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 amino acid sequences of the kainate binding proteins (KBPs) from frog and chicken brain are homologous with the carboxy terminal half of the rat brain AMPA receptors. In this study, we have characterized the oligosaccharide side chains present on the KBPs from chicken and frog brain, and the AMPA receptors (GluR1, GluR2, and GluR3) from rat brain. Deglycosylation of the asparagine-linked carbohydrates present on the chicken, frog, and rat receptor subunits with N-glycanase, resulted in decreases in the relative molecular weights (M(r)) of 3.4, 3.4, and 5.1 kDa respectively. Thus the percent of asparagine linked carbohydrate (based on M(r) values derived from SDS polyacrylamide gels) of the 49 kDa chicken, the 48 kDa frog, and the 107 kDa receptor rat subunits is 6.9, 7.1, and 4.8 percent respectively. No shifts in the M(r) were detected after treatment with neuraminidase indicating that sialic acid does not appear to be a major component of these receptors. Lectin binding studies demonstrated that both asparagine-linked and serine/threonine-linked oligosaccharides were present in the chicken, frog, and rat proteins. The data indicate that at least one of the asparagine linked oligosaccharide side chains appear to be of the complex or non-bisected hybrid type in all three species. The similarities in the glycosyl moieties of the chicken and frog kainate KBPs and the rat brain AMPA receptors suggests that the homology in the amino acid sequences between these proteins may extend to homology in their oligosaccharide sides chains as well.
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PMID:Characterization of the oligosaccharide side chains on kainate binding proteins and AMPA receptors. 133 Feb 12

Toxocara canis infective stage larvae continually produce excretory-secretory (TES) glycoproteins in long-term in vitro culture. The kinetics of synthesis and secretion were studied by metabolic labelling with radioactive [35S]methionine, [14C]serine and [14C]threonine. Maximal incorporation rates required overnight pre-incubation of parasites in medium depleted of the appropriate amino acid. Larvae rapidly incorporated isotope into their somatic tissues, but there was a minimum delay of 10 h before secretion of labelled antigens. Labelling with [14C]serine and [14C]threonine demonstrated a relative abundance of these amino acids in the major surface/secreted glycoproteins of this nematode (TES-32 and 120). Pulse-chase experiments suggested that TES-120 may be derived from a 58 kDa precursor, reflecting extensive posttranslational glycosylation. Inhibition of N-glycosylation with tunicamycin and digestion with N-glycanase provided evidence of N-glycosylation in the lower molecular weight ES components (TES-32, 55 and 70). These agents had no effect on the higher molecular weight components (TES-120 and 400) implying that for these molecules glycosylation is predominantly O-linked. The largest ES component (TES-400) was unusual, in incorporating serine and threonine but not methionine, and by exhibiting increased apparent molecular weight following pronase digestion; it is suggested that this molecule is a proteoglycan.
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PMID:Biosynthesis and glycosylation of serine/threonine-rich secreted proteins from Toxocara canis larvae. 145 27

Analysis of the Sephacryl S-200 fractionated type IV collagen domains from bovine and human glomerular basement membranes (GBM) and calf anterior lens capsule (ALC) indicated that Asn-linked oligosaccharides are primarily or exclusively localized in the 7 S region, whereas the hydroxylysine-linked Glc alpha 1----2Gal disaccharides (Glc-Gal-Hyl) are present in all the major segments of the molecule (7 S, NC1, and helical domain); no Ser/Thr-linked saccharide were detected. The Asn-linked carbohydrate units observed in the 7 S domain (Mr approximately 300,000) occurred in a number equal to the 12 polypeptide chains constituting this cross-linked region, and this was consistent with lectin blots of the reduced electrophoretically resolved 7 S components. Fractionation of the N-glycanase and endo-beta-N-acetylglucosaminidase-released oligosaccharides by concanavalin A affinity and high performance liquid chromatography indicated that the Asn-linked carbohydrate occurred predominantly in the form of complex tri- and biantennary units, although submolar amounts of polymannose variants (Man5-7GlcNAc2) were also present in calf ALC and bovine GBM. Structural studies of the complex N-linked oligosaccharides employing hydrazine/nitrous acid fragmentation and glycosidase digestions indicated a pattern in which there was complete fucosylation of the innermost GlcNAc residue of the Man3GlcNAc2 core but only sparse substitution with capping groups of the nonrepeating N-acetyllactosamine branches. Whether tri- or biantennary, the oligosaccharides from bovine GBM contained only one capping residue, in the form of either NeuAc or alpha-D-Gal, whereas those from ALC had only a single alpha-D-Gal and no NeuAc; human GBM oligosaccharides were devoid of both NeuAc and alpha-D-Gal. The absence of terminal alpha-D-Gal in the human 7 S domain was reflected in its lack of reactivity with Bandeiraea simplicifolia I and from its failure to yield Gal alpha 1----3Gal beta 1----4 [3H]anhydromannitol after hydrazine/nitrous acid/NaB3H4 treatment. Application of the latter procedure to the collagen domains yielded, in addition to fragments from the N-linked oligosaccharides, a disaccharide (Glc alpha 1----2[3H]galactitol) derived from the Glc-Gal-Hyl units. The localization of Asn-linked carbohydrate units in the evolutionarily conserved 7S domain of type IV collagens suggests that these oligosaccharides may play a role in the assembly of the collagen network of basement membranes.
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PMID:Localization and structure of the asparagine-linked oligosaccharides of type IV collagen from glomerular basement membrane and lens capsule. 185 26

The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.
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PMID:gp160 of HIV-I synthesized by persistently infected Molt-3 cells is terminally glycosylated: evidence that cleavage of gp160 occurs subsequent to oligosaccharide processing. 189 96

Two beta-glucosidases (I and II) were isolated from Schizophyllum commune, and their physical and chemical properties studied. The two enzymes have very similar sequences, as shown by HPLC analysis of tryptic digests and partial amino acid sequencing. As judged by their circular dichroism spectra, they have almost identical secondary structure. The estimates for alpha-helix, beta-sheet, and other structures were 21%, 40% and 39%, respectively, for beta-glucosidase I and 27%, 32% and 41% for beta-glucosidase II. Their near-ultraviolet spectra were identical. beta-Glucosidase I was more highly glycosylated than beta-glucosidase II, having 2 mol N-acetylglucosamine/mol enzyme 36, mol mannose/mol enzyme and 1.2 mol glucose/mol enzyme vs 1.2, 17 and 3 mol/mol, respectively, in beta-glucosidase II. The native glycosylated form of beta-glucosidase I had a molecular mass of 102 kDa, and that of beta-glucosidase II, 96 kDa. As estimated from sensitivity to N-glycanase, beta-glucosidase II sugars were mainly asparagine linked, but much of the sugar in beta-glucosidase I was not removed by this treatment and was apparently serine or threonine linked. Kinetic analysis showed that both forms had similar Km values (0.3-2.1 mM) for oligosaccharides of 2-6 residues, but the kcat values of beta-glucosidase II were lower by 30-75% than those of beta-glucosidase I. The substrate dependence of kcat/Km indicated that both enzymes had binding sites for three glucose residues. The pH optimum of beta-glucosidase I was higher than that of beta-glucosidase II (5.8 vs 5.1). Both had similar specificities for several (R)-beta-D-glucosides tested. Both enzymes were competitively inhibited by their glucose product, but beta-glucosidase II was consistently less inhibited than beta-glucosidase I. Cellobiase activity was much more markedly inhibited than the activity with higher oligosaccharides, and the result of this, plus the lower hydrolytic rate with cellobiose, resulted in an accumulation of cellobiose as higher oligosaccharides were digested. Glucono-delta-lactone inhibited both enzymes and the hydrolysis of all oligosaccharide substrates similarly (Ki = 4 microM). We conclude that the catalytic site is identical in both enzymes, but subtle structural differences are reflected in a differential activity on the higher oligosaccharides and in the differential effects of the glucose product as an inhibitor. Furthermore, ethanol had a stimulatory effect on beta-glucosidase I but inhibited beta-glucosidase II, which presumably reflects differential effects of ethanol on the conformations of the two species.
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PMID:Kinetics and specificities of two closely related beta-glucosidases secreted by Schizophyllum commune. 211 5

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor. 216 Apr 49

The sexuality-inducing glycoprotein of Volvox carteri f. nagariensis was purified from supernatants of disintegrated sperm packets of the male strain IPS-22 and separated by reverse-phase HPLC into several isoforms which differ in the degree of O-glycosylation. Total chemical deglycosylation with trifluoromethanesulphonic acid yields the biologically inactive core protein of 22.5 kDa. This core protein possesses three putative binding sites for N-glycans which are clustered in the middle of the polypeptide chain. The N-glycosidically bound oligosaccharides were obtained by glycopeptidase F digestion and were shown by a combination of exoglycosidase digestion, gaschromatographic sugar analysis and two-dimensional HPLC separation to possess the following definite structures: (A) Man beta 1-4GlcNAc beta 1-4GlcNAc; (B) (Man alpha)3 Man beta 1-4GlcNAc beta 1-4GlcNAc Xyl beta; (C) (Man alpha)2 Man beta 1-4GlcNAc beta 1-4GlcNAc; (D) (Man)2Xyl(GlcNAc)2. Xyl beta Two of the three N-glycosidic binding sites carry one B and one D glycan. The A and C glycans are shared by the third N-glycosylation site. The O-glycosidic sugars, which make up 50% of the total carbohydrate, are short (up to three sugar residues) chains composed of Ara, Gal and Xyl and are exclusively bound to Thr residues.
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PMID:The oligosaccharides of the glycoprotein pheromone of Volvox carteri f. nagariensis Iyengar (Chlorophyceae). 240 Dec 94

Limited proteolysis of human alpha 2-macroglobulin (alpha 2M) by a novel bacterial proteinase resulted in the isolation of a soluble 20-kDa domain. The isolated fragment contained the receptor recognition site, expressed on alpha 2M complexes, as it competed effectively with alpha 2M-trypsin for binding to the receptor on skin fibroblasts. The fragment also reacted with two monoclonal antibodies which define epitopes that are part of the receptor recognition site. Characterization of the 20-kDa domain showed it to contain an intact disulfide bridge, while its susceptibility to N-glycanase and reaction with concanavalin A indicated the presence of N-linked carbohydrate. The NH2-terminal sequence (Glu-Glu-Phe-Pro-Phe-Ala-Leu-Gly-Val-Glu-Thr-Leu-Pro-Glu-Thr-Cys-Asp-Glu -Pro) proved this fragment to constitute the COOH terminus of human alpha 2M. Proteolysis occurred at Lys1313-Glu which together with the observation that tosyllysine chloromethyl ketone was an effective inhibitor of the bacterial proteinase, would indicate the latter to hydrolyze preferentially peptide bonds carboxyl-terminal to lysine residues.
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PMID:The receptor-binding domain of human alpha 2-macroglobulin. Isolation after limited proteolysis with a bacterial proteinase. 242 72

The Maackia amurensis leukoagglutinin has been shown to react specifically with the Neu5Ac (alpha 2,3) Gal sequence of asparagine-linked complex type oligosaccharides. We report here the preparation of Maackia amurensis lectin-gold complexes and their application for light and electron microscopic detection of the Neu5 Ac (alpha 2,3) Gal sequence in various tissues. The use of the lectin directly gold labeled was superior to a two-step cytochemical affinity technique using a fetuin-gold complex. The Maackia amurensis lectin-gold staining was inhibited by pre-incubation of the lectin-gold complexes with 50 mM alpha 2,3 sialyllactose, whereas alpha 2,6 sialyllactose up to concentrations of 1 M had no effect, thus demonstrating the high specificity of the histochemical staining. In addition to N-glycanase-sensitive asparagine-linked oligosaccharides, beta-elimination-sensitive serine/threonine-linked oligosaccharides could be detected. Data are presented which show that cellular staining patterns obtained with Maackia amurensis lectin-gold complexes may differ from those with elderberry bark lectin-gold, which detects the Neu5 Ac (alpha 2,6) Gal/GalN Ac sequence. Electron microscopic double labeling for direct study of the differential distribution of the Neu5 Ac (alpha 2,3) Gal and Neu5 Ac (alpha 2,6) Gal sequences is reported. Therefore, the availability of two sialic acid binding lectins with different linkage specificity for histochemistry provides the first opportunity to study tissue and cell type expression of these terminal sequences of glycoproteins.
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PMID:Detection of the Neu5 Ac (alpha 2,3) Gal (beta 1,4) GlcNAc sequence with the leukoagglutinin from Maackia amurensis: light and electron microscopic demonstration of differential tissue expression of terminal sialic acid in alpha 2,3- and alpha 2,6-linkage. 247 13

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.
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PMID:Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence. 264 96


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