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)

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by beta-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-l agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal beta Gal residues, Gal beta 1-3 GalNAc, Man alpha 1-3 Man, sialic acid alpha 2-6 linked to Gal or GalNAc; and sialic acid alpha 2-3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and alpha-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting.
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PMID:CBP70, a glycosylated nuclear lectin. 925 93

Mannose in N-linked oligosaccharides is assumed to be derived primarily from glucose through phosphomannose isomerase (PMI). The discovery of mammalian mannose-specific transporters that function at physiological concentrations suggested that mannose might directly contribute to oligosaccharide synthesis. To determine the relative contribution of glucose and mannose, human fibroblasts were labeled with either [2-3H]mannose or [1,5,6-3H]glucose at the same specific activity, and the N-linked chains were released by PNGase F digestion. Most of the trichloroacetic acid-precipitable [3H]mannose label was released by this digestion, but only about 10% of the trichloroacetic acid-precipitable material was released from cells labeled with [1,5,6-3H]glucose. Both sugars labeled a similar array of oligosaccharides, and acid hydrolysis of these chains showed that [2-3H]mannose contributed 65-75% of the [3H]mannose in cells labeled for 1 h, despite the 100-fold higher concentration of exogenous glucose. Mannose consumption and [2-3H]mannose utilization were within the range of rates expected for mannose transport via the mannose-specific transporter. About 7-14% of the [2-3H]mannose is used for glycosylation, while the rest (86-93%) is catabolized to 3H2O via PMI. Increasing the exogenous mannose concentration beyond mannose transporter saturation results in the conversion of >99% of [2-3H]mannose into 3H2O. Long term labeling of cells with [2-3H]mannose showed that the specific activity of mannose in glycoproteins reached 77% of the specific activity of [2-3H]mannose added to the medium. These results show that when fibroblasts are provided with physiological concentrations of mannose, they use the mannose-specific transporter to supply the majority of mannose needed for glycoprotein synthesis. PMI may normally be used to catabolize excess mannose rather than to primarily supply Man-6-P for glycoprotein synthesis.
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PMID:Human fibroblasts prefer mannose over glucose as a source of mannose for N-glycosylation. Evidence for the functional importance of transported mannose. 928 14

D-Glucose entry into erythrocytes from adult dolphins (Tursiops truncatus) was rapid, showed saturation at high substrate concentrations, and demonstrated a marked stimulation by intracellular D-glucose. Kinetic parameters were estimated from the concentration dependence of initial rates of tracer entry at 6 degrees C: for zero-trans entry, Michaelis constant (K(m)) was 0.78 +/- 0.10 mM and maximal velocity (Vmax) was 300 +/- 9 mumol.l cell water-1.min-1; for equilibrium exchange entry, K(m) was 17.5 +/- 0.6 mM and Vmax was 8,675 +/- 96 mumol.l cell water-1.min-1. Glucose entry was inhibited by cytochalasin B, and mass law analysis of reversible, D-glucose-displaceable, cytochalasin B binding gave values of 0.37 +/- 0.03 nmol/mg membrane protein for maximal binding and 0.48 +/- 0.10 microM for the dissociation constant. Dolphin glucose transporter polypeptides were identified on sodium-dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots [using antibodies that recognized human glucose transporter isoform (GLUT-1)] as two molecular species, apparent relative molecular weights of 53,000 and 47,000. Identity of these polypeptides was confirmed by D-glucose-sensitive photolabeling of membranes with [3H]cytochalasin B. Digestion of both dolphin and human red blood cell membranes with glycopeptidase F led to the generation of a sharp band of relative molecular weight 46,000 derived from GLUT-1. Trypsin treatment of human and dolphin erythrocyte membranes generated fragmentation patterns consistent with similar polypeptide structures for GLUT-1 in human and dolphin red blood cells.
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PMID:GLUT-1 mediation of rapid glucose transport in dolphin (Tursiops truncatus) red blood cells. 945 6

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat-derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life-cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose, and N-acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species-specificity of glycoprotein glycosylation, preparations of human-derived P. carinii (comprised of mixed life-cycle stages) were also examined and found to contain the same sugars as those found in rat-derived organisms. Most of the purified rat-derived glycoproteins bound Concanvalin A, which was abolished by treatment with N-glycanase. This suggested that the majority of the oligosaccharides were N-linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host.
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PMID:Cell wall antigens of Pneumocystis carinii trophozoites and cysts: purification and carbohydrate analysis of these glycoproteins. 962 93

The effect of ammonium chloride was determined on a culture of CHO cells transfected with the human erythropoietin (EPO) gene. Cell growth was inhibited above a culture concentration of 5 mM NH(4)Cl with an IC-50 determined to be 33 mM. The specific production of EPO increased with the addition of NH(4)Cl above 5 mM. At 10 mM NH(4)Cl, the final cell density after 4 days in culture was significantly lower but the final yield of EPO was significantly higher. This appeared to be due to continued protein production after cell growth had ceased. The metabolic effects of added NH(4)Cl included higher specific consumption rates of glucose and glutamine and an increased rate of production of alanine, glycine, and glutamate. The EPO analyzed from control cultures had a molecular weight range of 33-39 kDa and an isoelectric point range of 4.06-4.67. Seven distinct isoforms of the molecule were identified by two-dimensional electrophoresis. This molecular heterogeneity was ascribed to variable glycosylation. Complete enzymatic de-glycosylation resulted in a single molecular form with a molecular mass of 18 kDa. Addition of NH(4)Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased molecular weight and pI range. Enzymatic de-sialylation of the EPO from the ammonia-treated and control cultures resulted in identical electrophoretic patterns. This indicated that the effect of ammonia was in the reduction of terminal sialylation of the glycan structures which accounted for the increased pI. Selective removal of the N-glycan structures by PNGase F resulted in two bands identified as the O-glycan linked structure (19 kDa) and the completely de-glycosylated structure (18 kDa). The proportion of the O-linked glycan structure was reduced, and its pI increased in cultures to which ammonia was added. Thus, the glycosylation pattern altered by the presence of ammonia included a reduction in terminal sialylation of all the glycans and a reduction in the content of the O-linked glycan. The addition of a sialidase inhibitor to the cultures had no effect on the ammonia-induced increase in EPO heterogeneity. Also, the effect of ammonia on glycosylation could not be mimicked using the weak base chloroquine in our system.
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PMID:Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. 1074 5

D-Glucose entry into erythrocytes from adult grey-headed flying fox fruit bats (Pteropus poliocephalus) was rapid and showed saturation at high substrate concentrations. Kinetic parameters were estimated from the concentration dependence of initial rates of zero-trans D-glucose entry at 5.5 degrees C as Michaelis constant (K(m)) 1. 64+/-0.56 mM, and maximal velocity (V(max)) 1162+/-152 micromol.l. cell water(-1).min(-1). D-Glucose entry was inhibited by cytochalasin B; mass law analysis of D-glucose-displaceable cytochalasin B binding gave values of K(d) 37.1+/-5.0 nM and B(max) 361.2+/-9.1 pmol/mg membrane protein. Entry of 2-deoxy-D-glucose, and 3-O-methyl-D-glucose, into P. poliocephalus red cells was rapid, entry of D-fructose was very slow. Glucose transporter polypeptides were identified on immunoblots as a band M(r) 47000-54000 and their identity confirmed by D-glucose-sensitive photolabeling of membranes with [3H]-cytochalasin B. Peptide-N-glycanase F digestion of both human and bat erythrocyte membranes generated GLUT-1-derived bands M(r) 37000. Trypsin digestion of human and fruit bat erythrocyte membranes generated fragmentation patterns consistent with similar GLUT-1 polypeptide structures in both species. Erythrocytes from adult Australian ghost bats (Macroderma gigas), a carnivorous microchiropteran bat, also expressed high levels of GLUT-1.
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PMID:Rapid GLUT-1 mediated glucose transport in erythrocytes from the grey-headed fruit bat (Pteropus poliocephalus). 1090 51

We have cloned and characterized a novel glucose transporter (GLUT11) that is alternatively spliced. The GLUT11 gene maps to chromosome 22q11.2 and consists of 13 exons. The long form (GLUT11-L) cDNA uses 13 exons to produce a protein containing 503 amino acids. The short form of GLUT11 (GLUT-11) cDNA is missing exon 2 and produces a protein of 496 amino acids with a 14 amino acid N-terminal difference compared to the long form. GLUT11 has significant similarity to known GLUTs and contains 12 putative membrane-spanning helices along with sugar transporter signature motifs that have previously been shown to be essential for transport activity. The putative glycosylation site of GLUT11 is present in loop 1. Northern blot analysis showed that GLUT11 mRNA is expressed in a number of tissues and most abundantly in the skeletal muscle and heart. RT-PCR assay showed that GLUT11 is alternatively spliced and the two isoforms are distributed differently in various tissues. Immunofluorescence microscopy demonstrated that GLUT11-L resides on the plasma membrane when overexpressed in HEK293T cells. Western blot analysis revealed that GLUT11-L runs as a broad band of approximately 42 kDa that was converted to a 38 kDa polypeptide by PNGase F digestion. Furthermore, a liposome reconstitution functional assay showed that GLUT11-L has glucose transport activity.
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PMID:Cloning and characterization of glucose transporter 11, a novel sugar transporter that is alternatively spliced in various tissues. 1217 79

Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.
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PMID:Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei. 1249 6

The pancreatic/duodenal homeobox-1 protein (PDX-1, also called STF-1, IPF-1) is a transcription factor that plays an important role in pancreatic function and development. Here, we have overexpressed and purified PDX-1 from baculovirus/sf-9 cells, transiently transfected Cos-7 cells and native Min6 cells and demonstrated that the protein is posttranslationally modified by O-linked N-acetylglucosamine (O-GlcNAc). The approaches we used include binding of the protein to the lectin WGA, labeling with galactosyltransferase and UDP-[(3)H]gal and probing with the O-GlcNAc-specific antibody, RL-2. PNGase F treatment and structural analysis indicate that the carbohydrate is beta-linked O-GlcNAc. Mapping of [(3)H]gal-labeled tryptic peptides indicates that PDX-1 has two major sites for O-GlcNAcylation. In Min6 cells, elevated glucose concentration leads to an increase in protein O-GlcNAcylation and this hyperglycosylation correlates with an increase in DNA binding activity of PDX-1 and insulin secretion. On the other hand, the GFAT inhibitor azaserine reduces intracellular O-GlcNAc levels and profoundly attenuates glucose-stimulated insulin secretion. These data suggest that O-GlcNAcylation may be involved in the regulation of PDX-1 DNA binding activity and in glucose-stimulated insulin secretion in beta-cells.
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PMID:The transcription factor PDX-1 is post-translationally modified by O-linked N-acetylglucosamine and this modification is correlated with its DNA binding activity and insulin secretion in min6 beta-cells. 1283 37

The presence of mannose (Man) in the glycoconjugates of primordial germ cells (PGCs) of Xenopus embryos was elucidated by lectin histochemistry with Concanavalin A (Con A) and snowdrop (Galanthus nivalis) bulb lectin (GNA), in combination with deglycosylative pretreatments: beta-elimination, which removes O-linked oligosaccharides, and incubation with Peptide N glycosidase F (PNGase F), which removes N-linked glycan chains. In addition, histochemistry with Con A, which binds to Man and glucose (Glc), was also performed after glucose-oxidase incubation, which converts Glc into gluconic acid, and GNA was carried out after acid hydrolysis, which removes terminal sialic acid (NeuAc) moieties. PGCs were analyzed during their migration over the mesentery until the genital ridge, and after colonization of this gonad anlage. The results showed that for both lectins: (1) the PGCs and other surrounding tissue showed a similar binding pattern, and (2) the staining in the PGCs was similar in the developmental stages studied. Labeling with Con A was due to Man, and not to Glc, as shown after incubation with glucose-oxidase, and it was assumed that Man was in N-linked oligosaccharides. However, GNA labeling was mainly due to O-linked oligosaccharides, because the pretreatment of beta-elimination turned cells negative. Moreover, acid hydrolysis pretreatment gave rise to a stronger GNA-staining, suggesting that either Man was also in subterminal position to NeuAc or some Man-containing glycans were unmasked after removal of NeuAc from other oligosaccharide chains.
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PMID:Identification of mannose moieties in N- and O-linked oligosaccharides of the primordial germ cells of Xenopus embryos. 1672 33


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