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 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

The soluble histo-blood group A glycosyltransferase (Fuc alpha 1----Gal alpha 1----3-N-acetylgalactosaminyltransferase) was purified approximately 600,000-fold to homogeneity from human lung tissue. The enzyme was solubilized in 1% Triton X-100, partially purified by affinity chromatography on Sepharose 4B, and eluted with UDP. Final purification was obtained by twice repeated fast protein liquid chromatography ion exchange (Mono STM) with NaCl gradient elution and reverse-phase chromatography (proRPC) with acetonitrile gradient elution. Identity of the purified protein was established by (i) demonstration of the putative A transferase protein only in affinity-purified extracts of A but not O individuals, and (ii) specific immunoprecipitation of enzyme activity and putative protein with monoclonal antibodies. Sodium dodecyl sulfate electrophoresis revealed a single protein band with apparent Mr of approximately 40,000 under both reducing and nonreducing conditions. Digestion with N-glycanase yielded a reduction in Mr of approximately 6,000 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), suggesting that the A transferase is a glycoprotein with N-linked carbohydrate chains. Amino acid composition and N-terminal amino acid sequence of the intact transferase, as well as of peptides released by endolysyl peptidase digest or cyanogen bromide cleavage, are presented.
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PMID:Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase from human lung tissue. 210 27

A 3,000-base pair EcoRI fragment containing the Flavobacterium meningosepticum gene for peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was cloned into the Bluescript plasmid vector and expressed in Escherichia coli. The gene consists of an open reading frame of 1,062 base pairs coding for a 354-amino acid protein; the first 40 amino acids are presumed to be the natural secretory signal sequence, with the remaining 314 amino acids (34,779 Da) representing the catalytically active protein. The deduced amino acid sequence was verified independently by direct microsequencing of over 94% of the pure protein (Flavobacterium peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase) as tryptic and cyanogen bromide peptides. Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was not secreted by E. coli; molecular weight analysis of the partially purified recombinant enzyme suggested incomplete processing of the putative leader sequence.
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PMID:Molecular cloning and amino acid sequence of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from flavobacterium meningosepticum. 218 34

Two protein antigens were isolated from excretory-secretory products of Trichinella spiralis by biochemical methods and characterized with respect to their chemical and immunological properties. One antigen, of apparent Mr 43,000, is an abundant secreted protein of infective L1 larvae, while the other, of 45-50 kDa, is present in smaller amounts. Yields, extinction coefficients, isoelectric points, amino acid compositions, and partial N-terminal amino acid sequences for each are reported. Partial amino acid sequences of peptides derived from the 43-kDa protein by cyanogen bromide cleavage have been determined. Treating a reduced-pyridylethylated derivative of the 43-kDa protein with glycopeptidase F (N-glycanase) resulted in formation of a transient product of 37 kDa followed by a stable polypeptide of 32 kDa (by SDS-PAGE), suggesting the presence of two N-linked carbohydrate groups. A similar result was obtained with the 45-50-kDa protein, which gave a transient doublet of 38 and 40 kDa and a final, stable product of 33 kDa, with a minor component of 35 kDa. Two glycosylation sites of the 43-kDa protein and one site of the 45-50-kDa protein can be identified in the amino acid sequences. Polyclonal antibodies prepared against the two proteins cross-reacted extensively, but failed to react with the doubly deglycosylated polypeptides in Western blots. The dominant epitopes present in the reduced-pyridylethylated polypeptides are, therefore, N-linked carbohydrate, although the presence of peptide epitopes in the native proteins cannot be excluded.
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PMID:Partial characterization of two antigens secreted by L1 larvae of Trichinella spiralis. 239 16

Recombinant murine interleukin-4 (muIL-4) expressed in COS-7 monkey kidney cells was purified to homogeneity by sequential CM-Sepharose, Sephadex G-100 chromatography and mono-S FPLC to a specific activity of 6.10(7) units per mg of protein based on an in vitro HT-2 cell proliferation assay. Two electrophoretic variants, designated a and b, which migrated on SDS-PAGE as a closely spaced doublet with Mr 19,000, were present in the final product. Gas phase sequencing of the purified protein revealed the presence of an N-terminus corresponding to the mature protein predicted from the cDNA sequence and sequencing of a cyanogen bromide digest confirmed 75 of the 120 predicted amino acids. Elution behavior on gel filtration corresponded to that of a monomer of Mr 19,000. Since there are three potential sites of N-glycosylation predicted by the cDNA sequence, the contribution of glycosylation to the observed heterogeneity was examined by treatment with endoglycosidases. Variant b was digested by either endo-beta-N-acetylglucosaminidase H (endo H) or endo-beta-N-acetylglucosaminidase F (endo F) to protein of Mr 15,000 on SDS-PAGE but was unaffected by treatment with endo-beta-N-acetylglucosaminidase D (endo D), thus indicating the presence of high mannose type of N-glycan. In contrast, variant a was resistant to endo H, F and D. Complete conversion of a mixture of variants a and b to a single protein of Mr 15,000 on SDS-PAGE was obtained only after treatment with N-glycanase. Both variants were resistant to neuraminidase and O-glycanase treatment. These data show that the microheterogeneity observed in purified muIL-4 preparations is due to differences in the nature of the N-linked oligosaccharides. The availability of purified recombinant muIL-4 and a methodology for both total and selective deglycosylation provides a basis for the initiation of structure-function studies of this novel T-cell lymphokine.
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PMID:Multiple forms of recombinant murine interleukin-4 expressed in COS-7 monkey kidney cells. 278 92

The low molecular weight proteoglycan fraction extracted from articular discs with 4 M guanidinium chloride was found to consist predominantly of an iduronate-rich dermatan sulphate proteoglycan, together with chondroitin sulphate-containing material. The dermatan sulphate proteoglycan was purified by ion-exchange and gel-filtration chromatography and its core protein isolated after digestion with chondroitinase ABC. The amino acid composition and pattern of cyanogen bromide peptides obtained from this core were closely similar to those of the protein core of bovine skin proteodermatan sulphate. Four monoclonal antibodies raised against bovine skin proteodermatan sulphate also reacted with the disc protein core and its cyanogen bromide peptides. Results of digestion with glycopeptidase F demonstrated the presence of three N-linked oligosaccharides. The combined size of these oligosaccharides appeared to be somewhat less than the size of those on skin proteodermatan sulphate. The glycosaminoglycan chain released by digestion with cathepsin C had a higher molecular weight than that from skin. These differences in glycosylated structures may be responsible for the different effects on collagen fibrillogenesis in vitro; whereas skin proteodermatan sulphate only reduced the rate of fibril growth, disc dermatan sulphate proteoglycan also increased the length of the lag-phase and the final opacity.
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PMID:Proteoglycans of the articular disc of the bovine temporomandibular joint. II. Low molecular weight dermatan sulphate proteoglycan. 279 47

Galactosyltransferase (GT) (EC 2.4.1.38) was purified to homogeneity from human ovarian tumor effusion fluid and normal human serum by chromatography on alpha-lactalbumin and anti-human immunoglobulin affinity (to selectively absorb contaminating IgG) columns. Both preparations showed a single, broad band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis centered at a molecular weight of 48,000, but nondenaturing polyacrylamide gel electrophoresis of GT isolated from tumor effusion fluid revealed the presence of a series of oligomeric proteins possessing GT activity, which were barely detectable in normal human serum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of N-glycanase- and O-glycanase-treated GT revealed that each endoglycanase removed carbohydrate with an approximate molecular weight of 3,000, revealing the presence of both N-linked and O-linked oligosaccharide substitutions on GT. Purified GT (containing a mixture of GT isoenzymes) was used to immunize BALB/c mice for monoclonal antibody (MAb) preparation. Four of the MAb isolated reacted with GT. MAb 3872 (patent pending; an IgG1) was determined to be specific for a cancer-associated GT isoenzyme (GT-II) by immunostaining of Western blots and nondenaturing polyacrylamide gel electrophoresis of GT specifically eluted from a MAb 3872 affinity column. Two 125I-labeled cyanogen bromide peptides (Mr 8,400 and 7,400) prepared from 125I-GT were specifically bound and eluted from a MAb 3872 affinity column, demonstrating that the MAb 3872 GT-II-specific antigenic epitope resides on these peptides. MAb 3872 was immobilized on 1,1'-carbonyldiimidazole-activated trisacryl GF-2000 and used to specifically assay serum GT-II levels in 29 individual normal human serum samples and 77 serum samples from 38 patients with advanced ovarian tumors. The normal serum GT-II level was found to be 85.3 +/- 30.9 milliunits/ml, with a range of 17 to 160 milliunits/ml. Of the 38 tumor patients, 33 showed GT-II values in excess of 200 milliunits/ml, with a range of 216 to 8,469 milliunits/ml. Serial samples obtained from the ovarian tumor patients suggested that the serum GT-II level reflected the tumor burden of the patient.
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PMID:Characterization and immunoassay of human tumor-associated galactosyltransferase isoenzyme II. 313 19

A hybrid heterodimeric alkaline phosphatase expressed in KB cells, consisting of placental and intestinal (fetal) subunits, was purified by use of two different immunoaffinity columns using the monoclonal antibodies 2HIMS-1 and HPMS-1. The closely related subunits were found to yield a dimeric active enzyme glycosylated as the mature heterodimeric forms. This enzyme displays intermediate properties to the placental and intestinal (fetal) isozymes with regard to heat stability, inhibition patterns with amino acids and amino acid derivatives, as well as reactivity with monoclonal antibodies specific for human alkaline phosphatase isozymes. Peptide fragments obtained from the hybrid enzyme after cyanogen bromide cleavage belong to either the placental or intestinal (meconial) isozyme as evaluated by SDS polyacrylamid gel electrophoresis, and the N-terminal amino acid sequences, corresponding to the placental and intestinal subunits, can be identified in the peptide fragments. By N-glycanase digestion or tunicamycin treatment, the molecular mass of the subunits was reduced to 62 kDa compared to 69 kDa for the native ones. The results confirm that some cell lines can synthesize hybrid alkaline phosphatases.
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PMID:Expression of a heterodimeric (placental-intestinal) hybrid alkaline phosphatase in KB cells. 801 16

We report the complete structures of the N-linked oligosaccharides and the site-specificity of the N-glycosylation of recombinant gp120 (rgp120) of the HIV-1 BH8 isolate produce by a baculovirus expression system. Glycopeptides derived from the tryptic digests of intact rgp120 or of cyanogen bromide-generated fragments of rgp120 were isolated by their binding to concanavalin A-Sepharose and were purified by reversed-phase HPLC. The isolated glycopeptides were treated with PNGase F, releasing the carbohydrate moiety while converting Asn to Asp, and identified by amino acid analysis and/or peptide sequencing. Our results indicate that all 22 potential N-glycosylation sites in the rgp120 sequence are utilized. We did not detect N-acetylgalactosamine in rgp120, indicating that the glycoprotein lacks typical O-linked oligosaccharides. To investigate the oligosaccharide structures at the sites of glycosylation, we determined the carbohydrate composition for each site and characterized the oligosaccharides by 1H-NMR spectroscopy and by oligosaccharide mapping using high pH anion-exchange chromatography. Mannose and N-acetylglucosamine were the only sugars observed in the intact rgp120 and likewise in individual glycopeptides. All glycopeptides derived from rgp120 contained high mannose-type N-linked oligosaccharides, ranging from GlcNAc2Man5 to GlcNAc2Man9. However, different glycosylation sites showed varied degrees of processing of the high mannose-type oligosaccharides, as characterized by the ratio of GlcNAc2Man8-9 to GlcNAc2Man5-7. These results demonstrate that N-glycosylation of rgp120 in the baculovirus expression system occurs at all potential sites and is site specific in terms of oligosaccharide structures.
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PMID:Site-specific N-glycosylation and oligosaccharide structures of recombinant HIV-1 gp120 derived from a baculovirus expression system. 821 72

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).
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PMID:The major site of photoaffinity labeling of the gamma-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the alpha subunit. 862 79


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