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)

A new murine IgA mAb (JKT.M1), developed against Jurkat T cells chronically infected with HIV IIIB induces in vitro homotypic aggregation in several hemopoietic cell lines. The JKT.M1 Ag is expressed on a wide variety of cell types including human lymphocytes, monocytes, platelets, RBC, human umbilical vein endothelial cells, many T cell lines, myelomonocytic cell lines, and a primate kidney cell line. The JKT.M1 Ag shows differential expression on myelomonocytic cells; it is present on K562 and HL60 cell lines, which represent precursors of E and monocytes, respectively, but is not expressed on the surface of U937 and THP-1 cell lines, which appear to represent intermediate cell types of the monocytic cell lineage. However, the JKT.M1 Ag is expressed on mature peripheral blood monocytes and the MonoMac cell line. Immunoprecipitation from cell lysates (Jurkat, SupT1, PBMC, MonoMac) with the JKT.M1 mAb yields a 20-kDa Ag with few if any carbohydrate residues as determined by N-glycanase and neuraminidase treatments. The pI appears acidic by two-dimensional gel analysis, and the nonreduced form migrates more slowly than the reduced form when analyzed by SDS-PAGE suggesting the presence of intramolecular disulfide bridge(s). JKT.M1 mAb-induced cell adhesion is shown to be divalent cation- and temperature-dependent. The adhesion induced by JKT.M1 mAb is inhibited by 20 microM cytochalasin B and also by 2 mM 2-deoxyglucose plus 10 mM sodium azide suggesting that cytoskeletal changes and metabolic energy are required. Aggregation induced by JKT.M1 appears to be independent of CD43, CD44, and VLA4 (CD29/CD49d), mAb against which have also been shown to induce homotypic cell adhesion. Anti-CD18 mAb have been shown to inhibit homotypic aggregation in other studies but failed to do so in the present study. Thus JKT.M1-induced adhesion also appears to be independent of CD18, the beta-chain of leukocyte integrins. However, like mAb against LFA-1, immobilized JKT.M1 stimulates a T cell line to undergo dramatic morphologic changes which could be enhanced by the addition of phorbol ester. These data suggest that the novel 20-kDa molecule recognized by the JKT.M1 mAb may trigger cell adhesion through a previously undescribed mechanism.
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PMID:A monoclonal antibody against a novel 20-kDa protein induces cell adhesion and cytoskeleton-dependent morphologic changes. 138 18

A recent study by C.F. Burant et al. (13) demonstrates that GLUT5 is a high-affinity fructose transporter with a much lower capacity to transport glucose. To characterize the potential role of GLUT5 in fructose and glucose transport in insulin-sensitive tissues, we investigated the distribution and insulin-stimulated translocation of the GLUT5 protein in human tissues by immunoblotting with an antibody to the COOH-terminus of the human GLUT5 sequence. GLUT5 was detected in postnuclear membranes from the small intestine, kidney, heart, four different skeletal muscle groups, and the brain, and in plasma membranes from adipocytes. Cytochalasin-B photolabeled a 53,000-M(r) protein in small intestine membranes that was immunoprecipitated by the GLUT5 antibody; labeling was inhibited by D- but not L-glucose. N-glycanase treatment resulted in a band of 45,000 M(r) in all tissues. Plasma membranes were prepared from isolated adipocytes from 5 nonobese and 4 obese subjects. Incubation of adipocytes from either group with 7 nM insulin did not recruit GLUT5 to the plasma membrane, in spite of a 54% insulin-stimulated increase in GLUT4 in nonobese subjects. Thus, GLUT5 appears to be a constitutive sugar transporter that is expressed in many tissues. Further studies are needed to define its overall contribution to fructose and glucose transport in insulin-responsive tissues and brain.
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PMID:Human small intestine facilitative fructose/glucose transporter (GLUT5) is also present in insulin-responsive tissues and brain. Investigation of biochemical characteristics and translocation. 139 12

Candida albicans GDH 2346 produces extracellular polymeric material (EP) which contains a mannoprotein adhesin with a lectin-like affinity for fucose-containing glycosides. EP isolated from culture supernatants of this strain was used as starting material for purification of the adhesin. The purification protocol involved a stepwise treatment of EP with N-glycanase, papain, and dilute alkali to cleave the protein and carbohydrate portions of the mannoprotein molecule. Fucoside-binding protein fragments were then recovered by affinity adsorption with the trisaccharide determinant of the H (type 2) blood group antigen which terminates in a residue of L-fucose. The purified adhesin was devoid of carbohydrate and inhibited yeast adhesion to buccal epithelial cells 221 times more efficiently, on a protein weight basis, than did EP. Adhesion inhibition reached a maximum of 78 to 80% at an adhesin concentration of 10 micrograms ml-1. Our results indicate that this protein is the major adhesin of yeast-phase cells of C. albicans GDH 2346 but that one or more secondary adhesion mechanisms may operate.
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PMID:Characterization of a fucoside-binding adhesin of Candida albicans. 139 83

Tn polyagglutinability syndrome is an acquired condition where erythrocytes express Tn neo-antigen and become susceptible to hemagglutination by the naturally occurring anti-Tn present in normal sera. Early studies had indicated that O-linked N-acetyl galactosamine was the sole serologic Tn determinant, but more recently O-linked NeuNAc alpha 2, 6GalNAc also has been implicated as a Tn antigen (sialosyl-Tn). However, none of these studies were performed on purified glycoproteins. In this report we examine oligosaccharides of glycophorins A and B purified from Tn erythrocytes of two affected individuals to establish how N- and O-linked saccharides differ from normal. Analysis of carbohydrate composition and treatment with N-glycanase showed that the Asn-linked unit of glycophorin A was not affected. O-linked oligosaccharides were obtained by beta-elimination in the presence of tritiated sodium borohydride. The reduced radiolabeled products were fractionated by Bio-Gel P-2 chromatography, and their structures were investigated by comparison with standards, by monosaccharide quantification, and by neuraminidases of known specificities. The results show that Tn glycophorins from both donors contain intact and truncated forms of trisaccharide and tetrasaccharide NeuNAc alpha 2,3Gal beta 1,3GalNAc and NeuNAc alpha 2,3Gal beta 1,3- (NeuNAc alpha 2,6)GalNAc usually present in glycophorins A and B. The truncated forms include the protein O-linked monosaccharide, GalNAc and disaccharide, NeuNAc alpha 2,6GalNAc (major isomer). The presence of intact glycans in the total population of Tn erythrocytes was confirmed by their susceptibility to T activation after treatment with neuraminidase. The proportion of the four species was not identical in glycophorins of these two donors but, in both, the truncated units predominated and the amount of the disaccharide was approximately one half of that of the monosaccharide. The data are consistent with alterations in UDPGal:GalNAc beta 1,3galactosyl transferase that may have multiple molecular origins and with induction of a specific GalNAc protein alpha 2,6 sialosyl transferase in Tn hematopoietic precursor cells. The molecular basis for these alterations awaits further study.
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PMID:O-linked oligosaccharides of glycophorins A and B in erythrocytes of two individuals with the Tn polyagglutinability syndrome. 142 10

The binding in pre-colonoscopic effluent of Adnab-9, a monoclonal antibody raised against colonic adenomas, was evaluated for specificity in the diagnosis of colorectal cancer. A heterogeneous group of 58 patients was evaluated by ELISA. Effluent samples and tissue extracts were subjected to Western blotting or ELISA to confirm specificity. Immunohistochemistry was performed on the cancer tissue sections. The proportion of positive effluent binding was higher in the cancer when compared to the normal group (P = 0.036). A dominant 87 M(r) band was found in adenoma extracts and some effluent samples. Adnab-9 binding in effluent samples predominated in membrane-bound fractions. Immunohistochemistry showed no specific staining in the cancer cells. The antigen recognised is a glycoprotein shown by effects of N-glycanase digestion and not cross-reactive with carcinoembryonic antigen. Non-gastro-intestinal tissue extracts did not bind Adnab-9. The major 87 M(r) adenoma-derived antigen may be found in effluent material, particularly in the membrane-bound fraction.
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PMID:Adenoma-derived antibody, Adnab-9 recognizes a membrane-bound glycoprotein in colonic tissue and effluent material from patients with colorectal neoplasia. 142 46

B4-2-1 cells (Lec15 cells) are Chinese hamster ovary cells deficient in mannosylphosphoryldolichol synthase activity. They synthesize the truncated lipid intermediate Man5GlcNAc2-P-P-dolichol rather than the Glc3Man9GlcNAc2-P-P-dolichol synthesized by wild-type cells. In this report we present evidence that these cells did synthesize glucosylated Man5GlcNAc2-P-P-dolichol, but this species represented only a minor fraction of the labeled oligosaccharide-lipid. On the other hand, glucosylated oligosaccharides were a major species transferred to protein in these cells, showing that in vivo, glucosylated oligosaccharides are preferentially transferred to protein. The truncated oligosaccharides found in B4-2-1 cells were removed from the protein by N-glycanase treatment, since they were resistant to both endo-beta-N-acetylglucosaminidase H and F activity. B4-2-1 cells processed the glucosylated, truncated oligosaccharides transferred to G protein of vesicular stomatitis virus, leading to infectious virus.
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PMID:Lec15 cells transfer glucosylated oligosaccharides to protein. 144 60

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.
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PMID:Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit. 144 27

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

This report describes the N-glycosylation site mapping of human serotransferrin (h-STF). Reduced and S-carboxymethylated h-STF was digested with trypsin or chymotrypsin. Glycopeptides in the proteolytic digests were isolated by serial concanavalin A (Con A), Sambucus nigra agglutinin (SNA), and Phaseolus vulgaris leukoagglutinin (LPHA) affinity chromatography and subjected to preliminary analysis by 1H NMR spectroscopy. The glycopeptide fractions were then individually digested with N-glycanase. One part of the digest of each fraction was analyzed by fast atom bombardment-mass spectrometry (FAB-MS) to identify the peptide sequences of the glycosylation sites. The other part was used to isolate the oligosaccharide by the corresponding lectin affinity chromatography and to characterize the structures of the isolated oligosaccharides by 1H NMR spectroscopy and FAB-MS. The oligosaccharides in the Con A-bound fraction were shown to have bi-alpha(2-->6)-sialyl, diantennary structures. The SNA-bound fraction was shown to contain trisialyl, triantennary structures. Di- and triantennary oligosaccharides were found to occur on each of the two N-glycosylation sites of h-STF (Asn413 and Asn611) in the ratio of approximately 85:15. The SNA-bound glycopeptides were further fractionated by LPHA affinity chromatography. Two different oligosaccharides were characterized, namely, a trisialyl 2,4-triantennary and a trisialyl 2,6-triantennary glycan. The ratio of 2,4-triantennary vs 2,6-triantennary oligosaccharides attached to glycosylation site Asn413 was found to be approximately 5:1, whereas the two isomeric triantennary oligosaccharides were found to be attached to glycosylation site Asn611 in the ratio approximately 1:1.
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PMID:N-glycosylation site mapping of human serotransferrin by serial lectin affinity chromatography, fast atom bombardment-mass spectrometry, and 1H nuclear magnetic resonance spectroscopy. 145 41

The Na-K-Cl cotransporter mediates the coupled transport of Na, K, and Cl across the plasma membrane of many animal cell membranes. It is inhibited by loop diuretics such as furosemide, bumetanide, and benzmetanide. We have developed a panel of monoclonal antibodies directed against the 195-kDa shark rectal gland Na-K-Cl cotransport protein. Four representative antibodies (J3, J4, J7, and J25), each of which recognizes a discrete structural domain, were selected for detailed characterization. When a radiolabeled loop diuretic is bound to the cotransporter prior to solubilization, each antibody immunoprecipitates the same diuretic-protein complex. Of the four antibodies, J4 favors the native protein over the denatured one and does not bind well to proteolytic fragments; in contrast, J7 recognizes the cotransporter only after it has been solubilized. J3, J7, and J25 each recognize a unique ensemble of proteolytic fragments of the 195-kDa protein; analysis of the patterns of recognition has yielded a tentative assignment of the approximate location of the epitopes within the peptide. When the cotransport protein is treated with N-glycanase to remove N-linked oligosaccharides, its apparent mass decreases to approximately 135 kDa. The deglycosylated form is recognized by each of the antibodies except J25; this suggests that the J25 epitope is within the oligosaccharide component or in a peptide domain whose folding is disturbed by carbohydrate removal. An immunoaffinity matrix constructed with the J4 antibody permits single-step purification of the 195-kDa protein; other proteins copurify with the large glycoprotein, but none of these appear to be subunits of a stoichiometric complex. The amino acid sequence of four fragments of the 195-kDa cotransport protein is reported. Immunofluorescence and immunoelectron microscopy demonstrates, in agreement with physiological evidence, that the 195-kDa protein is distributed along the basolateral membrane and excluded from the apical membrane of the rectal gland secretory cell.
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PMID:The Na-K-Cl cotransport protein of shark rectal gland. I. Development of monoclonal antibodies, immunoaffinity purification, and partial biochemical characterization. 146 38


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