Phylogeny and Molecular Characterisation of PRNP in Red-Tailed Phascogale (Phascogale calura)

<b>Background/Objectives</b>: The normal cellular prion protein (PrP^C) is a cell-surface glycoprotein, mainly localised in neurons of the central nervous system (CNS). The human <i>PRNP</i> gene encodes 253 amino acid residues of precursor PrP^C. Several studies that investigated the role of <i>PRNP</i> and PrP^C in placental mammals, such as humans and mice, failed to reveal its exact function. <b>Methods</b>: In this study, we sequenced and characterised the <i>PRNP</i> gene and PrP^C of the marsupial, <i>P. calura</i>, as a strategy to gain molecular insights into its structure and physicochemical properties. Placentals are separated from marsupials by approximately 125 million years of independent evolution. <b>Results</b>: Standard Western blotting analysis of PrP^C phascogale displayed the typical un-, mono-, and di-glycosylated bands recognized in placentals. Furthermore, we showed that phascogale <i>PRNP</i> gene has two exons, similar to all the marsupials and placentals of the <i>PRNP</i> genes studied. Of note, the phascogale <i>PRNP</i> gene contained distinctive repeats in the PrP^C tail region comparable to the closely related Tasmanian devil (<i>Sarcophilus harrisii</i>) and more distantly related to the grey short-tailed opossum (<i>Monodelphis domestica</i>), common wombat (<i>Vombatus ursinus</i>), and Tammar wallaby (<i>Macropus eugenii</i>); however, its specific composition and numbers were different from placentals. Of importance, comparisons of the phascogale's PrP^C physicochemical properties with other monotremes, marsupials, and placentals confirmed the Monotremata-Marsupialia-Placentalia evolutionary distance. We found that the protein instability index, a method used to predict the stability of a protein in vivo (Stable: <40; Instable >40), showed that the PrP^C of all marsupials tested, including phascogale, were highly stable compared with the birds, reptiles, amphibians, and fish that were shown to be highly unstable. However, the instability index predicted that all placental species, including human (<i>Homo sapiens</i>), mouse (<i>Mus musculus</i>), bank vole (<i>Myodes glareolus</i>), rhinoceros (<i>Rhinocerotidae</i>), dog (<i>Canis lupus familiaris</i>), flying fox (<i>Pteropus vampyrus</i>), whale (<i>Physeter catodon</i>), cattle (<i>Bos taurus</i>), and sheep (<i>Ovis aries</i>), were either slightly unstable or nearly unstable. Further, our analysis revealed that despite their predicted high PrP^C stability, <i>P. calura</i> exhibited substantial N-terminal disorder (53.76%), while species with highly unstable PrP^Cs based on their instability index, such as Danio rerio, Oryzias latipes, and Astyanax mexicanus, displayed even higher levels of N-terminal disorder (up to 75.84%). These findings highlight a discrepancy between overall predicted stability and N-terminal disorder, suggesting a potential compensatory role of disorder in modulating prion protein stability and function. <b>Conclusions</b>: These results suggest that the high stability of marsupial prion proteins indicates a vital role in maintaining protein homeostasis; however more work is warranted to further depict the exact function.