Regular article
Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein 1

https://doi.org/10.1006/jmbi.1999.2831Get rights and content

Abstract

Prion diseases are fatal neurodegenerative disorders in man and animal associated with conformational conversion of a cellular prion protein (PrPc) into the pathologic isoform (PrPSc). The function of PrPcand the tertiary structure of PrPScare unclear. Various data indicate which parts of PrP might control the species barrier in prion diseases and the binding of putative factors to PrP. To elucidate these features, we analyzed the evolutionary conservation of the prion protein. Here, we add the primary PrP structures of 20 ungulates, three rodents, three carnivores, one maritime mammal, and nine birds. Within mammals and birds we found a high level of amino acid sequence identity, whereas between birds and mammals the overall homology was low. Various structural elements were conserved between mammals and birds. Using the CONRAD space-scale alignment, which predicts conserved and variable blocks, we observed similar patterns in avian and mammalian PrPs, although 130 million years of separate evolution lie in between. Our data support the suggestion that the repeat elements might have expanded differently within the various classes of vertebrates. Of note is the N-terminal part of PrP (amino acid residues 23-90), which harbors insertions and deletions, whereas in the C-terminal portion (91-231) mainly point mutations are found. Strikingly, we found a high level of conservation of sequences that are not part of the structured segment 121-231 of PrPcand of the structural elements therein, e.g. the N-terminal region from amino acid residue 23-90 and the regions located upstream of α-helices 1 and 3.

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.

References (61)

  • H Schätzl et al.

    Why is the codon 129 of PrP polymorphic in humans but not in animals?

    Lancet

    (1997)
  • M Scott et al.

    Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques

    Cell

    (1989)
  • M Scott et al.

    Propagation of prion with artifical properties in transgenic mice expressing chimeric PrP genes

    Cell

    (1993)
  • G.C Telling et al.

    Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another factor

    Cell

    (1995)
  • R.G Will et al.

    A new variant of Creutzfeldt-Jakob disease in the UK

    Lancet

    (1996)
  • O Windl et al.

    A candidate marsupial PrP gene reveals two domains conserved in mammalian PrP proteins

    Gene

    (1995)
  • L.C Bartz et al.

    Transmissible mink encephalopathy species barrier effects between ferret and minkPrP gene and protein analysis

    J. Gen. Virol.

    (1994)
  • M Billeter et al.

    Prion protein NMR structure and species barrier for prion diseases

    Proc. Natl Acad. Sci. USA

    (1997)
  • D.R Borchelt et al.

    Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells

    J. Cell Biol.

    (1990)
  • D Brown et al.

    The cellular prion protein binds copper in vivo

    Nature

    (1997)
  • M.E Bruce et al.

    Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent

    Nature

    (1997)
  • H Bueler et al.

    Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein

    Nature

    (1992)
  • F.E Cohen et al.

    Structural clues to prion replication

    Science

    (1994)
  • D.G Donne et al.

    Structure of the recombinant full-length hamster prion protein PrP(29-231)the N-terminus is highly flexible

    Proc. Natl Acad. Sci. USA

    (1997)
  • J.P Estibeiro

    Multiple roles for PrP in the prion diseases

    Trends Neurosci. Sci.

    (1996)
  • M Fischer et al.

    Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie

    EMBO J.

    (1996)
  • J.-M Gabriel et al.

    Molecular cloning of a candidate chicken prion protein

    Proc. Natl Acad. Sci. USA

    (1992)
  • M Gasset et al.

    Predicted α-helical regions of the prion protein when synthesized as peptides form amyloid

    Proc. Natl Acad. Sci. USA

    (1992)
  • C.J Gibbs et al.

    Normal isoform of amyloid protein (PrP) in brains of spawning salmon

    Mol. Psychiat.

    (1997)
  • D.A Harris et al.

    A prion-like protein from chicken brain copurifies with an acetylcholine receptor- inducing activity

    Proc. Natl Acad. Sci. USA

    (1991)
  • Cited by (0)

    1

    Edited by A. R. Fersht

    2

    †F.W. and G.W. contributed equally to this work.

    View full text