title: Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity.
abstract: Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.
authors.affiliation: Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Sang-Wook
authors.initials: SW
authors.lastname: Park
authors.forename: Noah M
authors.initials: NM
authors.lastname: Stevens
authors.forename: Jorge M
authors.initials: JM
authors.lastname: Vivanco
mesh_terms: D002384:Catalysis; D004798:Enzymes / Q000378:metabolism / Q000494:pharmacology*; D005658:Fungi / Q000187:drug effects* / Q000378:metabolism; D005659:Fungicides, Industrial / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D039282:Mirabilis / Q000378:metabolism; D010940:Plant Proteins / Q000494:pharmacology; D012335:RNA, Ribosomal / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism / Q000494:pharmacology*; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000187:drug effects* / Q000378:metabolism; D012276:Ricin / Q000494:pharmacology; D029750:Saponaria / Q000378:metabolism; D000076985:Saporins; D013379:Substrate Specificity
chemical_list: D004798:Enzymes; D005659:Fungicides, Industrial; D010940:Plant Proteins; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D012276:Ricin; D000076985:Saporins