Characterization of KPAP, a Novel Pokeweed Antiviral Protein Isoform

Phytolacca americana is a perennial plant that harbors the pokeweed antiviral protein (PAP) genes which belong to the N-glycosidase family. These proteins are called ribosome inactivating proteins (RIPs) which remove an adenine base from the sarcin/ricin loop (SRL) of the 28S rRNA in eukaryotes. This depurination results in protein translation inhibition leading to a concentration-dependent cytotoxic consequence. Many RIPs in the plant kingdom are being discovered with new genomic sequencing technologies but, many of them remain uncharacterized. Recently, our lab discovered a new RIP called Novel PAP/KPAP in the sequenced pokeweed genome. KPAP transcript levels did not behave the same way as other isoforms under phytohormone stimulated biotic stress, however, they showed a slight decrease in drought. In this study, I provide preliminary groundwork on the KPAP gene model, expression in abiotic stresses, protein structure, depurination activity and potential binding arrangement on the SRL. My results show the presence of predicted upstream open reading frames (uORFs) in the long leader intron, perhaps belonging to retrotransposons. The enzymatic activity recorded from KPAP in an in vitro translation assay resembles the activity of PAP-I. Moreover, the exclusive expression of KPAP in leaves compared to other isoforms and its downregulation in drought may indicate a function related to photosynthesis, carbon metabolism, plant growth or leaf expansion. Structurally the predicted protein was docked in silico to an adenine base and showed a similar disposition of the active site compared to PAP-I. Lastly, KPAP was docked in silico to the E.coli SRL and residue interactions are discussed in the context of other RIPs. This early research shows KPAP’s similarities and differences compared to the other isoforms, however, much remains to be learned from this isoform and RIPs in general.