Prion Strains Differ in Susceptibility to Photodynamic Oxidation

Abstract

Prion disorders, or transmissible spongiform encephalophaties (TSE), are fatal neurodegenerative diseases affecting mammals. Prion-infectious particles comprise of misfolded pathological prion proteins (PrP^TSE). Different TSEs are associated with distinct PrP^TSE folds called prion strains. The high resistance of prions to conventional sterilization increases the risk of prion transmission in medical, veterinary and food industry practices. Recently, we have demonstrated the ability of disulfonated hydroxyaluminum phthalocyanine to photodynamically inactivate mouse RML prions by generated singlet oxygen. Herein, we studied the efficiency of three phthalocyanine derivatives in photodynamic treatment of seven mouse adapted prion strains originating from sheep, human, and cow species. We report the different susceptibilities of the strains to photodynamic oxidative elimination of PrP^TSE epitopes: RML, A139, Fu-1 > mBSE, mvCJD > ME7, 22L. The efficiency of the phthalocyanine derivatives in the epitope elimination also differed (AlPcOH(SO₃)₂ > ZnPc(SO₃)_1-3 > SiPc(OH)₂(SO₃)_1-3) and was not correlated to the yields of generated singlet oxygen. Our data suggest that the structural properties of both the phthalocyanine and the PrP^TSE strain may affect the effectiveness of the photodynamic prion inactivation. Our finding provides a new option for the discrimination of prion strains and highlights the necessity of utilizing range of prion strains when validating the photodynamic prion decontamination procedures.

Keywords: PDI; PrP; TSE; photodynamic; phthalocyanine; prion; prion inactivation; protein folding; singlet oxygen; strain.

Figure 1

Structure of sulfonated metal phthalocyanines, where M represents coordinated central metal, and R₁ = R₂ = R₃ indicates the position of SO₃ substituents of peripheral benzene rings.

Figure 2

Phthalocyanine absorption spectra, aggregation state and O₂(¹∆_g) production. (a) Absorption spectra of Pc derivatives in PBS (10 µg mL⁻¹; red, green and black lines) and emission spectrum of LED light (gray peak) normalized to the maximal Pc absorption. (b) Absorption maxima of Pc derivatives in PBS and in DMSO (n = 3). (c) Relative production of O₂(¹∆_g) during photoactivation of Pc derivatives in iodide solution (n = 3).

Figure 3

Amino acid sequence of mouse prion protein (UniProt P04925) with the location of epitopes of monoclonal antibodies DC2 (aa35-46), AG4 (aa37-50), BE12 (aa50-99), 6D11 (aa93-109), D18 (aa132-156), 6H4 (aa144-152), AH6 (aa159-174) and GE8 (aa183-191). Underlined letters mark signal sequences which are cleaved off. The amino acid residues prone to oxidation by O₂(¹∆_g) are depicted in bold.

Figure 4

Photodynamic elimination of PrP^C and/or PrP^TSE epitopes proved by failure of antibodies binding. RML brain homogenate with 0.1 or 1 µg mL⁻¹ ZnPc(SO₃)_1-3 or AlPcOH(SO₃)₂ was illuminated for 90 min (red numbers) and control aliquots kept in dark either non-treated or with 10 µg mL⁻¹ of the Pc derivative (black numbers). One aliquot of each sample was cleaved with proteinase K (PK; +) to remove protease sensitive PrP^C and visualize PrP^TSE. PK treatment cleaves off the N-terminal part of PrP^TSE, resulting in its higher electrophoretic mobility and removal of DC2 and AG4 epitopes. The second aliquot was left intact (-) and contained both PrP^C and PrP^TSE. The samples were analyzed by Western blot with antibodies against different parts of PrP molecule (DC2, AG4, BE12, 6D11, D18, 6H4, AH6, GE8). The photodynamic elimination of the epitopes was documented for all tested antibodies. The figure is a representative of three independent experiments.

Figure 5

Photodynamic elimination of PrP^TSE signal by Pc derivatives. (a) Western blot analysis of photodynamic elimination of PrP^TSE signal induced by photoactivation with AlPcOH(SO₃)₂, ZnPc(SO₃)_1-3 and SiPc(OH)₂(SO₃)_1-3. PrP^TSE in the brain homogenates infected by different prion strains (ME7, 22L, mBSE, 139A, mvCJD, Fu-1, RML) was detected using the antibody 6D11 as proteinase K-resistant fragments PrPres. S; molecular weight standard. The Western blots are representative of three independent experiments. (b) Densitometry evaluation of the PrPres signal on Western blot membranes (n = 3). Dotted line represents the background signal. (c) Discrimination of the prion strains based on their sensitivity to photodynamic treatment by Pc derivatives. The strain was considered sensitive (+) when the average signal of PrPres on the Western blot reduced by SD is lower than the background.