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, 26 (10), 1312-1322

Pangolin Genomes and the Evolution of Mammalian Scales and Immunity

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Pangolin Genomes and the Evolution of Mammalian Scales and Immunity

Siew Woh Choo et al. Genome Res.

Abstract

Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.

Figures

Figure 1.
Figure 1.
Case studies of pseudogenized genes in pangolins. (A) Three tooth development-related genes were pseudogenized and may be related to the lack of teeth in pangolins. There was a frameshift deletion in positions c1311–c1324, a single base pair deletion in c1476, and an insertion of AGAT at position c1621, resulting in another premature stop codon in the AMBN gene. The AMELX gene contains a large deletion at position c438–c497. (B) Two genes were pseudogenized and may be related to the poor vision of pangolins. Blue = insertion, green = deletion, pink = stop codon.
Figure 2.
Figure 2.
Multiple sequence alignment of all mammalian IFNE genes. Seventy-three mammalian species have available IFNE sequences, used for alignment. (A) Nucleotide sequence alignment of IFNE genes across different mammalian species. Blue = insertion, green = deletion, pink = stop codon. Protein sequence alignment of IFNE genes across different mammalian species is also shown. We identified an insertion (blue) in IFNE starting from nucleotide position 195 but not in other mammalian species, indicating that this insertion is specific to pangolins and possibly a marker to differentiate pangolins and other mammalian species. A premature stop codon or frameshift (orange) in the IFNE gene was consistently detected in both pangolin genomes. These mutations were validated by Sanger sequencing in eight Malayan pangolins. Protein sequences highlighted in orange are the frameshift mutations and premature stop codon. (B) Comparison between pangolin and human (reference) IFNE protein sequences. Pangolins have a short putative protein sequence because of a premature stop codon. Predicted functional domains and signatures in the human IFNE gene are represented by colored boxes. Yellow and pink boxes represent the predicted binding residues to IFNAR2 and IFNAR1, respectively, and which are bounded by interferons, which we obtained from a previosuly published paper (Fung et al. 2013). IFabd (SM00076) is a conserved functional domain in known interferons. The main conserved structural feature of interferons is a disulfide bond. INTERFERONAB (PR00266) is a three-element fingerprint that provides a signature for alpha, beta, and omega interferons. The elements 1 and 3 contain Cys residues involved in disulfide bond formation.
Figure 3.
Figure 3.
Comparative analysis between pangolins and mammals. (A) Venn diagram showing the unique and shared gene families among pangolins and their closest relatives (cat, dog, and giant panda). (B) Heat map showing the expression level of pangolin-specific genes across different pangolin organs, represented by FPKM values. Any FPKM values >5 were set to 5 in the heat map for visualization purposes. Only genes expressed (FPKM ≥ 0.3) in at least one organ were shown. (C) GO enrichment analysis of 1152 pangolin-specific genes. Significantly enriched GO terms are shown for the categories of cellular compartment (blue), molecular function (yellow), and biological process (light orange). (D) Phylogenetic tree and gene family expansion and contraction. Expanded gene families are indicated in blue, whereas contracted gene families are indicated in red. The proportion of expanded and contracted gene families is also shown in pie charts. (E) Phylogenetic tree showing significant contraction of the interferon gene family in pangolins.
Figure 4.
Figure 4.
Evolution of immunity-related pathways and scale-related genes in the pangolin ancestor. Genes under positive selection in the pangolin lineage in hematopoietic cell lineage (A) and cytosolic DNA-sensing pathway (B) are highlighted in red. (C) Several critical pangolin-specific mutations were identified in the functionally relevant Fibronection Type III signatures (INTERPRO ID = IPR003961) of CSF3R, the Integrin alpha-2 signatures (IPR013649) of ITGAM, and the Stimulator of Interferon Genes Protein region (INTERPRO ID = IPR029158) of TMEM173. For the CSF3R, ITGAM, and TMEM173, P-values of the detection of positive selection were 1.58 × 10−2, 3.75 × 10−2, and 2.92 × 10−5, respectively. (D) Several critical pangolin-specific amino acid changes were detected in hair-/scale-related keratin proteins, KRT36 and KRT75, which are located in functionally relevant regions that may affect protein functions. For the KRT36 and KRT75, P-values of the detection of positive selection were 2.24 × 10−2 and 1.33 × 10−5, respectively. We also examined whether African species (M. tricuspis, M. tetradactyla, and M. temminckii) have the critical pangolin-specific amino acid changes that we observed in the Asian pangolins. Circles at the bottom indicate our preliminary results: Red circle = all African species that we examined have the identical amino acid change, yellow circle = not all African species that we examined have the identical amino acid change (but they all have amino changes/deletion that likely affect protein function as predicted by PROVEAN), brown circle = all African species that we examined have the same amino acids like the human reference sequence, white circle = data not available. The alignment results are shown in Supplemental Figure S7.5.

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