NR ALKN
AU Taraboulos,A.; Serban,D.; Prusiner,S.B.
TI Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells
QU Journal of Cell Biology 1990 Jun; 110(6): 2117-32
PT journal article
AB 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.
IN Das zellulär Prionprotein PrPc ist ein auf der Zellmembranaußenseite über Glycosylphosphatidylinositol verankertes Sialoglycoprotein. Scrapie-Infektionen führen zu einer Akumulation der Scrapie-typischen Isoform des Prionproteins PrPsc. Die Autoren isolierten aus Zellkulturen PrPsc durch Proteolyse und Verdauung mit der Phosphatidylinositol-spezifischen Phospholipase C und steigerten seine in-situ-Immunoreaktivität durch Denaturierung. So konnten sie aus einer Scrapie-infizierten, neuen Hamsterhirnzelllinie (HaB), PrPsc-produzierende Klone isolieren und stellten eine exzellente Korrelation zwischen PrPsc-Gehalt und Infektiosität fest. Sie stellten fest, dass PrPsc in kultivierten, Scrapie-infizierten Hamsterhirn- (HaB) und Mausneuroblastomzellen hauptsächlich intrazellulär akumuliert.
MH Animal; Brain/*cytology/metabolism/microbiology; Cells, Cultured; Cytoplasm/*metabolism/microbiology/ultrastructure; Epitopes/immunology; Fluorescent Antibody Technique; Hamsters; Mesocricetus; Mice; Microscopy/methods; Microscopy, Electron/methods; PrPsc Proteins; Prions/isolation & purification/ultrastructure; Protein Denaturation; Scrapie/metabolism/pathology; Sheep; Support, Non-U.S. Gov't; Support, U.S. Gov't, P.H.S.; Temperature; Viral Proteins/immunology/*metabolism
AD Department of Neurology, University of California, San Francisco 94143.
SP englisch
PO USA
ZF grobe Zusammenfassung von Roland Heynkes