Journal of Molecular Biology
Regular articleAnalysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein 1
Introduction
Prion diseases are neurodegenerative disorders in humans and animals associated with a proteinaceous infectious pathogen designated prion (Prusiner, 1982). The nature of the transmissible pathogen has been obscure but now there is convincing evidence that prions are composed largely, if not entirely, of the scrapie isoform of the prion protein, PrPScPrusiner 1991, Prusiner 1997. A chromosomal gene was identified that encodes the normal cellular prion protein, PrPc(Oesch et al., 1985), whose function is unclear. After synthesis, it is transported to the cell surface where it is attached to the membrane by a glycoinositol phospholipid (GPI) anchor. PrPcis converted into the pathologic PrPScisoform by a post-translational process Borchelt et al 1990, Borchelt et al 1992, Taraboulos et al 1990. Attempts to reveal post-translational chemical modifications featuring in the synthesis of PrPSchave been unsuccessful (Stahl et al., 1993). Initial spectroscopic studies have shown that PrPcis rich in α-helices, whereas PrPSchas a high β-sheet content (Pan et al., 1993). This gave rise to the implication that prion diseases are disorders associated with abnormal protein isoforms (Cohen et al., 1994), representing the prototype of a novel molecular disease entity. Since then, several proteins in yeast and fungi have been described as being capable of similar self-perpetuating structural changes (Wickner, 1994).
Initial studies could not find phenotypic alterations in transgenic mice devoid of PrPc(PrP0/0), which would be indicative for the possible function of this protein (Bueler et al., 1992). Several recent studies that questioned these results revealed subtle phenotypic alterations (reviewed byEstibeiro, 1996). Similarly, the tertiary structure of the PrPScisoform is still under debate Huang et al 1994, Huang et al 1995, whereas the tertiary structures of murine and hamster PrPcwere finally determined by NMR Riek et al 1996, Riek et al 1997, James et al 1997, Donne et al 1997.
An important aspect of prion diseases is the species barrier phenomenon. Typically, the transfer of prions from one species into another results in a prolongation of incubation time and in a change in the PrPScdeposition pattern in the brain (Pattison, 1965). Studies that used transgenic animals and “classical” prion strains demonstrated that this feature might be encoded mainly by the degree of homology between the prion proteins of host and recipient species Scott et al 1989, Scott et al 1993. Further data were added by detailed analysis of the primary structures of the prion proteins of primates (Schätzl et al., 1995). Unfortunately, the overall degree of homology alone cannot always predict whether the barrier between two species is high or low, as some not clearly identified regions of PrP seem to dominate the species barrier. Recent data with bovine transgenes in mice argue that amino acid residues 184, 186, 203, and 205 form an epitope that is involved in the control of the species barrier (Scott et al., 1997).
The BSE epidemic in Great Britain demonstrated that the transfer of prion diseases into other ungulate and carnivore species not previously appreciated is possible (Kirkwood & Cunningham, 1994). The appearance of the new variant of Creutzfeldt-Jakob disease in Great Britain (nvCJD;Will et al., 1996) raises the question of whether prions from domestic and other animals could pose a danger to humans. Some evidence, not finalized, has been reported Hill et al 1997, Bruce et al 1997. The detailed genotype analysis of PrP genes from a variety of species will be helpful in gaining greater insight into structural and functional as well as species barrier aspects of prion proteins. We present here a PrP analysis including 27 novel PrP sequences of mammalian species consisting of ungulate, rodent, carnivore and a maritime species, and of nine bird species. Our study represents the most comprehensive analysis of mammalian and non-mammalian prion proteins in the context of structure-function relationships and species barrier aspects.
Section snippets
PrP gene variation in ungulates
In total, 20 new PrP sequences from ungulate species were analyzed. For most species the entire amino acid sequence of the mature PrP (residues 23-231) could be deduced (Figure 1; the numbering system used correlates to the human PrP), although from several species the sequences of the N and C-terminal peptides are missing (1-22 and 232-253, respectively). Seventeen ungulate species we analyzed belong to the super-order artiodactyla, and most of these species to the order ruminantia (ruminants)
Discussion
The results reported here add a total of 27 mammalian and nine avian PrP sequences to the previously analyzed PrPs of about 45 species and widely expand the number of known PrP gene sequences. Given the fact that the function of PrP is not known, the location and patterns of amino acid variation across this spectrum of aligned sequences provide further insight into various putative structural and functional aspects of PrPs.
Biological materials
Peripheral blood leukocytes (PBL) were prepared by the Ficoll-hypaque procedure or collected upon lysis of erythrocytes. If available, we used cell line and tissue materials in addition. From birds the whole blood pellet was used for DNA preparation. Usually DNA from two or more samples per species was prepared. High molecular mass genomic DNA was prepared by proteinase K/SDS treatment (0.04 % (w/v) and 0.4 % (w/v), respectively), extracted three times with phenol and precipitated with alcohol.
Acknowledgements
We greatly appreciate the cooperation of the following institutions: Zoological Gardens at San Francisco (G. Hedberg; Dr A. Bennett); Zoological Gardens Munich (Dr Hektor); Zoological Gardens Augsburg (Dr Gorgas, Dr J. Erben, Dr R. Fritz); the Department of Microbiology, the Faculty of Veterinary Medicine, University of Munich (Professor O. Kaaden, Dr U. Truyen, Dr Wolf); the Department of Surgery (Dr U. Matis, Dr H. Gerhards); the Society for Prevention of Cruelty to Animals in Munich (Dr.
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