Characterization of Two Novel Type I Ribosome-Inactivating Proteins from the Storage Roots of the Andean CropMirabilis expansa1

Abstract

Two novel type I ribosome-inactivating proteins (RIPs) were found in the storage roots of Mirabilis expansa, an underutilized Andean root crop. The two RIPs, named ME1 and ME2, were purified to homogeneity by ammonium sulfate precipitation, cationexchange perfusion chromatography, and C 4 reverse-phase chromatography. The two proteins were found to be similar in size (27 and 27.5 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their isoelectric points were determined to be greater than pH 10.0. Amino acid N-terminal sequencing revealed that both ME1 and ME2 had conserved residues characteristic of RIPs. Amino acid composition and western-blot analysis further suggested a structural similarity between ME1 and ME2. ME2 showed high similarity to the Mirabilis jalapa antiviral protein, a type I RIP. Depurination of yeast 26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity. Because these two proteins were isolated from roots, their antimicrobial activity was tested against root-rot microorganisms, among others. ME1 and ME2 were active against several fungi, including Pythium irregulare, Fusarium oxysporum solani, Alternaria solani, Trichoderma reesei, and Trichoderma harzianum, and an additive antifungal effect of ME1 and ME2 was observed. Antibacterial activity of both ME1 and ME2 was observed against Pseudomonas syringae, Agrobacterium tumefaciens, Agrobacterium radiobacter, and others.

The N-Glycosidase Activity of the Ribosome-inactivating Protein ME1 Targets Single-stranded Regions of Nucleic Acids Independent of Sequence or Structural Motifs

“…35) and ME 2 (27 kDa; Ref. 30), two RIPs previously found to be present in M. expansa roots. ME 1 was further purified by cation-exchange chromatography, and its RNA Nglycosidase activity was confirmed with ribosomes prepared from S. cerevisiae as described previously (36).…”

“…This antibody, which was raised previously in our laboratory (30), was used as a ligand to generate an affinity matrix via coupling to CNBr-activated Sepharose. Total root proteins of M. expansa were applied to the column, and immunobound proteins were eluted with pH linear gradients as outlined under "Experimental Procedures."…”

“…Because viral genomes must form single-stranded templates that are largely free of coat protein at some stage during the infection process, depurination of virus-related nucleic acids could provide direct protection against infection. The presence of high concentrations of RIPs within the cell wall (30,36,59) could facilitate RIP-viral nucleic acid contact; alternatively, RIPs could enter the cytoplasm together with the virions via mechanical damage to the cell wall and plasma membrane and act on viral RNA during cotranslational disassembly (62,63). Because viroids lack the protein capsid that protects almost all conventional viruses, one might expect them to be particularly sensitive to RIPs.…”

“…A polyclonal antibody raised previously in our laboratory against reverse-phase high pressure liquid chromatography-purified ME 1 (30) was coupled to CNBr-activated Sepharose at pH 8.0. Total root proteins of M. expansa were equilibrated with 75 mM Tris-HCl, pH 8.0, to facilitate protein-ligand interaction, and immunobound proteins were then eluted with 100 mM glycine-HCl, pH 2.7, containing 0.5 M NaCl.…”

“…Heat Treatment and Depurination Analysis-Partial denaturation of DNA and RNA substrates was carried out for 30 s at various temperatures (30,45,60,75, and 90°C). Each reaction (15 l) contained ϳ150 nM either DNA in incubation buffer PS-1 (10 mM Tricine-KOH, pH 8.7, 2.5 mM KCl, 3 mM MgCl 2 , 3.75 g/ml bovine serum albumin, 0.005% (v/v) Tween 20) or RNA transcripts in buffer PS-2 (1.25 mM Tris-HCl, pH 7.4, 2.5 mM KCl, 1.25 mM MgCl 2 , 0.01 mM EDTA).…”