Proteinase K-Resistant Material in ARR/VRQ Sheep Brain Affected with Classical Scrapie Is Composed Mainly of VRQ Prion Protein

Abstract

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP C ) to the scrapie-associated form (PrP Sc ) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP res , the proteinase K-resistant PrP Sc core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP res was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP res yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP res were detected. Enhanced 171R res proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP res of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.

PRNP variants in goats reduce sensitivity of detection of PrPSc by immunoassay

“…Antibody F89 successfully detected rec-PrP C when expressed as wild type, S146, H154, or K222, whereas the presence of M142 or R143 abolished the signal. Detection with 2 additional antibodies, L42 (detecting aa 148-153, YEDRYY 23 ) or SAF84 (detecting aa 166-172, YRPVDQY; deduced for goat based on the sequence recognized in bovine prion protein 18 ) was not altered by any of the allelic variants when compared with wild type.…”

Caprine PrP variants harboring Asp-146, His-154 and Gln-211 alleles display reduced convertibility upon interaction with pathogenic murine prion protein in scrapie infected cells

“…However, substantial evidence attributes reduced conversion efficiency to resistant PrP variants, as the highly protective Ala-136,Arg-154,Arg-171 variant was found to constitute only a minor fragment of the PrP SC molecules detected in the brains of scrapie-affected sheep carrying the Ala-136,Arg-154,Arg-171/Val-136,Arg-154,Gln-171 genotype. 61,62 Further, susceptibility to prion diseases is linked with the propensity of PrP variants to transit from 400 E. Kanata et al…”

Prion Type-Dependent Deposition of PRNP Allelic Products in Heterozygous Sheep

“…Immunochemical quantification of PrP res was subsequently performed by fluorimetric detection monitored in a three-laser-beam imager (Typhoon Trio variable-mode imager; Amersham Biosciences) (38). For estimation of the ARR-and VRQ-PrP fractions in PrP res , a mixture of two antibodies was applied, one of which (SAF84) binds only if the 171Q polymorphism is present (VRQ-PrP or ARQ-PrP) while the other binds equally well to VRQ-, ARQ-, and ARR-PrP (33,38,39). Two different mixtures with SAF84 were used: SAF84 with L42 (L42/SAF84 combination) and SAF84 with Sha31 (Sha31/SAF84 combination).…”

“…PrP res was prepared from 10% (wt/ vol) brain stem homogenates prepared in lysis buffer, digested with PK at 37°C, and further partially purified by precipitation with 1-propanol as described previously (38). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured samples in loading buffer (with lithium-dodecyl sulfate and ␤-mercaptoethanol) was performed in 17-well gels (33). Detection of PrP res on blot membranes was carried out in our triplex Western blotting system, but for this study a mixture of only two primary antibodies instead of three was used.…”

“…In a previous study we reported that in scrapie-infected ARR/VRQ sheep, the VRQ-PrP in PrP res was highly overrepresented, with 91 to 100% VRQ-PrP product (33,34). Yet the expression levels of the PrP C alleles in heterozygous animals are considered equal (34,35), which means that during PrP Sc formation in ARR/VRQ scrapie-infected animals, there occurs a selective incorporation of the VRQ-PrP allotype.…”