Studies of deletion mutants of deletion mutants of fluorescently labeled prion protein (PrP) in cell culture

Abstract

The prion protein has been implicated in a number of neurodegenerative diseases of the transmissible spongiform encephalopathy (TSE) type. During disease development, the cellular prion protein (PrPC) undergoes a conformational change to the infective scrapie isoform (PrPSc). In normal brain and in several other tissues, as well as in cultured cell lines, PrPC is to a variable extent proteolytically processed at a putative major cleavage site, generating N- and C-terminal fragments called N1 and C1. The cleavage site lies adjacent to an unstructured N-terminal domain and a globular C-terminal domain. In man, and in some other mammalian species, the PrPC cleavage site is typically near amino acid 113. Manipulations in the corresponding region of ovine PrPC (fused with green fluorescent protein GFP) uncovered a remarkably robust processing activity, which was largely independent of the primary structure near amino acid 113. In this study, the PrP::GFP fusion protein has been investigated further with respect to processing and intracellular transport. Preliminary studies conducted in this thesis showed that PrP::GFP is similarly processed in MDCK II, N2a, and SH-SY5Y cells. Three new variants of ovine PrPC fused to GFP with different deletions near or around the proposed cleavage site were made. A PrP::GFP variant where amino acids 115-125 were deleted, gave similar cleavage products as observed for PrP::GFP, with two cleaved fragments detected by the prion-specific P4 antibody in Western blots. MS analysis revealed that these two fragments, of slightly different molecular mass, both were derived from the N-terminal portion of PrP::GFP, but the reason for the mass difference could not be determined at present. Two PrP::GFP variants where amino acids 104-115 or 104-125 were deleted displayed only one N-terminal fragment after intracellular cleavage, the one of the lower molecular mass. Additional analyses are required to further understand the proteolytic mechanism underlying the formation of the cleavage products and the cellular localization where cleavage of PrP::GFP variants takes place.