Data_Sheet_1_First report of a novel polymorphism and genetic characteristics of the leporine prion protein (PRNP) gene.docx

Transmissible spongiform encephalopathies (TSEs) have been reported in a broad spectrum of hosts. The genetic polymorphisms and characteristics of the prion protein (PRNP) gene have a vital impact on the development of TSEs. Notably, natural TSE infection cases have never been reported in rabbits, and genetic variations of the leporine PRNP gene have not been investigated to date. To identify leporine PRNP gene polymorphism, we performed amplicon sequencing in 203 rabbits. We report a novel single nucleotide polymorphism on the leporine PRNP gene. In addition, we performed a comparative analysis of amino acid sequences of prion protein (PrP) across several hosts using ClustalW2. Furthermore, we evaluated the effect of changes of unique leporine PrP amino acids with those conserved among various species using Swiss-Pdb Viewer. Interestingly, we found seven unique leporine amino acids, and the change of unique leporine amino acids with those conserved among other species, including S175N, Q221K, Q221R, A226Y, A230G, and A230S, was predicted to reduce hydrogen bonds in leporine PrP.</p

Image_1_First report of structural characteristics and polymorphisms of the prion protein gene in raccoon dogs: The possibility of prion disease-resistance.tif

Prion diseases are fatal degenerative encephalopathies caused by misfolded prion protein (PrPSc) converted from normal prion protein (PrPC). Previous studies have reported that genetic polymorphisms of the prion protein gene (PRNP) play a critical role in susceptibility to prion diseases. In addition, prion disease-resistant animals showed unique structural features of prion protein (PrP) related to species-specific amino acids. However, investigations of genetic polymorphisms of the PRNP gene and structural characteristics of PrP have not been performed in raccoon dogs thus far. We investigated genetic polymorphisms of PRNP in 87 raccoon dogs using amplicon sequencing and analyzed the genotype, allele, haplotype frequencies, and linkage disequilibrium (LD) using Haploview version 4.2. In addition, we performed phylogenetic analysis and multiple sequence alignment (MSA) using MEGA X version 10.1.8 and Clustal X version 2.1, respectively. We estimated the impact of raccoon dog and Canidae family-specific amino acids using PolyPhen-2, PROVEAN, and AMYCO. Furthermore, we analyzed the effect of raccoon dog and Canidae family-specific amino acids using the AlphaFold2 and Swiss-PdbViewer programs. We found 4 novel single nucleotide polymorphisms (SNPs) of the raccoon dog PRNP gene. In addition, the raccoon dog PrP showed 99.61% identity and the closest genetic distance to dog PrP. Among the substitutions of Canidae-specific amino acids with interspecific amino acids, D163N showed increased amyloidogenic propensity, and R181H showed alterations of hydrogen bonds. Furthermore, electrostatic potentials were changed according to the substitutions of D163N and R181H. By comparing PrP between raccoon dogs and raccoons, R168K and K224R were found to be related to changes in hydrogen bonds, and K224R altered the electrostatic potential of raccoon dog PrP. In the present study, we first reported 4 novel synonymous SNPs of the raccoon dog PRNP gene. We also identified that the PrP of raccoon dog has high homology (99.61%) with PrP of dog, which is a prion-resistant animal. In addition, raccoon dog PrP-specific amino acids are related to low amyloid propensity and inherent characteristics of 3D structure of raccoon dog PrP compared to the PrP of prion-susceptible species.</p

Data_Sheet_1_First report of structural characteristics and polymorphisms of the prion protein gene in raccoon dogs: The possibility of prion disease-resistance.docx

Prion diseases are fatal degenerative encephalopathies caused by misfolded prion protein (PrPSc) converted from normal prion protein (PrPC). Previous studies have reported that genetic polymorphisms of the prion protein gene (PRNP) play a critical role in susceptibility to prion diseases. In addition, prion disease-resistant animals showed unique structural features of prion protein (PrP) related to species-specific amino acids. However, investigations of genetic polymorphisms of the PRNP gene and structural characteristics of PrP have not been performed in raccoon dogs thus far. We investigated genetic polymorphisms of PRNP in 87 raccoon dogs using amplicon sequencing and analyzed the genotype, allele, haplotype frequencies, and linkage disequilibrium (LD) using Haploview version 4.2. In addition, we performed phylogenetic analysis and multiple sequence alignment (MSA) using MEGA X version 10.1.8 and Clustal X version 2.1, respectively. We estimated the impact of raccoon dog and Canidae family-specific amino acids using PolyPhen-2, PROVEAN, and AMYCO. Furthermore, we analyzed the effect of raccoon dog and Canidae family-specific amino acids using the AlphaFold2 and Swiss-PdbViewer programs. We found 4 novel single nucleotide polymorphisms (SNPs) of the raccoon dog PRNP gene. In addition, the raccoon dog PrP showed 99.61% identity and the closest genetic distance to dog PrP. Among the substitutions of Canidae-specific amino acids with interspecific amino acids, D163N showed increased amyloidogenic propensity, and R181H showed alterations of hydrogen bonds. Furthermore, electrostatic potentials were changed according to the substitutions of D163N and R181H. By comparing PrP between raccoon dogs and raccoons, R168K and K224R were found to be related to changes in hydrogen bonds, and K224R altered the electrostatic potential of raccoon dog PrP. In the present study, we first reported 4 novel synonymous SNPs of the raccoon dog PRNP gene. We also identified that the PrP of raccoon dog has high homology (99.61%) with PrP of dog, which is a prion-resistant animal. In addition, raccoon dog PrP-specific amino acids are related to low amyloid propensity and inherent characteristics of 3D structure of raccoon dog PrP compared to the PrP of prion-susceptible species.</p