Page 9 - L. Genetics and genomics
P. 9
The first report of genetic and structural diversities in the SPRN gene in the horse,
an animal resistant to prion disease
Sae-Young Won, Byung-Hoon Jeong
Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Republic of Korea
Department of Bioactive Material Sciences, Jeonbuk National University, Jeonju 54896, Republic of Korea
ABSTRACT INTRODUCTION
Prion diseases are fatal neurodegenerative diseases and are Previous studies have reported that the stability of prion protein (PrP)
Sc
characterized by the accumulation of abnormal prion protein (PrP ) in and species-specific amino acids of PrP are related to prion disease
the brain. During the outbreak of the bovine spongiform susceptibility. The horse has a distinctive amino acid, S167, that
encephalopathy (BSE) epidemic in the United Kingdom, prion contributes to the stability of horse PrP and non-toxicity in the
diseases in several species were reported; however, horse prion transgenic Drosophila model. However, spontaneous PrP aggregation
disease has not been reported thus far. In previous studies, the was observed in the transgenic mouse model expressing mouse PrP
shadow of prion protein (Sho) has contributed to an acceleration of with a horse-specific amino acid substitution of D167S. This result
C
Sc
conversion from normal prion protein (PrP ) to PrP , and the shadow indicates that the protective effect of horse-specific amino acid, 167S is
of prion protein gene (SPRN) polymorphisms have been significantly elusive and suggests there is another factor related to resistance of
associated with the susceptibility of prion diseases. We investigated
the genotype, allele and haplotype frequencies of the SPRN gene prion disease in horse besides the properties of the PrP sequence
using direct sequencing. In addition, we analyzed linkage encoded in the prion protein gene (PRNP). The SPRN gene encodes
disequilibrium (LD) and haplotypes among polymorphisms. We also shadow of prion protein (Sho) and an N-glycosylation site and a
investigated LD between PRNP and SPRN single nucleotide glycosylphosphatidylinositol (GPI) anchor like PrP. The hydrophobic
polymorphisms (SNPs). To perform Sho protein modeling, we utilized domain of the SPRN gene is very similar to that of the PRNP gene. The
SWISS-MODEL and Swiss-PdbViewer programs. We found a total of Sho protein is mainly expressed in the brain and contributes to the
Sc
four polymorphisms in the equine SPRN gene; however, we did not conversion of PrP C to PrP . In a previous study, in/del (AAAG)
observe an in/del polymorphism, which is correlated with the polymorphisms in the hydrophobic region of bovine Sho protein were
susceptibility of prion disease in prion disease-susceptible animals. found in atypical BSE-infected cattle. In goats, 602_606insCTCCC of the
The SPRN SNPs showed weak LD value with PRNP SNP. In addition, 3’ untranslated region (UTR) was significantly different between healthy
we found 12 horse-specific amino acids of Sho protein that can and scrapie-infected cases. This evidence suggests that Sho protein
induce significantly distributional differences in the secondary may affect the susceptibility to prion disease. In this study, we
structure and hydrogen bonds between the horse and several prion investigated polymorphisms of the equine SPRN gene and analyzed LD
disease-susceptible species. To the best of our knowledge, this is the and haplotypes among polymorphisms. We also investigated LD
first report regarding the genetic and structural characteristics of the between SPRN and PRNP SNPs. Finally, we performed structural
equine SPRN gene. modeling of equine Sho protein.
RESULTS
Fig. 1. Gene map and polymorphisms of the equine shadow of prion protein gene Fig. 2. Comparisons of amino acid sequences of the Sho protein among several species. Fig. 3. Homology-based modeling of Sho protein among several species. Upper
(SPRN) in chromosome 1. (a) The open reading frame (ORF) in exon 2 is Comparison of amino acid sequences of sho protein in homo sapiens (NP_001012526.2), panels indicate secondary structure predicted by the Swiss-PdbViewer.
marked with a black block, and the white blocks represent the 5’ and 3’ Bos Taurus (AAY83885.1), Ovis aries (NP_001156033.1), Capra hircus (AGU17009.1), Middle panels indicate three dimensional structures of Sho protein analyzed
untranslated regions (UTRs). The edged horizontal bar indicates the Cervus elaphus (ACG274724.1) and Equus caballus (XP_023492126.1). Amino acids were by the SWISS-MODEL program. Lower panels indicate the ball-and-stick
sequenced regions. The four novel polymorphisms found in this study are aligned by the ClustalW2 program. Colors indicate the chemical properties of amino model and hydrogen bonds of the Sho protein analyzed by Swiss-PdbViewer.
indicated with vertical lines above the gene. (b) Electropherograms of the 4 acids as follows: blue: acidic, red: nonpolar aliphatic, magenta: basic, and green: (a), human; (b), cattle; (c), sheep; (d), goat; (e), red deer; (f), dog; (g), horse.
novel polymorphisms that were found: c.87G>C, c.502C>T, c.696C>T and hydroxyl, sulfhydryl, amine and glycine. Asterisks indicate differences in amino acids The red dotted lines indicate α-helices. Green dotted lines indicate hydrogen
c.728C>T. between the horse and other species. bonds. Underlines indicate binding site of PrP. Bold text indicates horse-
specific amino acids. C: coil; H: apha-helix.
Table 1. Genotype and allele frequencies of shadow of prion protein gene (SPRN) Table 2. Linkage Disequilibrium (LD) among four polymorphisms of SPRN in Table 5 Linkage Disequilibrium (LD) between single nucleotide polymorphisms
2
polymorphisms in Thoroughbred horses Thoroughbred horses (SNPs) of SPRN and PRNP gene with D’ and r value in Thoroughbred horses
|D’ |
Genotype frequency, n (%) Allele frequency, n (%) HWE |D’ |
r 2 c.87G>C c.502C>T c.696C>T c.728C>T
GG GC CC G C
c.87G>C 0 PRNP SPRN SPRN SPRN SPRN
191 (98.5) 0 (0) 3 (1.5) 382 (98.5) 6 (1.5) c.87G>C - 1.0 1.0 1.0 r 2
c.525G>C c.87G>C c.502C>T c.696C>T c.728C>T
CC CT TT C T c.502C>T 0.21 - 0.919 0.238
c.502C>T 0.0223
170 (87.6) 21 (10.9) 3 (1.5) 361 (93.1) 27 (6.9)
c.696C>T 0.194 0.782 - 0.138 PRNP
CC CT TT C T - 0.102 0.314 0.195 0.037
c.696C>T 0.4665 c.525G>C
168 (86.6) 23 (11.9) 3 (1.5) 359 (92.5) 29 (7.5) c.728C>T 0.036 0.01 0.004 -
CC CT TT C T SPRN
c.728C>T 0 Table 3. Haplotype frequency of four SPRN polymorphisms in Thoroughbred horses as 0.001 - 1.0 1.0 1.0
108 (55.7) 54 (27.8) 32 (16.5) 270 (69.6) 118 (30.4) c.87G>C
predicted by Haploview
Table 4 Detailed information of Sho protein in several species analyzed by SWISS-MODEL Haplotype Thoroughbred (n=388) SPRN 0.022 0.247 - 0.905 0.289
and Swiss-PdbViewer programs c.502C>T
GCCC 251 (0.647)
GCCT 105 (0.271) SPRN
Common Scientific Location of α- Number of GTTC 14 (0.036) 0.009 0.226 0.748 - 0.167
GenBank ID Location of hydrogen bonds hydrogen c.696C>T
name name helix structure bonds CTTT 6 (0.016)
GTTT 5 (0.013)
SPRN
GCTC 4 (0.010) 0.001 0.042 0.014 0.005 -
c.728C>T
Human Homo sapiens NP_001012526.2 Not found A73-A75, A85-S87 2 Others 3 (0.007)
Cattle Bos taurus AAY83885.1 Not found A65-A67, A77-S79 2 CONCLUSIONS
Sheep Ovis aries NP_001156033.1 Not found G66-A69, G66-A70, A79-S81 3 ⚫ We found a total of 4 SNPs in the SPRN gene: 1 synonymous SNP and 3 SNPs in the 3’ UTR. In addition, SPRN SNPs showed weak LD with PRNP SNP.
⚫ Next, we found a total of 12 horse-specific amino acids via protein alignment.
⚫ Finally, significant structural differences, such as secondary structures and hydrogen bonds, were identified by comparison analysis of the 3D structure
Goat Capra hircus AGU17009.1 Not found A65-A67, A77-S79 2
of Sho protein among several species. To the best of our knowledge, this was the first study regarding the SPRN gene in horses.
Red deer Cervus elaphus ACG274724.1 Not found A65-A67, A77-S79 2
REFERENCE
L62-V64, L62-A65, V64-A67,
Canis lupus Codons 64-69, ⚫ Peletto, S.; Bertolini, S.; Maniaci, M. G.; Colussi, S.; Modesto, P.; Biolatti, C.; Bertuzzi, S.; Caramelli, M.; Maurella, C.; Acutis, P. L. Association of an indel
Dog XP_022267623.1 V64-G68, A67-A70, A70-G72, 8
familiaris 75-80
G74-G78, A75-G78 polymorphism in the 3'UTR of the caprine SPRN gene with scrapie positivity in the central nervous system. J. Gen. Virol. 2012, 93, 1620-1623.
⚫ Mesquita, P.; Garcia, V.; Marques, M. R.; Santos Silva, F.; Oliveira Sousa, M. C.; Carolino, I.; Pimenta, J.; Fontes, C. M.; Horta, A. E.; Prates, J. A.; Pereira,
R. M. The prion-related protein (testis-specific) gene (PRNT) is highly polymorphic in Portuguese sheep. Anim. Genet. 2016, 47, 128-132.
Codons 64-68, L62-V64, L62-A65, V64-A67, ⚫ Wilson, I. A.; Haft, D. H.; Getzoff, E. D.; Tainer, J. A.; Lerner, R. A.; Brenner, S. Identical short peptide sequences in unrelated proteins can have different
Horse Equus caballus XP_023492126.1 6
72, 77-80 C64-G68, A67-A70, G75-G78
conformations: a testing ground for theories of immune recognition. Proc. Natl. Acad. Sci. U S A. 1985, 82, 5255-5259.
