Genetic predictions of prion disease susceptibility in carnivore species based on variability of the prion gene coding region.

Mammalian species vary widely in their apparent susceptibility to prion diseases. For example, several felid species developed prion disease (feline spongiform encephalopathy or FSE) during the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, whereas no canine BSE cases were detected. Whether either of these or other groups of carnivore species can contract other prion diseases (e.g. chronic wasting disease or CWD) remains an open question. Variation in the host-encoded prion protein (PrP(C)) largely explains observed disease susceptibility patterns within ruminant species, and may explain interspecies differences in susceptibility as well. We sequenced and compared the open reading frame of the PRNP gene encoding PrP(C) protein from 609 animal samples comprising 29 species from 22 genera of the Order Carnivora; amongst these samples were 15 FSE cases. Our analysis revealed that FSE cases did not encode an identifiable disease-associated PrP polymorphism. However, all canid PrPs contained aspartic acid or glutamic acid at codon 163 which we propose provides a genetic basis for observed susceptibility differences between canids and felids. Among other carnivores studied, wolverine (Gulo gulo) and pine marten (Martes martes) were the only non-canid species to also express PrP-Asp163, which may impact on their prion diseases susceptibility. Populations of black bear (Ursus americanus) and mountain lion (Puma concolor) from Colorado showed little genetic variation in the PrP protein and no variants likely to be highly resistant to prions in general, suggesting that strain differences between BSE and CWD prions also may contribute to the limited apparent host range of the latter.

Alternative translation initiation generates cytoplasmic sheep prion protein.

Cytoplasmic localization of the prion protein (PrP) has been observed in different species and cell types. We have investigated this poorly understood phenomenon by expressing fusion proteins of sheep prion protein and green fluorescent protein ((GFP)PrP) in N2a cells, with variable sequence context surrounding the start codon Met(1). (GFP)PrP expressed with the wild-type sequence was transported normally through the secretory pathway to the cell surface with acquisition of N-glycan groups, but two N-terminal fragments of (GFP)PrP were detected intracellularly, starting in frame from Met(17). When (GFP)PrP was expressed with a compromised Kozak sequence ((GFP)PrP*), dispersed intracellular fluorescence was observed. A similar switch from pericellular to intracellular PrP localization was seen when analogous constructs of sheep PrP, without inserted GFP, were expressed, showing that this phenomenon is not caused by the GFP tag. Western blotting revealed a reduction in glycosylated forms of (GFP)PrP*, whereas the N-terminal fragments starting from Met(17) were still present. Formation of these N-terminal fragments was completely abolished when Met(17) was replaced by Thr, indicating that leaky ribosomal scanning occurs for normal sheep PrP and that translation from Met(17) is the cause of the aberrant cytoplasmic localization observed for a fraction of the protein. In contrast, the same phenomenon was not detected upon expression of similar constructs for mouse PrP. Analysis of samples from sheep brain allowed immunological detection of N-terminal PrP fragments, indicating that sheep PrP is subject to similar processing mechanisms in vivo.

Dynamic expression of the prion-like protein Doppel in ovine testicular tissue.

Transgenic knockout of the gene encoding the prion-like protein Doppel (Dpl) leads to male infertility in mice. The precise role of Dpl in male fertility is still unclear, but sperm from Dpl-deficient mice appear to be unable to undergo the normal acrosome reaction that is necessary to penetrate the zona pellucida of the ovum. We have investigated the expression pattern and some biochemical properties of Dpl in sheep testicular tissue and spermatozoa. Neither the Dpl protein nor its mRNA was detected in pre-pubertal sheep testis. This was in contrast to the findings in adult rams where both Dpl mRNA and protein were present. The molecular mass and glycosylation pattern of sheep Dpl were similar to that of mice Dpl. The Dpl protein was detected in the seminiferous epithelium during the two final (7 and 8) and the two initial (1 and 2) stages of the spermatogenic cycle in a characteristic pattern. In stage 8, an intense brim of granular Dpl-immunoreactivity associated with maturation phase spermatids was observed, while after the release of spermatozoa in stages 1 and 2, the Dpl-staining was disseminated more diffusely in the epithelium, reaching the basal lamina. From stage 3 to stage 6, Dpl-immunoreactivity could not be detected, indicating that the Dpl protein had disappeared between stages 2 and 3. Dpl was not detected on ejaculated spermatozoa. These patterns of staining indicate that Dpl is enriched in residual bodies, which are phagocytosed and destroyed by Sertoli cells after release of sperm into the lumen of the seminiferous tubule.