Proteolytic processing of the ovine prion protein in cell cultures

Characterization of the prion protein 3F4 epitope and its use as a molecular tag

“…This is most likely due to the mutational exchange of residues 203, 205, 206, 208, just C-terminal to the N-glycosylation site at aspargine 200. As previously pointed out by others and us (Chen et al, 1995;Pan et al, 2002;Tveit et al, 2005), the traditional use of the 3F4 antibody suffers somewhat by the fact that the epitope is affected by the normal proteolytic processing of PrP, resulting in detection of full length PrP mainly. This problem can now be overcome in transgenic experiments by moving the epitope to another carefully selected location in PrP.…”

“…The failure of 3F4 to detect any other PrP molecules except full length molecules was first demonstrated in human brain, and verified through protein sequencing showing that the amino-terminal end of the major PrP cleavage fragment (C1) contained part of the 3F4 epitope (Chen et al, 1995). This limits to some degree the suitability of the 3F4 antibody because both in brain and other tissues as well as in cell culture, a considerable portion of the PrP molecules have undergone proteolytic processing (Pan et al, 1992(Pan et al, , 2002Mange et al, 2004;Tveit et al, 2005). Thus, the 3F4 antibody will only detect a sub-population of the PrP molecules present.…”

Unexpected Tolerance of α-Cleavage of the Prion Protein to Sequence Variations

“…Other studies also suggested that α-cleavage is not affected by the removal of the octapeptide region [15], [18], [20] or by the insertion of supernumerary octapeptide repeats [20]. α-cleavage of PrP C mutants was also assessed in cells expressing ovine PrP (ovPrP) with GFP inserted into the N-proximal region [21]. In this system, the point mutations ovPrP K113R , ovPrP K113D , ovPrP K113A and ovPrP KHV113–115AAA did not appear to affect cleavage, with the respective mutant proteins processed similarly to the fusion protein of wild-type ovPrP C with GFP.…”

Alternative Translation Initiation Generates Cytoplasmic Sheep Prion Protein

“…This nucleotide sequence, known as the Kozak sequence, has been comprehensively studied for its influence on protein translation (31). A peculiarity of the constructs that lack an intact Kozak sequence was that they resulted in a shift in cellular localization of PrP, with a dispersed, intracellular, fluorescence signal, accompanied by a characteristic doublet of bands in Western blots ( GFP PrP*), containing two N-terminal fragments of PrP (36). Further analysis using a polyclonal anti-SP antibody (43), identified full-length PrP and an N-terminal truncation doublet, neither of which had been processed by the ER residing signal peptidase.…”

“…Construction of Plasmids-The construction of plasmids coding for PrP* and GFP PrP* has been described previously (36). The PrP and the GFP PrP, which contain the naturally occurring sheep Kozak sequence (5Ј-GTC ATC preceding the start codon ATG, otherwise similar to PrP and GFP PrP*), were generated using the PrP* and the GFP PrP* constructs as templates, standard cloning techniques, and the following primers: forward, 5Ј-TAGCCTCGAGGTCATCATGGTGAAAAGCCACAT-AGG-3Ј; and reverse, 5Ј-CGACTCTAGAGTACTATCCTAC-TATGAGAAAAATGAGG-3.…”