Artigo 15- Sequence Analysis of the Prion Protein Gene in Mongolian Gazelles (Procapra Gutturosa

8/6/2019 Artigo 15- Sequence Analysis of the Prion Protein Gene in Mongolian Gazelles (Procapra Gutturosa

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Sequence analysis of the prion protein gene in Mongolian gazelles( Procapra gutturosa)

Yiqin Wang Æ Zhenkui Qin Æ Yonggan Bao ÆJunwen Qiao Æ Lifeng Yang Æ Deming Zhao

Received: 6 March 2009 / Accepted: 17 June 2009/ Published online: 5 July 2009

Ó Springer Science+Business Media, LLC 2009

Abstract Prion diseases are a group of human and animal

neurodegenerative conditions, which are caused by thedeposition 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 asso-

ciated with the incubation period, susceptibility, and spe-

cies 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 Thomson’s 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 octa-

peptide repeat at the N-terminal octapeptide repeat region.

The polymorphisms of gPrnp will assist the study of prion

disease pathogenesis, resistance, and cross speciestransmission.

Keywords Mongolian gazelle (Procapra gutturosa) Á

Prnp Á Sequence analysis Á Gene polymorphism Á

Prion disease Á Species barrier

Introduction

The prion protein is implicated in various types of neuro-

degenerative spongiform encephalopathies, including Cre-

utzfeldt–Jakob 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 configuration [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, sus-

ceptibility, and species barrier to the scrapie disease in

sheep [2, 4–7].

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 Creutzfeldt–Jakob

Y. Wang Á J. Qiao Á L. Yang (&) Á D. Zhao (&)

National Animal Transmissible Spongiform Encephalopathies

Laboratory, College of Veterinary Medicine, China Agricultural

University, Beijing 100193, Chinae-mail: [email protected]

D. Zhao

e-mail: [email protected]

Y. Wang Á Y. Bao

Erlianhot Entry-Exit Inspection and Quarantine,

Inner Mongolia 011100, China

Z. Qin

Chinese Academy of Inspection and Quarantine,

Beijing 100025, China

123

Virus Genes (2009) 39:273–278

DOI 10.1007/s11262-009-0383-9



disease (vCJD), which was different from CJD in incuba-

tion 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 Mongoliangazelles 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 ana-lyzed. The findings of PrP polymorphisms in Mongolian

gazelles will assist the study of TSE pathogenesis and cross

species transmission.

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 Geno-

mic DNA Rapid Isolation Kit (BioDev-Tech, China) and

amplified 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 Gen-

Bank (EU 224471): forward primer 50-ATGGTGAAA

AGCCACATAGGCAGTTG-3 0; reverse primer 50-CTATC

CTACTATGAGAAAAATGAGGAAAG-3 0. 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 94°C

for 5 min, 36 cycles of denaturation at 94°C for 30 s,

annealing at 61°C for 40 s and extension at 72°C for 40 s,

and a final extension at 72°C for 8 min. The amplified

target DNA was purified using the E.Z.N.A. Gel Extraction

Kit (Omega, USA). After purification, 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

the dye-terminator cycle method on an ABI Prism 377

automated DNA sequencer (Applied Biosystems, Foster

City, USA). Amino acid alignment analysis and phyloge-

netic 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, and221 (Table 1). Of the 26 animalsstudied, 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 87–94.

Amino acid alignment of 13 mammalian prion proteins is

shown in Fig. 1. The gPrnp sequence shared high identity

with the prion sequences of Thomson’s 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 Substitution Homozygotes Heterozygotes Frequency

119 N ? S 0 6 6/26

143 S ? G 0 6 6/26

160 Y ? H 0 6 6/26

172 V ? A 0 1 1/26

182 N ? S 0 2 2/26

221 V ? A 0 1 1/26

87-94 Deletion 0 4 4/26

274 Virus Genes (2009) 39:273–278

123



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), Thomson’s

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

Virus Genes (2009) 39:273–278 275

123



common octapeptide allele has six repeats in bovine Prnp,

whereas there are five repeats in the Prnp of other mam-

malian species. In our study, the majority (84.6%) of

Mongolian gazelles had the 6:6 genotype in the Prnp N-

terminal octapeptide repeat region, 15.4% carried the 6:5

genotype, and no animals expressed the 5:5 genotype

(Table 2).

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

of prion diseases is determined by the degree of identity of

the prion proteins shared between the recipient and host

species [12–14]. Although the region of the PrP sequence

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. Thomson’s gazelle (Gazella

thomsonii, EU 032301), 10. Blackbuck ( Antilope cervicapra,

AY720706), 11. Wildebeest (Connochaetes, EF165086), 12. Mongo-

lian gazelle (Procapra gutturosa, AB473611), 13. Mongolian gazelle

(Procapra gutturosa, AB473604)

Amino Acid Substitutions (x100)

05.7 24

Fallow deerWestern roe deerMongolian gazelle 2BlackbuckMongolian gazelle 1Thomson's gazelleSheepGoatWildebeestCattleElandHuman.Mouse.

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), Thomson’s gazelle (Gazella thomsonii, EU

032301), Blackbuck ( Antilope cervicapra, AY720706), Wildebeest

(Connochaetes, EF165086), Mongolian gazelle (Procapra gutturosa,

AB473611), Mongolian gazelle (Procapra gutturosa, AB473604)

Table 2 Octapeptide repeats in gPrnp

Codon Repeat Amino acid sequence

54–62 R1 PQGGGGWGQ

63–70 R2 PHGGG–WGQ



87–94 R5 PHGGG–WGQ (Deletion)

95–103 R6 PHGGGGWGQ

The octapeptide repeat between R4 and R6 was deleted

276 Virus Genes (2009) 39:273–278

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responsible for the species barrier has not been identified, 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 respon-

sible 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, 17–19]. Susceptibility to the disease is appar-

ently influenced 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

field and laboratory conditions [20–23]. 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 associ-

ated 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 indi-

viduals [6, 25]. A study with scrapie-infected mouse neu-

roblastoma 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 five repeats in ovine. Inherited prion

diseases such as familial Creutzfeldt–Jakob disease and

Gerstmann–Straussler–Scheinker syndrome in humans are

associated with the insertion of additional octapeptide

repeats in the N-terminal region of PrP [27–29]. 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 Thomson’s 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 glob-

ular structural domain. The polymorphisms of gPrnp will

assist the study of TSE pathogeneses, prion disease resis-

tance, and cross species transmission.

Acknowledgments The authors thank Mrs. L. Tian and Mr. HuaiboWei (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 financially sup-

ported by the National Key Technology Research and Development

Program (2006BAD06A13) and the National Science Foundation of

China (30871854).

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Artigo 15- Sequence Analysis of the Prion Protein Gene in Mongolian Gazelles (Procapra Gutturosa