Reduced PrP^C solubility is an effect specific to the binding of certain metal ions.

<p>Protein suspensions from brain homogenates derived from C57BL wild-type mice were reacted with increasing concentrations of ZnCl₂, CuCl₂ and MgCl₂ as indicated. Proteins were centrifuged resulting in a separation of PrP^C into a fraction of high solubility in the supernatant and a fraction of low solubility in the pellet. Following SDS-PAGE and immunoblotting, PrP^C signals were visualized using mabs SAF34 (A) and SAF70 (B) followed by chemiluminescence substrate development. PrP^C specific bands are indicated with d (diglycosylated), m (monoglycosylated), n (nonglycosylated) and C1 (truncated glycosylated C1 fragment). Neuron-specific enolase (NSE) was used as control (C).</p

The PrP^C–Zn²⁺ phenotype dominated in PrP^C interactions after simultaneous and serial CuCl₂ and ZnCl₂ incubation.

<p>(A) Brain homogenate proteins (10%) derived from the C57BL wild-type and T182N transgenic mice were suspended in TBS with OGP (2%) followed by simultaneous incubation with ZnCl₂ (Zn) and CuCl₂ (Cu) in concentrations of 100 and 1000 µM as indicated. Proteins were separated by centrifugation into a protein fraction of high solubility represented as supernatant (S) and low solubility PrP^C in the pellet (P). (B)Aliquots of highly soluble proteins from C57BL wild-type and T182N transgenic mice were pre-treated with zinc (Zn) and copper (Cu) ions (1 mM) each. As a result of centrifugation, proteins were separated into a pellet fraction (P1) and into highly soluble protein fractions, which were incubated additionally with ZnCl₂ (+ Zn) and CuCl₂ (+ Cu; 1 mM each) or in the absence of ions (-) as controls. Highly soluble proteins were detected in the supernatant S2, whereas poorly soluble PrP^C were detected in the pellet P2 after centrifugation. Copper ions were unable to alter the PrP^C–Zn²⁺ phenotype, which would have shown a conversion of pelleted PrP^C to a fraction of highly soluble proteins. Protein suspensions were separated by SDS-PAGE, immunoblotted and PrP^C were detected using mabs SAF34 and SAF70 as indicated. Signals were visualized after chemiluminescence reaction on an imager.</p

Amino-terminally truncated PrP^C isoforms are unable to bind metal ion.

<p>Proteins from pooled wild-type C57BL mouse brain homogenate were treated enzymatically with bromelain (50 µg/ml, 37°C, 60 min) generating a carboxy-terminal core protein lacking the octapeptide region to which divalent cations might bind. After incubation in the presence of CuCl₂ (Cu), ZnCl₂ (Zn) and MgCl₂ (Mg) in concentrations of 1 mM each or in the absence of metal ions (no metal), proteins were separated by centrifugation into fractions of high and low solubility, respectively, represented as supernatants (S) and pellets (P). Proteins were separated by SDS-PAGE, immunoblotted and PrP^C signals were visualized using mab SAF70 and chemiluminescence substrate development. Untreated protein (PrP^C) was used as control.</p

Reduced solubility due to ZnCl₂ and CuCl₂ is reversed by EDTA and SDS application.

<p>Pooled brain tissues derived from C57BL wild-type mice (A) and bovine (B) homogenized in TBS and 2% N-octyl-β-D-glucopyranoside were supplemented with ZnCl₂ (1 mM) and CuCl₂ (1 mM) followed by incubation in the absence (0) or presence of EDTA in concentrations indicated or 1% SDS. Following centrifugation, proteins were separated into fractions of high solubility in the supernatant and low solubility in the pellet. PrP^C signals were detected using mab SAF34 and visualized by chemiluminescence substrate development.</p

The glycosylation states influenced changes in solubility of mouse prion isoforms as a result of metal-binding.

<p>Pooled mouse brains from C57BL wild-type and transgenic T182N mice were homogenized (10%) and supplemented with 2% N-octyl-β-D-glucopyranoside (OGP) followed by centrifugation to obtain supernatant (S1) and sedimented protein (P1). Highly soluble proteins from the S1 fraction were pre-incubated in the absence or presence of metal ions such as CuCl₂ (Cu), ZnCl₂ (Zn), MgCl₂ (Mg) and CaCl₂ (Ca), 1 mM each. Proteins were once again separated by centrifugation into fractions of high and low solubility represented as supernatants (S2) and pellets (P2), respectively. After immunoblotting, PrP^C proteins were identified using mab SAF70 and signals were visualized by chemiluminescence substrate development. Glyosylated full-length PrP^C proteins of both mouse types were detected in the pellet fraction when bound to zinc ions. A considerably lower effect was observed in the T182N PrP^C interaction with copper ions, whereas other metals played no role in structural changes resulting in sedimentation. When metal ions were bound, mainly full length glycosylated PrP^C changed into the pellet fraction.</p

Interaction of metals with PrP^C induced a change to low solubility.

<p>Immunoblot analysis of brain homogenates (10%) derived from C57BL wild-type mice, bovine, human and sheep is as indicated. Homogenates were pre-incubated with various metal ions (1 mM) followed by centrifugation to generate a protein fraction of high solubility in the supernatant and of low solubility in the pellet. The pellet fractions were re-suspended in homogenate buffer back to the original volume. Following the addition of sample buffer, proteins were denatured by heating, and identical volumes were loaded for separation on SDS-PAGE and subsequent immunoblotting. PrP^C specific signals were detected on immunoblots by mab SAF34 and visualized using a chemiluminescence substrate. Unlike other metals, the interaction of ZnCl₂ with PrP^C led to reduced protein solubility in samples from all four species.</p

Comparison of PrP^Sc distribution patterns in C-type BSE affected cattle after oral versus intracerebral challenge.

<p>Data from orally challenged animals IT09, IT22, IT38, IT49 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067599#pone.0067599-Hoffmann1" target="_blank">[21]</a> were assembled with data from intracerebrally challenged animals termed case no. 8, 12, 15, 16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067599#pone.0067599-Fukuda1" target="_blank">[23]</a>. Signal intensities described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067599#pone.0067599-Fukuda1" target="_blank">[23]</a> and determined in this study were quantified as follows: – no PrP^Sc detectable, +: moderate PrP^Sc depositions, ++ strong PrP^Sc depositions.</p><p>i.c.  =  intracerebral inoculation.</p>*<p>Fukuda et al., 2012 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067599#pone.0067599-Fukuda1" target="_blank">[23]</a>.</p>**<p>Obex.</p>***<p>Accumbens.</p

PrP^Sc distribution pattern determined by IDEXX HerdChek® EIA.

<p>Results as determined for the different brain areas using the approved BSE rapid test, performed according to the manufacturer’s instructions. Testing was done in duplicate and the arithmetic means were calculated for the individual BSE types. Cranial medulla was set as the reference (100%) and the other brain regions were set in relation to it. CB – cerebellum, MB – midbrain including pons, crM – cranial medulla oblongata, caM – caudal medulla oblongata, RH – rhinencephalon (olfactory bulb), FC – frontal cortex, PC – parietal cortex, OC – occipital cortex, BN – basal nuclei, TH – thalamus.</p

PrP^Sc distribution pattern determined by PTA-WB.

<p>Western blots analysis using mab L42 as the detection antibody of ten brain regions of cattle clinically affected with C-, L- or H-type BSE. The figure displays three representative experimental animals (one of each BSE form), all in the final clinical stage of the disease (++ moderate to +++ severe clinical signs). CB – cerebellum, MB – midbrain including pons, crM – cranial medulla oblongata, caM – caudal medulla oblongata, RH – rhinencephalon (olfactory bulb), FC – frontal cortex, PC – parietal cortex, OC – occipital cortex, BN – basal nuclei, TH – thalamus.</p

Susceptibility to PK digestion.

<p>PK susceptibility was analyzed by PTA-WB. (A) Influence of final PK concentrations: 0, 25, 50, 250, 500 and 1000 µg/ml. The intensity of the Western blot signals were determined, whereby 50 µg/ml, which is the standard PK concentration used in our protocol, was set as a reference point (100%). PK digestion was performed for 60 min at 55°C. (B) Degradation of the PrP^Sc in accordance to the length of PK digestion between 1 and 26 hours. 50 µg/ml PK was applied in all of the experiments. Signal intensity was measured and the signal obtained after 60 min PK digestion (standard protocol) was set as the reference point (100%).</p