Positively charged lysine residues, particularly located at codons 24 and 27, are importnat for the pre-OR residues 23–31 to form a PK-resistant structure in prion-infected N2a cells.

(A) Amino acid sequences of the pre-OR residues 23–31 in mutant proteins. Bold residues indicate substituted residues. (B) Western blotting of N2aC24L1-3 cells transfected with control pcDNA3.1(+) and expression vectors encoding each mutant protein using 3F4 anti-PrP antibodies. The cell lysates were treated with PK at 5 µg/ml. All of the mutant proteins were converted into PK-resistant isoforms in N2aC24L1-3 cells, and all of the mutant isoforms gave rise to doublet non-glycosylated and mono-glycosylated bands. The doublet bands of moPrP(3F4)^ScΔ32–88(K23A), moPrP(3F4)^ScΔ32–88(K24A) and moPrP(3F4)^ScΔ32–88(K27A) were similar in molecular size to those of moPrP(3F4)^ScΔ32–88. However, moPrP(3F4)^ScΔ32–88(K24,27A) gave rise to the doublet band with the upper band migrating very closely to the lower band, similarly to moPrP(3F4)^ScΔ32–88(3K3A). MoPrP(3F4)^ScΔ32–88(K23,24A) and moPrP(3F4)^ScΔ32–88(K23,27A) showed the upper band with an intermediate molecular size. MoPrP(3F4)^ScΔ32–88(3K3R) giving rise to doublet bands with similar molecular size to those of moPrP(3F4)^ScΔ32–88. (C) IBL-N antibodies recognized all of the PK-resistant isoforms except for moPrP(3F4)^ScΔ32–88(3K3A) and moPrP(3F4)^ScΔ32–88(K24,27A).