Isolation of Proteinase K-Sensitive Prions Using Pronase E and Phosphotungstic Acid

Disease-related prion protein, PrPSc, is classically distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrPSc using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrPC, while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrPC

PK and pronase E digestion of mouse brain homogenate.

<p>10% (w/v) brain homogenate from uninfected CD-1 mice (CD-1) and RML prion-infected CD-1 mice (RML) were analysed by immunoblotting in the absence of protease digestion (−) or after digestion (+) with PK (50 µg/ml, 37°C, 1 h) (A) or pronase E (Pro) (100 µg/ml, 37°C, 30 min) (B) or after sequential digestion with pronase E (100 µg/ml, 37°C, 30 min) and PK (50 µg/ml, 37°C, 30 min) (C). Immunoblots were probed with anti-PrP monoclonal antibody ICSM35. The blot shown in panel C has been over-exposed to film to show residual PrP^C. Apparent molecular masses are indicated in kDa.</p

RML prion infectivity and PrP content following digestion with PK, thermolysin or pronase E.

<p>10% (w/v) RML prion-infected brain homogenate was incubated at 37°C with (A) PK (50 µg/ml); (B) thermolysin (100 µg/ml) or (C) pronase E (100 µg/ml) for varying incubation times. For each time point, PrP (filled circles) was measured by ELISA and expressed as a percentage of total PrP present in the untreated sample; mean ± S.E.M. (<i>n</i> = 3, PK and thermolysin; <i>n</i> = 5, pronase E). RML prion infectivity (open circles) was measured by the Scrapie Cell Assay and expressed as a percentage of total infectivity present in the untreated sample; mean ± S.E.M. (<i>n</i> = 3, PK and thermolysin; <i>n</i> = 5, pronase E). Immunoblots of the corresponding protease and incubation time point are shown in panels to the right. Blots were probed with anti-PrP monoclonal antibody ICSM35. Apparent molecular masses are shown in kDa. Data in panels A and B have been published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015679#pone.0015679-Cronier1" target="_blank">[25]</a>.</p

RML prion infectivity following digestion with 1 mg/ml pronase E.

<p>10% (w/v) RML brain homogenate was incubated with pronase E (1 mg/ml at 37°C) for varying incubation times. For each time point RML prion infectivity was measured by the Scrapie Cell Assay and expressed as a percentage of total infectivity present in the untreated sample; mean ± S.E.M. (<i>n</i> = 5).</p

Immunoblot analysis of pronase E digested and NaPTA precipitated mouse brain homogenate.

<p>10% (w/v) homogenates from normal CD-1 brain (CD-1) or RML prion-infected brain (RML) were either untreated (Pro −, NaPTA −), digested by pronase E at 100 µg/ml, 37°C for 30 min (Pro +, NaPTA −), precipitated by NaPTA (Pro −, NaPTA +) or sequentially digested by pronase at 100 µg/ml, 37°C for 30 min and precipitated by NaPTA (Pro +, NaPTA +). Equivalent aliquots of each sample were analysed by immunoblotting using anti-PrP monoclonal antibody ICSM35. Apparent molecular masses are indicated in kDa.</p

Pronase E digested and NaPTA precipitated mouse brain homogenate evaluated by silver stain SDS-PAGE.

<p>10% (w/v) homogenates from normal CD-1 brain (CD-1) or RML prion-infected brain (RML) were either untreated (Pro −, NaPTA −), digested by pronase E at 100 µg/ml, 37°C for 30 min (Pro +, NaPTA −), precipitated by NaPTA (Pro −, NaPTA +) or sequentially digested by pronase E at 100 µg/ml, 37°C for 30 min and precipitated by NaPTA (Pro +, NaPTA +). The equivalent of 2 µl 10% (w/v) brain homogenate was loaded in each lane and the gel was stained with silver nitrate.</p

Pronase E-resistant RML prion infectivity is precipitated by NaPTA.

<p>10% (w/v) homogenates from normal CD-1 brain (CD-1, white bars) or RML prion-infected brain (RML, black bars) were either untreated (whole homogenate), incubated at 37°C for 90 min and 20°C for 30 min (temperature control), digested with pronase E at 100 µg/ml, 37°C for 30 min (pronase), precipitated by NaPTA (NaPTA) or sequentially digested by pronase E at 100 µg/ml, 37°C for 30 min and precipitated by NaPTA (pronase + NaPTA). The concentration of PrP in all samples was measured by ELISA (A) and is expressed as a percentage of the total PrP present in the untreated RML samples; mean ± S.E.M. (<i>n</i> = 5). RML prion infectivity was measured by the Scrapie Cell Assay (B) and expressed as total infectivity present in the samples; mean ± S.E.M. (<i>n</i> = 5).</p