Prion protein biosynthesis in scrapie-infected and uninfected neuroblastoma cells.
Numerous studies have indicated that a modified proteinase K-resistant form of an endogenous brain protein, prion protein (PrP), is associated with scrapie infection in animals. This scrapie-associated PrP modification appears to occur posttranslationally in brain, but its molecular nature is not known. To learn about the normal PrP biosynthesis and whether it is altered by scrapie infection in vitro, we did metabolic labeling experiments with uninfected and scrapie-infected mouse neuroblastoma tissue culture cells. Pulse-chase labeling experiments indicated that, in both cell types, two major PrP precursors of 28 and 33 kilodaltons (kDa) were processed to mature 30- and 35- to 41-kDa forms. Endoglycosidase H, tunicamycin, and phospholipase treatments revealed that the 28- and 33-kDa precursors resulted from the addition of high-mannose glycans to a 25-kDa polypeptide containing a phosphatidylinositol moiety and that maturation of the precursors involved the conversion of the high-mannose glycans to hybrid or complex glycans. Treatments of the live cells with trypsin and phosphatidylinositol-specific phospholipase C indicated that the mature PrP species were expressed solely on the cell surface, where they were anchored by covalent linkage to phosphatidylinositol. Once on the cell surface, the major PrP forms had half-lives of 3 to 6 h. No differences in PrP biosynthesis were observed between the scrapie-infected versus uninfected neuroblastoma cells.
Caughey
B
B
Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana 59840.
Race
R E
RE
Ernst
D
D
Buchmeier
M J
MJ
Chesebro
B
B
D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000378:metabolism; D009447:Neuroblastoma; D009474:Neurons / Q000382:microbiology*; D018622:PrP 27-30 Protein; D011233:Precipitin Tests; D011328:Prions / Q000502:physiology*; D011498:Protein Precursors / Q000096:biosynthesis* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
D009419:Nerve Tissue Proteins; D011328:Prions; D011498:Protein Precursors; D018622:PrP 27-30 Protein; D014415:Tunicamycin; D010738:Type C Phospholipases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin