Citrullination Was Introduced into Animals by Horizontal Gene Transfer from Cyanobacteria

Abstract

Protein post-translational modifications (PTMs) add great sophistication to biological systems. Citrullination, a key regulatory mechanism in human physiology and pathophysiology, is enigmatic from an evolutionary perspective. Although the citrullinating enzymes peptidylarginine deiminases (PADIs) are ubiquitous across vertebrates, they are absent from yeast, worms and flies. Based on this distribution PADIs were proposed to have been horizontally transferred, but this has been contested. Here, we map the evolutionary trajectory of PADIs into the animal lineage. We present strong phylogenetic support for a clade encompassing animal and cyanobacterial PADIs that excludes fungal and other bacterial homologues. The animal and cyanobacterial PADI proteins share functionally relevant primary and tertiary synapomorphic sequences that are distinct from a second PADI type present in fungi and actinobacteria. Molecular clock calculations and sequence divergence analyses using the fossil record estimate the last common ancestor of the cyanobacterial and animal PADIs to be less than one billion years old. Additionally, under an assumption of vertical descent, PADI sequence change during this evolutionary time frame is anachronistically low, even when compared to products of likely endosymbiont gene transfer, mitochondrial proteins and some of the most highly conserved sequences in life. The consilience of evidence indicates that PADIs were introduced from cyanobacteria into animals by horizontal gene transfer (HGT). The ancestral cyanobacterial PADI is enzymatically active and can citrullinate eukaryotic proteins, suggesting that the PADI HGT event introduced a new catalytic capability into the regulatory repertoire of animals. This study reveals the unusual evolution of a pleiotropic protein modification.

The virtues and vices of protein citrullination

“…The distribution of PADIs across the tree of life is highly unusual and the PADI sequence has been subject to extensive losses, modifications and duplications across evolution [25]. PADIs are present in some bacteria and fungi and, although their functions in these organisms are completely unknown, they have been shown to be catalytically active [25][26][27]. Paradoxically, PADIs are absent from yeast, worms and flies, but are ubiquitous across vertebrates.…”

“…Ray-finned fish have a single PADI gene, but duplications down the vertebrate lineage have resulted in five paralogues in mammals. At least two different PADI types can be found across life [ 25 ], the animal-type three-domain PADI and the fungal-type, two-domain PADI. The animal-type PADI emerged during cyanobacterial evolution, while the fungal-type PADI can be found in actinobacteria and other bacteria.…”

“…The animal-type PADI emerged during cyanobacterial evolution, while the fungal-type PADI can be found in actinobacteria and other bacteria. This and other complementary pieces of evidence, including phylogeny and sequence evolution rate analyses, indicate that the ancestral cyanobacterial gene was introduced into animals by horizontal gene transfer [ 25 ]. The cyanobacterial PADI is catalytically active and can citrullinate mammalian proteins including histones, which are absent from bacteria, suggesting that the horizontal transfer event introduced citrullination as a new catalytic activity in animals.…”

“…Two other types of enzymes can catalyse citrullination: pPAD, an extended agmatine deiminase found in the human pathogen Porphyromonas gingivalis [ 28 ] and gADI, an extended free L-arginine deiminase found in the human parasite Giardia Lamblia [ 29 ]. These have evolved independently from PADIs and are found in bacteria and some eukaryotic organisms [ 25 ]. Furthermore, a recent study identified a protein with peptidylarginine deiminase activity in the plant Arabidopsis thaliana [ 30 ] .…”

“…Protein At5g08170 was identified after searching the Arabidopsis thaliana genome for a protein motif generated by aligning the catalytic core sequences of bacterial deiminases, after the authors identified citrullinated proteins within the Arabidopsis proteome. It is unclear whether At5g08170 is a PADI homologue, since a comprehensive search for PADI orthologues across the tree of life identified no PADI homologues in plants [ 25 ]. Regardless of this, however, At5g08170 was shown to act in a calcium-dependent manner, similarly to PADIs, to citrullinate proteins with nucleic acid functions, upon cold stress.…”