A C-terminal protease-resistant prion fragment distinguishes ovine "CH1641-like" scrapie from bovine classical and L-Type BSE in ovine transgenic mice

Abstract

The protease-resistant prion protein (PrP(res)) of a few natural scrapie isolates identified in sheep, reminiscent of the experimental isolate CH1641 derived from a British natural scrapie case, showed partial molecular similarities to ovine bovine spongiform encephalopathy (BSE). Recent discovery of an atypical form of BSE in cattle, L-type BSE or BASE, suggests that also this form of BSE might have been transmitted to sheep. We studied by Western blot the molecular features of PrP(res) in four "CH1641-like" natural scrapie isolates after transmission in an ovine transgenic model (TgOvPrP4), to see if "CH1641-like" isolates might be linked to L-type BSE. We found less diglycosylated PrP(res) than in classical BSE, but similar glycoform proportions and apparent molecular masses of the usual PrP(res) form (PrP(res) #1) to L-type BSE. However, the "CH1641-like" isolates differed from both L-type and classical BSE by an abundant, C-terminally cleaved PrP(res) product (PrP(res) #2) specifically recognised by a C-terminal antibody (SAF84). Differential immunoprecipitation of PrP(res) #1 and PrP(res) #2 resulted in enrichment in PrP(res) #2, and demonstrated the presence of mono- and diglycosylated PrP(res) products. PrP(res) #2 could not be obtained from several experimental scrapie sources (SSBP1, 79A, Chandler, C506M3) in TgOvPrP4 mice, but was identified in the 87V scrapie strain and, in lower and variable proportions, in 5 of 5 natural scrapie isolates with different molecular features to CH1641. PrP(res) #2 identification provides an additional method for the molecular discrimination of prion strains, and demonstrates differences between "CH1641-like" ovine scrapie and bovine L-type BSE transmitted in an ovine transgenic mouse model.

Figure 1. Western blot analysis of PrP^res from “CH1641-like” isolates in TgOvPrP4 mice detected by Bar233 monoclonal antibody.

CH1641 and natural “CH1641-like” (06-017, 05-825, TR316211, and O104) scrapie isolates are compared with experimental ovine BSE (Ov BSE), L-type (L-BSE), or classical (C-BSE) BSE in cattle. (A) and (B) show results in TgOvPrP4 mice at first passage and (C) and (D) at second passage. PrP^res was analysed before ([A] and [C]) or after ([B] and [D]) PNGase deglycosylation.

Figure 2. Glycoform ratios of PrP^res from “CH1641-like” isolates in TgOvPrP4 mice.

Glycoform ratios of PrP^res in individual mice inoculated with ovine or bovine isolates (first passage) are shown in (A) and (B), respectively. In (A), CH1641 is shown in red, and natural isolates 06-017 and 05-0825 in blue. In (B), Classical and L-type BSE are shown in red and green, respectively. PrP^res was detected by Bar233 antibody.

Figure 3. Western blot analysis of PrP^res from “CH1641-like” isolates in TgOvPrP4 mice detected by SAF84 monoclonal antibody.

CH1641 and natural “CH1641-like” (06-017, 05-825, TR316211, and O104) scrapie isolates are compared with ovine BSE (Ov BSE), L-type (L-BSE), or classical (C-BSE) natural BSE in cattle and transmissible mink encephalopathy experimentally transmitted to cattle (TME bov) . (A) and (B) show results in TgOvPrP4 mice at first passage and (C) and (D) at second passage. PrP^res was analysed before ([A] and [C]) or after ([B] and [D]) PNGase deglycosylation.

Figure 4. Western blot analysis of PrP^res from BSE sources and from “non-CH1641-like” isolates in TgOvPrP4 mice.

BSE sources shown in (A–C) include BSE from cattle (lane 2), sheep homozygous for the A₁₃₆R₁₅₄Q₁₇₁ (lane 3) or A₁₃₆R₁₅₄R₁₇₁ (lane 4) prnp allele, goat (lane 5), and cheetah (lane 6). “Non-CH1641-like” natural scrapie isolates include O171, O59, O69, O87, and O111 scrapie isolates. PrP^res in TgOvPrP4 mice (first passage) was analysed before ([A,B,D,E]) or after ([C] and [F]) PNGase deglycosylation. PrP^res was detected by Bar233 antibody in (A) and (D) and by SAF 84 antibody in (B), (C), (E), and (F).

Figure 5. Western blot analysis of PrP^res from experimental scrapie and BSE sources in TgOvPrP4 mice.

Scrapie sources include SSBP1 and CH1641 experimental scrapie isolates and Chandler, 87V, 79A, and C506M3 scrapie strains in TgOvPrP4 mice (second passage). BSE is derived from C57Bl/6 mice infected with classical BSE (Mu BSE) (second passage in TgOvPrP4 mice). PrP^res was analysed before (A, B) or after (C) PNGase deglycosylation and detected using Bar233 (A) or SAF84 (B, C).

Figure 6. Proportions of PrP^res #2 from scrapie sources transmitted to TgOvPrP4 mice.

Proportions of PrP^res #2 were evaluated by repeated Western blot analyses of the samples after PNGase deglycosylation and detection using SAF84 antibody. In the case of O104-infected mice, the mice are numbered as indicated in Figure 7 showing Western blot results.

Figure 7. Western blot analysis of PrP^res from O104 natural scrapie isolate in TgOvPrP4 mice.

PrP^res was analysed in TgOvPrP4 mice (first passage) by Western blot after PNGase deglycosylation and detected using Bar233, P4, and SAF84 monoclonal antibodies in (A), (B), and (C), respectively. PrP^res from individual mice infected with O104 isolate (first passage) is shown, with BSE derived from BSE-infected C57Bl/6 mice (Mu BSE) and C506M3 scrapie controls in TgOvPrP4 mice (second passage). O104-infected mice are numbered as indicated in Figure 5 showing the proportions of PrP^res #2.

Figure 8. Enrichment of PrP^res #2 by differential immunoprecipitation.

Western blot analysis of PrP^res from TgOvPrP4 infected with a “CH1641-like” isolate (TR316211). PrP^res released from beads after each capture cycle is shown for 5 successive cycles using Sha31-coated beads (lanes 3 to 7, respectively), then from the following capture cycle using SAF84-coated beads (lane 8). PrP^res controls from C506M3 strain and TR316211 are shown on lanes 1 and 2, respectively. PrP^res was detected using SAF84 antibody that recognizes both PrP^res #1 and PrP^res #2 forms.