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)

Mouse teratocarcinoma F9 cells were induced to primitive endoderm differentiation with retinoic acid, and poly-N-acetyllactosamine-containing surface glycoproteins were identified by radiolabelling endo-beta-galactosidase-cleavable glycans with galactosyltransferase and radiolabelled UDP-galactose. One major radiolabelled band with an apparent size of 250-500 kDa was identified which differed from the known poly-N-acetyllactosamine-containing glycoproteins laminin, fibronectin, lysosome-associated membrane protein (LAMP)-1 and LAMP-2. This acidic glycoprotein, resistant to glycosaminoglycan-degrading enzymes and proteases, was purified by extraction and phase partition with Triton X-114, octyl Sepharose and Helix pomatia lectin chromatography. The purified glycoprotein could be digested by endo-beta-galactosidase and glycopeptide N-glycosidase F to an apparent size of 160-240 kDa. During retinoic-acid-induced differentiation into primitive endoderm cells, the glycoprotein showed a several-fold increase and a broadening to an apparent size of 200- > 700 kDa. The glycoprotein was no longer detected in retinoic-acid and dibutyryl-cAMP-treated cells which had undergone further differentiation to parietal endoderm cells, nor in the permanently differentiated parietal endoderm line F9-AC. The results suggest that the glycoprotein is a major carrier of poly-N-acetyllactosamine chains on differentiating teratocarcinoma F9 cells, and that its expression as revealed by the poly-N-acetyllactosamine labelling method is regulated by the stage of cellular differentiation.
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PMID:Identification of a major poly-N-acetyllactosamine-containing cell-surface glycoprotein of mouse teratocarcinoma cells. Appearance on cells induced to primitive endoderm but not parietal endoderm differentiation. 812 95

We report our discovery that many glycoproteins synthesized by Chinese hamster ovary (CHO) cells contain fucose in O-glycosidic linkage to polypeptide. To enrich for the possible presence of O-linked fucose, we studied the lectin-resistant mutant of CHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferase I and are therefore unable to synthesize complex-type N-linked oligosaccharides. Lec1 cells were metabolically radiolabelled with [6-3H]fucose and total glycoproteins were isolated. Glycopeptides were prepared by proteolysis and fractionated by chromatography on a column of concanavalin A (Con A)-Sepharose. The sets of fractionated glycopeptides were treated with mild base/borohydride to effect the beta-elimination reaction and release potential O-linked fucosyl residues. The beta-elimination produced [3H]fucitol quantitatively from [3H]fucose-labelled glycopeptides not bound by Con A-Sepharose, whereas none was generated by treatment of glycopeptides bound by the lectin. The total [3H]fucose-labelled glycoproteins from Lec1 cells were separated by SDS-PAGE and detected by fluorography. Treatment of selected bands of detectable glycoproteins with mild base/borohydride quantitatively generated [3H]fucitol. Pretreatment of the glycoproteins with N-glycanase prior to the SDS-PAGE method of analysis caused an enrichment in the percentage of radioactivity recovered as [3H]fucitol. Trypsin treatment of [3H]fucose-labelled intact CHO cells released glycopeptides that contained O-linked fucose, indicating that it is present in surface glycoproteins. These findings demonstrate that many glycoproteins from CHO cells contain O-linked fucosyl residues and raise new questions about its biosynthesis and possible function.
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PMID:O-linked fucose in glycoproteins from Chinese hamster ovary cells. 813 Mar 91

The angiotensin II receptors of human myometrial tissue were characterized using ligand binding, cross-linking with radioactive label, detergent solubilization and partial purification by lectin-affinity chromatography. Human myometrial membrane preparations contained variable amount (5-650 fmol/mg protein) of high affinity (Kd = 44-65 pM) binding sites for 125I-CGP42112, a ligand specific for the AT2 subtype of angiotensin II receptors. Competition studies with AT1-specific and AT2-specific compounds indicated that angiotensin II receptors on these membranes were exclusively of the AT2 subtype. The binding sites for 125I-CGP42112 were efficiently solubilized by the detergent Chaps, albeit with a marked decrease in affinity (Kd = 1.2 nM). The proteins in the myometrial membrane preparation were cross-linked to 125I-[Sar1, Ile8]angiotensin II (Sarile) with disuccinimidyl suberate. When low concentrations of cross-linker were used, a single radiolabelled band of about 66-70 kDa was revealed on SDS/PAGE. At higher concentrations additional bands of about 105-120 kDa and 200 kDa were labelled. The 66-70-kDa and 105-120-kDa bands were separated by electroelution of SDS/PAGE gel slices and submitted to trypsin cleavage. The tryptic-peptide maps were identical for both products, suggesting that the additional bands are homodimers and trimers of the labelled polypeptide. The Chaps-solubilized receptor was retained on wheat-germ-agglutinin-Sepharose and specifically eluted by the competing sugar triacetylchitotriose, leading to a fivefold purification factor. Treatment of the 125I-Sarile-labelled protein with N-glycanase caused a shift in its apparent molecular mass on SDS/PAGE from 66-70 kDa to 33 kDa.
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PMID:Characterization of a membrane glycoprotein having pharmacological and biochemical properties of an AT2 angiotensin II receptor from human myometrium. 814 46

Cranin is a 120 kDa integral membrane glycoprotein which binds laminin under conditions of physiologic ionic strength in a calcium-dependent manner. Here, binding of cranin to laminin has been characterized using both ligand-blotting assays and laminin affinity bead assays. Binding was specifically inhibited by anti-laminin antibodies against the A chain terminal domain G, but not by several other region-specific antibodies. Dextran sulfate, fucoidin, and sulfatide were potent inhibitors of binding (50% inhibition at 0.03, 0.5, and 1.7 micrograms/ml, respectively); heparin was a weaker inhibitor (50% inhibition approximately 5 micrograms/ml), and mannan and chondroitin sulfate were not inhibitory at 100 micrograms/ml. Binding was not inhibited by lactose or the A chain peptide PA22-2. The mobility of the broad, fuzzy cranin band was shifted after digestion with neuraminidase, N-glycanase, and O-glycanase, though none of these treatments decreased band heterogeneity nor destroyed the ability to bind laminin. Cranin bound to Jacalin lectin, which recognizes the Gal beta 1-3GalNAc linkage expressed in certain classes of mucins. These findings indicate that cranin binds at or near the high affinity sulfatide-binding site previously mapped to the E3 domain of laminin, which is known to exhibit bioactivity for neural cells. In view of the extremely low abundance of cranin in brain membranes (approximately 0.005%), its avid laminin-binding activity is remarkable, and strongly suggests that cranin may play a physiologic role in regulating specific neural cell interactions.
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PMID:Cranin interacts specifically with the sulfatide-binding domain of laminin. 814 89

New sialic acid-specific lectin has been isolated from culture supernatant of the protozoan Tritrichomonas mobilensis. It was purified by adsorption by erythrocytes or bovine submaxillary gland mucin (BSM)-Sepharose affinity chromatography. The T. mobilensis lectin (TML) does not require bivalent cations for activity and agglutinates all human erythrocytes. The lectin forms multimeric complexes with molecular mass 556 and 491 kDa as determined by size-exclusion chromatography. SDS/PAGE under reducing conditions disclosed a large band of 343 kDa and three bands of 246, 265 and 286 kDa which, after denaturation with urea, were split into three subunits of 56, 61 and 66 kDa; under non-reducing conditions there were two bands, of 360 and 260 kDa. Western blots performed with anti-TML monoclonal antibodies revealed bands identical with those in the silver-stained gels, suggesting homogeneity of the BSM -Sepharose-purified lectin. TML is a highly glycosylated protein with approx. 8% of N-linked glycosides found by protein-N-glycanase F treatment; the total amount of saccharides revealed by chemical deglycosylation was 20%. Haemagglutination-inhibition studies documented exclusive specificity for sialic acid (NeuAc). Both (alpha 2-->6)- and (alpha 2-->3)-linked and free NeuAc were eight times more potent inhibitors than N-glycolylneuraminic acid. The lectin does not require O-acetyl groups on NeuAc for recognition. A spectrum of mono- and oligo-saccharides other than sialic acid had no inhibitory effect at 200 mM. Anti-TML monoclonal antibodies strongly inhibited the lectin activity. TML was stable at temperatures below 4 degrees C and lyophilized with 3% (w/w) glycerol.
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PMID:Purification and characterization of a sialic acid-specific lectin from Tritrichomonas mobilensis. 817 92

In vitro released products of the adult stage of the bovine lungworm. Dictyocaulus viviparus, were characterized according to their SDS-PAGE profile, glycosylation pattern, in vitro synthesis and antigenicity in the context of infection and vaccination with irradiated larvae. Biosynthetic labelling experiments with 35S-methionine indicated active synthesis of ES throughout this time. There was, however, little incorporation of 3H-glucosamine into ES products, and lectin affinity chromatography and glycopeptidase F digestion identified only one glycosylated component. Immunoprecipitation of 125I-labelled ES products with sera from calves patently infected with D. viviparus demonstrated that all of these, with the exception of two components, are antigenic to the bovine host. One of those not immunoprecipitated was shown to be host serum albumin carried over into culture. A limited degree of cross-reactivity between nematode species was observed, with a D. viviparus female-specific antigen of 290 kDa being recognized by serum antibody from calves infected with the gastrointestinal nematodes Cooperia oncophora and Ostertagia ostertagi. Calves vaccinated with irradiated larvae of D. viviparus, despite not being exposed to the adult stage of the parasite, also showed some recognition of adult ES products. This might suggest that vaccination with irradiated larvae operates against both pre-pulmonary and pulmonary stages of the infection.
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PMID:Characterization of excretory-secretory products of adult Dictyocaulus viviparus and the antibody response to them in infection and vaccination. 831 10

In an approach to examine the lectin-hypothesis in the pathogenesis of coeliac disease, the presence of lectin-like components in three wheat gluten preparations known to induce coeliac disease, gliadin, Frazer fraction III and an acetic acid/ethanol extract of gluten, was investigated. Lectin-like components in these wheat gluten preparations were traced in binding studies employing a variety of model glycoproteins glycosylated with the different types of N-linked oligosaccharides, i.e., those of the high mannose-, complex- and hybrid-type. Binding affinity of wheat proteins to these glycoproteins was analyzed by affinity dotting and blotting techniques and was compared to that of the well characterized lectins Galanthus nivalis agglutinin, Concanavalin A and wheat germ agglutinin. Though the three wheat gluten preparations exhibited binding reactivity for distinct model glycoproteins, no correlation was found between the type of N-glycosylation of the model glycoproteins and their binding capability to the different wheat gluten preparations. Moreover, binding of the three gluten preparations to the model glycoproteins could not be inhibited by competitive saccharides (methyl-alpha-D-mannopyranoside, N-acetyl-D-glucosamine, mannan). Enzymatic deglycosylation of the ligand glycoproteins with endo-beta-N-acetylglucosaminidase H (Endo H, EC 3.2.1.96) or peptide N-glycosidase F (PNGase F, EC 3.5.1.52) abolished their binding reactivity for the plant lectins, but did not affect binding of the wheat gluten preparations. These results give no evidence for the presence of lectin-like components in wheat gluten preparations and do question the 'lectin hypothesis' of coeliac disease.
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PMID:Studies on the aetiology of coeliac disease: no evidence for lectin-like components in wheat gluten. 831 50

The major envelope glycoproteins gp120 and gp41 of human immunodeficiency virus type 1, the causative agent for human AIDS, contain numerous N-linked oligosaccharides. We report here our discovery that N-acetylglucosamine residues within the complex-type N-linked oligosaccharides of both gp120 and its precursor, gp160, are sulfated. When human Molt-3 cells persistently infected with human T-cell leukemia virus IIIB were metabolically radiolabeled with 35SO4, gp160, gp120, and to some extent gp41 were radiolabeled. The 35SO4-labeled oligosaccharides were quantitatively released by N-glycanase treatment and were bound by immobilized Ricinus communis agglutinin I, a lectin that binds to terminal beta-galactosyl residues. The kinetics of release of sulfate upon acid hydrolysis from 35SO4-labeled gp120 indicate that sulfation occurs in a primary sulfate ester linkage. Methylation analysis of total glycopeptides from Molt-3 cells metabolically radiolabeled with [3H]glucosamine demonstrates that sulfation occurs at the C-6 position of N-acetylglucosamine. Fragmentation of the gp120-derived 35SO4-labeled glycopeptides by treatment with hydrazine and nitrous acid and subsequent reduction generated galactosyl-anhydromannitol-6-35SO4, which is the expected reaction product from GlcNAc-6-sulfate within a sulfated lactosamine moiety. Charge analysis of the [3H]galactose- and [3H]glucosamine-labeled glycopeptides from gp120 and gp160 indicates that approximately 14% of the complex-type N-linked oligosaccharides are sulfated.
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PMID:Complex-type N-linked oligosaccharides of gp120 from human immunodeficiency virus type 1 contain sulfated N-acetylglucosamine. 841 50

The H and M antigens of Histoplasma capsulatum are glycoproteins, and both possess epitopes found on the C antigen, a cross-reactive galactomannan shared by the major genera of systemic dimorphic fungi. We modified the H and M glycoproteins by chemical and enzymatic digestion to determine the relative contributions of the carbohydrate and protein moieties to the immunological reactivities and the apparent molecular weights of these antigens. Endoglycosidases with known action patterns were used to determine the nature of the glycopeptide bonds in the H and M antigens. The effects of these treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lectin binding, and enzyme-linked immunoelectrotransfer blots probed with polyclonal and monoclonal antibodies (MAbs). Oxidation with 100 mM periodate destroyed the common fungal epitope recognized by MAb CA1-CB4 and nearly all of the concanavalin A-binding sites on both the H and M antigens; it also caused the molecular mass of the M antigen to shift from 94 to 88 kDa. Treatment of samples with O-glycanase had little, if any, effect on the H and M glycoproteins. On the other hand, treatments with endo-beta-N-acetylglucosaminidase H, and particularly peptide N-glycoproteins F (PNGase F), produced pronounced shifts in the M(r) but did not completely eliminate concanavalin A- or MAb CA1-CB4-binding sites. PNGase F treatment caused the molecular mass of the H antigen to shift from 116 to 94 kDa and that of the M antigen to shift from 94 to 74 kDa. The susceptibilities of the H and M glycoproteins to endo-N-acetyl-beta-D-glucosaminidases suggest that their glycosidic moieties are N linked.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Immunochemical analysis of the H and M glycoproteins from Histoplasma capsulatum. 855 2

The deduced amino acid sequence of an estrogen-dependent sheep oviductal glycoprotein (M(r) 90,000-116,000) revealed the presence of several potential sites for glycan substitution on a protein backbone of M(r) approximately 66,500, and identity with chitinases. In order to further define the nature of the secreted glycoprotein, the objectives of the present study were 1) to devise a method to significantly enrich for the glycoprotein from oviductal secretions, 2) to biochemically characterize the glycoprotein by use of lectin blotting and enzymatic and chemical digestion, and 3) to determine whether unfractionated and enriched fractions containing the glycoprotein have chitinase activity. Oviducts were obtained from ovariectomized ewes treated with estradiol for 6 days and explant-cultured for 24 h. The oviductal glycoprotein was enriched approximately 80-85% from explant culture media by Maackia amurensis agglutinin (MAA) lectin affinity chromatography. Enriched fractions containing the oviductal protein were separated on SDS gels, transferred to polyvinyl difluoride, and probed with digoxigenin-labeled lectins. Lectin blotting revealed that the glycoprotein contained the carbohydrate moieties N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, and sialic acid both in alpha(2,3) and alpha(2,6) linkages, typical of sialomucins. Enzymatic digestion with neuraminidase and N-glycanase indicated that approximately 20% and approximately 6% of the molecular weight of the oviductal glycoprotein can be accounted for by sialic acid and N-linked glycans, respectively. The oviductal glycoprotein was resistant to digestion with O-glycanase alone and chondroitinase ABC, with the latter indicating that it was not a proteoglycan. Treatment with trifluoromethanesulfonic acid resulted in a deglycosylated product of M(r) approximately 66,000 immunoreactive with antibodies to the oviductal glycoprotein. No chitinase activity could be detected for unfractionated culture medium proteins or enriched fractions containing the M(r) 90,000-116,000 oviductal glycoprotein when the substrate methylumbelliferyl chitotriose was used. These data show that 1) MAA lectin chromatography can significantly enrich for the M(r) 90,000-116,000 glycoprotein from oviductal secretions, 2) the secreted glycoprotein contains saccharide residues typical of sialomucins, and 3) despite primary amino acid sequence identity, the oviductal glycoprotein does not share an enzymatic relationship with chitinases.
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PMID:An estrogen-dependent sheep oviductal glycoprotein has glycan linkages typical of sialomucins and does not contain chitinase activity. 856 10


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