Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells.
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.
Borchelt
D R
DR
Department of Neurology, University of California, San Francisco 94143.
Scott
M
M
Taraboulos
A
A
Stahl
N
N
Prusiner
S B
SB
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002472:Cell Transformation, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D015151:Immunoblotting; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D018620:PrPSc Proteins; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D007106:Immune Sera; D018620:PrPSc Proteins; D011328:Prions; D014764:Viral Proteins; D008715:Methionine