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Effect of transition metals (Mn, Cu, Fe) and deoxycholic acid (DA) on the conversion of PrP^C to PrP^res

Nam-Ho Kim^*,, Jin-Kyu Choi^*, Byung-Hoon Jeong^*, Jae-Il Kim, Myung-Sang Kwon, Richard I. Carp and Yong-Sun Kim^*,1

^* Ilsong Institute of Life Science, Anyang, Kyounggi-do, South Korea;
Department of Veterinary Medicine, Kangwon National University, Chuncheon, Kangwon-do, South Korea; and
New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, USA

¹ Correspondence: Ilsong Institute of Life Science and Department of Microbiology, College of Medicine, Hallym University, Ilsong Building, 1605-4 Gwanyang-dong, Dongan-gu Anyang, Kyounggi-do 431-060, South Korea. E-mail: yskim{at}hallym.ac.kr

SPECIFIC AIMS

Transition metals, which have a redox capacity, have been implicated in metal-catalyzed oxidation (MCO) of proteins, which can lead to structural alteration or aggregation of these proteins. We hypothesized that transition metals such as Mn and molecules oxidized by MCO would be cofactors for PrP^res formation. This concept was supported by our finding that the level of Mn was significantly increased in brain homogenates, mitochondrial fractions of brain and PrP^Sc-enriched fractions obtained from scrapie-positive animals. Thus, we focused on Mn in the formation of PrP^res by several cycles of incubation-sonication; we tested the effect of addition of Cu and Fe to the incubation-sonication reaction. The aim of this study was to investigate whether transition metals and deoxycholic acid (DA), an oxidized molecule, can induce PrP^res amplification in normal brain homogenate in vitro.

PRINCIPAL FINDINGS

1. Increase of Mn level in the scrapie-infected hamster brain
Total Mn levels in whole brain of 263K scrapie-infected hamster brains were significantly higher than in control brain preparations (33.8% higher, P<0.01). In mitochondrial fractions from 263K brains, Mn levels were higher than in control mitochondrial fractions (32.8% higher, P<0.01). The Mn levels were also higher in sarkosyl supernatants from scrapie brain preparations compared with preparations from normals (34.5%, P<0.05). In contrast, there was no difference in the Mn levels in NaCl-treated preparations between scrapie and control. Sarkosyl extracts of scrapie brain are rich in scrapie-associated fibrils (SAF), whereas SAF are not found in supernatants after NaCl treatment.

2. PrP^res induced by modified PMCA and inhibition of PrP^res formation by EDTA
We applied the new technique of protein misfolding cyclic amplification (PMCA) to PrP^C using cycles of incubation-sonication with Mn-treated normal hamster brain homogenate but without the PrP^Sc seed used in standard PMCA. We used several cycles of incubation-sonication plus an additional incubation of Mn-treated normal brain homogenate; this protocol is termed modified PMCA. In incubation-only cycles (0 cycles), PrP^res in the homogenate treated with 350 mM MnCl₂ was detected with proteinase K (PK) doses of up to 50 µg/mL; PrP^res was not detected at lower levels of MnCl₂. In contrast, using four cycles of modified PMCA in the presence of 350 mM MnCl₂, PrP^res was resistant to PK at doses of up to 250 µg/mL. At lower MnCl₂ doses, using one or four cycles of modified PMCA at different power amplitudes (20, 30, and 60%), PrP^res was detected, whereas no PrP^res was found after 0 cycles. By performing in vitro deglycosylation with PNGase F, we found that PrP^res produced in the presence of Mn by modified PMCA was glycosylated. The banding pattern seen in Cu- or Fe-treated samples was different from that found in Mn-treated samples after one cycle of modified PMCA. The major difference is that with Cu- or Fe-treated samples there was a band consistently found at 29–30 kDa that was not routinely found with Mn treatment. The induction of PrP^res by modified PMCA with Mn was reversed by EDTA, a metal chelater, suggesting PrP^res production results from Mn. The effect of Mn in the modified PMCA was not a function of the acidity of the MnCl₂ solution used. Induction of PrP^res by Mn occurred at incubation temperatures of 37°C and 65°C.

3. Effect of DA on the Mn-induced PrP^res by modified PMCA
We examined whether the composition of the immunoprecipitation (IP) buffer might influence Mn-induced PrP^res formation in the modified PMCA. We compared one cycle of modified PMCA using various doses of Mn with different buffers. Solutions such as PBS and distilled water did not permit PrP^res formation. We found that elimination of DA from the IP buffer yielded markedly reduced levels of PrP^res formation whereas in IP buffers without Triton X-100 or NaCl, there was not a profound effect (Fig. 1 A). The Mn-induced PrP^res signal increased in IP buffer as a function of increasing concentrations of DA (Fig. 1B ), whereas PrP^res signal was not found using DA alone (i.e., without MnCl₂ or at a pH range (6.657.87) comparable to the pH of the DA solution) (Fig. 1C ). Acids such as DA or HEPES are needed in Mn-induced PrP^res formation by modified PMCA.

View larger version (23K): [in this window] [in a new window]   Figure 1. Induction of PrPres by modified PMCA with Mn using various buffers and various concentrations of DA and Mn. A) Normal hamster brain homogenates in different buffers (listed to the right of each Western blot) containing various concentrations of MnCl2·4H2O were subjected to 1 cycle of modified PMCA. B) Normal hamster brain homogenates in IP buffer and in buffer with NaCl and Triton X-100 (NT buffer) were supplemented with various concentrations of DA or of DA + MnCl2. C) Normal hamster brain homogenates in IP buffer or NT-buffer were treated with MnCl2 at various pH levels corresponding to the pH of the DA solutions used. Samples of brain homogenates containing 5.46 mM MnCl2·4H2O were subjected to 1 cycle of the modified PMCA. All samples were incubated for 5 h at 37°C and sonicated at 30% power using 25 pulses of 4 s each. Samples were digested with PK (50 µg/mL at 37°C for 30 min) and immunoblotted with 3F4 anti-PrP monoclonal antibody.

4. The PrP^res produced by modified PMCA in the presence of Mn and DA can act as a template for further PrP^res production
Putative PrP^res templates were produced by modified PMCA in the presence of Mn plus DA. These preparations were diluted in normal hamster brain homogenates and these mixtures were subjected to 10 cycles of incubation-sonication (standard PMCA but without the PrP^Sc seed). PrP^res signals after 10 PMCA cycles could be diluted further than preparations incubated without sonication (Fig. 2 ). PrP^res amplification was detected even after a 2560-fold dilution of template, whereas the end point for the incubation-only samples was 320-fold. We also found that EDTA added to the solution used in the modified PMCA reaction eliminated the template effect of the preparation (Fig. 2) .

View larger version (49K): [in this window] [in a new window]   Figure 2. Template activity of Mn-induced PrPres produced by modified PMCA. PrPres templates were made by 1 cycle of incubation (37°C, 5 h) -sonication (40% power using 25 pulses of 4 s each) and one additional incubation of normal hamster brain homogenates in IP buffer containing 4% DA with 43.7 mM MnCl2·4H2O. For PrPres templates plus EDTA, normal hamster brain homogenates in IP buffer containing 4% DA with 43.7 mM MnCl2·4H2O were treated with 125 mM EDTA and subjected to 1 cycle of incubation (37°C, 5 h) and sonication (40% power with 25 pulses of 4 s each) plus an additional incubation at 37°C for 5 h. These templates were diluted serially into 10% normal hamster brain homogenate substrate. The dilutions of templates were 1 to 10, 20, 40, 80, 160, 320, 640, 1280, and 2560. Diluted samples were subjected to 10 PMCA cycles in the absence of a PrPSc spike (37°C, 3 h incubation, sonication condition: 40% power using 10 pulses of 1 s each). Control samples were treated identically but without sonication. All samples were digested with PK (50 µg/mL at 37°C for 30 min) and immunoblotted with 3F4 anti-PrP monoclonal antibody.

CONCLUSIONS AND SIGNIFICANCE

Certain cofactors may play a role in the conformational change of PrP isomers that occurs during the course of scrapie pathogenesis. Studies of PrP^res complex bound to metals by MCO are expected to provide new insight into the PrP^C PrP^Sc conversion mechanism in prion diseases. In the present study, PrP^res amplification by modified PMCA required both Mn and oxidized molecules such as DA (Fig. 1) . In scrapie-infected hamsters, the level of Mn was increased in whole brain, mitochondria, and SAF-enriched fraction compared with the values in preparations from the control group.

On the basis of our results, we suggest that PK-resistant PrP^C-Mn complex is formed through binding of PrP^C to Mn in the presence of DA in IP buffer. This PrP-metal complex would be partially resistant to PK and could provide a template for further PrP^res formation in a PMCA-type reaction (Fig. 3 ).

View larger version (21K): [in this window] [in a new window]   Figure 3. Diagrammatic representation of the modified PMCA technique in which Mn and DA are added to the mixture used for incubation-sonication.

Carboxyl acids such as 3-aminophenylacetic acid and valproic acid are oxidized molecules similar to DA; these molecules have been shown to exacerbate amyloid-like fibril formation or accumulation. In our study, PrP^res formation was not found in DA alone, but required Mn plus DA. Mn and oxidized molecules such as carboxyl acids may be involved in the mechanism of PrP^C conversion to PrP^Sc.

MCO may be involved in human neurological disorders and, as described by others, may cause structural alteration, dysfunction, and aggregation of proteins. In prion diseases, previous studies have shown that PrP is prone to MCO, resulting in conformational changes or aggregation in vitro. These findings suggest that PrP^C may be converted to an oxidized form by transition metals and that this is an important pathogenic mechanism in prion diseases. The precise mechanism of metal-catalyzed PrP oxidation remains to be determined.

Use of the modified PMCA technique may provide insight into the roles of metals and oxidized molecules in PrP^res amplification.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2117fje;

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