Structural characterization of dioicin 1 from Phytolacca dioica L. gains novel insights into phylogenetic relationships of Phytolaccaceae type 1 RIPs

Ribosome-inactivating proteins are plant cytotoxic enzymes, also present in fungi, algae and bacteria, mainly known for their ability to inhibit protein synthesis. We previously purified and structurally characterized three type 1 RIPs (PD-S1-3) from Phytolacca dioica seeds and four type 1 RIPs (PD-L1-4) from adult plant leaves. Two additional RIPs, named dioicin 1 and dioicin 2, were isolated from leaves of young plants and developing leaves of adult plants. The evidence that P. dioica synthesizes and accumulates these RIPs isoforms suggests that these proteins have been conserved during evolution. Though several aspects of P. dioica type 1 RIP characterization have been studied, some important questions remain to be answered especially with respect to Phytolaccaceae RIP evolution. One of the major problems encountered in approaching RIPs phylogeny concerns the availability of their sequences. In this study, we report the characterization of biological and structural properties of dioicin 1, including the determination of its primary structure by using a combined approach based on Edman degradation, de novo sequencing by ESI-Q-TOF-MS/MS and peptide mapping by MALDI-TOF MS. Knowledge of dioicin 1 primary structure provide us a mean to deepen Phytolaccaceae's RIPs phylogeny. We speculate that both dioicins 1 and 2 share common ancestors with PAP-II and PAP icos-II and that dioicin 1 is not closely related to other members of this clade, thus shedding lights on evolutionary relationships among type 1 RIPs from Phytolaccaceae.