted secretion of the carboxypeptidase extracellularly.
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PMID:The carbohydrate moiety of the acid carboxypeptidase from Aspergillus saitoi. 776 37

We evaluated LAK cell cytotoxicity toward a sensitive B cell lymphoma and several resistant EBV-transformed cell lines in order to explore the mechanism by which some cells are preferentially recognized as targets. Cytolysis of the sensitive cells was inhibited by monoclonal antibodies against the surface proteins LFA-1 and ICAM-1; however, surface expression of ICAM-1 was similar on the resistant and sensitive cell lines. Prevention of post-translational addition of N-linked oligosaccharides by treatment of the resistant cells with tunicamycin resulted in a dramatic enhancement in LAK cell cytotoxicity which was partially inhibited by antibodies against LFA-1 and ICAM-1. Treatment of the resistant cells with the endoglycosidase N-glycanase also increased LAK cell sensitivity. Tunicamycin treatment caused a decrease in the molecular weight of ICAM-1 from approximately 95,000 to 50,000 Da. Conjugate formation between the LAK cells and the sensitive and resistant target cells was similar before and after deglycosylation. We conclude that carbohydrate modification of ICAM-1 or an alternative glycoprotein confers resistance to LAK cell cytotoxicity in some cell lines.
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PMID:The role of N-linked carbohydrate residues in lymphokine-activated killer cell-mediated cytolysis. 790 99

The GLP-1 receptor on RINm5F cells is a glycoprotein with a M(r) of 63,000. Treatment of the receptor with glycopeptidase F generated a protein with a M(r) of 51,000, indicating that the GLP-1 receptor contains N-linked glycans. Tunicamycin pretreatment concentration-dependently decreased GLP-1 binding to RINm5F cells due to a decreased receptor number without change of receptor affinity. Tunicamycin exerted no effect on the GLP-1 receptor mRNA expression. The stimulation of cAMP production was decreased in tunicamycin-treated cells. Our data show that glycosylation of the GLP-1 receptor is a precondition for regular receptor function.
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PMID:Glycosylation of the GLP-1 receptor is a prerequisite for regular receptor function. 793 45

Because of the limited information available about the synthesis of N-linked glycoproteins in nerve cells, in regard to both processing steps and enzyme characterization, the biosynthetic processing of Thy-1 of the rat neuronal tumor cell line BN-1010-1 has been investigated using several inhibitors of biosynthesis and transport. (i) Tunicamycin completely inhibited mannose incorporation into Thy-1. Unglycosylated Thy-1 was not transported to the cell surface and was probably degraded rapidly following synthesis. (ii) Brefeldin A completely inhibited the transport of all [3H]mannose-labeled proteins releasable by phosphatidylinositol-specific phospholipase C, including Thy-1, to the surface of BN-1010-1 cells. Removal of the inhibitor led to rapid reversal of the inhibition. Pulse-chase experiments demonstrated that approximately 50% of Thy-1 was degraded after 4 h in the presence of brefeldin A. (iii) Castanospermine treatment slowed the appearance of Thy-1 on the cell surface. The surface Thy-1 contained mainly normal Man5, Man6, and Man7 oligosaccharides, suggesting that Golgi endo-alpha-D-mannosidase effected the removal of glucose. (iv) Treatment with deoxymannojirimycin resulted in the synthesis of Thy-1 that contained Man8 and Man9 oligosaccharides compared to Man5, Man6, and Man7 in the control. Neither the rate of appearance nor the level of surface expression was affected by the drug. (v) Swainsonine did not affect either the rate of appearance or the level of surface expression of Thy-1. The HPLC elution profile of neutral oligosaccharides resulting from Endo-H digestion of Thy-1 synthesized in the presence of swainsonine was indistinguishable from controls. The lack of an effect of swainsonine is explained by the unexpected absence of a complex type oligosaccharide in Thy-1 of BN-1010-1 cells, as shown in experiments with a variety of lectins as well as digestions with Endo-H or glycopeptidase F, or digestions with both enzymes in sequence. The fact that, after [3H]fucose-labeling, Endo-H digestion produced Thy-1 still labeled with fucose indicates that hybrid oligosaccharide is present in Thy-1.
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PMID:Biosynthetic and structural studies on Thy-1 in a rat neuronal tumor cell line. 809 81

The carrier protein for methotrexate and tetrahydrofolate cofactors (GP-MTX) in CCRF-CEM human lymphoblastic leukemia cells in a 117 kDa glycoprotein containing both N- and O-linked oligosaccharides (Matherly et al., J Biol Chem 267: 23253-23260, 1992). Tunicamycin, an inhibitor of N-glycosylation, was used to investigate the roles of asparagine-linked oligosaccharides in the structure, intracellular routing, and transport function of GP-MTX. Tunicamycin was growth inhibitory toward CCRF-CEM cells (IC50-0.80 micrograms/mL) and caused a potent suppression of [3H]mannose incorporation into nascent glycoproteins. From 1-3 micrograms/mL, inhibition of [3H]mannose incorporation was 66-87%, exceeding that for [35S]methionine incorporation by 2 to 4-fold. Tunicamycin (1 and 2 micrograms/mL) exposures decreased the median molecular masses of GP-MTX on immunoblots (to 82 and 67 kDa, respectively) and were accompanied by reduced maximal rates of methotrexate uptake (31 and 37%, respectively, of control levels). Conversely, the Ki values for methotrexate binding to the transporter were unaffected by tunicamycin treatments. The effects of tunicamycin on methotrexate influx closely correlated with lower levels of immunoreactive GP-MTX in plasma membranes and specific [3H]methotrexate binding to intact cells, suggesting that the transport effect was due to decreased numbers of carrier proteins at the membrane surface. The reduced molecular mass values for GP-MTX, which accompanied tunicamycin exposures, were further decreased (to 55 and 50 kDa at 1 and 2 micrograms/mL, respectively) by digestions with N-glycanase. Hence, despite the large loss of N-glycan from GP-MTX in tunicamycin-treated cells, residual core oligosaccharides remained. The sizes of hypoglycosylated GP-MTX following both treatments were similar to that of the functionally homologous methotrexate membrane carrier previously identified in L1210 murine leukemia cells.
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PMID:Role of N-glycosylation in the structure and function of the methotrexate membrane transporter from CCRF-CEM human lymphoblastic leukemia cells. 814 10

Autoantibodies to a 64-kD protein and a 40-kD tryptic fragment from pancreatic islets have been detected at high frequency in the sera of patients with insulin-dependent diabetes mellitus (IDDM). IA-2, a newly isolated transmembrane protein tyrosine phosphatase, is a major islet cell autoantigen in IDDM and the precursor of a 40-kD tryptic fragment. To express large quantities of recombinant IA-2 protein and analyse post-translational modifications we expressed full-length human IA-2 in baculovirus-infected Sf-9 cells. IA-2 expression was analysed by Western blot and by immunoprecipitation of 35S-methionine-radiolabelled proteins with rabbit antisera or IDDM sera. A 120-kD IA-2 protein was detected during the early, but not the late, phase of the infection. Pulse-chase experiments showed that the 120-kD protein was processed into fragments of 64 kD and smaller fragments of approximately 50 kD, 38 kD and 32 kD. The 64-kD fragment appeared as a doublet. Tunicamycin and PNGase F treatment down-shifted the 120-kD protein and the 64-kD doublet into lower molecular weight bands, suggesting that both were glycosylated. Trypsin treatment converted the 120-kD protein and the 64-kD doublet into a 40-kD fragment. Baculovirus-expressed IA-2 was as sensitive or slightly more sensitive than in vitro translated IA-2 in detecting autoantibodies to IA-2: 66% of sera from newly diagnosed IDDM patients reacted with baculovirus-expressed IA-2 compared with 59% of the same sera which reacted with in vitro translated IA-2. It is concluded that baculovirus-expressed IA-2 is a good source of autoantigen and that a number of lower molecular weight fragments with which IDDM autoantibodies react are derived from the 120-kD full-length IA-2 molecule.
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PMID:Expression, characterization, processing and immunogenicity of an insulin-dependent diabetes mellitus autoantigen, IA-2, in Sf-9 cells. 973 64

Polypeptide growth factors mediate their cellular responses by binding to and activating specific cell surface receptors. Monoclonal antibody (MAb) VBS-1, produced against native fibroblast growth factor receptor-1 (FGFR-1), inhibited the binding of fibroblast growth factor-2 (FGF-2) to its receptor on coronary venular endothelial cells (CVECs) as determined by 125I-FGF-2 Scatchard analysis and [3H]thymidine uptake assays (ED50 = 80 ng/mL). Enzyme studies demonstrated that MAb VBS-1 binds to a protein epitope. Proteolytic mapping of the CVEC-FGFR established that a 52 kDa doublet contained the FGF binding site and the MAb VBS-1 antigenic epitope. N-glycanase digestion suggested the presence of a 50 kDa core protein for the CVEC-FGFR. Tunicamycin treatment resulted in the loss of expression of the core protein and the mature receptor, indicating the importance of CVEC-FGFR n-linked glycosylation. By Northern blot analysis, it was determined that CVECs express fgfr-1 and not fgfr-2. VBS-1 recognized FGFR-1 (140 kDa) and crossreacted weakly with FGFR-2 (135 kDa). Using a combination of affinity crosslinking, proteolytic mapping and Mab VBS-1 binding studies, we have located the FGF binding site near the NH2-terminal domain of the receptor close to the highly acidic box.
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PMID:Partial characterization of endothelial FGF receptor functional domain by monoclonal antibody VBS-1. 1216 40

Pregnancy-associated glycoproteins (PAGs) are products of the ruminant placenta that belong to the aspartic proteinase family. Extensive glycosylation may account for the size and heterogeneity of their molecules. To assess this we investigated the effect of glycosidase and tunicamycin treatments on native (n) and mammalian-cell generated recombinant (r) bovine PAGs. Native PAG came from explant culture conditioned medium (150 days pregnancy) while rPAG was obtained by transfection of HEK 293 cells with the bPAG-1 gene employing the PRcRSV expression vector. The undigested nPAG gave a homogenous band at 67 kDa after one-dimensional SDS-PAGE, silver staining and Western blotting, but rPAG gave dual bands at 54 and 52 kDa. PNGase F digestion of nPAG gave five bands ranging from 60 to 37 kDa and digestion of rPAG gave three bands ranging from 54 to 37 kDa. On two-dimensional electrophoresis, the undigested pI ranges of n- and rPAGs were 4.7-5.6 and 7.3-8.8, respectively. The digested isoforms of n- and rPAGs had pI ranges from 5.1 to 8.5 and 7.9-8.5, respectively. Tunicamycin treatment had no effect on the mobility of nPAG but it had a pronounced time-dependant effect on the mobility of rPAG. Our findings indicate that both n- and rPAGs have principally N-linked oligosacharides.
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PMID:Characterization of native and recombinant bovine pregnancy-associated glycoproteins. 1527 71


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