Similar biochemical signatures and prion protein genotypes in atypical scrapie and Nor98 cases, France and Norway

Abstract

Isolates of atypical scrapie recently identified in sheep and goats in France were compared with Nor98 isolates reported in Norway. Western blot methods for characterization of the protease-resistant prion protein showed that all these isolates shared a unique biochemical signature: 5 groups of bands, including a characteristic band of apparent low molecular weight (11 kDa). This pattern could originate from the presence of 3 different protease cleavage products, including the 11 kDa most likely cleaved at both N- and C-sides of the protein. Genetic data, which strongly suggested the higher susceptibility of AHQ and AF141RQ animals in French cases, resembled earlier data from Nor98 scrapie.

Figure 1

Atypical scrapie and Nor98 isolates PrPres Western blot pattern. Western blot (WB) profile in atypical (A, lane 2) and classic (B, lane 3) scrapie isolates with curves of chemiluminescence measured along the lane and corresponding apparent MWs (MWs), assessed by Bio-Rad Quantity One software analysis after signal capture using Versa Doc5000. Molecular weight (MW) standard (lanes 1). WB profiles of French atypical isolates in sheep (n = 17) and goats (n = 2) were compared with those of a Nor98 isolate. Apparent molecular masses (C) and proportions (D) of bands I to V were assessed from 3 independent runs for each sample by Bio-Rad Quantity One software analysis after signal capture using Versa Doc5000. Apparent MWs are measures for each of the atypical scrapie isolates, and proportions of bands are the means and standard deviations in the 19 atypical scrapie isolates. WB profiles of PrPres after PNGase deglycosylation with curves of chemiluminescence in atypical (E, lane 4) and classic (F, lane 5) scrapie isolates. Apparent MWs were estimated by comparison with a MW standard (lanes 1) from 10 independent runs.

Figure 2

Western blot profiles of PrPres in an atypical scrapie isolate (lane 2) detected by using N-terminal (4F2, P4), central (Sha31), or C-terminal (99/97.6.1) monoclonal antibodies. Molecular weight (MW) standard (lane 1). Immunoreactivities obtained with each antibody on 10 different atypical scrapie isolates are indicated (+, strong, ±, low, –, absent).

Figure 3

A) Schematic representation of ovine PrPc with location of epitopes recognized by the monoclonal antibodies used during the study and approaching sizes of PrPres fragments in atypical scrapie and Nor98 isolates. Theoretical apparent MWs of PrP fragments were calculated, by using those of each amino acid included in the known ARQ sheep PrP sequence, according to Sambrook and Russell (17). B) Interpretation of PrPres Western blot (WB) profiles in atypical scrapie and Nor98 isolates. Theoretical WB profile shows the expected apparent molecular masses of glycosylated PrP forms estimated by addition of 3.8 (*, monoglycosylated) or 7.9 (**, diglycosylated) kDa to the apparent molecular masses of A and B PrPres forms observed after PNGaseF deglycosylation. Values of 3.8 and 7.9 kDa were estimated from comparisons of glycosylated and unglycosylated forms in a classic scrapie isolate. The Sha31 WB profile included the mean apparent MWs assessed from highest resolution WB analysis (n = 32) and showed 2 separate peaks of maximal intensity in pictograms of signal intensities of bands II and III (19 sheep scrapie isolates).