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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cellular prion protein (PrPC) is a sialoglycoprotein anchored to the external surface of cells by a glycosyl phosphatidylinositol moiety. During scrapie, an abnormal PrP isoform designated PrPSc accumulates, and much evidence argues that it is a major and necessary component of the infectious prion. Based on the resistance of native PrPSc to proteolysis and to digestion with phosphatidylinositol-specific phospholipase C as well as the enhancement of PrPSc immunoreactivity after denaturation, we devised in situ immunoassays for the detection of PrPSc in cultured cells. Using these immunoassays, we identified the sites of PrPSc accumulation in scrapie-infected cultured cells. We also used these immunoassays to isolate PrPSc-producing clones from a new hamster brain cell line (HaB) and found an excellent correlation between their PrPSc content and prion infectivity titers. In scrapie-infected HaB cells as well as in scrapie-infected mouse neuroblastoma cells, most PrPSc was found to be intracellular and most localized with ligands of the Golgi marker wheat germ agglutinin. In one scrapie-infected HaB clone, PrPSc also localized extensively with MG-160, a protein resident of the medial-Golgi stack whereas this colocalization was not observed in another subclone of these cells. Whether the sites of intracellular accumulation of PrPSc are limited to a few subcellular organelles or they are highly variable remains to be determined. If the intracellular accumulation of PrPSc is found in the cells of the central nervous system, then it might be responsible for the neuronal dysfunction and degeneration which are cardinal features of prion diseases.
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PMID:Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells. 169 23

The cellular prion protein (PrPc) is a host-encoded sialoglycoprotein bound to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor. A posttranslationally modified PrP isoform (PrPSc) is a component of the infectious particle causing scrapie and the other prion diseases. mAb have been raised against the protease-resistant core of Syrian hamster (SHa) PrPSc designated PrP 27-30. To map the epitopes within PrP reacting to these antibodies, we have expressed wild-type, chimeric mouse (Mo)/SHa and mutant MoPrP genes using recombinant vaccinia virus systems. The fidelity of the expression of recombinant PrPC was examined using vaccinia viruses expressing SHa-PrPC. It is full length, possesses Asn-linked carbohydrates and is attached to the external surface of the cell membrane by a glycosyl phosphatidylinositol anchor that is sensitive to cleavage by phosphatidylinositol-specific phospholipase C. We have tested 18 mAb for their ability to bind to chimeric prion proteins on immunoblots. Three distinct epitopes were identified that mapped to amino acid differences between SHa and MoPrP sequences. The first epitope, recognized by three of the antibodies tested, was defined by methionines at amino acids 108 and 111 in the mouse protein. The second epitope was dependent upon the presence of asparagines at positions 154 and 174 in MoPrP and was recognized by four of the antibodies tested. The third epitope mapped to a single amino acid substitution at residue 138 in MoPrP. mAb raised against SHaPrP 27-30 specific for this epitope are able to bind MoPrPC which has a single amino acid change (Ile to Met) at position 138. Eleven of the 18 antibodies tested mapped to this immunodominant epitope. It is located within a postulated amphipathic helix, a structure associated with immunodominant Ag. Inasmuch as PrPC, in its native form on the cell surface, is detected by the mAb 13A5 (a prototypic antibody of the immunodominant third epitope class), it is likely that this epitope is accessible in the native conformation of this protein.
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PMID:Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system. 171 82

Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrPc and PrPSc are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrPC can be distinguished from PrPSc by limited proteolysis under conditions where PrPC is hydrolyzed and PrPSc is resistant. We report here that PrPC can be released from the surface of both normal-control and scrapie-infected murine neuroblastoma (N2a) cells by phosphatidylinositol-specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrPSc was neither released by PIPLC nor labeled with sulfo-NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrPC while incorporation into PrPSc molecules was observed only during the chase period. While PrPC is synthesized and degraded relatively rapidly (t1/2 approximately 5 h), PrPSc is synthesized slowly (t1/2 approximately 15 h) and appears to accumulate. These results are consistent with several observations previously made on rodent brains where PrP mRNA and PrPC levels did not change throughout the course of scrapie infection, yet PrPSc accumulated to levels exceeding that of PrPC. Our kinetic studies demonstrate that PrPSc is derived from a protease-sensitive precursor and that the acquisition of proteinase K resistance results from a posttranslational event. Whether or not prolonged incubation periods, which are a cardinal feature of prion diseases, reflect the slow synthesis of PrPSc remains to be established.
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PMID:Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. 196 66

The abnormal isoform of the scrapie prion protein PrPSc is both a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the normal cellular isoform PrPC have different physical properties that apparently arise from a posttranslational event. Both PrP isoforms are covalently modified at the carboxy terminus by a glycoinositol phospholipid. Using preparations of dissociated cells derived from normal and scrapie-infected hamster brain tissue, we find that the majority of PrPC is released from membranes by phosphatidylinositol-specific phospholipase C (PIPLC), while PrPSc is resistant to release. In contrast, purified denatured PrP 27-30 (which is formed from PrPSc during purification by proteolysis of the amino terminus) is completely cleaved by PIPLC. Incubation of the cell preparations with proteinase K cleaves PrPSc to form PrP 27-30, demonstrating that PrPSc is accessible to added enzymes. We have also developed a protocol involving biotinylation that gives a quantitative estimate of the fraction of a protein exposed to the cell exterior. Using this strategy, we find that a large portion of PrPSc in the cell preparations reacts with a membrane-impermeant biotinylation reagent. Whether alternative membrane anchoring of PrPSc, inaccessibility of the glycoinositol phospholipid anchor to PIPLC, or binding to another cellular component is responsible for the differential release of prion proteins from cells remains to be determined.
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PMID:Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C. 197 60

The scrapie (PrPSc) and cellular (PrPC) prion proteins are encoded by the same gene, and their different properties are thought to arise from posttranslational modifications. We have found a phosphatidylinositol glycolipid on both PrPC and PrP 27-30 (derived from PrPSc by limited proteolysis at the amino terminus). Ethanolamine, myo-inositol, phosphate, and stearic acid were identified as glycolipid components of gel-purified PrP 27-30. PrP 27-30 contains 2.8 moles of ethanolamine per mole. Incubation of PrP 27-30 with a bacterial phosphatidylinositol-specific phospholipase C (PIPLC) releases covalently bound stearic acid, and allows PrP 27-30 to react with antiserum specific for the PIPLC-digested glycolipid linked to the carboxyl terminus of the trypanosomal variant surface glycoprotein. PIPLC catalyzes the release of PrPC from cultured mammalian cells into the medium. These observations indicate that PrPC is anchored to the cell surface by the glycolipid.
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PMID:Scrapie prion protein contains a phosphatidylinositol glycolipid. 244 40

Inherited forms of prion disease have been linked to mutations in the gene encoding PrP, a neuronal and glial protein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. One familial form of Creutzfeldt-Jakob disease is associated with a mutant PrP containing six additional octapeptide repeats. We report here our analysis of cultured Chinese hamster ovary cells expressing a murine homologue of this mutant PrP. We find that, like wild-type PrP, the mutant protein is glycosylated, GPI-anchored, and expressed on the cell surface. Surprisingly, however, cleavage of the GPI anchor using phosphatidylinositol-specific phospholipase C fails to release the mutant PrP from the surface of intact cells, suggesting that it has an additional mode of membrane attachment. The phospholipase-treated protein is hydrophobic, since it partitions into the detergent phase of Triton X-114 lysates; and it is tightly membrane-associated, since it is not extractable in carbonate buffer at pH 11.5. Whether membrane attachment of the mutant PrP involves integration of the polypeptide into the lipid bilayer, self-association, or binding to other membrane proteins remains to be determined. Our results suggest that alterations in the membrane association of PrP may be an important feature of prion diseases.
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PMID:A mutant prion protein displays an aberrant membrane association when expressed in cultured cells. 759 79

Chronic (2 day) exposure of human neuroblastoma cells to the organophosphate pesticide phosmet induced a marked concentration-dependent increase in the levels of PrP present on the cell surface as assessed by biotin labelling and immunoprecipitation. Levels of both phospholipase C (PIPLC)-releasable and non-releasable forms of PrP were increased on the plasma membrane. These increases appear to be due to post-transcriptional mechanisms, since PrP mRNA levels as assessed by Northern blotting were unaffected by phosmet treatment. These data raise the possibility that phosmet exposure could increase the susceptibility to the prion agent by altering the levels of accessible PrP.
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PMID:Phosmet induces up-regulation of surface levels of the cellular prion protein. 963 35

Exposure of susceptible neuroblastoma N2a cells to mouse scrapie prions leads to infection, as evidenced by the continued presence of the scrapie form of the prion protein (PrP(Sc)) and infectivity after 300 or more cell doublings. We find that exposure to phosphatidylinositol-specific phospholipase C (PIPLC) or to the monoclonal anti-prion protein (PrP) antibody 6H4 not only prevents infection of susceptible N2a cells but also cures chronically scrapie-infected cultures, as judged by the long-term abrogation of PrP(Sc) accumulation after cessation of treatment. A nonpassaged, stationary infected culture rapidly loses PrP(Sc) when exposed to the antibody or PIPLC, indicating that the PrP(Sc) level is determined by steady state equilibrium between formation and degradation, and that depletion of the cellular form of PrP can interrupt the propagation of PrP(Sc). These findings encourage the belief that passive immunization may provide a therapeutic approach to prion disease.
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PMID:Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody. 1147 Aug 93

The molecular mechanism of neurodegeneration in transmissible spongiform encephalopathies (TSEs) remains unclear. Using radioactive copper ((64)Cu) at physiological concentration, we showed that prion infected cells display a marked reduction in copper binding. The level of full-length prion protein known to bind the metal ion was not modified in infected cells, but a fraction of this protein was not releasable from the membrane by phosphatidylinositol-specific phospholipase C. Our results suggest that prion infection modulates copper content at a cellular level and that modification of copper homeostasis plays a determinant role in the neuropathology of TSE.
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PMID:Prion infection impairs copper binding of cultured cells. 1263 48

1 Sulphonated aluminium phthalocyanine (SALPC) photodynamic action induces amylase secretion and permanent calcium oscillation in rat pancreatic acinar cells, because of the activation of phospholipase C or signalling proteins upstream. The aim of the present study was to investigate the involvement of muscarinic acetylcholine and cholecystokinin (CCK) receptors. 2 Muscarinic receptor antagonist atropine (10 micro M) blocked amylase secretion induced by bethanechol (100 micro M), and CCK(1) receptor antagonist (S)-N-[1-(2-fluorophenyl)-3,4,6,7-tetrahydor-4-oxo-pyrrolo-[3,2,1-jk][1,4] benzodiazepine-3yl]-1H-indole-2-carboxamide (FK480) (1 micro M) blocked amylase secretion induced by CCK (100 pM). 3 Amylase secretion induced by SALPC photodynamic action was not inhibited when atropine and FK480 were present during photodynamic action. However, addition of FK480 1 micro M after initiation of photodynamic action inhibited photodynamic amylase secretion. Bethanechol (10, 100 micro M) added after photodynamic action resulted in a full secretory response. 4 Atropine (10 nM) abolished calcium oscillation induced by bethanechol (5 micro M), and FK480 (10 nM) blocked calcium oscillation induced by CCK (10 pM). 5 Atropine up to 10 micro M was without effect on Ca(2+) oscillation triggered by photodynamic action, but these oscillations were abolished by FK480 (10 nM). FK480 (10 nM) had no effect on calcium oscillations induced by bethanechol (5 micro M). Bethanechol 5 micro M, added after FK480 blockade of photodynamic calcium oscillation, still triggered regular calcium oscillation. 6 It is concluded that SALPC photodynamic action selectively and permanently activates CCK receptor in rat pancreatic acini. Such permanent and selective modulation of signalling proteins has important implications for the treatment of pancreatitis, prion diseases, and neurodegenerative disorders.
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PMID:Selective activation by photodynamic action of cholecystokinin receptor in the freshly isolated rat pancreatic acini. 1281 11


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