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 report a carbohydrate-dependent supramolecular architecture in the extracellular giant hemoglobin (Hb) from the marine worm Perinereis aibuhitensis; we call this architectural mechanism carbohydrate gluing. This study is an extension of our accidental discovery of deterioration in the form of the Hb caused by a high concentration of glucose. The giant Hbs of annelids are natural supramolecules consisting of about 200 polypeptide chains that associate to form a double-layered hexagonal structure. This Hb has 0.5% (wt) carbohydrates, including mannose, xylose, fucose, galactose, glucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). Using carbohydrate-staining assays, in conjunction with two-dimensional polyacrylamide gel electrophoresis, we found that two types of linker chains (L1 and L2; the nomenclature of the Hb subunits followed that for another marine worm, Tylorrhynchus heterochaetus) contained carbohydrates with both GlcNAc and GalNAc. Furthermore, two types of globins (a and A) have only GlcNAc-containing carbohydrates, whereas the other types of globins (b and B) had no carbohydrates. Monosaccharides including mannose, fucose, glucose, galactose, GlcNAc, and GalNAc reversibly dissociated the intact form of the Hb, but the removal of carbohydrate with N-glycanase resulted in irreversible dissociation. These results show that carbohydrate acts noncovalently to glue together the components to yield the complete quaternary supramolecular structure of the giant Hb. We suggest that this carbohydrate gluing may be mediated through lectin-like carbohydrate-binding by the associated structural chains ("linkers").
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PMID:Carbohydrate gluing, an architectural mechanism in the supramolecular structure of an annelid giant hemoglobin. 763 98

The carbohydrate moiety of the envelope glycoprotein gp51 of bovine leukemia virus, American strain, was studied. The virus was grown in ovine, bovine, porcine, bat and rat cells of various organ specificities. The gp51 was purified by immunoaffinity chromatography from virions of ten different virus-producing cells derived from various body organs of different species. Highly purified glycoproteins (single band in PAGE) were compared for their electrophoretic mobility, for the presence of epitopes by a battery of monoclonal antibodies, and for the glycosylation pattern by lectin blot analysis. Electrophoretic analysis of all tested glycoproteins deglycosylated by glycopeptidase F detected the same polypeptide backbone according to PAGE. The glycoproteins produced in rat cells migrated faster in PAGE, as detected in cells or in virions, than those produced in ovine cells. The pattern of their glycosylation was found to be dependent on the type of cells used for virus production. The differences in glycosylation were most pronounced when comparing the glycoprotein produced in ovine cells versus bat or rat cells. Changes in epitope expression were also detected. The differences in the patterns of glycosylation and in the accessibility of epitopes owing to the virus production in various kind of cells are discussed from virus infectivity and vaccine points of view.
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PMID:Envelope glycoprotein gp51 of bovine leukemia virus is differently glycosylated in cells of various species and organ origin. 768 44

Mannoproteins of approximately 50 kDa from blastoconidia and 60 kDa from hyphae of Candida albicans reacted in Western blots (immunoblots) with either a polyclonal rabbit antiserum (CA-7) or a monoclonal antibody (CA-A) to the C. albicans C3d-binding protein (complement receptor type 2). The glycosylated nature of these proteins was demonstrated by their reactivity with concanavalin A and by selective labeling with the biotin-hydrazide reagent following periodate oxidation. Differences in the oligosaccharides of these proteins were observed in regard to their reactivity with lectin-peroxidase reagents and sensitivity to glycosidases such as N-glycanase or endoglycosidase F (but not endoglycosidase H). The 60-kDa mannoprotein reacted with wheat germ agglutinin, while the 50-kDa mannoprotein did not. Treatment of the 60-kDa mannoprotein with the glycosidases mentioned above resulted in its conversion into a species of 40 to 45 kDa. Enzyme treatment had no obvious effect on the electrophoretic mobility of the 50-kDa species from blastoconidia. Both the 50- and 60-kDa glycoproteins remained immunoreactive after treatment with the glycosidases. Reactivities of the two mannoproteins to neuraminidase also differed. Finally, the 50-kDa (blastoconidia) and the 60-kDa (hyphae) mannoproteins were purified by using ion-exchange chromatography and electroelution. The purified proteins differed in net charge, the 60-kDa species having a more acidic pI. Functional activity of the purified mannoproteins was demonstrated, as each inhibited the rosetting of antibody-sensitized sheep erythrocytes conjugated with iC3b or C3d by hyphae. Thus, an epitope(s) common to both a mycelial and blastoconidial mannoprotein is associated with a structurally different oligosaccharide for each growth form.
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PMID:Analysis of mannoproteins from blastoconidia and hyphae of Candida albicans with a common epitope recognized by anti-complement receptor type 2 antibodies. 769 55

Among a panel of mouse mAbs generated to a human T cell clone, one mAb, V7.1, inhibited T cell activation in the mixed lymphocyte reaction and was studied further. V7.1 reacted strongly with Ag-specific T cell clones, in addition to freshly isolated monocytes and granulocytes. However, the mAb reacted weakly with freshly isolated PBLs (T cells, B cells, and NK cells), T cells stimulated with phytohemagglutinin, or Con A, and did not stain the vast majority of transformed cell lines of hemopoietic origin. Stimulation of T cells with anti-CD3, or the combination of anti-CD3 and PMA, or anti-CD3, PMA and ionomycin, markedly increased V7.1 surface staining. The mAb precipitated a single polypeptide chain of approximately 135 kDa from alloactivated T cells or monocytes, which was reduced to approximately 110 kDa after treatment with N-glycanase. The proliferative response of T cells to allogeneic monocytes or B lymphoblastoid cells was inhibited by V7.1, and inhibition was maximal when the mAb was present at the initiation of culture. V7.1 also exhibited dose-dependent inhibition of the T cell response to immobilized anti-CD3 Ab in the absence of APCs, indicating that the inhibitory effect of this Ab occurs at the T cell level. Expression of CD25 (IL-2R) on anti-CD3-activated T cells and secretion of IL-2 induced with anti-CD3 and PMA were inhibited by V7.1, whereas the Ab had no effect on T cell proliferation induced by PHA or Con A or on T cell-mediated cytotoxicity. These results indicate that V7.1 recognizes a novel leukocyte surface glycoprotein, designated V7, that is up-regulated on Ag but not lectin-activated T cells, and appears to play a role in TCR/CD3-dependent T cell activation. In an accompanying study, the gene encoding the V7 Ag is described and the molecule is shown to be a novel member of the Ig superfamily.
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PMID:V7, a novel leukocyte surface protein that participates in T cell activation. I. Tissue distribution and functional studies. 772 99

A 56K protein co-purified with bovine milk fat globule membrane (MFGM) proteins bound to Wisteria floribunda agglutinin (WFA) like most MFGM glycoproteins. Treatment with N-glycanase or beta-N-acetylhexosaminidase abolished the lectin binding to the protein. Amino acid sequence and immunoblot analyses revealed that the 56K protein is an IgG heavy chain. Lectin column chromatography of the oligosaccharides released by hydrazinolysis from the purified IgG heavy chains revealed that 0.08% of the total N-linked sugar chains bind to a WFA-agarose column, suggesting that they contain the beta-N-acetylgalactosaminylated structure.
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PMID:A bovine IgG heavy chain contains N-acetylgalactosaminylated N-linked sugar chains. 775 1

The isolation and partial characterization of PAS-4 glycoprotein (78 kDa) from bovine milk fat globule membrane (MFGM) is described. PAS-4 was selectively extracted with Triton X-114 nonionic detergent and then fractionated on DEAE-Sepharose at pH 7.5. The PAS-4 fraction that was not bound on DEAE-Sepharose gave a single band by SDS-PAGE. The recovery of PAS-4 was 57.4% from MFGM. An amino acid analysis found a high percentage of nonpolar residues. Approximately 7.2% of carbohydrate from PAS-4 was composed of mannose, galactose (Gal), N-acetylglucosamine, N-acetylgalactosamine (GalNAc), and sialic acid, most of the Gal and GalNAc in PAS-4 being released after mild alkaline hydrolysis. This indicated that PAS-4 contained both N- and O-linked sugar chains in concordance with the results of lectin affinity. PAS-4 had apparent isoelectric points of 7.45, 7.41, and 7.32, but these were shifted to pI 7.47 by a neuraminidase treatment. The apparent molecular weight of PAS-4 after deglycosylation with N-glycanase was approximately 57,000 by SDS-PAGE.
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PMID:Rapid and simple procedure for purifying PAS-4 glycoprotein from bovine milk fat globule membrane. 778 99

Envelope glycoproteins of human immunodeficiency virus (gp120 and gp41) occur as oligomers. Here, we show by gel filtration analysis that gp120 oligomerization in vitro is calcium- and temperature-dependent. Recombinant gp120 (rgp120) species were recovered as monomers at 20 degrees C in the absence of calcium, but as tetramers at 37 degrees C in 10 mM CaCl2. Under the latter condition, N-glycanase-deglycosylated rgp120 formed hexamers. Relative to intact rgp120, which has been reported to display carbohydrate-binding properties for N-acetyl-beta-D-glucosaminyl and mannosyl residues, deglycosylation enhanced rgp120 specific binding to mannose-divinylsulfone-agarose, para-aminophenyl-beta-D-GlcNAc-agarose and fetuin-agarose matrices. Taken together, these results rule out the role of homologous lectin-carbohydrate interactions via N-linked glycans in the rgp120 oligomerization, even though its lectin properties may also be calcium-dependent. Deglycosylation may unmask domains of rgp120 polypeptide backbone that independently play a role either in rgp120 lectin activity or in calcium-dependent oligomerization.
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PMID:The role of calcium and N-linked glycans in the oligomerization and carbohydrate binding properties of human immunodeficiency virus external envelope glycoprotein. 780 9

Previous studies from this laboratory have identified rat epididymal luminal fluid acid beta-D-galactosidase activity which also optimally hydrolyses a glycoprotein substrate at neutral pH [Skudlarek, Tulsiani and Orgebin-Crist (1992) Biochem. J. 286, 907-914]. We have now separated the luminal fluid beta-D-galactosidase into two molecular forms by ion-exchange chromatography on a column of DE-52. The separated enzyme activities were purified to an apparent homogeneity by molecular-sieve chromatography followed by affinity chromatography on a column of immobilized p-nitrophenyl beta-D-thiogalactopyranoside. The purified forms, when resolved by SDS/PAGE under reducing conditions, showed apparent molecular masses of 84 and 97 kDa. Kinetic studies, including a pH-dependent substrate preference and pH-dependent association/dissociation, disclosed no differences between these two forms. The two forms had identical N-terminal amino acid sequences. However, the 97 kDa form contained much more total carbohydrate and sialic acid than the 84 kDa form. The carbohydrate moieties in the two forms were assessed by comparing their size on SDS/PAGE before and after treatment with endo-enzymes. The removal of N-linked glycans by treatment with N-glycanase or endoglycosidase F generated de-N-glycosylated polypeptides of an apparent molecular mass of 70 kDa, and indicated that the two forms contained varying amounts of asparagine (N)-linked high mannose/hybrid-type and biantennary complex-type oligosaccharides. This result and the fact that the two molecular forms had identical N-terminal amino acid sequences indicated that the two forms probably have identical or very similar polypeptides. The potential role of the enzyme in modification of sperm plasma membrane (PM) glycoproteins was examined by resolving caput sperm PM proteins (before and after treatment in vitro of the membranes with the purified beta-D-galactosidase) on SDS/PAGE, followed by staining with peanut agglutinin (PNA), a lectin which preferentially binds to Gal beta 1,3GalNAc-linkages found in O-linked glycoproteins. The evidence presented in this report has indicated that a PNA-positive glycoprotein of an apparent molecular mass of 135-150 kDa present on the caput (but not cauda) sperm PM is degalactosylated by the digestion in vitro of the membranes with purified luminal fluid beta-D-galactosidase. This result suggests a possible role for the epididymal luminal fluid beta-D-galactosidases.
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PMID:Purification and characterization of two forms of beta-D-galactosidase from rat epididymal luminal fluid: evidence for their role in the modification of sperm plasma membrane glycoprotein(s). 782 52

Myelomonocytic lineage cells express an M(r) 60,000 mannose specific lectin, MR60 (Pimpaneau et al. (1991), Carbohydr Res 213: 95-108). Under non-reducing conditions, this protein migrates as a 120,000 protein. MR60 does not contain any N-glycan moiety cleavable by the action of N-glycanase. MR60 induces a sugar selective aggregation of beads coated with glycosylated albumin: beads bearing alpha-D-mannosyl residues are aggregated while beads bearing alpha-D-glucosyl residues are not. A monoclonal antibody Lec101B, specific for MR60, recognizes a single M(r) 60,000 protein by Western blotting. This monoclonal antibody does not label the cell surface of cells expressing MR60, but decorates intracellular vesicles upon permeabilization of these cells.
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PMID:Characterization and cellular localization by monoclonal antibodies of the 60 kDa mannose specific lectin of human promyelocytic cells, HL60. 787 29

We have previously isolated and characterized two dengue (DEN) 2 viruses mutant in their fusion-from-within (FFWI) phenotype in the insect cell line C6/36. Both viruses lost a potential glycosylation site (Asn-153) in the envelope (E) glycoprotein. To determine whether the change in FFWI phenotype was due to a change in E-glycoprotein glycosylation, we characterized the patterns of glycosylation on the E-glycoprotein of wild-type DEN 1 and DEN 2 viruses. The E-glycoproteins were isolated from purified virus grown in Aedes albopictus C6/36 cells, by use of high-performance size-exclusion chromatography. The tryptic maps of wild-type glycosylated and enzymatically (PNGase F) deglycosylated E-glycoproteins were compared by reverse-phase high-performance liquid chromatography. The DEN 1 virus E-glycoprotein was found to have two peaks in the tryptic map that exhibited shifts after deglycosylation, whereas the DEN 2 virus E-glycoprotein had only one. Besides the potential glycosylation site at Asn-153, both DEN 1 and DEN 2 virus E-glycoproteins have another potential site located at Asn-67. Amino-terminal sequencing of the shifted peaks revealed that DEN 2 virus E-glycoprotein is glycosylated only at Asn-67; however, DEN 1 virus E-glycoprotein is glycosylated at both Asn-67 and Asn-153. These DEN virus serotypes are thus heterogeneous in their use of glycosylation sites. We also determined by a lectin-binding assay that the attached carbohydrates for both viruses were likely to be of the high-mannose type.
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PMID:The envelope glycoproteins of dengue 1 and dengue 2 viruses grown in mosquito cells differ in their utilization of potential glycosylation sites. 805 48


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