Isolation and Characterization of Ribosome-Inactivating Proteins from Cucurbitaceae

Due to their RNA-N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in biomedical and agricultural research. We have isolated and characterized two such proteins, foetidissimin II and texanin, from two Cucurbitaceae species. Foetidissimin II, obtained from the roots of Cucurbita foetidissima, was identified as a type-2 RIP, with a molecular weight of 61 kDa, as estimated by gel electrophoresis. It is composed of two chains, a 29-kDa chain A, and a 32-kDa chain B. Texanin, isolated from the fruits of Cucurbita texana, is a type-I RIP, with a single chain of molecular weight 29.7 kDa, as estimated by MALDI-TOF-MS. Both proteins exhibit RNA-N-glycosidase activity, with aniline playing a critical role in rRNA cleavage. The IC₅₀ value of foetidissimin II, determined by cell-free protein-synthesis inhibition, was 0.251 μM. In an in vitro cytotoxicity assay, foetidissimin II exhibited IC₅₀ values of ca. 70 nM to both adenocarcinoma and erythroleukemia cells. Texanin exhibited a weaker anticancer activity against erythroleukemia cells, with an IC₅₀ value of 95 μM, but no activity against adenocarcinoma cells. The N-terminal sequences of both proteins were compared with those of reported RIPs.