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)

E-selectin is a cytokine-inducible membrane glycoprotein capable of mediating adhesion of leukocytes to endothelial cells. It is highly glycosylated, containing 11 sites for N-linked glycosylation. N-Glycosylation of E-selectin was analyzed by endoglycosidase treatment. Analysis of immunoprecipitated E-selectin from human umbilical vein endothelial cells (HUVEC) by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that E-selectin was completely resistant to endoglycosidase H, but sensitive to peptide N-glycanase F digestion. This suggested that all N-linked oligosaccharide chains were of the complex type. The role of N-linked glycosylation in surface expression and secretion of E-selectin was studied using interleukin-1-stimulated HUVEC, cultured in the presence of the soluble glycosylation inhibitors tunicamycin or castanospermine. Cell surface expression was analyzed by indirect flow cytometry. N-Glycosylation was blocked by tunicamycin, and resulted in a significantly reduced surface expression of E-selectin, whereas castanospermine only marginally reduced E-selectin expression. The deglycosylated forms of E-selectin were also found to be fully capable of mediating adhesion of HT-29 cells in vitro. In conclusion, these studies show that E-selectin is heavily glycosylated with complex type N-linked oligosaccharides and that N-glycosylation is important for expression of E-selectin on human endothelial cells.
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PMID:Role of N-linked glycosylation in expression of E-selectin on human endothelial cells. 758 10

The 32-kDa glycoprotein of Chlamydia trachomatis was shown to have a pI of 6.2 to 6.4 which distinguished this protein from the chlamydial histone-like protein of similar molecular mass that has a pI of > 10. The initial interaction of the glycan of 32 kDa glycoprotein and HeLa cells was also investigated. Glycan was cleaved from the protein backbone by N-glycanase and radiolabeled with tritium by sodium borohydride reduction. Competition assays showed the binding of glycan to HeLa cells was inhibited by galactose, mannose, and N-acetylglucosamine but not by sedoheptulose and fructose. Untreated and UV-treated organisms inhibited the binding, while heat-inactivated organisms did not. Binding was blocked by rabbit antiserum against whole organisms but not by rabbit anti-155-kDa antiserum or monoclonal antibodies against the lipopolysaccharide and major outer membrane protein.
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PMID:The 32-kDa glycoprotein of Chlamydia trachomatis is an acidic protein that may be involved in the attachment process. 798 77

Carbonic anhydrase (CA) was purified from the gills of the shore crab Carcinus maenas using affinity chromatography and HPLC. The predominantly membrane-bound CA was found to share several features with mammalian CA IV. Its apparent molecular weight of 36 kDa was reduced to 33 kDa by treatment with PNGase F, suggesting that crab CA is a glycoprotein with one N-linked oligosaccharide chain. More than half of the membrane-bound crab CA was released from membranes by treatment with a phosphatidylinositol-specific phospholipase C, indicating that the branchial CA is anchored to membrane surfaces by a phosphatidylinositol-glycan linkage. The enzyme also resembles mammalian CA IV in its relative sensitivity to inhibition by sulfonamides and the resistance to inhibition by halide ions. Amino acid composition of the HPLC-purified crab CA was examined and CNBr cleavage was carried out followed by N-terminal amino acid sequencing. Amino-terminal sequence of the native enzyme differed considerably from those of mammalian isozymes (human CA I and CA II, bovine CA III, human and rat CA IV). However, antisera raised against rat CA IV, CA II, and CA I all cross-reacted weakly with crab CA. Unlike mammalian CA IVs, crab gill CA was sensitive to 0.2% sodium dodecyl sulfate, suggesting that although crab gill CA is like mammalian CA IVs in many ways, it is less stabilized by intramolecular disulfide bonds.
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PMID:Membrane-associated carbonic anhydrase from the crab gill: purification, characterization, and comparison with mammalian CAs. 803 56

The sodium-independent GLUT1 glucose transporter is expressed in high density in human erythrocytes and in tissues which serve a barrier function. In the polarized endothelial cells of the brain capillaries, which comprise the blood-brain barrier (BBB), GLUT1 is expressed on both apical and basolateral membranes; however, in the epithelium of the choroid plexus, GLUT1 expression is restricted to the basolateral surface. The present study examined whether these differences in subcellular localization of GLUT1 at the BBB and choroid plexus could be correlated with differential N-linked or O-linked glycosylation of the protein. Western blot analysis of solubilized brain capillaries (BC) and choroid plexus (CP) revealed that while the BC GLUT1 had an average molecular mass identical to that of the purified human erythrocyte transporter (54 kDa), the CP GLUT1 was of lower molecular mass (47 kDa). Treatment of brain capillaries and choroid plexus with N-glycanase resulted in a shift in the mobility of the GLUT1 of both samples to a lower molecular mass of 42 kDa; however, in contrast, treatment with O-glycanase produced no change in the mobility patterns of GLUT1, but did result in O-linked deglycosylation of another BBB marker, gamma-glutamyl transpeptidase. In conclusion, BBB and choroid plexus GLUT1 are subject to differential N-linked glycosylation with the protein having an N-linked carbohydrate side chain of higher molecular mass at the BBB in comparison to the choroid plexus.
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PMID:Differential glycosylation of the GLUT1 glucose transporter in brain capillaries and choroid plexus. 803 91

PNGase F is an amidase that hydrolyzes the beta-aspartylglucosylamine bond of asparagine-linked glycopeptides and glycoproteins. Enzymatic activity of PNGase F requires the recognition of both the peptide and the carbohydrate moiety. Crystals of PNGase F were grown by sitting drop vapor diffusion methods at 10 degrees C. The precipitating buffer contains both polyethylene glycol 3350 and (NH4)2SO4 in sodium acetate buffer at pH 4.3. The crystals belong to the orthorhombic space group C222(1) with cell dimensions: a = 87.16 A, b = 125.10 A, c = 79.33 A and diffract to 1.8 A resolution.
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PMID:Crystallization and preliminary crystallographic analysis of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase PNGase F. 805 83

Previously, we have shown that galaptin, an endogenous beta-galactoside-binding lectin, is present in extracellular matrix where it may participate in the adhesion of A121 human ovarian carcinoma cells to extracellular matrix via interaction with specific cell surface carbohydrate receptors. We now report that A121 cells adhere to polystyrene plates coated with polymerized human splenic galaptin. The carbohydrate-mediated specificity of this adhesive interaction was demonstrated by inhibition with lactose. Additionally, treatment of A121 cells with neuraminidase increased cellular adherence by 30%, while beta-galactosidase treatment of cells decreased adherence by 65%. These findings prompted us to isolate and identify the cell surface galaptin receptor. In a Western blot of A121 cell extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 125I-labeled polymerized galaptin bound [corrected] to a unique cellular protein having a molecular mass of 110 kDa. This receptor was enriched by affinity chromatography using polymerized galaptin-Sepharose. Treatment of this material with N-glycanase ablated its galaptin-binding activity. In related studies, A121 cells metabolically labeled with [3H]glucosamine demonstrated a radiolabeled polymerized galaptin-binding protein with an identical molecular mass of 110 kDa. These studies confirmed the glycoprotein nature of this putative endogenous cellular galaptin receptor. Further studies with antibodies directed against two lysosomal associated membrane proteins, lamp-1 and lamp-2, demonstrated specific reactivity in Western blots with the 110-kDa glycoprotein. Additionally, 125I-polymerized galaptin recognized a 110-kDa protein in Western blots of material immunoprecipitated from A121 cell lysates by lamp-1 and lamp-2 antibodies. Finally, indirect immunofluorescence using antibodies directed against lamps detected cell surface antigenicity. Therefore, lamp-1 and/or lamp-2 appear to be the putative cell surface receptors involved in the adhesion of ovarian carcinoma cells to extracellular matrix mediated by galaptin.
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PMID:Galaptin-mediated adhesion of human ovarian carcinoma A121 cells and detection of cellular galaptin-binding glycoproteins. 834 96

Affinity-purified antibodies against the C-terminal region of the Na+/H+ exchanger (NHE-1) were used to analyse the carbohydrate moiety of the protein. The Na+/H+ exchanger in human placental brush-border membranes has an apparent molecular mass of 105 kDa. Incubation of intact or detergent-solubilized membranes with glycopeptidase F removed the carbohydrate moiety and increased the apparent mobility of the exchanger. Digestion with endoglycosidase-F caused a similar change in mobility, but endoglycosidase-H had no effect, suggesting that the placental Na+/H+ exchanger is a glycoprotein of the biantennary complex type. Removal of the carbohydrate moiety with glycopeptidase F had no effect on the ability of the protein to promote the exchange of Na+ for H+, and had no detectable effect on the sensitivity of the exchanger to trypsin. Limited digestion with glycopeptidase F and neuraminidase indicated the presence of two intermediate forms between the fully glycosylated and the deglycosylated protein. This suggests the presence of at least two, and possibly three, N-linked carbohydrate moieties.
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PMID:Multiple carbohydrate moieties on the Na+/H+ exchanger. 838 44

Calcitonin gene-related peptide (CGRP) immunoreactivity is widely distributed in the central nervous system and gastrointestinal (GI) tract, and specific receptors have been described on many GI tissues. In the present study, we compared CGRP receptors on gastric smooth muscle cells with those on pancreatic acini from guinea pig with the use of chemical cross-linking techniques combined with various enzymatic digestions. 125I-labeled rat CGRP-I demonstrated temperature-dependent saturable binding to both acinar and gastric smooth muscle cell membranes. After binding, membranes were incubated with 1 mM disuccinimidyl suberate (DSS), solubilized with sodium dodecyl sulfate (SDS), and subjected to SDS-polyacrylamide gel electrophoresis. Cross-linked radioactivity was analyzed by autoradiography. A single broad radioactive band [molecular weight (M(r)) 57,000] was seen on cell membranes from both tissues and after cross-linking to intact cells. These bands were not altered by addition of dithiothreitol. This radioactive band was not detected without DSS present or with addition of 10 microM rCGRP-I. rCGRP-I inhibited cross-linking with half-maximal inhibition of 32 nM with membranes from both tissues, and there was a close correlation between its ability to inhibit binding and to inhibit cross-linking. Cross-linking was not inhibited by non-CGRP related peptides. With membranes from both tissues, N-glycanase digestion increased the mobility of the original band. Neuraminidase digestion only slightly increased the mobility of the original band; however, the subsequent addition of O-glycanase showed no additional effect on both membranes. Endoglycosidase H digestion had no effect in either tissue. The present results demonstrate that on both tissues the cell membrane receptor for CGRP is an N-linked sialoglycoprotein. The apparent M(r) of this sialoglycoprotein is 57,000, and this polypeptide does not contain disulfide-linked subunits or O-linked carbohydrates.
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PMID:Structural analysis of CGRP receptors on gastric smooth muscle and pancreatic acinar cells. 839 10

Three subtype-specific antisera were generated against peptides corresponding to portions of the amino terminus, interdomain 1-2, and carboxy terminus of the rH1 sodium channel primary sequence to confirm the expression of this protein in the adult rat heart and to determine selected biochemical properties of this protein that might contribute to its subtype-specific characteristics. All three antisera identify a 240-kD band on Western blots of partially purified cardiac membrane proteins and by immunoprecipitation of iodinated partially purified membrane proteins. Unlike other characterized mammalian sodium channels, no beta subunit is detected in association with the rH1 alpha subunit. The rH1 alpha subunit is a complex sialoglycoprotein as evidenced by its interaction with wheat germ agglutinin-Sepharose and by reduction in its apparent molecular weight after treatment with neuraminidase; deglycosylation with N-glycanase confirms that the rH1 protein contains significantly less carbohydrate than other sodium channel proteins characterized to date (5% versus 25% to 30%). Consistent with electrophysiological studies indicating a role of phosphorylation in channel regulation, the rH1 alpha subunit can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase A. The possible functional significance of these findings is discussed.
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PMID:Partial characterization of the rH1 sodium channel protein from rat heart using subtype-specific antibodies. 839 5

Growth factors for rat primary glial cells were identified in conditioned medium of a human glioma-derived cell line. The factors, designated glia-activating factors (GAFs), were purified to homogeneity by a combination of heparin affinity chromatography, gel filtration, and high performance liquid chromatography on a heparin affinity column and a C4 reversed-phase column. GAFs could be resolved into three peaks by C4 column chromatography. The M(r) values of these three proteins were estimated to be 30,000, 29,000, and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. These M(r) values were in good agreement with the value of 26,000 +/- 3,000 estimated from the elution volume upon gel filtration chromatography under nondenaturing conditions. These data suggested that each of the GAFs consists of a single polypeptide chain and has no subunit structures. These three purified GAFs had almost the same growth-stimulating effect on glial cells in vitro, and the half-maximal dose was around 10(-11) M. Concanavalin A staining and glycopeptide N-glycosidase treatment of GAFs indicated that an asparagine-linked oligosaccharide chain(s) was attached to these three kinds of GAFs. Microsequencing of each GAF revealed a single amino-terminal sequence with no significant homology to any known protein, and the amino-terminal sequence of the 30-kDa GAF included that of the 29-kDa GAF. GAFs also stimulated the cell growth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c3T3 fibroblasts, and PC-12 cells but not that of human umbilical vein endothelial cells.
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PMID:Novel secretory heparin-binding factors from human glioma cells (glia-activating factors) involved in glial cell growth. Purification and biological properties. 842 60


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