The Two Domains of the Avian Double-β-Defensin AvBD11 Have Different Ancestors, Common with Potential Monodomain Crocodile and Turtle Defensins

SIMPLE SUMMARY: Vertebrate defensins are a multigene family of antimicrobial peptides that evolved following a series of gene duplication and divergence events during the expansion of vertebrates. In birds, the repertoire of avian defensins contains an atypical defensin, namely AvBD11 (avian beta-defensin 11), which consists of two repeated but divergent defensin units (or domains) while most vertebrate defensins only possess one unit. In this study, we investigated the evolutionary scenario leading to the formation of this double defensin in birds by comparing each defensin unit of AvBD11 with other defensins from birds and closely related reptiles (crocodile, turtles) predicted to have a single defensin unit. Our most outstanding results suggest that the double defensin AvBD11 probably appeared following a fusion of two ancestral genes or from an ancestral double defensin, but not from a recent internal duplication as it can be observed in other types of proteins with domain repeats. ABSTRACT: Beta-defensins are an essential group of cysteine-rich host-defence peptides involved in vertebrate innate immunity and are generally monodomain. Among bird defensins, the avian β-defensin 11 (AvBD11) is unique because of its peculiar structure composed of two β-defensin domains. The reasons for the appearance of such ‘polydefensins’ during the evolution of several, but not all branches of vertebrates, still remain an open question. In this study, we aimed at exploring the origin and evolution of the bird AvBD11 using a phylogenetic approach. Although they are homologous, the N- and C-terminal domains of AvBD11 share low protein sequence similarity and possess different cysteine spacing patterns. Interestingly, strong variations in charge properties can be observed on the C-terminal domain depending on bird species but, despite this feature, no positive selection was detected on the AvBD11 gene (neither on site nor on branches). The comparison of AvBD11 protein sequences in different bird species, however, suggests that some amino acid residues may have undergone convergent evolution. The phylogenetic tree of avian defensins revealed that each domain of AvBD11 is distant from ovodefensins (OvoDs) and may have arisen from different ancestral defensins. Strikingly, our phylogenetic analysis demonstrated that each domain of AvBD11 has common ancestors with different putative monodomain β-defensins from crocodiles and turtles and are even more closely related with these reptilian defensins than with their avian paralogs. Our findings support that AvBD11′s domains, which differ in their cysteine spacing and charge distribution, do not result from a recent internal duplication but most likely originate from a fusion of two different ancestral genes or from an ancestral double-defensin arisen before the Testudines-Archosauria split.