N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state.
Scrapie and related transmissible spongiform encephalopathies result in the accumulation of a protease-resistant form of an endogenous brain protein called PrP. As an approach to understanding the scrapie-associated modification of PrP, we have studied the processing and sedimentation properties of protease-resistant PrP (PrP-res) in scrapie-infected mouse neuroblastoma cells. Like brain-derived PrP-res, the neuroblastoma cell PrP-res aggregated in detergent lysates, providing evidence that the tendency to aggregate is an intrinsic property of PrP-res and not merely a secondary consequence of degenerative brain pathology. The PrP-res species had lower apparent molecular masses than the normal, protease-sensitive PrP species and were not affected by moderate treatments with proteinase K. This suggested that the PrP-res species were partially proteolyzed by the neuroblastoma cells. Immunoblot analysis of PrP-res with a panel of monospecific anti-PrP peptide sera confirmed that the PrP-res species were quantitatively truncated at the N terminus. The metabolic labeling of PrP-res in serum-free medium did not prevent the proteolysis of PrP-res, showing that the protease(s) involved was cellular rather than serum-derived. The PrP-res truncation was inhibited in intact cells by leupeptin and NH4Cl. This provided evidence that a lysosomal protease(s) was involved, and therefore, that PrP-res was translocated to lysosomes. When considered with other studies, these results imply that the conversion of PrP to the protease-resistant state occurs in the plasma membrane or along an endocytic pathway before PrP-res is exposed to endosomal and lysosomal proteases.
Caughey
B
B
Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840.
Raymond
G J
GJ
Ernst
D
D
Race
R E
RE
D000818:Animals; D002449:Cell Aggregation; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D009447:Neuroblastoma; D018620:PrPSc Proteins; D011328:Prions / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D018620:PrPSc Proteins; D011328:Prions; D010450:Endopeptidases