Virus Genes (2009) 39:273278 DOI 10.

1007/s11262-009-0383-9

Sequence analysis of the prion protein gene in Mongolian gazelles (Procapra gutturosa)
Yiqin Wang Zhenkui Qin Yonggan Bao Junwen Qiao Lifeng Yang Deming Zhao

Received: 6 March 2009 / Accepted: 17 June 2009 / Published online: 5 July 2009 Springer Science+Business Media, LLC 2009

Abstract Prion diseases are a group of human and animal neurodegenerative conditions, which are caused by the deposition of an abnormal isoform prion protein (PrPSc) encoded by a single copy prion protein gene (Prnp). In sheep, genetic variations of Prnp were found to be associated with the incubation period, susceptibility, and species barrier to the scrapie disease. We investigated the sequence and polymorphisms of the prion protein gene of Mongolian gazelles (gPrnp). gPrnp gene sequence analysis of blood samples from 26 Mongolian gazelles showed high identity within species. The gPrnp gene was closely related to the Prnp genes of Thomsons gazelle, blackbuck, and cattle with 100, 100, and 98.5% identity, respectively, whereas the gPrnp gene with a deletion was closely related to the Prnp genes of wildebeest, Western roe deer, and sheep with 99.3, 99.3, and 98.9% identity, respectively. Polymorphisms of the open reading frame of Prnp as amino acid substitutions were detected at codons 119(N ? S), 143(S ? G) or 160(Y ? H), 172(V ? A), 182(N ? S) and 221(V ? A). There was also deletion of one octapeptide repeat at the N-terminal octapeptide repeat region.
Y. Wang J. Qiao L. Yang (&) D. Zhao (&) National Animal Transmissible Spongiform Encephalopathies Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China e-mail: yanglf@cau.edu.cn D. Zhao e-mail: zhaodm@cau.edu.cn Y. Wang Y. Bao Erlianhot Entry-Exit Inspection and Quarantine, Inner Mongolia 011100, China Z. Qin Chinese Academy of Inspection and Quarantine, Beijing 100025, China

The polymorphisms of gPrnp will assist the study of prion disease pathogenesis, resistance, and cross species transmission. Keywords Mongolian gazelle (Procapra gutturosa) Prnp Sequence analysis Gene polymorphism Prion disease Species barrier

Introduction The prion protein is implicated in various types of neurodegenerative spongiform encephalopathies, including CreutzfeldtJakob Disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle [1], all of which are generally referred to as transmissible spongiform encephalopathies (TSE) or prion diseases. Prion diseases are caused by the deposition of an abnormal isoform prion protein (PrPSc) encoded by a single copy prion protein gene (Prnp). Prion protein is attached to the cell membrane through a glycosylphosphatidylinosytol (GPI) anchor, and the entire open reading frame (ORF) is located within a single exon [2]. Compared to the normal cellular prion protein (PrPC), PrPSc is a variant prion with the same gene and the same amino acid sequence but a different tertiary conguration [3]. Although the molecular mechanisms of prion disease pathogenesis remains unclear, single-nucleotide polymorphisms (SNPs) of Prnp were found to be associated with the incubation period, susceptibility, and species barrier to the scrapie disease in sheep [2, 47]. TSE has been reported in a wide range of animal species and humans. Besides sheep, cattle, and humans, big cats, macaques, cats, and exotic ungulates were also found to be infected with TSE [8, 9]. Variant CreutzfeldtJakob

123

274

Virus Genes (2009) 39:273278

disease (vCJD), which was different from CJD in incubation period and pathology, was found to be consistent with BSE, strongly suggesting that human vCJD is transmitted from bovine [2]. Mongolian gazelles (Procapra gutturosa), an ungulate animal species, are found in Mongolia, Russia, and China. They are herd living animals, and migrate in large groups in search of the best grassland. The meat of Mongolian gazelles is edible and consumed by Mongolian residents, and the horns are used as valuable medicines. Therefore, prion diseases, if present, in Mongolian gazelles could be transmitted to humans and domestic animals. The gPrnp encodes a precursor protein of 264/256 amino acids, including a signal peptide of 24 amino acids in the N-terminal and a 22 amino acid GPI signal peptide in the C-terminal [3, 10, 11]. At present, there have been no reports of sequence and polymorphism analyses of Prnp in Mongolian gazelles. In the present study, blood samples were taken from Mongolian gazelles for Prnp gene cloning and sequencing, and polymorphisms of Prnp were analyzed. The ndings of PrP polymorphisms in Mongolian gazelles will assist the study of TSE pathogenesis and cross species transmission.

the dye-terminator cycle method on an ABI Prism 377 automated DNA sequencer (Applied Biosystems, Foster City, USA). Amino acid alignment analysis and phylogenetic tree inference were conducted using the codon usage table by the DNAMAN program (Version 5.2.2) and DNAStar (Lasergene 7.0), respectively, based on the obtained DNA sequences.

Results The ORF region of gPrnp was cloned and sequenced and found to possess either 795 or 771 bp, encoding 264 or 256 amino acids. The sequence was posted in the GenBank database with accession numbers from AB473602 to AB473615. Sequence alignment of the prion protein using DNAMAN and DNAStar revealed that the PrP sequences from the 26 Mongolian gazelles shared high identity (98.89%). Polymorphisms of the ORF were found at codons 119, 143, 160, 172, 182, and 221 (Table 1). Of the 26 animals studied, 6 had amino acid substitutions at 119(N ? S), 143(S ? G), or 160(Y ? H), two at 182(N ? S) or 221 (V ? A), and one at 172(V ? A). Four animals exhibited a deletion of one octapeptide repeat at codons 8794. Amino acid alignment of 13 mammalian prion proteins is shown in Fig. 1. The gPrnp sequence shared high identity with the prion sequences of Thomsons gazelle (EU 032301, 100%), blackbuck (AY720706, 100%), cattle (EU224471, 98.5%), eland (EF165082, 98.1%), western roe deer (AY639096, 96.3%), sheep (M31313, 95.9%), goats (EU032305, 95.9%), and Fallow deer (AY639094, 95.6%), and had 86.9% homology with the human sequence (NM 183079) and 84.3% with the mouse sequence (NM011170). The gPrnp gene with a deletion was closely related to the Prnp genes of wildebeest, Western roe deer, and sheep, with 99.3, 99.3, and 98.9% identity, respectively (Fig. 2). The phylogenetic tree of 13 mammalian prion proteins is shown in Fig. 3. Length variation of the Prnp N-terminal octapeptide repeat region has been reported in bovine breeds. The most
Table 1 Frequencies of Mongolian gazelle Prnp polymorphisms Number of animals Codon 119 143 160 172 182 221 87-94 Substitution N?S S?G Y?H V?A N?S V?A Deletion Homozygotes 0 0 0 0 0 0 0 Heterozygotes 6 6 6 1 2 1 4 Frequency 6/26 6/26 6/26 1/26 2/26 1/26 4/26

Materials and methods Blood samples were collected into EDTA tubes from 26 healthy, 2- to 3-year-old Mongolian gazelles in the Huhhot Wildlife Zoo, Inner Mongolia, China. DNA was extracted from blood samples using a Genomic DNA Rapid Isolation Kit (BioDev-Tech, China) and amplied by PCR. PCR primer pairs were designed using Primer Premier 5.0 based on the ORF region of a Bos taurus prion protein gene sequence deposited in the GenBank (EU 224471): forward primer 50 -ATGGTGAAA AGCCACATAGGCAGTTG-30 ; reverse primer 50 -CTATC CTACTATGAGAAAAATGAGGAAAG-30 . The primers amplify a 795/771-bp fragment of the ORF of Prnp, and the fragment encodes 264/256 amino acids. The PCR reaction mixture (25 ll) consisted of 200 ng DNA template, 0.5 lM each of primers, 200 lM dNTP, 5 unit of Taq Plus DNA Polymerase (Takara, Japan), 0.1 M 10X ammonium buffer, and 17 ll double distilled water. PCR reaction parameters were set as follows: initial denaturation at 94C for 5 min, 36 cycles of denaturation at 94C for 30 s, annealing at 61C for 40 s and extension at 72C for 40 s, and a nal extension at 72C for 8 min. The amplied target DNA was puried using the E.Z.N.A. Gel Extraction Kit (Omega, USA). After purication, the PCR products were sequenced directly, or cloned into the pGEM-T easy vector (Promega, USA) following standard protocols. Three to four clones from each sample were sequenced by

123

Virus Genes (2009) 39:273278

275

Fig. 1 Amino acid alignment of 13 mammalian prion proteins. The deduced amino acid sequence of Human (Homo sapiens, NM183079), Mouse (Mus musculus, NM011170), Cattle (Bos taurus, EU224471), Eland (Tragelaphus oryx, EF165082), Fallow deer (Cervus dama, AY639094), Sheep (Ovis aries, M31313), Goat (Capra hircus, EU032305), Western roe deer (Capreolus, AY639096), Thomsons

gazelle (Gazella thomsonii, EU 032301), Blackbuck (Antilope cervicapra, AY720706), and Wildebeest (Connochaetes, EF165086) were compared with Mongolian gazelles (Procapra gutturosa, AB473611 and AB473604). Deletions are indicated by dashes and sites identical to the consensus sequence are denoted by dots

123

276

Virus Genes (2009) 39:273278

Fig. 2 Sequence identity of 13 mammalian prion proteins. 1. Human (Homo sapiens, NM183079), 2. Mouse (Mus musculus, NM011170), 3. Cattle (Bos Taurus, EU224471), 4. Eland (Tragelaphus oryx, EF165082), 5. Fallow deer (Cervus dama, AY639094), 6. Sheep (Ovis aries, M31313), 7. Goat (Capra hircus, EU032305), 8. Western

roe deer (Capreolus, AY639096), 9. Thomsons gazelle (Gazella thomsonii, EU 032301), 10. Blackbuck (Antilope cervicapra, AY720706), 11. Wildebeest (Connochaetes, EF165086), 12. Mongolian gazelle (Procapra gutturosa, AB473611), 13. Mongolian gazelle (Procapra gutturosa, AB473604)
Fallow deer Western roe deer Mongolian gazelle 2 Blackbuck Mongolian gazelle 1 Thomson's gazelle Sheep Goat Wildebeest Cattle Eland Human. Mouse.

5.7
4 2 0

Amino Acid Substitutions (x100)

Fig. 3 Phylogenetic tree of 13 mammalian prion proteins. Human (Homo sapiens, NM183079), Mouse (Mus musculus, NM011170), Cattle (Bos Taurus, EU224471), Eland (Tragelaphus oryx, EF165082), Fallow deer (Cervus dama, AY639094), Sheep (Ovis aries, M31313), Goat (Capra hircus, EU032305), Western roe deer (Capreolus, AY639096), Thomsons gazelle (Gazella thomsonii, EU 032301), Blackbuck (Antilope cervicapra, AY720706), Wildebeest (Connochaetes, EF165086), Mongolian gazelle (Procapra gutturosa, AB473611), Mongolian gazelle (Procapra gutturosa, AB473604)

common octapeptide allele has six repeats in bovine Prnp, whereas there are ve repeats in the Prnp of other mammalian species. In our study, the majority (84.6%) of Mongolian gazelles had the 6:6 genotype in the Prnp Nterminal octapeptide repeat region, 15.4% carried the 6:5 genotype, and no animals expressed the 5:5 genotype (Table 2).

of prion diseases is determined by the degree of identity of the prion proteins shared between the recipient and host species [1214]. Although the region of the PrP sequence
Table 2 Octapeptide repeats in gPrnp Codon 5462 6370 7178 7986 8794 95103 Repeat R1 R2 R3 R4 R5 R6 Amino acid sequence PQGGGGWGQ PHGGGWGQ PHGGGWGQ PHGGGWGQ PHGGGWGQ (Deletion) PHGGGGWGQ

Discussion Interspecies transmission of prion diseases occurs for some forms of TSE (e.g., BSE), while other forms of TSE are transmitted readily only within species. The species barrier

The octapeptide repeat between R4 and R6 was deleted

123

Virus Genes (2009) 39:273278

277

responsible for the species barrier has not been identied, a study of transgenic mice with the bovine and human prion transgenes suggests that an epitope consisting of amino acid residues 187, 189, 206, and 208 is probably responsible for prion transmission across species [15, 16]. In Mongolian gazelles, amino acid polymorphisms were detected at codons 119(N ? S), 143(S ? G), 160 (Y ? H), 182(N ? S), 221(V ? A), and 172(V ? A) of the ORF within the PrP C-terminal globular structural domain. These polymorphisms may contribute to species barrier between Mongolian gazelles and other animal species or humans. It is believed that SNPs of the Prnp gene are associated with susceptibility and incubation period to the scrapie disease [5, 1719]. Susceptibility to the disease is apparently inuenced by amino acid residues at codons 136, 154, and 171. Sheep with amino acid residues V, R, and Q at codons 136, 154, and 171 (VRQ), respectively, in the protein sequence were highly susceptible to scrapie and had a short survival period after challenge with scrapie; whereas animals with amino acid residues A, R, and R (ARR) at these codons were resistant to the disease under eld and laboratory conditions [2023]. Polymorphisms of Prnp in North American deer and elks were also associated with relative susceptibility to the chronic wasting disease (CWD). Elks with methionine at codon 132 were associated with increased susceptibility to CWD [24]. Human Prnp codon 129 homozygous for methionine was linked to human vCJD [6, 25]. In 15 Polish CJD cases 73.3% were homozygous for methionine, 13.3% homozygous for valine, and 13.3% heterozygous (Met/Val), compared to 45% homozygous for methionine in 109 unaffected individuals [6, 25]. A study with scrapie-infected mouse neuroblastoma cells transfected with chimeric human/mouse PrP genes showed that amino acid substitutions at codons 167, 171, 214, or 218 prevented PrPSC formation [26]. It appears that polymorphisms in the C-terminal globular structural domain of Prnp in animals and humans are associated with susceptibility to prion diseases. It is yet to be determined whether the gPrnp with polymorphisms in the C-terminal globular region are associated with a greater susceptibility (or resistance) to prion disease compared to the Prnp of other species. The majority (85.4%) of Mongolian gazelles had the 6:6 genotype in the Prnp N-terminal octapeptide repeat region, with 14.6% of the animals having the 6:5 genotype. The most common octapeptide allele carries six repeats in bovine Prnp and ve repeats in ovine. Inherited prion diseases such as familial CreutzfeldtJakob disease and GerstmannStrausslerScheinker syndrome in humans are associated with the insertion of additional octapeptide repeats in the N-terminal region of PrP [2729]. A total number of repeats above eight is associated with an

increased risk to the CJD [28], and prion diseases with small octapeptide repeat insertions (OPRI) in the Prnp appear to have a later age of onset and shorter survival time after infection relative to those with the larger repeat insertions [27, 29]. However, an association of octapeptide repeats with prion disease susceptibility has not yet been demonstrated in animals [28]. No prion disease has been detected in Mongolian gazelles. In conclusion, gPrnp shares high intra-species identity. Compared with 13 mammalian prion protein sequences, amino acids were substituted at codons 119, 143, 160, 172, 182, and 221. The gPrnp sequence shared 100% identity with that of Thomsons gazelle and blackbuck and over 98% with that of cattle and eland. In addition, gPrnp has a deletion of one octapeptide repeat in the C-terminal globular structural domain. The polymorphisms of gPrnp will assist the study of TSE pathogeneses, prion disease resistance, and cross species transmission.
Acknowledgments The authors thank Mrs. L. Tian and Mr. Huaibo Wei (Erlianhot Entry-Exit Inspection and Quarantine) for providing Mongolian gazelle blood samples. We are grateful to Dr. Jin Zhu (Therapeutic Goods Administration, Australia) for his assistance in the preparation of the manuscript. This study was nancially supported by the National Key Technology Research and Development Program (2006BAD06A13) and the National Science Foundation of China (30871854).

References
1. S.B. Prusiner, Molecular biology and pathogenesis of prion diseases. Trends Biochem. Sci. 21(12), 482487 (1996) 2. A.F. Hill, M. Desbruslais, S. Joiner, K.C. Sidle, I. Gowland, J. Collinge, L.J. Doey, P. Lantos, The same prion strain causes vCJD and BSE. Nature 389(6650), 448500, 526 (1997) 3. S.B. Prusiner, Prions. Proc. Natl Acad. Sci. USA 95(23), 13363 13383 (1998) 4. E.A. Asante, J.M. Linehan, M. Desbruslais, S. Joiner, I. Gowland, A.L. Wood, J. Welch, A.F. Hill, S.E. Lloyd, J.D. Wadsworth, J. Collinge, BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein. EMBO J. 21(23), 63586366 (2002) 5. A. Bossers, B.E. Schreuder, I.H. Muileman, P.B. Belt, M.A. Smits, PrP genotype contributes to determining survival times of sheep with natural scrapie. J. Gen. Virol. 77(Pt 10), 26692673 (1996) 6. J. Collinge, K.C. Sidle, J. Meads, J. Ironside, A.F. Hill, Molecular analysis of prion strain variation and the aetiology of new variant CJD. Nature 383(6602), 685690 (1996) 7. D. Westaway, V. Zuliani, C.M. Cooper, M. Da Costa, S. Neuman, A.L. Jenny, L. Detwiler, S.B. Prusiner, Homozygosity for prion protein alleles encoding glutamine-171 renders sheep susceptible to natural scrapie. Genes Dev. 8(8), 959969 (1994) 8. J.K. Kirkwood, A.A. Cunningham, Epidemiological observations on spongiform encephalopathies in captive wild animals in the British Isles. Vet. Rec. 135(13), 296303 (1994) 9. S. Lezmi, A. Bencsik, E. Monks, T. Petit, T. Baron, First case of feline spongiform encephalopathy in a captive cheetah born in France: PrP(sc) analysis in various tissues revealed unexpected

123

278 targeting of kidney and adrenal gland. Histochem. Cell Biol. 119(5), 415422 (2003) Y. Kuroda, Y. Maeda, S. Sawa, K. Shibata, K. Miyamoto, T. Nakagawa, Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy. J. Pept. Sci. 9(4), 212220 (2003) A.R. Walmsley, N.M. Hooper, Distance of sequons to the C-terminus inuences the cellular N-glycosylation of the prion protein. Biochem. J. 370(Pt 1), 351355 (2003) S.B. Prusiner, Molecular biology of prion diseases. Science 252(5012), 15151522 (1991) H.M. Schatzl, M. Da Costa, L. Taylor, F.E. Cohen, S.B. Prusiner, Prion protein gene variation among primates. J. Mol. Biol. 245(4), 362374 (1995) M. Scott, D. Foster, C. Mirenda, D. Serban, F. Coufal, M. Walchli, M. Torchia, D. Groth, G. Carlson, S.J. DeArmond, D. Westaway, S.B. Prusiner, Transgenic mice expressing hamster prion protein produce species-specic scrapie infectivity and amyloid plaques. Cell 59(5), 847857 (1989) M.R. Scott, J. Safar, G. Telling, O. Nguyen, D. Groth, M. Torchia, R. Koehler, P. Tremblay, D. Walther, F.E. Cohen, S.J. DeArmond, S.B. Prusiner, Identication of a prion protein epitope modulating transmission of bovine spongiform encephalopathy prions to transgenic mice. Proc. Natl Acad. Sci. USA 94(26), 1427914284 (1997) F. Wopfner, G. Weidenhofer, R. Schneider, A. von Brunn, S. Gilch, T.F. Schwarz, T. Werner, H.M. Schatzl, Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of exible regions of the prion protein. J. Mol. Biol. 289(5), 11631178 (1999) M. Baylis, W. Goldmann, F. Houston, D. Cairns, A. Chong, A. Ross, A. Smith, N. Hunter, A.R. McLean, Scrapie epidemic in a fully PrP-genotyped sheep ock. J. Gen. Virol. 83(Pt 11), 2907 2914 (2002) W. Goldmann, N. Hunter, J.D. Foster, J.M. Salbaum, K. Beyreuther, J. Hope, Two alleles of a neural protein gene linked to scrapie in sheep. Proc. Natl Acad. Sci. USA 87(7), 24762480 (1990) N. Hunter, W. Goldmann, E. Marshall, G. ONeill, Sheep and goats: natural and experimental TSEs and factors inuencing incidence of disease. Arch. Virol. Suppl. (16), 181188 (2000)

Virus Genes (2009) 39:273278 20. P.B. Belt, I.H. Muileman, B.E. Schreuder, J. Bos-de Ruijter, A.L. Gielkens, M.A. Smits, Identication of ve allelic variants of the sheep PrP gene and their association with natural scrapie. J. Gen. Virol. 76(Pt 3), 509517 (1995) 21. F. Houston, W. Goldmann, A. Chong, M. Jeffrey, L. Gonzalez, J. Foster, D. Parnham, N. Hunter, Prion diseases: BSE in sheep bred for resistance to infection. Nature 423(6939), 498 (2003) 22. N. Hunter, L. Moore, B.D. Hosie, W.S. Dingwall, A. Greig, Association between natural scrapie and PrP genotype in a ock of Suffolk sheep in Scotland. Vet. Rec. 140(3), 5963 (1997) 23. S. McCutcheon, N. Hunter, F. Houston, Use of a new immunoassay to measure PrP Sc levels in scrapie-infected sheep brains reveals PrP genotype-specic differences. J. Immunol. Methods 298(12), 119128 (2005) 24. K.I. ORourke, T.E. Besser, M.W. Miller, T.F. Cline, T.R. Spraker, A.L. Jenny, M.A. Wild, G.L. Zebarth, E.S. Williams, PrP genotypes of captive and free-ranging Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease. J. Gen. Virol. 80(Pt 10), 27652769 (1999) 25. J. Bratosiewicz, P.P. Liberski, J. Kulczycki, R. Kordek, Codon 129 polymorphism of the PRNP gene in normal Polish population and in Creutzfeldt-Jakob disease, and the search for new mutations in PRNP gene. Acta Neurobiol. Exp. 61(3), 151156 (2001) 26. K. Kaneko, L. Zulianello, M. Scott, C.M. Cooper, A.C. Wallace, T.L. James, F.E. Cohen, S.B. Prusiner, Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation. Proc. Natl Acad. Sci. USA 94(19), 1006910074 (1997) 27. L.G. Goldfarb, P. Brown, W.R. McCombie, D. Goldgaber, G.D. Swergold, P.R. Wills, L. Cervenakova, H. Baron, C.J. Gibbs Jr., D.C. Gajdusek, Transmissible familial Creutzfeldt-Jakob disease associated with ve, seven, and eight extra octapeptide coding repeats in the PRNP gene. Proc. Natl Acad. Sci. USA 88(23), 1092610930 (1991) 28. W. Goldmann, PrP genetics in ruminant transmissible spongiform encephalopathies. Vet. Res. 39(4), 30 (2008) 29. S. Mead, Prion disease genetics. Eur. J. Hum. Genet. 14(3), 273 281 (2006)

10.

11.

12. 13.

14.

15.

16.

17.

18.

19.

123