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 myelin/oligodendrocyte glycoprotein (MOG) is found exclusively in the CNS, where it is localized on the surface of myelin and oligodendrocyte cytoplasmic membranes. The monoclonal antibody 8-18C5 identifies MOG. Several studies have shown that anti-MOG antibodies can induce demyelination, thus inferring an important role in myelin stability. In this study, we demonstrate that MOG consists of two polypeptides, with molecular masses of 26 and 28 kDa. This doublet becomes a single 25-kDa band after deglycosylation with trifluoromethanesulfonic acid or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that there are no or few O-linked sugars and that the doublet band represents differential glycosylation. Partial trypsin cleavage, which also gave a doublet band of lower molecular weight, confirmed this idea. MOG was purified by polyacrylamide gel electrophoresis, followed by electroelution. Three N-terminal sequences of eight to 26 amino acids were obtained. By western blot analysis, no binding was found between MOG and cerebellar soluble lectin. MOG does not seem to belong to the signal-transducing GTP-binding proteins. Reduced MOG concentrations were observed in jimpy and quaking dysmyelinating mutant mice, giving further support to its localization in compact myelin of the CNS.
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PMID:Purification and partial structural and functional characterization of mouse myelin/oligodendrocyte glycoprotein. 137 75

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.
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PMID:Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies. 161 9

The biochemical and immunochemical characteristics of T. spiralis molecules (group II antigens) sharing an immunodominant epitope were examined. Six major proteins, ranging from 43-68 kDa, and from pI 5.0-6.3, express the determinant. Together, they account for at least 3% by weight of the total protein in L1 larval homogenate. The antigens are glycosylated. Following periodate oxidation, they reacted with biotin aminocaproyl hydrazide, and treatment with trifluoromethanesulfonic acid decreased their Mr. Deglycosylated group II antigens lost immunoreactivity with a monoclonal antibody specific for the determinant, and oligosaccharides released by treatment with mild base blocked binding of the monoclonal antibody to native antigens. The determinant on one of the group II antigens (43 kDa) was removed by N-glycanase. Neither phosphorylcholine nor antibody to phosphorylcholine interfered with monoclonal antibody binding to native group II antigens. Together, these results suggest that the immunodominant group II antigen epitope is associated with N- and O-linked oligosaccharides, and that it is not phosphorylcholine.
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PMID:Characterization of Trichinella spiralis antigens sharing an immunodominant, carbohydrate-associated determinant distinct from phosphorylcholine. 169 36

Bovine corneal keratan sulfate proteoglycan (KSPG) contains two core proteins, 37 and 25 kDa, if fully deglycosylated, but 47 and 35 kDa, respectively, after endo-beta-galactosidase (Funderburgh, J. L., and Conrad, G. W. (1990) J. Biol Chem. 265, 8297-8303). Chicken corneal KSPG released a single core protein of 47 kDa after endo-beta-galactosidase, and of 35 and 36 kDa, if deglycosylated with N-glycanase or trifluoromethanesulfonic acid. Affinity purified rabbit antibodies against each KSPG recognized only the intact proteoglycan or its core proteins in immunoblots of unfractionated guanidine-HCl extracts of whole cornea after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity purified antibody to a synthetic peptide duplicating the NH2-terminal sequence of the 37-kDa bovine core protein showed little reactivity with untreated corneal extract but reacted with the 47-kDa bovine protein in endo-beta-galactosidase-treated extracts. RNA was isolated from bovine and chick corneal stromas and used for in vitro translation. Antibody against bovine KSPG immunoprecipitated two proteins of 56-53 kDa and a protein of 41 kDa after translation of bovine RNA. Translation of chick RNA produced a double band of 38-39 kDa and a single band of 25 kDa precipitating with antibody against chicken KSPG. Homologous unlabeled KSPG competed for binding of antibodies to these translation products. These data suggest that in vertebrate corneas, the multiple KSPG core protein isoforms may arise as products of separate mRNAs, rather than from proteolytic processing of a large polypeptide precursor.
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PMID:Cell-free translation and characterization of corneal keratan sulfate proteoglycan core proteins. 171 81

The breast cancer-associated epitope (mammary serum antigen or MSA) defined by monoclonal antibody (Mab) 3E1.2 is a neuraminidase-sensitive carbohydrate expressed on MUC-1-encoded molecules. However, the reactivity of Mab 3E1.2 is also reduced by protease treatment of the mucin, which suggests that 3E1.2 binds to multimers of the sialylated carbohydrate in a protein conformation-dependent manner. The common N-acetyl derivative of neuraminic acid (5-acetylneuraminic acid) is not involved in the epitope, since lectins specific for 5-acetylneuraminic acid (linked to GalNAc or Gal) are nonreactive with MSA-positive molecules. However, the N-glycolyl derivative, 5-glycolylneuraminic acid (Neu5Gc), forms a major part of the epitope since both free Neu5Gc and porcine stomach mucin (greater than 90% neuraminic acid as Neu5Gc) inhibit the binding of Mab 3E1.2, while bovine or ovine submaxillary mucins, fetuin, bovine gangliosides, and other carbohydrates do not. Indeed, the presence of Neu5Gc on human tumor mucin was confirmed by electrospray mass spectrometry. Neu5Gc is attached to an O-linked carbohydrate, since the expression of MSA by MCF-7 breast cancer cells is inhibited by the O-glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide, but not by the N-glycosylation inhibitor tunicamycin, and the epitope is removed by treatment with O-glycanase but not N-glycanase F, endoglycosidase F, or endoglycosidase H, which are specific for N-linked glycans. This is likely to be a core glycan since 3E1.2 reacts after treatment of the mucin with trifluoromethanesulfonic acid, which removes most backbone and peripheral carbohydrates. Treatment with galactosidase or N-acetyl glucosaminidase enhances the binding of Mab 3E1.2, indicating that the Neu5Gc is not attached to galactose or N-acetyl galactosamine. Furthermore, the susceptibility of MSA to treatment with Arthrobacter urea-faciens neuraminidase [which is specific for alpha (2-6)-linked NeuNAc] and the loss in reactivity of GalNAc-specific lectins after periodate oxidation [alpha (2-3)-linked but not alpha (2-6)-linked NeuNAc protects GalNAc from periodate oxidation] indicate that the Neu5Gc may be attached alpha (2-6) to peptide-linked GalNAc. These results show that MSA is a Neu5Gc-containing O-linked core glycan, which represents a unique tumor-associated epitope not previously identified on human mucins.
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PMID:The breast tumor-associated epitope defined by monoclonal antibody 3E1.2 is an O-linked mucin carbohydrate containing N-glycolylneuraminic acid. 171 85

Biochemical properties of the concanavalin A-binding 43-kDa glycoprotein (gp43) of Paracoccidioides brasiliensis and its deglycosylated form were compared. Deglycosylation was achieved by treatment with trifluoromethanesulfonic acid, endoglycosidase H, N-glycanase, or metabolically, by growing cells with tunicamycin. The resulting antigen in all cases had Mr 38,000, and probably derived from the gp43 by loss of N-linked high-mannose oligosaccharide chains. The presence of galactopyranose units in the carbohydrate chains was suggested by antigen binding to peanut lectin. Pulse and chase experiments using [35S]methionine metabolic labeling of P. brasiliensis growing in the presence of tunicamycin showed that the N-linked chains of gp43 are not required for antigen secretion. The 38-kDa antigen was more susceptible than the native antigen to the action of papain and pronase, thus indicating a protective role of the carbohydrate moiety against proteolysis. Both forms are equally resistant to endogenous proteases at neutral pH. The gp43, itself, has a proteolytic activity at pH 5-6, but not at neutral pH. Deglycosylation with endoglycosidase H or tunicamycin preserved epitopes in the 38-kDa molecule reactive with (a) antibodies from patients with paracoccidioidomycosis, or rabbit immunized with the gp43 and (b) mouse monoclonal antibodies against the gp43 antigen. The present results provide a basis for the understanding of diagnostic reactions and fungal virulence involving the gp43 exocellular antigen of P. brasiliensis.
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PMID:The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: excretion and susceptibility to proteolysis. 189 73

Post-translational modification of the scrapie prion protein (PrP) is thought to account for the unusual features of this protein. Molecular cloning of a PrP cDNA identified two potential Asn-linked glycosylation sites. Both the scrapie (PrPSc) and cellular (PrPC) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PNGase F digestion of PrPC yielded two proteins of Mr26K and 28K; however, the 26-k species was only a minor component. In contrast, PNGase F digestion of PrPSc yielded equimolar amounts of two proteins of Mr26K and 28K. The significance of this altered stoichiometry between the 26- and 28-kDa deglycosylated forms of PrP during scrapie infection remains to be established. Both isoforms as well as PrP 27-30, which is produced by limited proteolysis of PrPSc, exhibited a reduced number of charge isomers after PNGase F digestion. The molecular weight of PrP 27-30 was reduced from 27K-30K by PNGase F digestion to 20K-22K while anhydrous hydrogen fluoride or trifluoromethanesulfonic acid treatment reduced the molecular weight to 19K-21K and 20K-22K, respectively. Denatured PrP 27-30 was radioiodinated and then assessed for its binding to lectin columns. PrP 27-30 was bound to wheat germ agglutinin (WGA) or lentil lectins and eluted with N-acetylglucosamine or alpha-methyl-mannoside, respectively. Digestion of PrP 27-30 with sialidase prevented its binding to WGA but enhanced its binding to Ricinus communis lectin. These findings argue that PrP 27-30 probably possesses Asn-linked, complex oligosaccharides with terminal sialic acids, penultimate galactoses, and fucose residues attached to the innermost N-acetyl-glucosamine. Whether differences in Asn-linked oligosaccharide structure between PrPC and PrPSc exist and are responsible for the distinct properties displayed by these two isoforms remain to be established.
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PMID:Asparagine-linked glycosylation of the scrapie and cellular prion proteins. 250 74

A weakly metastatic wheat-germ-agglutinin-resistant mutant Wa4-b1 was previously shown to be less adherent to endothelial cell extracellular matrix than the more metastatic parental B-16 melanoma cells. This report describes reduced adhesion and spreading of Wa4-b1 cells on the cell-binding domain of fibronectin (CBD) and laminin (LN). Cell surface receptors which mediate such interactions are members of the integrin family of membrane glycoproteins. An antibody that recognizes the beta 1 integrin subunit inhibited spreading on both the CBD and LN. The integrins of the mutant cells immunoprecipitated by the antibody appeared to be structurally altered, showing a greater electrophoretic mobility. The mobility difference between the parent and the mutant receptors was abolished following removal of the glycan moieties of the receptors enzymatically using glycopeptidase F, or chemically using trifluoromethanesulfonic acid, suggesting that the structural alteration of the mutant receptors is in glycosylation. The altered receptors may be responsible for the observed decrease in cell adhesion and spreading of the mutant cells to the CBD and LN. Such a decrease in Wa4-b1 cell interaction with extracellular matrix components may play a role in their decreased metastatic potential.
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PMID:Reduced cell adhesion to fibronectin and laminin is associated with altered glycosylation of beta 1 integrins in a weakly metastatic glycosylation mutant. 278 45

Maltase-glucoamylase (MGA) was immunoprecipitated from detergent extracts of brush border membranes of the human small intestinal mucosa. Electrophoretic analysis of the precipitates under denaturing conditions revealed a single polypeptide of Mr = 335,000 in the presence or absence of reducing agents. Cross-linking of brush border membranes with the homobifunctional reagent dithiobis(succinimidylpropionate) did not result in considerable changes in the electrophoretic pattern of MGA. In contrast, aminopeptidase N, used in these studies as a control glycoprotein of the brush border membrane revealed dimeric structures of its single subunit in the presence of dithiobis(succinimidylpropionate). These data suggest that MGA is expressed in the human small intestinal brush border as a monomeric polypeptide. The biosynthesis of MGA was studied by pulse-labeling of human intestinal biopsy specimens or mucosal explants in organ culture. Continuous labeling with [35S]methionine for 30 min revealed a single polypeptide high mannose precursor of Mr = 285,000 (MGAh) which matures after 4 h of labeling to the Mr = 335,000 as judged by the susceptibility of these two forms to endo-beta-N-acetylglucosaminidase H. Owing to the absence of pancreatic secretions in the culture medium and the isolation of an identical species from nonlabeled mucosa, this result indicates that the Mr = 335,000 does not undergo an in situ extracellular cleavage by intraluminal proteases. Further, biosynthetically labeled, intracellularly cleaved polypeptides corresponding to the high mannose precursor or mature forms of MGA were not detected. The mature form of MGA (MGAm) bears in addition to N-linked glycans also O-glycosidically linked oligosaccharides. In fact, endo-beta-N-acetylglucosaminidase F/glycopeptidase F treatment of MGAm followed by chemical deglycosylation with trifluoromethanesulfonic acid revealed approximately 35,000 daltons of O-linked sugars. Furthermore, MGAm as well as its N-linked sugars-depleted form bound to Helix pomatia lectin which has specificity toward Gal-GalNAc structures. In addition, the data were suggestive of a post-translational O-glycosylation of the molecule since (i) the high mannose precursor of MGA did not bind to H. pomatia lectin and (ii) its endo-beta-N-acetylglucosaminidase H or endo-beta-N-acetylglucosaminidase F/glycopeptidase F form displayed an apparent molecular weight similar to that obtained upon endo-beta-N-acetylglucosaminidase F/glycopeptidase F/trifluoromethanesulfonic acid deglycosylation. Finally, pulse-chase experiments revealed a relatively slow rate of post-translational processing of MGA in comparison to aminopeptidase N.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structure, biosynthesis, and glycosylation of human small intestinal maltase-glucoamylase. 314 29

The biosynthesis and maturation of human sucrase-isomaltase (SI, EC 3.2.1.48-10), was studied in cultured small intestinal biopsy specimens and mucosa explants. Pulse-chase experiments with [35S]methionine revealed one high mannose intermediate of Mr = 210,000 (pro-SIh) which was processed at a slow rate to an endo H-resistant, mature form of Mr = 245,000 (pro-SIc). The fully core-glycosylated form (Mr = 212,000) was detected only when 1-deoxynojirimycin was added to the culture medium, thus indicating that the core sugars undergo rapid processing by rough endoplasmic reticulum membrane-bound glycosidases. The data presented showed that trypsin specifically and instantaneously (within 1 min) cleaves pro-SIc to two subunits Ic (Mr = 145,000) and Sc (Mr = 130,000). Elastase and chymotrypsin are not effective. Enzymic and chemical deglycosylations of SI with endo-beta-N-acetylglucosaminidase F/glycopeptidase F and trifluoromethanesulfonic acid (TFMS) as well as probing for the binding capacity of SI to Helix pomatia lectin demonstrated that pro-SIc, Ic, and Sc are N- and O-glycosylated. Furthermore, the results were indicative of a posttranslational O-glycosylation of pro-SI, since (i) the earliest detectable precursor form, pro-SIh, did not bind to H. pomatia lectin and (ii) its deglycosylation products with both endo-beta-N-acetylglucosamidase H and TFMS were identical. Both the Sc and Ic subunits contain eight N-linked glycan units, at least one of which is of the high mannose type and found on Sc. Finally, Sc, but not Ic, was shown to display at least four populations varying in their content of O-linked glycans. The heterogeneous O-glycosylation pattern of Sc could be correlated with the distal position of this subunit (and its O-glycosylation sites) within the pro-SI molecule, thus affecting the extent of O-linked oligosaccharide processing and their subsequent presentation on the mature molecule.
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PMID:Biosynthesis of the human sucrase-isomaltase complex. Differential O-glycosylation of the sucrase subunit correlates with its position within the enzyme complex. 336 77


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