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)

Endogenous acceptors in a Golgi apparatus-enriched subcellular fraction from rat liver were labeled with UDP-[3H]GalNAc. The great majority of these acceptors were protected from protease degradation in the absence of detergent. These molecules are therefore present in intact vesicles of the correct topological orientation, which are likely to be similar to the Golgi compartments of the intact cell. Several distinct glycoproteins are labeled, but most are different from those labeled with UDP-[3H]GlcNAc. The enzyme peptide-N4(N-acetyl-beta-glucosiminyl)asparagine amidase releases label from a few specific proteins, indicating that [3H]GalNAc is transferred to N-linked oligosaccharides. Both neutral and anionic N-linked oligosaccharides are found, the great majority of which do not bind to ConA-Sepharose. Most of the [3H]GalNAc found in neutral oligosaccharides is terminal and beta-linked. The negative charge on the anionic molecules is due to sialic acid, and phosphate. A major portion of the [3H] GalNAc in this fraction is acid labile, and is released with kinetics consistent with it being in a phosphodiester linkage. These results show the existence of a whole new class of GalNAc-containing N-linked oligosaccharides, and demonstrates that this in vitro approach can detect previously undescribed structures. O-linked oligosaccharide biosynthesis was also studied in the same labeled rat liver Golgi apparatus preparations. beta-Elimination releases approximately 95% of the peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F)-resistant label which, in the absence of other added nucleotides, is almost exclusively [3H] GalNAcitol. If other unlabeled sugar nucleotides and adenosine 3'-phosphate,5'-phosphosulfate are added during the chase period two anionic O-linked oligosaccharides are synthesized, indicating that the UDP-GalNAc:peptide-N-acetylgalactosaminyltransferase is at least in part functionally co-localized with enzymes that extend and modify O-linked oligosaccharides.
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PMID:The biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked and O-linked glycans labeled by UDP-[6-3H]N-acetylgalactosamine. 834 1

Heparan sulfate 2-sulfotransferase, which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulfate to position 2 of L-iduronic acid residue in heparan sulfate, was purified 51,700-fold to apparent homogeneity with a 6% yield from cultured Chinese hamster ovary cells. The isolation procedure included a combination of affinity chromatography on heparin-Sepharose CL-6B and 3',5'-ADP-agarose, which was repeated twice for each, and finally gel chromatography on Superose 12 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 47 and 44 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin and mouse Engelbreth-Holm-Swarm tumor heparan sulfate were used as acceptors, the purified enzyme transferred sulfate to position 2 of L-iduronic acid residue but did not transfer sulfate to the amino group of glucosamine residue or to position 6 of N-sulfoglucosamine residue. Heparan sulfates from pig aorta and bovine liver, however, were poor acceptors. The enzyme showed no activities toward chondroitin, chondroitin sulfate, dermatan sulfate, and keratan sulfate. The optimal pH for the enzyme activity was around 5.5. The enzyme activity was minimally affected by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5'-phosphosulfate was 0.20 microM.
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PMID:Purification and characterization of heparan sulfate 2-sulfotransferase from cultured Chinese hamster ovary cells. 863 1

We have used the technique of adenovirus-mediated gene transfer to study the in vivo function of the very low density lipoprotein receptor (VLDLR) in low density lipoprotein receptor (LDLR) knockout mice. We generated a replication-defective adenovirus (AdmVLDLR) containing mouse VLDLR cDNA driven by a cytomegalovirus promoter. Transduction of cultured Hepa (mouse hepatoma) cells and LDLR-deficient CHO-ldlA7 cells in vitro by the virus led to high-level expression of immunoreactive VLDLR proteins with molecular sizes of 143 kDa and 161 kDa. Digestion of the cell extract with the enzymes neuraminidase, N-glycanase, and O-glycanase resulted in the stepwise lowering of the apparent size of the 161-kDa species toward the 143-kDa species. LDLR (-/-) mice fed a 0.2% cholesterol diet were treated with a single intravenous injection of 3 x 10(9) plaque-forming units of AdmVLDLR. Control LDLR (-/-) mice received either phosphate-buffered saline or AdLacZ, a similar adenovirus containing the LacZ cDNA instead of mVLDLR cDNA. Comparison of the plasma lipids in the 3 groups of mice indicates that in the AdmVLDL animals, total cholesterol is reduced by approximately 50% at days 4 and 9 and returned toward control values on day 21. In these animals, there was also a approximately 30% reduction in plasma apolipoprotein (apo) E accompanied by a 90% fall in apoB-100 on day 4 of treatment. By FPLC analysis, the major reduction in plasma cholesterol in the AdmVLDLR animals was accounted for by a marked reduction in the intermediate density lipoprotein/low density lipoprotein (IDL/LDL) fraction. Plasma VLDL, IDL/LDL, and HDL were isolated from the three groups of animals by ultracentrifugal flotation. In the AdmVLDLR animals, there was substantial loss (approximately 65%) of protein and cholesterol mainly in the IDL/LDL fraction on days 4 and 9. Nondenaturing gradient gel electrophoresis indicates a preferential loss of the IDL peak although the LDL peak was also reduced. When 125I-IDL was administered intravenously into animals on day 4, the AdmVLDLR animals cleared the 125I-IDL at a rate 5-10 times higher than the AdLacZ animals. We conclude that adenovirus-mediated transfer of the VLDLR gene induces high-level hepatic expression of the VLDLR and results in a reversal of the hypercholesterolemia in 0.2% cholesterol diet-fed LDLR (-/-, mice. The VLDLR overexpression appears to greatly enhance the ability of these animals to clear IDL, resulting in a marked lowering of the plasma IDL/LDL. Further testing of the use of the VLDLR gene as a therapeutic gene for the treatment of hypercholesterolemia is warranted.
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PMID:Reversal of hypercholesterolemia in low density lipoprotein receptor knockout mice by adenovirus-mediated gene transfer of the very low density lipoprotein receptor. 863 10

A sensitive and reproducible method for the routine mapping of oligosaccharides in a humanised immunoglobulin (IgG) is described. The method involves the enzymic release of intact glycans using the endoglycosidase glycopeptidase-F, and subsequent derivatisation with 1-phenyl-3-methyl-5-pyrazolone to facilitate analysis by high-performance liquid chromatography (HPLC). The heterogeneous oligosaccharide chains are separated by a phosphate buffer-acetonitrile gradient reversed-phase HPLC method and monitored by ultraviolet detection at 245 nm, allowing the detection of picomole amounts. A number of standard oligosaccharides are similarly derivatised to enable classification of the types of structures present from a comparison of retention times.
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PMID:High-performance liquid chromatographic mapping of the oligosaccharides released from the humanised immunoglobulin, CAMPATH 1H. 873 90

Based on complex formation of borate with carbohydrates in alkaline solutions, the oligosaccharide microheterogeneity of a monoclonal antibody was studied using capillary zone electrophoresis. In borate buffers characteristic separation patterns were found that could be attributed to the same antibody by their UV spectra, while in a phosphate buffer, under otherwise the same conditions, only a single peak was observed. N- and O-glycans were chemically hydrolyzed by trifluoromethane sulfonic acid, resulting in a completely deglycosylated protein; alternatively, N-glycans were enzymatically cleaved by incubation with peptide N-glycosidase F (PNGase F). In both approaches a changed antibody pattern was detected, indicating that the separation is due to carbohydrate microheterogeneity of the protein. Deglycosylation of the antibody by treatment with PNGase F was investigated by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A shift to lower molecular masses of approximately 1500 Da for the enzymatically treated protein, compared with the intact glycoprotein, was found. The separation method was validated for linearity and reproducibility of migration time and peak area and optimized in terms of buffer pH, capillary temperature and borate concentration. This technique is sensitive to analyze batch-to-batch consistency in production and to test the stability of galenical formulations. After antibody storage in glass vials for 3 months at 37 degrees C, the separation profile changed distinctly due to degradation at the carbohydrate or sialic acid moiety of the antibody, as indicated by MALDI-TOF-MS.
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PMID:Profiling of oligosaccharide-mediated microheterogeneity of a monoclonal antibody by capillary electrophoresis. 890 Sep 53

When crude extracts of Spirometra mansoni plerocercoid (sparganum) were analysed by SDS-polyacrylamide gel electrophoresis (PAGE)/immunoblot using patients' sera, IgE antibodies reacted specifically with 21, 27 and 53 kDa proteins. The 21 and 27 kDa proteins have been previously characterized as cysteine proteases. In this study, the 53 kDa protein was confirmed, by immunoprecipitation, to induce a specific IgE response. The protein was purified by affinity chromatography using an IgG1 (kappa 2) type mAb. The protein was partially sensitive to peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (endo F) digestion. It exhibited an endoproteinase activity in a thiol-dependent manner preferentially degrading benzoyloxycarboxyl-phenylalanyl-arginyl-4- methoxy-beta-naphthylamide (Z-phe-arg-MNA) of a panel of substrates tested. This endoprotease activity was maximal at pH 6.5 and in 0.1 M sodium phosphate. The proteolytic activity was inhibited by 10(-5) M L-trans-epoxysuccinyl-L- leucylamido-(4-guanidino)butane (E-64) and 1 mM iodoacetamide (IAA), and potentiated by dithiothreitol (DTT, 5 mM).
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PMID:A neutral cysteine protease of Spirometra mansoni plerocercoid invoking an IgE response. 907 45

A material of Mr 24,000 has been isolated from a cachexia-inducing mouse tumor (MAC16) and shown to initiate protein degradation in isolated gastrocnemius muscle. Biological activity was destroyed by preincubation with peptide N-glycosidase F (PNGase F) and endo-alpha-N-acetylgalactosaminidase (O-glycosidase) but not by neuraminidase or trypsin. Antibody reactivity was destroyed by treatment with periodate, indicating carbohydrate moieties to be the antigenic determinants. Antigenic activity was also reduced by treatment with PNGase F and O-glycosidase and was completely destroyed by treatment with chondroitinase ABC but was unaffected by treatment with either trypsin or chymotrypsin, confirming that the N- and O-linked sulfated oligosaccharide chains were both the antigenic and biological determinants. Biosynthetic labeling of MAC16 cells using a combination of [35S]sulfate and [6-3H]GlcN gave a single component of Mr 24,000 containing both radiolabels. Similar material could not be isolated from a cell line (MAC13) originating from a tumor that does not cause cachexia in vivo. Digestion of 3H/35S material with PNGase F produced two fragments of Mr 14,000 and 10,000 containing both radiolabels, and digestion with O-glycosidase produced three fragments of Mr 14,000, 6,000, and 4, 000, the first two contained both radiolabels and the third contained only 3H. Digestion of the fragment of Mr 14,000 released by PNGase F with O-glycosidase also gave fragments of Mr 6,000 and 4, 000. The products from both digestions were acidic as determined by anion exchange chromatography on DEAE-cellulose. The negative charge on the fragment of Mr 4,000 was removed by treatment with alkaline phosphatase. This suggests that the charge originated from phosphate residues, and this has been confirmed by biosynthetic labeling of MAC16 cells with [32P]orthophosphate, where radiolabel was incorporated into material of Mr 24,000 and into the fragment of Mr 4,000 after treatment with O-glycosidase. To determine the size of the polypeptide core MAC16 cells were biosynthetically labeled with L-[2,5-3H]His which after chemical deglycosylation produced a major component of Mr 4,000. These results suggest a model for the Mr 24, 000 material consisting of a central polypeptide chain of Mr 4,000 and with phosphate residues that may be attached to the polypeptide or a short oligosaccharide chain containing GlcN, one O-linked sulfated oligosaccharide chain containing GlcN, and of Mr 6,000 and one N-linked sulfated oligosaccharide chain of Mr 10,000 also containing GlcN. Neither chain was cleaved into disaccharides with chondroitinase ABC, suggesting that the material is a sulfated glycoprotein.
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PMID:Structural analysis of a tumor-produced sulfated glycoprotein capable of initiating muscle protein degradation. 913 70

Supernatants prepared from disrupted Coxiella burnetii possess acid phosphatase (ACP) activity that apparently accounts for the inhibition of the metabolic burst of formyl-Met-Leu-Phe(fMLP)-stimulated human neutrophils. Results are presented regarding purification and biochemical-biological characterization of the ACP. The highly purified enzyme, which exhibited an apparent M(r) of 91 K and optimal activity at pH 5.0, also inhibited neutrophils. The enzyme retained full activity at pH 4.5, 5.5, and 7.4, when incubated overnight at 0 degrees C and room temperature; at pH 5.5, it retained full activity after overnight incubation at 37 degrees C. Apparently, the enzyme contains asparagine-linked but not serine- or threonine-linked glycan residues since its treatment with N-glycosidase F (PNGase F) decreased its M(r) to 87 K and no changes were detected with O-glycosidase. The enzyme's capacity to hydrolyze phosphate from a number of phosphate-containing compounds was examined; five phosphocompounds were significantly hydrolyzed: 5'-CMP > fructose 1,6-diphosphate > tyrosine phosphate > 3'-AMP > 5'-AMP. The ACP also dephosphorylated (32)P-Raytide, a phosphotyrosine-containing peptide. Dephosphorylation of Raytide was inhibited by the following phosphatase inhibitors: sodium molybdate, potassium fluoride, sodium ortho-vanadate and D2, a heteropolymolybdate compound. These results indicate that C. burnetii ACP may play a role in disrupting tyrosine phosphorylation/dephosphorylation reactions associated with the signal transduction pathway culminating in the metabolic burst. Interestingly, Western blot analysis of ACP-inhibited neutrophils showed a marked increase in tyrosine phosphorylation of a 44 K protein as compared to uninhibited cells.
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PMID:Protein-tyrosine phosphatase activity of Coxiella burnetii that inhibits human neutrophils. 917 54

alpha-L-Iduronidase is a lysosomal hydrolase that is deficient in Hurler syndrome and clinically milder variants. Recombinant human alpha-L-iduronidase, isolated from secretions of an overexpressing Chinese hamster ovary cell line, is potentially useful for replacement therapy of these disorders. Because of the importance of carbohydrate residues for endocytosis and lysosomal targeting, we examined the oligosaccharides of recombinant alpha-L-iduronidase at each of its six N-glycosylation sites. Biosynthetic radiolabeling showed that three or four of the six oligosaccharides of the secreted enzyme were cleaved by endo-beta-N-acetylglucosaminidase H, with phosphate present on the sensitive oligosaccharides and L-fucose on the resistant ones. For structural analysis, tryptic and chymotryptic glycopeptides were isolated by lectin binding and reverse phase high pressure liquid chromatography; their molecular mass was determined by matrix-assisted laser desorption-time of flight mass spectrometry before and after treatment with endo- or exoglycosidases or with alkaline phosphatase. Identification of the peptides was assisted by amino- or carboxyl-terminal sequence analysis. The major oligosaccharide structures found at each site were as follows: Asn-110, complex; Asn-190, complex; Asn-336, bisphosphorylated (P2Man7GlcNAc2); Asn-372, high mannose (mainly Man9GlcNAc2, some of which was monoglucosylated); Asn-415, mixed high mannose and complex; Asn-451, bisphosphorylated (P2Man7GlcNAc2). The Asn-451 glycopeptide was unexpectedly resistant to digestion by N-glycanase unless first dephosphorylated, but it was sensitive to endo-beta-N-acetylglucosaminidase H and to glycopeptidase A. The heterogeneity of carbohydrate structures must represent the accessibility of the glycosylation sites as well as the processing capability of the overexpressing Chinese hamster ovary cells.
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PMID:Carbohydrate structures of recombinant human alpha-L-iduronidase secreted by Chinese hamster ovary cells. 927 35

We obtained two human cDNA clones encoding phosphatidic acid phosphatase (PAP) isozymes named PAP-2a (Mr = 32,158) and -2b (Mr = 35, 119), both of which contained six putative transmembrane domains. Both enzymes were glycosylated and cleaved by N-glycanase and endo-beta-galactosidase, thus suggesting their post-Golgi localization. PAP-2a and -2b shared 47% identical sequence and were judged to be the human counterparts of the previously sequenced mouse 35-kDa PAP(83% identity) and rat Dri42 protein (94% identity), respectively. Furthermore, the sequences of both PAPs were 34-39% identical to that of Drosophila Wunen protein. In view of the functions ascribed to Wunen and Dri42 in germ cell migration and epithelial differentiation, respectively, these findings unexpectedly suggest critical roles of PAP isoforms in cell growth and differentiation. Although the two PAPs hydrolyzed lysophosphatidate and ceramide-1-phosphate in addition to phosphatidate, the hydrolysis of sphingosine-1-phosphate was detected only for PAP-2b. PAP-2b was expressed almost ubiquitously in all human tissues examined, whereas the expression of PAP-2a was relatively variable, being extremely low in the placenta and thymus. In HeLa cells, the transcription of PAP-2a was not affected by different stimuli, whereas PAP-2b was induced (up to 3-fold) by epidermal growth factor. These findings indicate that despite structural similarities, the two PAP isozymes may play distinct functions through their different patterns of substrate utilization and transcriptional regulation.
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PMID:Cloning and characterization of two human isozymes of Mg2+-independent phosphatidic acid phosphatase. 930 23


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