Molecular Evolution of the Nuclear Factor (Erythroid-Derived 2)-Like 2 Gene Nrf2 in Old World Fruit Bats (Chiroptera: Pteropodidae)

Abstract

Mammals developed antioxidant systems to defend against oxidative damage in their daily life. Enzymatic antioxidants and low molecular weight antioxidants (LMWAs) constitute major parts of the antioxidant systems. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2, encoded by the Nrf2 gene) is a central transcriptional regulator, regulating transcription, of many antioxidant enzymes. Frugivorous bats eat large amounts of fruits that contain high levels of LMWAs such as vitamin C, thus, a reliance on LMWAs might greatly reduce the need for antioxidant enzymes in comparison to insectivorous bats. Therefore, it is possible that frugivorous bats have a reduced need for Nrf2 function due to their substantial intake of diet-antioxidants. To test whether the Nrf2 gene has undergone relaxed evolution in fruit-eating bats, we obtained Nrf2 sequences from 16 species of bats, including four Old World fruit bats (Pteropodidae) and one New World fruit bat (Phyllostomidae). Our molecular evolutionary analyses revealed changes in the selection pressure acting on Nrf2 gene and identified seven specific amino acid substitutions that occurred on the ancestral lineage leading to Old World fruit bats. Biochemical experiments were conducted to examine Nrf2 in Old World fruit bats and showed that the amount of catalase, which is regulated by Nrf2, was significantly lower in the brain, heart and liver of Old World fruit bats despite higher levels of Nrf2 protein in Old World fruit bats. Computational predictions suggest that three of these seven amino acid replacements might be deleterious to Nrf2 function. Therefore, the results suggest that Nrf2 gene might have experienced relaxed constraint in Old World fruit bats, however, we cannot rule out the possibility of positive selection. Our study provides the first data on the molecular adaptation of Nrf2 gene in frugivorous bats in compensation to the increased levels of LWMAs from their fruit-diet.

Fig 1. Unconstrained Nrf2 Bayesian phylogenetic tree and species tree.

(A) Unconstrained Bayesian phylogenetic tree based on Nrf2 coding sequences, under the GTR+G model. Values on the nodes are posterior probabilities. (B) Species tree of the 21 mammals used in this study based on their accepted relationships. Four branches tested by the two-ratio model tests and the branch-site model tests are marked as A, B, C, and D. The four species with orange labels are Old World fruit bats. The seven amino acid substitutions specific to Old World fruit bats are shown (A100D, N133D, D205F, D224G, T279I, T350R and N424S).

Fig 2. Omega (d_N/d_S) values of sites along the Nrf2 protein sequence on selected branches.

(A) Ancestral branch of Yinpterochiroptera insectivorous bats, (B) ancestral branch of Yangochiroptera, (C) ancestral branch of Old World fruit bats (OWFBs), and (D) branch of New World fruit bat (NWFB). The red arrows indicate the positions of the seven amino acid replacements in OWFBs with elevated ω values.

Fig 3. Distribution of the seven OWFBs-specific amino acid replacements in the secondary structure of Nrf2 protein.

The locations of the seven amino acid replacements in OWFBs are labeled in red line. The seven Neh domains are shown, Neh1 to Neh7. Neh1 domain (residue 435–561) is the CNC-bZIP domain. Neh2 domain (residue 27–82) is a redox-sensitive regulatory domain that binds with the repressor Keap1, and also contains one of the three nuclear localization sequences (NLS: residue 42–53, 494–511 and 587–593). Neh3 (residue 562–605), Neh4 (residue 112–134) and Neh5 (residue 183–201) domains are involved in Nrf2 transactivation activity. Neh6 domain (residue 338–388) is involved in Nrf2 stability. Neh7 domain (residue 209–316) is involved in interactions with RXRα and functions as a transcriptional repressor.

Fig 4. The levels of Nrf2 protein and catalase (CAT) in the brain, heart and liver of four bats species.

Four bats species were used in this study and are two insectivorous bats (M.ric: Myotis ricketti; R.fer: Rhinolophus ferrumequinum) and two Old World fruit bats (R.les: Rousettus leschenaultia; C.sph: Cynopterus sphinx). Protein level of (A) Nrf2 protein and (B) catalase in representative bat species and rats (positive control) were determined by Western blotting. Relative levels are presented as mean ± SD. Arrows indicate the predicted molecular weights (kDa) of the proteins. Statistical significance of the differences among species were determined by one-way ANOVA with post hoc Holm-Sidak tests: *P < 0.05, **P < 0.001.