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 monoclonal antibody (mAb), 2E12, against the neural cell adhesion molecule L1 recognized the 200 kDa component of L1. The epitope of L1 reacting with mAb 2E12 was localized in its carbohydrate chain, judging from the results of experiments on glycopeptidase F treatment. The physiological effect of adding mAbL1 (2E12) to cultured mouse dorsal root ganglion neurons was studied using patch-clamp techniques. The binding of mAbL1 (2E12) to the neurons expressing L1 molecule induced an inward current inhibited by calcium channel blockers such as nifedipine and Lanthanum. It was also found that the mAbL1 (2E12) leads to a rise in the intracellular Ca2+ concentration ([Ca2+]i) in cultured neurons. This rise seems to be due to an influx of extracellular Ca2+, since treatment with EGTA abolished those phenomena. L-type calcium channel blockers such as nifedipine and cadmium, as well as inward current, blocked the effect of mAbL1 (2E12). These results suggest that the carbohydrate chain of L1 glycoprotein is directly involved in the induction of calcium current, and that the L1 molecule may play a prominent role in regulation of the Ca2+ channel.
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PMID:Monoclonal antibody that recognizes the carbohydrate portion of cell adhesion molecule L1 influences calcium current in cultured neurons. 138 50

Previous studies have established the importance of a complex, N-linked oligosaccharide chain, recognized by a monoclonal antibody (mAb 1223), in the formation of sea urchin embryonic skeletal components known as spicules. To further investigate the function of this epitope, mAb 1223 was added to primary mesenchyme (PM) cell cultures prior to spiculogenesis. The antibody did not inhibit cell migration, cell attachment, or synthesis of the filapodial networks upon which the spicules are deposited. However, it did block deposition of mineralized CaCO3 along these filapodia, strongly supporting the previously proposed role for the 1223 epitope in calcium accumulation and/or deposition. Previously the 1223 epitope has been most extensively studied in association with a mesenchyme-specific protein of 130 kDa (msp 130). It has now been established, by Western blot analysis of whole embryo and PM cell extracts using mAb 1223, that two other proteins of 205 and 250 kDa contain the 1223 epitope. A study of the developmental profiles of expression of these glycoproteins revealed that all three were first expressed just prior to spiculogenesis, consistent with a role for any or all of these proteins in this process. Additionally all three proteins incorporated ethanolamine, myristate, and palmitate, the precursors of the glycosylphosphatidylinositol (GPI) anchor. Further labeling studies revealed differences in the metabolic lability of the GPI anchor in the three proteins; pulse-chase studies demonstrated that the ethanolamine moiety was stable in msp 130, but was rapidly chased from the 205-kDa protein (T1/2 = 14 hr). Phosphatidylinositol-specific phospholipase C partially released (50%) msp 130 from the PM cell surface, whereas it had no effect on release of the 205- and 250-kDa proteins. Studies with 35SO4 labeling and PNGase F treatment directly established that all three proteins are sulfated, and that most of the sulfate is attached to the N-linked oligosaccharide chains. Thus, the three major mAb 1223-reactive glycoproteins in PM cells are also the three major proteins containing both sulfated N-linked oligosaccharide chains and GPI anchors. Further investigation of this intriguing correlation may help to define the precise function of the 1223 epitope in the process of spicule formation.
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PMID:Characterization of post-translational modifications common to three primary mesenchyme cell-specific glycoproteins involved in sea urchin embryonic skeleton formation. 155 76

Pharmacological and biochemical characteristics of the partially purified gamma-aminobutyric acid (GABA)B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [3H]GABA binding to the purified GABAB receptor showed a linear relationship and the KD and Bmax values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg2+ did not affect on the GABAB receptor binding, Ca2+ significantly increased [3H]GABA binding to the purified GABAB receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca2+ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [3H]GABA, but phospholipase A2 had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and beta-galactosidase significantly decreased the binding of [3H]GABA to the purified GABAB receptor. These results suggest that purification of the solubilized GABAB receptor by the affinity column chromatography may result in the functional uncoupling of GABAB receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABAB receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A2 treatment may not be involved in the exhibition of the binding activity.
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PMID:Pharmacological and biochemical characteristics of partially purified GABAB receptor. 166 62

Time course digestion of intact human erythrocytes and right side-out vesicles with carboxypeptidase Y altered the Rh polypeptides and removed the 125I label that is normally incorporated by cell-surface radioiodination, but did not affect the RhD, Rhc, or RhE antigens. Under the same conditions, however, the LW antigens were rapidly destroyed. Digestion of inside-out and right side-out vesicles with aminopeptidase M was without any detectable effect on the Rh and LW antigens or polypeptides, although glycophorin A was degraded from right side-out but not from inside-out vesicles. These findings demonstrate that the C-terminal domain of the Rh and LW polypeptides is exposed at the external surface of human erythrocytes and indicate, in addition, that the LW antigens and tyrosine residue(s) of the LW and Rh proteins, respectively, are located close to the C termini of these polypeptides. Further studies using monoclonal and polyclonal antibodies showed that LW antigen expression is inhibited by treatment of red cells with EDTA and is selectively restored by Mg2+, but not by Mn2+ or Ca2+, whereas the Rh antigens were not affected under these conditions. In addition, O- and N-glycanase digestion of the LW glycoprotein removed its sugar chains, but did not alter significantly the epitopes recognized by the monoclonal anti-LW antibody.
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PMID:Surface orientation and antigen properties of Rh and LW polypeptides of the human erythrocyte membrane. 197 26

An alpha 1,2-mannosidase (Man9-mannosidase) involved in N-linked oligosaccharide processing has been purified about 16,000-fold from pig liver crude microsomes (microsomal fractions) by CM-Sepharose and DEAE-Sephacel chromatography, concanavalin A (Con A)-Sepharose chromatography and, as the key step of the procedure, affinity chromatography on immobilized N-5-carboxypentyl-l-deoxymannojirimycin (CP-dMM). On SDS/polyacrylamide-gel electrophoresis under reducing conditions, the isolated enzyme migrated as a single protein band with a molecular mass of 49 kDa. The enzyme does not bind Con A and is not susceptible to glycopeptidase F, indicating that it lacks N-linked oligosaccharides of the high-mannose or complex type. Purified Man9-mannosidase has a pH optimum close to 6.0 and requires bivalent cations for activity, with Ca2+ being most effective. The enzyme is inhibited strongly by basic sugar analogues of mannose such as 1-deoxymannojirimycin (dMM, Ki approximately 5 microM), N-methyl-dMM (Ki approximately 55 microM) and CP-dMM (Ki approximately 150 microM), whereas NN-dimethyl-dMM and the mannosidase II inhibitor swainsonine were hardly or not at all inhibitory. A homogeneous preparation of the 49 kDa enzyme cleaves specifically three of the four alpha 1,2-mannosidic linkages in the natural Man9-GlcNAc2 (M9) substrate. The relative rates by which the parent and intermediate structures are hydrolysed were found to be about 3:2:5 for M9, M8 and M7 respectively. The enzyme displays only marginal activity toward the remaining alpha 1,2-mannosidic linkages in the Man9-GlcNAc2 oligosaccharide (relative rate of M6 hydrolysis approximately 0.02) and is not active against nitrophenyl and methylumbelliferyl alpha-mannosides. This unique substrate specificity suggests that Man9-mannosidase processing differs from that catalysed by other trimming alpha 1,2-mannosidases hitherto reported. A polyclonal antibody raised against the denatured 49 kDa polypeptide not only recognizes a protein band of similar size in Western blots of crude microsomes, but also reacts strongly with a 65 kDa protein species. On trypsin treatment of detergent-solubilized microsomes, the 65 kDa protein is converted specifically into a stable 49 kDa fragment, indicating a precursor-product relationship between the two proteins. We conclude from this observation that the 65 kDa protein represents the intact form of Man9-mannosidase from which the 49 kDa enzyme which we have isolated has been generated, with retention of catalytic activity, by proteolysis during purification. Proteolytic studies with sealed microsomes suggest that the intact 65 kDa enzyme is a protein with a membrane-spanning domain, as well as a cytosolic polypeptide domain of size at least 3 kDa.
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PMID:Characterization of trimming Man9-mannosidase from pig liver. Purification of a catalytically active fragment and evidence for the transmembrane nature of the intact 65 kDa enzyme. 260 21

The structural features of the adult rat hepatocyte (ARH) forms of cell-CAM 105, a Mr 105,000 cell adhesion molecule, were compared using a variety of immunochemical and biochemical techniques with altered forms of more basic pI present on two transplantable hepatocellular carcinomas (THC 1682c and THC AS-30D). Immunoprecipitation analysis with polyclonal (anti-gp 105-2) and monoclonal (MAb) antibodies specific for cell-CAM 105 (MAb 362.50) demonstrated that ARH and THC cell-CAM 105 were indistinguishable in several respects including: (a) binding to wheat germ agglutinin; (b) labeling with NaIO4/NaB3H4; (c) susceptibility to digestion with endoglycosidases (endoglycosidase H and F and peptide N-glycosidase F N-glycanase); (d) rate of turnover on the cell surface; and (e) differential resistance of upper and lower forms to trypsin digestion in the presence or absence of calcium. Digestion with Clostridium perfringens or Vibrio cholerae neuraminidase did not equalize pI but instead decreased the size and increased the pI of both ARH and THC cell-CAM 105. Comparison of two-dimensional tryptic peptide maps, however, revealed five unique peptides in the THC AS-30D map and one peptide in the THC 1682c map, peptides which were only apparent in maps of deglycosylated ARH cell-CAM 105. Based on these results, it was concluded that there were significant differences in the glycosylation of ARH and THC cell-CAM 105. Biosynthetic labeling with 32PO4 and 35SO4 showed that both ARH and THC molecules were phosphorylated but not sulfated. Comparison of 32P-labeled peptides produced by digestion with V-8 protease revealed significant differences in the phosphorylation of the upper and lower forms from ARH and showed that the pattern of phosphorylation on THC cell-CAM 105 most closely resembled ARH upper form. Pulse-chase analysis of ARH cell-CAM 105 further indicated that only a subpopulation of the molecules labeled with [35S]methionine were phosphorylated.
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PMID:Comparison of the structural characteristics of cell-CAM 105 from hepatocytes with those of an altered form expressed by rat transplantable hepatocellular carcinomas. 281 19

Primary cultures of rat alveolar type II cells bind radiolabeled pulmonary surfactant protein A (SP-A) with high affinity. The binding of 125I-labeled SP-A is time- and temperature-dependent and is not accompanied by significant degradation. The binding process is saturable at low concentrations of SP-A (5 micrograms/ml), and unlabeled SP-A readily competes with labeled SP-A for cellular binding sites. Subsequent to binding, two pools of cell-associated 125I-labeled SP-A can be identified based upon sensitivity to trypsin at 0 degrees C. It is likely that the trypsin-sensitive pool comprises 125I-labeled SP-A bound to the cell surface and the trypsin-insensitive pool comprises the internalized protein. Scatchard analysis of cell surface binding of SP-A at 0.1-10 micrograms/ml shows positive cooperativity at concentrations between 0.1 and 1 micrograms/ml. Hill plots give nH = 1.34 +/- 0.08 with an apparent dissociation constant K'd = 1.02 +/- 0.32 micrograms/ml (which is 0.64 +/- 0.19 nM if the native molecular mass of oligomeric SP-A is assumed to be 1.6 MDa). The binding of SP-A to type II cells shows an absolute requirement for Ca2+. The putative receptor for SP-A is unaffected by treatment of type II cells with a variety of proteases and N-Glycanase (EC 3.5.1.52). Alveolar macrophages also exhibit high-affinity binding of SP-A, but rat lung fibroblasts and the alveolar epithelial cell line L2 exhibit only nonspecific binding.
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PMID:Alveolar type II cells express a high-affinity receptor for pulmonary surfactant protein A. 284 Jun 67

The glycoproteins responsible for calcium-dependent oligodendrocyte aggregation were purified and characterized. Using detergent extraction, lentil-lectin-Sepharose 4B affinity chromatography, and preparative gel electrophoresis, 3 proteins were purified to apparent homogeneity, with relative Mrs of 120,000, 140,000, and 180,000. The aggregation assay showed that all 3 proteins had the ability to block antibody-mediated inhibition of oligodendrocyte aggregation. The 120,000 protein was the most active of the three. Antisera were raised in rabbits to these 3 individual proteins. Western blot analyses showed that all three antisera recognized 120,000, 140,000, and 180,000 proteins, which indicated that the proteins were related. Western-blot analyses of cultured oligodendrocytes and purified rat myelin showed only the 120,000 protein. Immunoprecipitation of iodinated membrane proteins of cultured oligodendrocytes also indicated the presence of only the 120,000 Mr protein. Deglycosylation of the 120,000 protein by N-glycanase resulted in a 110,000 protein. The immunoblot pattern suggested some similarities between oligodendrocyte adhesion molecules and the neural cell adhesion molecule (N-CAM). Therefore, the 120,000, 140,000, and 180,000 Mr proteins were compared to N-CAM by Western-blot analysis, immunofluorescence staining, and by immunoprecipitation. The results suggest that oligodendrocytes contain a 120,000 membrane glycoprotein that is related to N-CAM.
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PMID:Oligodendrocyte cell adhesion molecules are related to neural cell adhesion molecule (N-CAM). 377 36

Envelope glycoproteins of human immunodeficiency virus (gp120 and gp41) occur as oligomers. Here, we show by gel filtration analysis that gp120 oligomerization in vitro is calcium- and temperature-dependent. Recombinant gp120 (rgp120) species were recovered as monomers at 20 degrees C in the absence of calcium, but as tetramers at 37 degrees C in 10 mM CaCl2. Under the latter condition, N-glycanase-deglycosylated rgp120 formed hexamers. Relative to intact rgp120, which has been reported to display carbohydrate-binding properties for N-acetyl-beta-D-glucosaminyl and mannosyl residues, deglycosylation enhanced rgp120 specific binding to mannose-divinylsulfone-agarose, para-aminophenyl-beta-D-GlcNAc-agarose and fetuin-agarose matrices. Taken together, these results rule out the role of homologous lectin-carbohydrate interactions via N-linked glycans in the rgp120 oligomerization, even though its lectin properties may also be calcium-dependent. Deglycosylation may unmask domains of rgp120 polypeptide backbone that independently play a role either in rgp120 lectin activity or in calcium-dependent oligomerization.
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PMID:The role of calcium and N-linked glycans in the oligomerization and carbohydrate binding properties of human immunodeficiency virus external envelope glycoprotein. 780 9

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


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