Biochemical and biological roles of ribosome -inactivating protein

Ribosome-inactivating proteins (RIPS) are a group of plant cytotoxic N-glycosidases that specifically cleave nucleotide N--C glycosidic bonds. In the present study, a novel type-1 RIP, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of Phytolacca americana, and biochemically characterized. PAP-H was determined to be 29.5 kD, and its full-length cDNA sequence has 1,125 nucleotides with an open reading frame of 1074 nucleotides representing 339 amino acids (Genebank, accession number AY071928). Further, PAP-H was found to be localized in the cell walls of hairy roots and root border cells using immuno-fluorescence microscopy, and was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. By secretion into the rhizosphere, PAP-H penetrates and inhibits the growth of soil-borne fungi by a synergetic combination of chitinase, beta-1,3-glucanase, and protease within root exudates. To further understand the antifungal mechanism of RIPS, I have examined three type I RIPS to determine their substrate specificities on fungal ribosomes, and I have correlated the data with their antifungal activity. The results provide experimental evidence that the enzymatic activity and specificity of RIPs are separated from their cytotoxicity. In addition, using protein-fluorescence labeling technology, I have found that RIPS selectively recognize, interact with, and are internalized into fungal cells to exert cytotoxicity.;The final stage of this research focused on investigating the enzymatic interaction of the RIP ME1 (from Mirabilis expansa) with non-ribosomal substrates upon their structural modification. The RIP-RNA/RIP-DNA interactions was found to depend on the secondary structure of substrates, and did not require any sequence or structure motif. RIP targeted only single-stranded regions of nucleic acid, removing adenine and, to a lesser extent, guanine residues. In addition, RIP was capable of recognizing and depurinating a pathogenic RNA transcript, completely abolishing its infectivity. Subsequently, we examined the activity of ME1 toward M. expansa ribosomes in the presence of increasing concentrations of small synthetic oligonucleotides (23SSO-A12), indicating that the long-considered only substrate of RIP, the ribosome, is unlikely be the primary target in plant systems.