Functional analysis of a RIP-like protein isolated from Agave sisalana leaves

Ribosome-inactivating proteins (RIPs) are plant proteins that are most known for their rRNA N-glycosidase activity among other biological functions. In the context of the present study, a RIP/RIP-type protein was purified in Agave sisalana leaves, and characterized with the help of biochemical, functional, and preliminary in silico techniques. The protein of about 27 kDa was isolated after protein extraction through ion exchange and size exclusion chromatography. Based on the functional analysis, it was found to have rRNA N-glycosidase activity and DNase-like activity, which validates its designation of RIP or RIP-like proteins. The purified protein had antibacterial effects on Staphylococcus aureus , Bacillus cereus, and Escherichia coli in a concentration dependent manner. Cytotoxic examination utilizing the MTT assay demonstrated lower viability against human cancer cell lines (MCF-7, MDA-MB-231, HepG2, and A549), while the non-cancerous HEK-293 cell line had a slightly higher vitality. Gene expression study in MCF-7 cells revealed upregulation in the expression of apoptosis-associated genes, such as BAX, p53, and Caspase-9 and downregulation of Bcl-2 expression. Nano LC-MS/MS de novo peptide sequencing did not provide a complete homology to known proteins, suggesting the protein that was isolated had not been previously described. Secondary and tertiary structure predictions proposed a mixed composition of alpha-helical and random coil, and low structural coverage. Combined, these findings provide experimental evidence that Agave sisalana holds a bioactive RIP-like protein.