Adaptive Evolution of Hepcidin Genes in Antarctic Notothenioid Fishes

Abstract

Hepcidin is a small bioactive peptide with dual roles as an antimicrobial peptide and as the principal hormonal regulator of iron homeostasis in human and mouse. Hepcidin homologs of very similar structures are found in lower vertebrates, all comprise approximately 20-25 amino acids with 8 highly conserved cysteines forming 4 intramolecular disulfide bonds, giving hepcidin a hairpin structure. Hepcidins are particularly diverse in teleost fishes, which may be related to the diversity of aquatic environments with varying degree of pathogen challenge, oxygenation, and iron concentration, factors known to alter hepcidin expression in mammals. We characterized the diversity of hepcidin genes of the Antarctic notothenioid fishes that are endemic to the world's coldest and most oxygen-rich marine water. Notothenioid fishes have at least 4 hepcidin variants, in 2 distinctive structural types. Type I hepcidins comprise 3 distinct variants that are homologs of the widespread 8-cysteine hepcidins. Type II is a novel 4-cysteine variant and therefore only 2 possible disulfide bonds, highly expressed in hematopoietic tissues. Analyses of d(N)/d(S) substitution rate ratios and likelihood ratio test under site-specific models detected significant signal of positive Darwinian selection on the mature hepcidin-coding sequence, suggesting adaptive evolution of notothenioid hepcidins. Genomic polymerase chain reaction and Southern hybridization showed that the novel type II hepcidin occurs exclusively in lineages of the Antarctic notothenioid radiation but not in the basal non-Antarctic taxa, and lineage-specific positive selection was detected on the branch leading to the type II hepcidin clade under branch-site models, suggesting adaptive evolution of the reduced cysteine variant in response to the polar environment. We also isolated a structurally distinct 4-cysteine (4cys) hepcidin from an Antarctic eelpout that is unrelated to the notothenioids but inhabits the same freezing water. Neighbor-Joining (NJ) analyses of teleost hepcidins showed that the eelpout 4cys variant arose independently from the notothenioid version, which lends support to adaptive evolution of reduced cysteine hepcidin variants on cold selection. The NJ tree also showed taxonomic-specific expansions of hepcidin variants, indicating that duplication and diversification of hepcidin genes play important roles in evolutionary response to diverse ecological conditions.

Multiple Hepcidins in a Teleost Fish, Dicentrarchus labrax: Different Hepcidins for Different Roles

“…These residues are involved in the formation of disulfide bonds, which give hepcidin the characteristic hairpin-like structure, believed to be essential for its activity, but there are reports of some fish hepcidins with as little as four cysteine residues (24,52) and still likely to be fully functional.…”

Molecular basis and genetic improvement of economically important traits in aquaculture animals

“…To identify candidate cold tolerance-related and cold change-related genes, a large-scale EST sequencing of multi-tissues including brain, liver, head kidney, and ovary had been performed in Antarctic notothenioid (Dissostichus mawsoni), and some important cold-specific expressed genes had been identified by comparative transcriptome analyses between D. mawsoni and non-notothenioid warm-water teleost fishes [133,134]. Moreover, the molecular origination of antifreeze protein III (AFP III) was demonstrated to come from a duplicate copy of sialic acid synthase (SAS) gene, supporting the evolutionary mechanism by neofunctionalization under escape from adaptive conflict [135].…”

A Novel Isoform of the Hepatic Antimicrobial Peptide, Hepcidin (Hepc-CB1), from a Deep-Sea Fish, the Spinyjaw Greeneye Chlorophthalmus bicornis (Norman, 1939): Molecular Characterisation and Phylogeny

“…The disulphide bonds which stabilise b-hairpin are highlighted with asterisks absence of QSHLS/DTHFP motif in the N-terminal region of the mature peptide, anionic nature of prepropeptide and propeptide further confirmed the identity of C. bicornis as HAMP2. Duplication of HAMP genes and retention of both HAMP1-and HAMP2-like lineages in acanthopterygian fishes and Antarctic notothenioid fishes could be favoured by the radiation of teleosts to different marine and brackish water environments and positive Darwinian selection [17,23,38]. The extreme habitats of the deep sea might have produced fascinating evolutionary events [33] like gene duplication and conservation of HAMP2-like genes in nonacanthopterygian fishes also.…”

“…Multiple copies of hepcidin isoforms were identified from many fishes including, Scophthalmus maximus [13], Acanthopagrus schlegelii [14], Lates calcarifer [15], Alphestes immaculatus [16], Pagrus auriga [17], Pseudosciaena crocea [18], Sparus aurata [19], Dicentrarchus labrax [20], Paralichthys olivaceus [21], Gadus morhua [8], Oreochromis mossambicus [22] and Antarctic notothenioid fishes [23]. Hepcidins are diverse in teleost fishes, due to the diversity of aquatic environments, that is, exposure to pathogens, oxygenation and iron concentration [23]. Apart from antimicrobial properties, hepcidin exhibits pivotal roles in immunomodulation and iron homeostasis [24,25].…”