Degradation of the disease-associated prion protein by a serine protease from lichens

Abstract

The disease-associated prion protein (PrP(TSE)), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE) inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE). Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE)-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE) and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

Figure 1. Extracts of the lichens P. sulcata, C. rangiferina and L. pulmonaria degrade PrP.

Equivalent amounts of PTA-enriched PrP^TSE (30 µg total protein) were incubated with the indicated lichen extracts (labels, 10 mg lichen equivalents) or with vehicle and were subjected to NuPAGE and immunoblotting. Extracts of lichens in panel (A) reduced PrP immunoreactivity compared to control, whereas extracts of lichens in panel (B) do not reduce PrP immunoreactivity. (C) Treatment of PrP^TSE using the method employed in panels (A) and (B) with extracts of isolated algae or cyanobacterium cultures (10 mg equivalents) do not degrade PrP. (D) Dilutions of control reactions containing PrP^TSE and no lichen extract indicate that PrP immunoreactivity was no longer detectable when dilution factors were greater than 100. (E) Adding P. sulcata extract to PrP^TSE, but not allowing it time to react (spiked) did not reduce PrP immunoreactivity compared to control (vehicle). Equivalent samples in which P. sulcata extract was allowed time to incubate (reacted) did not have detectable PrP immunoreactivity. (F–H) Methods to remove lichen compounds and DMSO from samples post-reaction, such as (F) washing remaining protein with PBS using a centricon filter column (10–12 kDa MWCO) or (G & H) precipitating protein using methanol (MeOH) or trichloroacetic acid (TCA) do not restore PrP immunoreactivity. All immunoblots (IB) used anti-PrP mAb 3F4, except for panel (A) which used both 3F4 and SAF84.

Figure 2. Lichen extracts degrade PrP^TSE in brain homogenate.

(A) Lichen extracts (10 mg lichen equivalents) were incubated with equal amounts (10 µL) of 10% brain homogenates from hamsters infected with Hyper (HY) TME, Drowsy (DY) TME or 263K scrapie strains of TSE agents and PrP degradation was assessed by immunoblotting. Treatment of each hamster brain homogenate with 50 µg·mL⁻¹ of PK demonstrates the presence of abnormal PrP in the starting material. Using the same conditions as in (A), lichen extracts cause degradation of PrP in (B) a CWD-infected white-tailed deer, (C) PK-treated HY hamster and (D) uninfected hamster brain homogenates. (E) Protein C5 of the 20S subunit of the proteasome, an unrelated protein, was also degraded by lichen extracts. Immunoblots (IB) used mAbs 3F4 (A, C and D), 6H4 (B) or pAb anti-C5 (E).

Figure 3. Semi-quantitative PMCA to assess RML PrP^TSE degradation.

(A) The extent of mouse RML PrP^TSE degradation was measured by PMCA analysis of dilutions (10⁻²–10⁻⁶) of vehicle or L. pulmonaria extract-treated RML. (B) Control reactions lacking PrP^TSE seed or sonication did not show amplification as measured by PK digestion. (C) RML samples were incubated with vehicle for 1 hr or with L. pulmonaria extract (reacted: 1 hr; spiked <10 sec) then diluted and subjected to PMCA. Adding L. pulmonaria extract in PMCA reactions without allowing time for reaction (spiked samples) does not appear to reduce PrP^TSE levels or affect amplification compared to control (vehicle). All immunoblots used mAb SAF83.

Figure 4. Effect of lichen extract dose on PrP degradation.

(A) Dose-response of the indicated lichen extracts (0–10 mg lichen equivalents) on degrading PTA-enriched PrP^TSE (30 µg total protein). (B) The indicated concentrations of PK (0–10000 µg·mL⁻¹) were incubated with PTA-enriched PrP^TSE (30 µg total protein) to assess degradation and for comparison with lichen extracts. Immunoblots used mAb 3F4.

Figure 5. Effect of pH on PrP degradation by P. sulcata and L. pulmonaria extracts.

Reaction solutions were buffered at the indicated pHs (5–8) and the effect of each indicated lichen extract (10 mg lichen equivalents) on degradation of PTA-enriched PrP^TSE (30 µg total protein) was observed by immunoblotting with mAb 3F4.

Figure 6. Common lichen chemicals do not affect PrP levels.

Samples of PTA-enriched PrP^TSE (30 µg total protein) were incubated with the indicated concentrations of lecanoric acid (LA), usnic acid, atranorin or vehicle followed by immunoblot analysis with mAb 3F4. P. sulcata extract (10 mg lichen equivalent) served as a positive control for PrP degradation.

Figure 7. Assessing enzymatic activity responsible for PrP degradation.

Reactions of PTA-enriched PrP^TSE (30 µg total protein) and the indicated lichen extracts (10 mg lichen equivalents) were prepared with enzyme inhibitors: (A) EDTA (1 or 10 mM) to inhibit metalloenzymes, (B) ascorbate (5 mM) as an antioxidant and chelator, (C) Cd²⁺ (80 mM) to inhibit laccases or (D) a broad-spectrum protease inhibitor cocktail (10 µL per reaction). The effect of each inhibitor on P. sulcata or L. pulmonaria extract-mediated degradation of PrP was measured by immunoblotting with mAb 3F4.

Figure 8. The serine protease inhibitor Pefabloc SC prevents PrP degradation.

Incubation of PTA-enriched PrP^TSE (30 µg total protein) with lichen extracts (10 mg lichen equivalents) were performed in the presence and absence of Pefabloc SC (14 mM). Reactions with the inhibitor had increased PrP immunoreactivity with mAb 3F4 (reduced PrP degradation).

Figure 9. Intact P. sulcata tissue or a water extract of the lichen degrades PrP.

(A) Freshly-collected P. sulcata or P. squarrosa (4 mg, each) were incubated with infected brain homogenate (100 µL of 10% v/w in distilled water) for 24 h. Following incubation, the lichen-treated brain homogenates (sup) and the lichen tissues (pel) were analyzed for PrP by immunoblotting with mAb 3F4. A reduction in PrP signal was associated with P. sulcata, but not with P. squarrosa. (B) PTA-enriched PrP^TSE (30 µg total protein) exposed to chitin beads is bound by the beads, but is extractable using NuPAGE sample buffer. Little immunoreactivity in the supernatant (sup) shows the protein binds chitin and treatment of bound PrP with sample buffer yields immunoreactivity approximately equal to PrP starting material. (C) A water extract from P. sulcata, but not P. squarrosa, causes PrP degradation. Immunoblotting comparison of PTA-enriched PrP^TSE (30 µg total protein) incubated in only water or exposed to a water extract of P. sulcata (100 mg lichen equivalent) with and without the serine protease inhibitor Pefabloc SC (10 mM). Water extract of P. sulcata induces a substantial reduction of immunoreactivity that is blocked by PefaBloc SC whereas a water extract of the related lichen P. squarrosa does not degrade PrP under the same conditions.