Gene 529 (2013) 345–350

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Short Communication

Molecular cloning and sequence analysis of prion protein gene in Xiji donkey in China

Zhuming Zhang a,b,1, Renli Wang b, Lihua Xu b, Fangzhong Yuan a, Xiangmei Zhou a,1, Lifeng Yang a,Xiaomin Yin a, Binrui Xu a,⁎, Deming Zhao a,⁎a State Key Laboratories for Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University,Beijing 100193, Chinab College of Agriculture, Ningxia University, Yinchuan 750021, China

Abbreviations: TSEs, transmissible spongiform encepJakob disease; vCJD, variant Creutzfeldt–Jakob diseases; BSlopathy; CWD, chronic wasting disease; FME, feline spomink spongiform encephalopathy; PRNP, prion proteinPrP; PrPC, cellular prion protein; PrP, prion protein; SNphisms; PCR, polyenzyme chain reaction; ORF, openphosphatidyl inositol.⁎ Corresponding authors. Tel./fax: +86 10 62732975.

E-mail address: demingzhao@yeah.net (D. Zhao).1 These authors equally contribute to this work.

0378-1119/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.gene.2013.08.019

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 3 August 2013Available online 15 August 2013

Keywords:Xiji donkeyPrion geneTSE

Prion diseases are a group of human and animal neurodegenerative disorders caused by the deposition of anabnormal isoform prion protein (PrPSc) encoded by a single copy prion protein gene (PRNP). Prion disease hasbeen reported in many herbivores but not in Equus and the species barrier might be playing a role in resistanceof these species to the disease. Therefore, analysis of genotype of prion protein (PrP) in these species may helpunderstand the transmission of the disease. Xiji donkey is a rare species of Equus not widely reared in Ningxia,China, for service, food and medicine, but its PRNP has not been studied. Based on the reported PrP sequencein GenBank we designed primers and amplified, cloned and sequenced the PRNP of Xiji donkey. The sequenceanalysis showed that the Xiji donkey PRNPwas consisted of an open reading frame of 768 nucleotides encoding256 amino acids. Amino acid residues unique to donkey as comparedwith some Equus animals,mink, cow, sheep,human, dog, sika deer, rabbit and hamster were identified. The results showed that the amino acid sequence ofXiji donkey PrP starts with the consensus sequence MVKSH, with almost identical amino acid sequence to thePrP of other Equus species in this study. Amino acid sequence analysis showed high identity within species andclose relation to the PRNP of sika deer, sheep, dog, camel, cow,mink, rabbit and hamsterwith 83.1–99.7% identity.The results provided the PRNP data for an additional Equus species, which should be useful to the study of theprion disease pathogenesis, resistance and cross species transmission.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Transmissible spongiform encephalopathies (TSEs), are a group offatal neurodegenerative disorders of animals and humans, also calledprion-related diseases, mainly including Creutzfeldt–Jakob disease(CJD), variant Creutzfeldt–Jakob diseases (vCJD) in humans, scrapie insheep, bovine spongiform encephalopathy (BSE or mad-cow disease)in cattle, chronic wasting disease (CWD) in deer (Lee et al., 2013;Prusiner, 1991), feline spongiform encephalopathy (FME) (Berthelin-Baker et al., 2000) and mink spongiform encephalopathy (TME)(Kretzschmar et al., 1992). These diseases are caused by the depositionof an abnormal isoform prion protein (PrPSc). Compared to the cellular

halopathies; CJD, Creutzfeldt–E, bovine spongiform encepha-ngiform encephalopathy; TME,gene; PrPSc, scrapie isoform ofPs, single-nucleotide polymor-reading frame; GPI, glycosyl-

ights reserved.

prion protein (PrPC), PrPSc is a variant prionwith the sameprion proteingene and the same amino acid sequence but a different tertiary config-uration (Borchelt et al., 1992; Prusiner, 1998). The molecular mecha-nism of prion disease pathogenesis remains unclear, while single-nucleotide polymorphisms (SNPs) of PRNPwere found to be associatedwith the incubation period, and species barrier (Asante et al., 2002; Hillet al., 1997;Weissmann, 2005; Zhang, 2011a). Prion species barrier is aphenomenon of one species being able to protect the prion infectionfrom other species. There are species particularity and strain particular-ity of prion in the process of PrPSc propagation. The species particularityis that one species can protect the prion attack from the other species,representing the prolonged preclinical period or even no occurrenceof the disease. The essential reason of species particularity is the differ-ence in primary and secondary structures of PrP between the recipientand host species. Variation or the polymorphism of PRNP in humanbeing, mouse, sheep, bovine and deer is considered to be associatedwith the prion disease (Liberski et al., 2001; Ning et al., 2005). Althoughpig, mice, macaques, and exotic ungulates were found to be infectedwith TSE (Kirkwood and Cunningham, 1994; Raeber et al., 1997;Wang et al., 2009), the process of transmission between differentspecies is either inefficient or completely ineffective (Westaway et al.,1994). Dogs, rabbits, and horses have been reported to be the few

Fig. 2. Electrophorus identification result of products by enzyme digestion (M: 5000 bpDNA marker. Lanes 1–2: Products of enzyme digestion with KpnI and EcoRV).

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mammalian species (Chianini et al., 2012; Zhang, 2011b) being resistantto infection from prion diseases before 2011. However, the speciesbarrier in rabbits was overcome by using the protein misfolding cyclicamplification (PMCA) technique in 2011 (Priola, 2013). Luckily, up tothe present there has been no report of the TSE cases in donkey aswell as in horse (Zhang, 2011c). It is remarkable considering that TSEsin other species tend to develop at advanced age and horses are oftenkept for a long lifetime.

Xiji donkey is one of the rare domestic donkey breeds in China,which are mainly raised in some small county villages in southernNingxia Hui Autonomous Region, and Gansu Province, northwest ofChina. According to the data from the Ningxia animal husbandry andveterinary bureau, it has a small population estimated at about 40 thou-sands, at the end of 2012. Xiji donkey is used for service and meat, itsskin is particularly important to use as the main ingredient of Ajiao, anessential blood-nourishing traditional Chinese medicine that has beencertified by FDA in the USA and other countries (Zhang and Zhao,2007). It is playing increasing important relationship to people's safetyand health with more international contacts and more informationabout TSEs in donkeys should be concerned.

Professional literatures about the prion protein and PRNP in Equusespecially donkeys are very limited compared to cattle, pigs, goats,sheep and deers. Within those research papers about Equus, most arefocused on the horses PRNP structure while a little about donkeys.There is no report about the prion protein and PRNP in Xiji donkey. Itis desirable to predict or estimate the susceptibilities or species barrierto prion infection between given species via comparison of theiramino acid homologies. In the present study, we cloned and sequencedthe PRNP of Xiji donkey and analyzed it to determine the variability ofthe PRNP gene. The polymorphisms of PRNP in Xiji donkeywill be usefulin the study of prion disease pathogenesis, resistance and cross speciestransmission.

2. Materials and methods

2.1. Sampling

Blood samples were collected by venipuncture into heparinizedvacuum tubes from 30 healthy, 2- to 3-year-old randomly selected Xijidonkeys from 4 unrelated flocks in the Xiji donkey breeding zone, locat-ed in Xiji County, Ningxia Hui Minority Autonomous Region, China. Allblood samples were frozen at−20 °C until use.

Fig. 1. PCR products of partial fragment of PRNP (M: 5000 bp DNA marker). Lanes 1–9:Partial PCR products of amplified PRNP of Xiji donkey.

2.2. DNA preparation and amplification

Genomic DNA of each blood sample was extracted using a Ge-nomic DNA Rapid Isolation Kit (BioDev-Tech, China) according to themanufacturer's instructions, and amplified by PCR. PCR primer pairswhich cover the entire open reading frame of the PRNP were designedusing Primer Premier 5.0 based on the Ovis aries PRNP sequence depositedin the GenBank (accession number HM803994). The forward and reverseprimers, 5′-TAGCTGATGCCACTGCTATG-3′ and 5′-AAGGTTGCCCCTATCCTACT-3′, respectively, were synthesized (Sangon Biotech, Shanghai,China) and stored at −20 °C. A 768 bp nucleotide of the donkey PRNPwas amplified in a 50 μL reaction mixture containing 15 pmol eachof the forward primer and the reverse primer, 50–100 ng of genomicDNA, 10 mM of each dNTP, 2.0 mMMgCl2, 3 U of Taq plus DNA Poly-merase (Takara, Japan) and 19 μL purified water. PCR reaction wasperformed in the Alpha Unit Block Assembly for DNA Engine Systems(Bio-Rad, USA) as follows: denaturation at 94 °C for 5 min, 35 ampli-fication cycles including denaturation at 94 °C for 30 s, annealing at58 °C for 40 s and extension at 72 °C for 40 s, followed by a final10-min extension at 72 °C. The amplified DNAs were confirmed byelectrophoresis on a 1% agarose gel and then purified using a DNAfragment gel purification and extraction kit (BioDev-Tech, China).

2.3. Cloning and sequencing

The purified DNAs were cloned into the pGEM-T easy vector(Promega, USA) according to the standard protocol. Next, the ligat-ed vectors were transformed into DH5α competent cells and theplasmid DNA was confirmed by enzyme digestion with KpnI andEcoRV. Purified plasmids were then sequenced on an ABI 3730XL auto-mated DNA sequencer (Applied Biosystems, USA) by a professionalgene sequencing company (Sunbiotech, Beijing, China). Each samplewas sequenced twice and all bases of both strands were sequenced.

2.4. DNA and protein sequence analysis

The sequence data were analyzed using DNASTAR (Version 7.0)and DNAMAN (Version 6.0), and aligned with the consensus se-quence of other known PRNP sequences of susceptible animals

Fig. 3.Amino acid alignment of 11mammalian prion proteins: Xiji donkey (Equus asinus, KC845010), horse (Equus caballus, NC_009165), sika deer (Cervus nippon, AY679695), sheep (Ovisaries, HM803994), dog (Canis lupus familiaris, NC_006606), camel (Camelus bactrianus, AY723285.1), cow (Bos taurus, AJ298878.1), human (Homo sapiens, NG_009087.1), mink (Mustelasp., S46825), rabbit (Oryctolagus cuniculus, NC_013672.1), and hamster (Mesocricetus auratus, M14054). Sites identical to the consensus sequence are denoted by dots, siteswith a deletionby short lines, and main polymorphisms in Xiji donkey were marked by boxes.

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(Xu et al., 2011a,b) and other equines in the GenBank database. Thealignment report for the amino acid, the phylogenetic trees, thenucleotide sequence identity and the amino acid sequences identi-ty were all constructed by Clustal V method of the Lasergene pack-age from DNASTAR (Version 7.0).

3. Results

The amplified PCR product of Xiji donkey PRNPwas about 768 bp insize (Fig. 1); it is consistent with the results of enzyme digestion (Fig. 2).

Sequencing of the amplified PCR fragments revealed a single openreading frame of 768 nucleotides encoding 256 amino acids. Partial

newly determined donkey PRNP sequences have been deposited in theGenBank database with accession numbers KC845010 to KC845023.

Complete nucleotide sequences of the open reading frames of the 30donkeys were obtained (not shown) with a high identity (99.87%). Thenucleotide substitutions were found at positions 153 (C → T), 237(T → C) and 354 (G → C) within Xiji donkey PRNP respectively,which all were synonymous substitutions and the PrP amino acidsequences showed no diversity; it has a 100% identity.

The deduced PrP sequences of the donkey PRNP and its specificregions such as N-terminal signal peptide, octapeptide and nonapeptiderepeat section,α-helix, β-sheet, GPI anchor position are shown in Fig. 3.Sequence analysis revealed that the amino acid sequence of the Xiji

Fig. 4. Nucleotide sequence identity of 11 mammalian prion proteins gene. 1 Xiji donkey (Equus asinus, KC845010), 2 horse (Equus caballus, NC_009165), 3 sika deer (Cervus nippon,AY679695), 4 sheep (Ovis aries, HM803994), 5 dog (Canis lupus familiaris, NC_006606), 6 camel (Camelus bactrianus, AY723285.1), 7 cow (Bos taurus, AJ298878.1), 8 human (Homo sapiens,NG_009087.1), 9 mink (Mustela sp., S46825), 10 rabbit (Oryctolagus cuniculus, NC_013672.1) and 11 hamster (Mesocricetus auratus, M14054).

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donkey PrP starts with the consensus sequence MVKSH, characteristicof most of the species PrP. A shorter variant, with the sequence MAN,is found in human, rabbit, and hamster (Murphy et al., 2001). Xiji don-key PrP had a N-terminal nonapeptides (PQGGGGWGQ), three repeatsof the octapeptides (PHGGGWGQ) and a C-terminal nonapeptides(PHGGGGWGQ) starting at position 95. Before the repeat region, similardeletions occur at positions 33 in donkey and horse.

When compared with other mammalian PrPs (Fig. 4), the PrPnucleotide sequence alignments of the Xiji donkey (KC845010)showed high identity with that of horse (KC845010, 99.7%), follow-ed by sika deer (AY679695, 88.5%), sheep (HM803994, 88.3%), dog(NC_006606, 87.6%), camel (AY723285.1, 87.4%), cow (AJ298878.1, 87.2%), human (NG_009087.1, 87.0%), mink (S46825, 86.3%), rab-bit (NC_013672.1, 82.9%) and hamster (M14054, 81.3%).

The phylogenetic tree of 11 mammalian prion proteins is shown inFig. 5, which characterized the genetic relationship of PrPs of humanand other animals. The initial pairwise matrix was used for multiplealignments. Xiji donkey and horse reside in the same group with smallvariations. Xiji donkey's amino acids showed high percent identity withthat of horse (99.6%), followed by sheep (93.0%), sika deer (91.8%), cow(90.2%), dog (89.5%), camel (88.7%),mink (88.6%), human (87.8%), rabbit(86.2%) and hamster (83.1%) (The table of sequence distances was notshown.). The phylogeny of PrP sometimes differs significantly fromwhat would be expected. The human PrP, hamster PrP and rabbit PrP

Fig. 5. Phylogenetic tree of 11 mammalian prion proteins. Cow (Bos taurus, AJ298878.1),dog (Canis lupus familiaris, NC_006606), sika deer (Cervus nippon, AY679695), hamster(Mesocricetus auratus,M14054), horse (Equus caballus, NC_009165), human (Homo sapiens,NG_009087.1), Xiji donkey (Equus asinus, KC845010), mink (Mustela sp., S46825), rabbit(Oryctolagus cuniculus, NC_013672.1), sheep (Ovis aries, HM803994), and camel (Camelusbactrianus, AY723285.1). The length of each pair of branches represents the distancebetween sequence pairs. The scale beneath the tree measures the distance betweensequences. Units indicate the number of substitution events.

are significantly more remote with Xiji donkey PrP. Also, the dog PrPand the mink PrP did not reveal an unexpected clustering.

In order to study the differences in the nucleotide sequence identity,the amino acid primary structure (not mentioned) and the homologydistance among Xiji donkey and other Equus species, similar alignmentsand analysis were taken.

Comparing with other Equus PrPs, the PrP nucleotide sequence andamino acid sequence alignments of the Xiji donkey showed high identi-ty (Figs. 6, 7) that of Dezhou donkey (99.6%, 100%), pony (99.4%, 99.0%),thoroughbred horse (99.3%, 99.6%), Equus przewalskii (99.2%, 99.5%),Equus kiang (99.1%, 98.8%), Zebra hartmannae (98.9%, 98.8%) andEquus asinus somalicus (98.9%, 99.2%), respectively.

4. Discussion

After alignments and analysis, although the deduced donkey prionprotein sequences contain several novel amino acid changes distributedin the variable regions across the entire sequence, the organization ofthe deduced Xiji donkey PrP sequence is similar to that of bovine,sheep, sika deer, dog, hamster, camel, human being, mink and otherspecies of Equus of the number of repeats or deletions of amino acid res-idue sites in the prion protein sequences. The PrP amino acid sequencesof 30 donkeys in our study showed 100% identity, although the resultmay be not exact because of the small number of sample.

Xiji donkey was unique in its PrP amino acids. There was an aspara-gine (codon AAT) at the position 169, while others were Ser (codonAGT) or Asp (codon GAT). Alignment also showed other polymorphismsat a number of residues, such as V6I, T17M, S97T, Y157H, E170Q, V183I,V205I, I217V, K222R, Y224S, E225Q, F227Y, I235V and I243V. Overall, thePrP of all species analyzed mainly varied in three regions: 1–20, 90–130and 170–246. Codons between 90 and 130 have a profound influence onthe transmissibility of prions from one species to another and 108–189are related to incubation (Schätzl et al., 1995). Codon 129 in human(131 in donkey and horse) is polymorphic for Met or Val in human PrPand modulates protease sensitivity of PrPSc (Parchi et al., 2000). Allcases to date of vCJD are homozygous for Met129, and heterozygosityat this site has been proposed to have a protective effect against sporadicand acquired prion diseases and in someof the inherited forms (Collinge,2001). We found the entire Xiji donkey PrP in present study was homo-zygous for Met131, since no prion cases have been reported in Equus(Fernández-Borges et al., 2013); therefore, the assumed protective effectof codon 129 heterozygosity is not a general feature. It's worthmention-ing that there are only two protein polymorphisms among Equus speciesmentioned in this study, such as N169S and V243I, less than sheep andcamels (Wang et al., 2008; Xu et al., 2011a,b; Yang et al., 2005; Yin and

Fig. 6.Nucleotide sequences identity of 8 Equus animals. 1 Equus przewalskii (EU887252), 2 Equus kiang (EF165076), 3Dezhou donkey (EF127815.1), 4 thoroughbredhorse (NC_009165), 5Equus asinus somalicus (EF165078), 6 Xiji donkey (KC845010), 7 Zebra hartmannae (EF165073), and 8 pony (AB195279.1).

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Zhao, 2006). Nucleotide sequence identity of 8 Equals animals showedthe Equus PRNP has less mutation but more hereditary stability thanmany species during mammalian evolution.

The analyzed amino acid sequences of PrP in Xiji donkeys in thisstudy contain three repeats of the octapeptide PHGGGWGQ, whichare enclosed by an N-terminal nonapeptide PQGGGGWGQ and a C-terminal nonapeptide PHGGGGWGQ in the region encompassingresidues 53 to 95. This feature is similar to other mammalian animals(horse, NC_009165, mink, S46825, sheep, HM803994, sika deer,AY679695) (Benestad et al., 2008; Goldmann, 2008). The species ofEquus in this study have the same repeats. Inherited prion diseasessuch as CJD are associated with the insertion of additional octapep-tide repeats in the N-terminal region of PrP (Goldfarb et al., 1991;Ponz et al., 2006). However, an association of octapeptide repeatswith prion disease susceptibility has not yet been verified in animalspecies.

For ovine PrP genetics the main polymorphic PRNP segmentsassociated with natural scrapie are codons 136 (T, A, V), 154 (R, H)and 171 (R, Q, H, K) (Goldmann et al., 1994; Gombojav et al., 2003;Tranulis, 2002; Tsunoda et al., 2010). The PrP allele ARR is associatedwith resistance to scrapie (Belt et al., 1995). In contrast, allele ARQ isassociated with high susceptibility to scrapie (Goldmann et al.,1990). The PrP allele VRQ is described as associated with a highersusceptibility to scrapie. Little is known about the association ofAHQ and ARH with susceptibility to scrapie (Bossers et al., 2000;Sweeney and Hanrahan, 2008; Tongue et al., 2004; Vaccari et al.,2001; Westaway et al., 1994). However, with the discovery of atyp-ical forms of scrapie more recently, it has become apparent that the

Fig. 7.Amino acid sequence identity of 8 Equus animals. 1 Equus przewalskii (EU887252), 2 EquuEquus asinus somalicus (EF165078), 6 Xiji donkey (KC845010), 7 Zebra hartmannae (EF165073

link between PrP genetics and disease susceptibility will have to bere-evaluated (Bossers et al., 1999). The genotypes of donkey andother Equus in our study are distinct from sheep. Human, zebra,donkey and horse usually encode glutamate at position 171 whencompared with sheep (Premzl and Gamulin, 2009). At the presenttime, there is no evidence that donkeys are susceptible to anyprion diseases, and transmission to them from infected cows, deer,or other species has not been noted, so we hypothesize that thePrP allele ARE in Equus is one of the determinants associated withresistance to scrapie or BSE.

Prion diseases have prolonged incubation periods and coding poly-morphisms in the prion protein gene are known to affect incubationtimes and susceptibility in humans, mice, and sheep (Moreno et al.,2008). However, not all individuals with similar PRNP alleles developthe disease, and if they do, they can present very different incubationperiods. In fact, in addition to the PRNP locus, other environmentaland genetic factors may act on the animal susceptibility. PrP aminoacid differences are not the sole genetic influence (Will et al., 2000),and other factors including additional genetic loci may contribute tothe observed variation. Some of these loci have been detected on chro-mosomes 2, 4, 6, 7, 8, and 11 (Moreno et al., 2003) in mice. In cattle,three marker loci (on chromosomes 5, 10, and 20) were associatedwith BSE (Hernandez-Sanchez et al., 2002). The sequence conservationbetween the donkey and susceptible animals' genome, such as sheep,deer, cow and human, suggests the possibility of prion disease suscepti-bility, but in fact, rabbits, dogs, and horses are the only mammalianspecies reported to be resistant to infection from prion diseases. Themechanism of resistant to prion diseases is not exactly clear. Whether

s kiang (EF165076), 3 Dezhou donkey (EF127815.1), 4 thoroughbred horse (NC_009165), 5) and 8 pony (AB195279.1).

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the QTL existed or not, it is still an enigmawhen the donkey was suffer-ing from prion diseases.

In conclusion, the analysis of PRNP sequence and deduced aminoacid in Xiji donkeys revealed that Xiji donkeys' PRNP shares homologywith that in other species of Equus. The polymorphisms of PRNP in Xijidonkeys were found mainly at codons 6, 17, 97, 157, 169, 170, 183,205, 217, 222, 224, 225, 227, 235 and 243. The present work providedvaluable data on the sequence of PRNP for future research on resistanceof Equus to TSE. Our findings supported the previous observations thatthe PRNP is highly conserved across species. Data are lacking on the cor-relation of the QTL with the resistant of the prion disease in Xiji donkeyas well as in Equus.

Conflict of interest

Deming Zhao and other co-authors have no conflict of interest.

Acknowledgment

This work was supported by the Natural Science Foundation of China(ProjectNo. 31001048,No. 31172293 andNo. 31272532), the SpecializedResearch Fund for the Doctoral Program of Higher Education (Project No.20100008120002), the Special Scientific Fund forNonprofit Public Indus-try (Agriculture), China (Project No. 200903027), the Beijing ScienceFoundation of China (Project No. 6101002), the Foundation of ChineseMinistry of Science and Technology (Project No. 2011BAI15B01), andthe Program for Cheung Kong Scholars and Innovative Research Teamin University of China (No. IRT0866).

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