Prion diseases are fatal, infectious neurodegenerative disorders caused by the misfolding of a benign prion protein (PrP^C) into an abnormal prion protein (PrP^Sc) (1). The exact process of converting PrP^C to PrP^Sc remains unclear, but several factors that play crucial roles in the conversion process have been identified (1, 2). The ability to infect some species and not others is a remarkable prion characteristic (3). A wide variety of species, including various members of mammalian families such as cattle, goats, deer, cats and other primate families, are susceptible to prion diseases (4–8). However, horses, dogs, and chickens are resistant to prion diseases (9–11). Previous studies have also indicated that genetic characteristics of the SPRN gene are significantly different between prion disease-susceptible species (cattle, goats) (12, 13) and -resistant species (horses, dogs, and chickens) (9, 10, 14). Therefore, the investigation of the prion disease-related genes in prion-resistant species has been of particular interest as it provides insights into the determinants of susceptibility.

Among the prion protein family, there are members such as shadow of prion protein (Sho) and PrP^C, which are mostly expressed in brain tissue (15, 16). These proteins have been reported to affect embryonic and mammary development (17). Previous studies suggest that the Sho protein, encoded by the shadow of prion protein gene (SPRN), interacts with PrP^C (18). This interaction speeds up the conversion of PrP^C to PrP^Sc (19). Since these two proteins interact and share similar characteristics, including protein expression profile and function (20, 21), it is essential to investigate the genetic characteristics of the SPRN gene to interpret the pathomechanism of prion diseases.

In the phylogenetic tree of the order Galliformes, chickens and pheasants have a close evolutionary relationship (22–24). Pheasants are members the genus Phasianus within the bird family (25). To date, prion infection has not been reported in birds and some other vertebrate species (26, 27). In our previous study, we reported 34 polymorphisms in the pheasant prion protein gene (PRNP), including 8 non-synonymous single nucleotide polymorphisms (SNPs) and 6 insertion/deletion polymorphisms (28). Furthermore, it has been reported that the pheasant PrP^C has a 3D structure similar to the chicken PrP^C; thus, pheasant PrP^C is predicted to have relatively prion-resistant feature (28). Genetic polymorphisms in prion-related genes contribute significantly to interindividual variation, so they have been investigated as useful biomarkers in medicine, as well as in the study of pathology, pharmacology, epidemiology, and clinical immunology (29). In addition, polymorphisms of the SPRN gene in prion disease-susceptible species play a crucial role in determining their susceptibility to prion diseases (12, 30, 31). The SPRN polymorphisms, which contribute to expression level or protein function, show strong associations with the pathogenesis of prion diseases in different species (32, 33). However, the SPRN gene sequence, genetic polymorphisms, and structural features of Sho in pheasants have not yet been investigated.

In this study, we amplified the SPRN gene sequence by polymerase chain reaction (PCR) and performed multiple sequence alignment of pheasant SPRN gene and Sho protein sequences with those of prion disease-susceptible and -resistant species. In addition, we performed amplicon sequencing of SPRN to identify genetic polymorphisms and investigated the genotype, allele and haplotype frequencies as well as linkage disequilibrium (LD) of SNPs of pheasant SPRN gene. Furthermore, we evaluated the change of Sho by non-synonymous SNPs in the pheasant SPRN gene using in silico tools. Lastly, we predicted the secondary and 3D structure of Sho in several prion disease-susceptible and -resistant species using AlphaFold2.

Considering that chickens are known to be resistant to prion diseases, it becomes crucial to investigate the genes associated with prion disease in pheasants. In the present study, we are the first to report pheasant SPRN gene sequences (Figure 1A). Chicken and pheasant have one nucleotide difference, but they share the same amino acid sequence. To identify the specific features of the pheasant Sho protein, we conducted a comparison of the amino acid sequences of the Sho from a variety of species and found that major components of Sho protein including prion interaction region and NXT glycosylation motif were conserved in pheasant like other prion-related species (Figure 1B).

In addition, we found 14 novel SNPs, including 7 non-synonymous and 4 synonymous SNPs, using amplicon sequencing (Figure 2). Previous studies have reported that prion disease-susceptible species, including cattle, goats, and sheep, have highly polymorphic SPRN genes (12, 13, 38, 39). In contrast, a prion disease-resistant species, horses and chickens have one polymorphism of the SPRN gene (9, 14, 40). Dogs, a prion disease-resistant animal, have one insertion/deletion polymorphism in SPRN gene (10). Interestingly, insertion/deletion polymorphisms have not been identified in the ORF of the pheasant SPRN gene. Although, phylogenetic analysis revealed that pheasants are closely related to chickens, a prion-resistant species (22–24), our results showed that pheasants have many polymorphisms. Further research is necessary to explore the SPRN gene in other species and to investigate the relationship between the number of SPRN gene polymorphisms and susceptibility to prion diseases. This is important because pathogenic mutations have a higher likelihood of occurring in SPRN genes that exhibit high polymorphism, as reported previously (10). Moreover, SPRN SNPs with low frequency have been discovered. Although the pheasant Sho sequence was identical to chicken Sho, the presence of a significant number of SNPs, albeit at a low frequency, can lead to structural abnormalities in the prion protein that encodes the prion-related protein. Therefore, further study is needed to comprehend how these characteristics could contribute to the progression of prion diseases and determine their impact on the prion pathomechanism.

To evaluate the impact of amino acid substitutions, we employed in silico programs (Table 5). The MutPred2 scores for all 7 non-synonymous SNPs were below 0.5, indicating no pathogenicity. We used MUpro to predict the effects of the SNPs on protein stability and found that a decrease in protein stability was associated with the 7 non-synonymous SNPs. Prion diseases are characterized by protein misfolding, and previous research has identified two important residues in PrP that promote stability in dogs and horses, animals known to be resistant to prion diseases (41). Those residues induce local changes that decrease the β-sheet content and enhance the structural stability of horse and dog PrP^C, allowing the changes to spread to nearby regions (41). Further research is needed to understand how the decreased stability of the protein, in addition to its association with accelerated prion formation, can be interpreted in relation to the intrinsic functions of the Sho protein, such as embryo development. In addition, we employed AMYCO to estimate the impact of the mutations on the aggregation propensity of the PrLDs in prion-like proteins. Protein aggregation is a characteristic observed in various neurodegenerative disorders (42). The pheasant Sho sequences, when substituted with 7 non-synonymous SNPs, exhibited a score of 0.0, suggesting that the 7 non-synonymous SNPs do not have any amyloid-prone features.

We analyzed the 3D structures of Sho among various species predicted by AlphaFold2. Interestingly, with the exception of chicken and pheasant Sho, all species displayed a similar 3D structure, and none of the Sho proteins from any species exhibited a β-sheet (Figure 4). Additionally, we analyzed pheasant Sho according to the haplotype of pheasant Sho (Figure 5). By comparing the 3D structures of the pheasant Sho with both the wild-type allele and an HT2 variant, we observed a decrease in the number of α-helices in the pheasant Sho with HT2. The presence of HT1 and HT3 did not seem to impact the structure of the Sho protein. Since Sho plays a vital role in embryonic development, gaining insights into its structure can greatly contribute to comprehending its functional mechanisms (17, 43). Research is needed to investigate how structural changes in the haplotype affect the general function of Sho and its ability to interact with PrP^C.

In this study, we first reported pheasant SPRN gene sequences and found 14 novel SNPs of the pheasant SPRN gene, including 7 non-synonymous and 4 synonymous in a total of 135 pheasants. In silico analysis, it was predicted that 7 non-synonymous SNPs induce decrease of protein stability. In addition, the secondary and tertiary structures of the wild-type pheasant Sho are identical to those of the chicken Sho. To the best of our knowledge, this is the first study on genetic polymorphisms of the pheasant SPRN gene.

The data presented in the study are deposited in the DRYAD repository https://datadryad.org/stash/share/9SErCsr_yxn1mmXQBsndJYntnvhR-sCJo-sjjnyumeE.

The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) at Jeonbuk National University (JBNU 2020-209). The study was conducted in accordance with the local legislation and institutional requirements.

D-IC: Conceptualization, Visualization, Writing – original draft. MZ: Formal analysis, Writing – review & editing. Y-CK: Formal analysis, Writing – review & editing. B-HJ: Conceptualization, Formal analysis, Writing – review & editing.