Abstract
The abnormally folded form of the prion protein (PrP(Sc)) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrP(Sc) N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrP(Sc) accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrP(Sc) proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrP(Sc) fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrP(Sc) and cell pathogenesis of prion infection.
Highlights
Agronomique. □S The on-line version of this article contains supplemental Figs
Centrifugation of cell lysates corroborated the presence of detergent-insoluble, truncated PrPSc, which was quantitatively recovered in the pellets, whereas normal PrPC did not sediment in these conditions
Our study first revealed that the efficiency of trimming varies dramatically depending on which cell or tissue supported replication of the infecting prion
Summary
Cell Culture—Rov cells (clone Rov9) and Rom cells are derived from the RK13 epithelial cell line and express the ovine or mouse PrP, respectively, in a doxycycline-dependent manner [25, 26]. CGN and CAS primary cultures from tg338 mice were exposed to 0.01% homogenate of 127S-infected tg338 mouse brain for 3 days and the accumulation of PrPSc was determined after 3 weeks as previously described [29]. CAD cells were incubated for 1 or 2 days with 0.5% brain homogenate of C57Bl/6 mice infected with 22L, 139A, or Chandler strains of mouse-adapted scrapie as described [38]. 50 l of IMAC column fractions were digested with 10 g/ml of PK for 1 h at 37 °C and denatured with 4ϫ sample buffer. 1 volume of crude lysates from uninfected brain or cell materials was mixed directly with 6 volumes of aqueous 48% hydrofluoric acid (Merck), incubated for 24 h at 4 °C, and vacuum-dried material was solubilized in 2ϫ SDS-PAGE sample buffer. For detection of PrP by immunoblotting, an enhanced chemiluminescence (ECL) detection system (Pierce or Roche) was used with goat anti-mouse IgG coupled to peroxidase as secondary antibodies
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