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Short Communication |
1 Roslin Institute and R(D)SVS, University of Edinburgh, Neuropathogenesis Division, Roslin, Midlothian, UK
2 National TSE Laboratory, College of Veterinary Medicine, China Agriculture University, Beijing, PR China
Correspondence
Wilfred Goldmann
wilfred.goldmann{at}roslin.ed.ac.uk
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EU559165 [GenBank] , EU559166 [GenBank] , EU595763 [GenBank] –EU595770 [GenBank] , EU596567 [GenBank] –EU596573 [GenBank] , EU605791 [GenBank] –EU605794 [GenBank] , GQ149482 [GenBank] and GQ174495 [GenBank] –GQ174500.
Two supplementary tables and a supplementary method are available with the online version of this paper.
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, 2004; Premzl & Gamulin, 2007; Watts & Westaway, 2007). PrP and Sho are highly expressed in the neurons of the central nervous system (Hu et al., 2007; Watts et al., 2007), whereas Dpl appears to be specific for the reproductive system (Genoud et al., 2003). There is overwhelming evidence that the PRNP gene is a crucial element in prion diseases (also known as transmissible spongiform encephalopathies, TSEs) such as scrapie, bovine spongiform encephalopathy (BSE) or Creutzfeldt–Jakob disease (CJD) (Chesebro, 1999). A recent study by Beck et al. (2008) demonstrated that the SPRN gene is associated with prion disease (sporadic and variant CJD). Intriguingly, Beck et al. (2008) showed that a frameshift mutation in SPRN, resulting in reduced expression of Sho, appears to increase disease risk for variant CJD, in contrast with the PRNP gene where reduced expression has the opposite effect on scrapie (Manson & Tuzi, 2001).
In most species that can be infected with prion diseases, the PRNP gene modulates disease susceptibility and pathogenesis. In sheep and goats, with their large number of PRNP alleles, the control of disease is linked in a complex way to specific PRNP genotypes. However, not all sheep with the same PRNP genotype are equally susceptible or develop pathology in the same way (Goldmann, 2008). This suggests the involvement of other host genes, amongst which the prion family proteins are primary candidates. Recently, Watts et al. (2007) hypothesized how the SPRN gene and Sho protein may be involved in prion disease, proposing that Sho and PrP bind to the same cellular receptor in their neuroprotective role and that perturbation of Sho expression during prion disease enhances neuronal death.
A close correlation between PRNP and SPRN expression in sheep brain tissue has been suggested by Lampo et al. (2009) and Gossner et al. (2009), emphasizing the need to understand the genetics of both genes. Assuming that Sho has a role in prion disease, genetic differences in ruminant SPRN genes may very likely contribute to variation of susceptibility to or pathogenesis of scrapie in sheep and goats, BSE in cattle and chronic wasting disease in deer.
Mouse Sho is post-translationally processed from an open reading frame (ORF) of 147 codons, it appears in cells as a 18 kDa GPI-anchored membrane protein with one N-linked carbohydrate (Watts et al., 2007). Lampo et al. (2007) published an ovine SPRN ORF encoding a protein of similar overall structure to mouse Sho. The most homologous region to PrP is the 20 aa hydrophobic sequence AAAGAAAGAAAGAAAGLAAG in sheep Sho protein. In PrP, a 16 aa hydrophobic sequence VAGAAAAGAVVGGLGG (sheep codons 115–130) is the most conserved part of the protein. Variants of this sequence are very rare, only 11 are known in almost 400 PrP variants from over 130 species. There are no insertions/deletions in this hydrophobic PrP sequence. It has been suggested that the hydrophobic core of PrP is the binding site for other proteins, such as stress-inducible protein 1 (STI1) (Zanata et al., 2002), that it modulates the formation of transmembrane PrP isoforms (Hegde et al., 1998) and that it is important for basolateral sorting of PrP in a cell (Uelhoff et al., 2005). Furthermore, the AGAAAAGA palindrome may play a part in the disease-specific PrPSc–PrPC interaction (Norstrom & Mastrianni, 2005): peptides containing this sequence are neurotoxic (Forloni et al., 1993) and amyloidogenic (Gasset et al., 1992), and the variant AGAAVAGA is linked to Gerstmann–Sträussler–Scheinker syndrome, a genetic form of human prion disease (Mallucci et al., 1999). The homology in this region between PrP and Sho implies that there is similar cellular sorting and ligands and that they use similar mechanisms to promote their neuroprotective properties (Chiarini et al., 2002; Watts et al., 2007). We investigated the degree of Sho sequence variation between species and the occurrence of Sho polymorphisms within species. The study focused on ruminants and rodents because these species are natural hosts and/or commonly used disease models of TSEs.
Full ORF sequences for Sho were derived from 807 samples from 12 species of the suborder Ruminantia and 19 samples from two species of the order Rodentia. DNA was prepared from blood samples, PCR-amplified with the primers shown in Supplementary Table S1 (available in JGV Online) and sequenced directly or after cloning into T-vector by using an AB3130 sequencer (details about the sample origins are given in Supplementary Table S2 and the full sequencing methods are available in JGV Online). A total of 24 different Sho sequences were found; 21 of these were novel. The overall genetic structure of Sho was conserved between all species, with the signal peptides and the N-glycosylation site maintained throughout (Fig. 1). There was no variation in the number of the basic Ala-Arg-Gly-type repeats within the ruminant Sho, but they differed from the rodent sequence (Premzl et al., 2003).
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, available in JGV Online, and Fig. 1
). There were five amino acid substitutions, three insertions, one deletion and three silent polymorphisms in the ORF. Based on the homology to bovine and human Sho, we regard haplotype 1 as the archetypal (phylogenetic wild-type) allele. The ovine Sho sequence described by Lampo et al. (2007) is represented here by haplotype 7 (Table 1). In six of the nine ovine Sho variants, the changes observed involve the amino acid alanine, which will result in a change in the hydropathy index of the protein of up to 50 %. Caprine SPRN was sequenced from 135 goats of different breeds and origins. Caprine Sho is 3 aa longer than ovine Sho due to an insertion of Leu-Arg-Pro at the very end of the ORF (Fig. 1). The four DNA polymorphisms 144G
A, 177GC, 300CT and 433CA (numbered relative to the ORF) were all silent mutations, no amino acid changes were detected.
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. A 4 aa deletion of AAAG (codons 67–70; Fig. 1) in cattle Sho was the equivalent of the polymorphism described for sheep SPRN haplotype 10. Additionally, the B. taurus sequences had three silent mutations: 37CT, 288AG and 360GA (numbered relative to the ORF). In B. frontalis, one allele was the same as the cattle wild-type sequence (Uboldi et al., 2006) except for a change to serine in codon 71. The other allele had a deletion of the amino acids SAAG (codons 71–74; Fig. 1). The B. grunniens sample revealed a heterozygous genotype; one allele was identical to the published cattle sequence at the amino acid level, the other allele, confirmed by cloning, showed two polymorphisms (125CT and 128GA) which led to amino acid changes A42V and R43K, respectively.
Nine samples were analysed from three other genera in the subfamilies Bovinae and Hippotraginae, which we here collectively refer to as antelopes. There were several distinctive differences between Sho from antelopes and Sho from the other ruminants (Fig. 1) but no change was common to all five species. None of the samples were heterozygous and no change was seen in the hydrophobic alanine region as described for sheep and bovine Sho sequences.
In 51 sequences analysed from red deer, one heterozygous animal showed the polymorphisms 25GA and 133GA on the same allele, resulting in the amino acid replacements G29S and R45H. In addition, there were four silent mutations, all mutually exclusive in 10 samples; they were 150CG, 231GA, 390GC and 408CT (numbered relative to the ORF). There were no amino acid polymorphisms or silent mutations in the ORF of the SPRN gene among the 79 wapiti. The most common Sho protein sequence from deer and wapiti differed from sheep in nine positions and from cattle in seven positions (Fig. 1).
We analysed Sho in two rodent species: house mouse (Mus musculus; n=10) and wood mouse (Apodemus sylvaticus; n=9). We did not find any polymorphisms in M. musculus and there were no differences to the published sequence (C57BL/6; GenBank accession no. NM183147) in any of the five laboratory strains or any of the four samples from free-ranging house mice. From nine wood mouse samples, two had a silent mutation 189GA. There was a 5 % difference at the DNA sequence level between house mouse and wood mouse but there was only one amino acid difference at codon 86, with the species having Arg and Lys, respectively.
Allele frequencies and their association with prion disease were analysed in Cheviot and Welsh Mountain sheep, which represented 73 % of the investigated breeds. They carried the single amino acid polymorphisms in codons 53 and 71 (haplotypes 3 and 11) and the insertions/deletions of haplotypes 6, 7, 9 and 10. In Cheviot sheep, haplotypes 3 and 11 were common, with frequencies of 18.5 and 11.7 %, respectively. Haplotype 7 was found in Cheviot sheep with a frequency of 6.5 %. The other modern sheep breeds in this study (23 %) were from UK flocks; they did not carry haplotype 3 and showed frequencies of 21.6 and 9 % for haplotypes 7 and 11, respectively. Haplotypes 6, 8 and 13 were found in only five animals. However, their frequencies may be higher in these breeds because haplotypes 6 and 8 were found in homozygosity. The frequency of haplotype 10 was 8 % but it was restricted to one flock of New Zealand-derived Cheviot sheep. In Soay sheep, which are one of the most ancient representatives of feral sheep in Europe and are similar to Mediterranean moufflon, only haplotypes 1, 3 and 7 were observed, with haplotype 7 having a frequency of 18.4 %.
The association between SPRN and scrapie susceptibility was analysed for a UK sheep flock that has a high level of classical scrapie linked to PRNP genotypes with the VRQ allele (flock 30, McIntyre et al., 2008). Ninety-five animals with VRQ/ARQ PRNP genotypes were selected, 42 were diagnosed as scrapie-positive and 43 as survivors at the end of the study. Haplotypes 9 and 12 were only found in this flock, but at a very low frequency, so no association with disease could be calculated. SPRN genotypes were established for haplotypes 3, 7 and 11 in both groups. The frequency of haplotype 3 was 6 and 7.5 % in scrapie-positive and -negative samples, respectively; for haplotype 11 it was 2.5 and 2.8 %, respectively. There was also no significant difference in the occurrence of haplotype 7 between scrapie-positive (14.3 %) and healthy (13.2 %) animals.
In humans, it has been shown that genetic variation of the SPRN gene is associated with differences in the onset of prion diseases. It is therefore of considerable importance to establish whether such an association also exists for prion diseases in animals, primarily scrapie in sheep and goats and chronic wasting disease in deer. To do this, it is useful to explore the extent and type of genetic variation of the SPRN gene ORF in ruminants.
Our data show that genetic variations in the SPRN ORF was detected in three of four species (sheep, goat, cattle and deer) for which we have analysed more than 100 samples, the exception being goats. There are 18 single amino acid differences between the wild-type alleles of the various ruminant species. Two regions within the protein are of special interest, the R/G-rich basic repeats (approx. codons 28–51) and the hydrophobic region (codons 59–78). There was no variation in the number of the basic repeats, but we found a few, mostly conservative, single amino acid substitutions (deer codons 29 and 45; yak codons 42 and 43) are mostly conserved in nature. The hydrophobic region of Sho shows the most homology with PrP and, as in PrP, it is remarkably conserved between the species. It is therefore of considerable interest to have found deletion and insertion variants in this region in both sheep and bovine species. The alanine insertion at codon 65 creates a perfect replica of the PrP palindromic amino acid sequence AGAAAAGA, which is proposed to form a binding site for protein–protein interaction and maybe even the interface between normal PrP and its disease-specific isoform PrPSc (Norstrom & Mastrianni, 2005). Whether the three ovine Sho variants with the sequences AGAAAGA, AGAAAAGA or AGAAAAAGA can all compete with or substitute PrP in these interactions remains an important question for future studies. The deletion of four amino acids (AAAG or SAAG) in Sho of four species (sheep, domestic cattle, gaur and human; Beck et al., 2008) may be the first indication that this motif can be regarded as a repeat unit, the number of which might vary within and between species. The instability of the Ala-rich sequence may be an important feature of the genetic variation of Sho in sheep, suggesting a different function from PrP in which this sequence is highly conserved and shows no insertion or deletion. Indeed, the double alanine insertion in sheep is not recent, as it was found in most breeds, including Soay, one of the oldest domestic breeds. Also, this insertion was not rare and appeared in homozygous genotypes in apparently healthy animals, making it unlikely to be a pathogenic mutation.
We did not detect amino acid polymorphisms in samples from goats and wapiti; domestic cattle and deer samples were also very stable, with three allelic variants amongst them. The same applies to laboratory mouse strains which, according to our analysis of the strains used for scrapie experiments, all express the same Sho. Therefore, it appears that the best model for SPRN genetic association studies is currently sheep.
In sheep, at least nine SPRN alleles present opportunities to analyse scrapie association. A first study of scrapie in a highly infected flock revealed no association of SPRN genotypes with disease susceptibility in VRQ/ARQ PRNP genotypes. Scrapie genetic association studies are notoriously difficult because parameters such as long incubation periods, different agent strains, subclinical infection and strong PRNP genetics influence outbreaks substantially. Therefore, this initial analysis does not yet prove that there is no association with susceptibility nor that SPRN could not modulate other aspects of disease such as pathology or subclinical infection.
It has been suggested that PRNP and SPRN have evolved from the same ancestral gene into genes that may still share some functions but may also have gained new biological roles (Premzl et al., 2004). Our study has shown that the genetic variation of SPRN is quite different from PRNP in ruminants, especially that the hydrophobic sequence motif that both proteins share is variable in one (Sho) but not the other (PrP) protein. Future studies will show whether this genetic difference determines mechanisms in function and prion disease.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 4 March 2009;
accepted 8 June 2009.
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