Polynucleotide:Adenosine Glycosidase Is the Sole Activity of Ribosome-Inactivating Proteins on DNA

Barbieri, Luigi; Valbonesi, Paola; Righi, F.; Zuccheri, Giampaolo; Monti, Federica; Gorini, P; Samorı̀, Bruno; Stirpe, Fiorenzo

doi:10.1093/oxfordjournals.jbchem.a022827

Cited by 52 publications

(23 citation statements)

References 2 publications

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“…Analysis of the in vivo activity of other saporin isoforms (such as L1) [63] will be required to assess whether multiple depurination events play any major role in the mammalian cell’s intoxication process. In addition to the PNAG activities reported above, concerning the DNase-like activities [52,57] proposed for the type I RIPs, several pieces of evidence indicate that the DNase activity associated with ricin, saporin (and possibly with other type I RIPs) may be instead due to nuclease contamination [6,54,65,66]. …”

Section: Biochemical Characteristicsmentioning

confidence: 99%

Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

Virgilio

Lombardi

Caliandro

et al. 2010

Toxins

146

128

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Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential.

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Section: Biochemical Characteristicsmentioning

confidence: 99%

Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

Virgilio

Lombardi

Caliandro

et al. 2010

Toxins

146

128

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“…The release of adenine from polynucleotides by RIPs has been reported (l, 2,5,14). In the present study, the adenine released by Stxl was reacted with chloroacetaldehyde to form the fluorescent sA (8)(9)(10), which was quantified and used as a measure of Stxl biological activity.…”

Section: Resultsmentioning

confidence: 83%

“…The BAA measured the ability of an RIP (Stxl or ricin) to remove adenine residues from a 2,551-bp DNA substrate as previously described (14). The release of adenine residues from polynucleotides has been used by previous investiga tors as an indicator of RIP biological activity (1,2,5). Results obtained with the ELISA were compared with those obtained with the BAA, and the correlation between the results was evaluated.…”

mentioning

confidence: 99%

Comparison of the Presence of Shiga Toxin 1 in Food Matrices as Determined by an Enzyme-Linked Immunosorbent Assay and a Biological Activity Assay

Lumor

Fredrickson

Ronningen

et al. 2012

Journal of Food Protection

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This study was conducted to compare the identification of Shiga toxin 1 (Stx1) based on its specific biological activity and based on results of a commercial enzyme-linked immunosorbent assay (ELISA) kit. Stx1 was thermally treated for various periods in phosphate-buffered saline, milk, and orange juice. The residual Stx1 concentration was determined with the commercial ELISA kit, and its residual enzymatic activity (amount of adenine released from a 2,551-bp DNA substrate) was determined with a biological activity assay (BAA). Regression analysis indicated that the inactivation of Stx1 as a function of time followed first-order kinetics. The half-lives determined at 60, 65, 70, 75, 80, and 85°C were 9.96, 3.19, 2.67, 0.72, 0.47, and 0.29 min, respectively, using the BAA. The half-lives determined by the ELISA with thermal treatments at 70, 75, 80, and 85°C were 40.47, 11.03, 3.64, and 1.40 min, respectively. The Z, Q(10), and Arrhenius activation energy values derived by both assays were dissimilar, indicating that the rate of inactivation of the active site of Stx1 was less sensitive to temperature change than was denaturation of the epitope(s) used in the ELISA. These values were 10.28°C and 9.40 and 54.70 kcal/mol, respectively, with the ELISA and 16°C and 4.11 and 34 kcal/mol, respectively, with the BAA. Orange juice enhanced Stx1 inactivation as a function of increasing temperature, whereas inactivation in 2% milk was not very much different from that in phosphate-buffered saline. Our investigation indicates that the ELISA would be a reliable method for detecting the residual toxicity of heat-treated Stx1 because the half-lives determined with the ELISA were greater than those determined with the BAA (faster degradation) at all temperatures and were highly correlated (R(2) = 0.994) with those determined with the BAA.

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“…Therefore, the saporin catalytic activity we measured is not contaminated with RNase activity. 44–46 …”

Section: Resultsmentioning

confidence: 99%

Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity

Yuan

Sturm

et al. 2015

Biochemistry

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Saporin L3 from Saponaria officinalis (soapwort) leaves is a type 1 ribosome-inactivating protein. It catalyzes the hydrolysis of oligonucleotide adenylate N-ribosidic bonds to release adenine from rRNA. Depurination sites include both adenines in the GAGA tetraloop of short sarcin-ricin stem-loops and multiple adenines within eukaryotic rRNA, tRNAs, and mRNAs. Multiple Escherichia coli vector designs for saporin L3 expression were attempted but demonstrated high toxicity even during plasmid maintenance and selection in E. coli nonexpression strains. Saporin L3 is >103 times more efficient at RNA deadenylation on short GAGA stem-loops than saporin S6, the saporin isoform currently used in immunotoxin clinical trials. We engineered a construct for the His-tagged saporin L3 to test for expression in Pichia pastoris when it is linked to the protein export system for the yeast α-mating factor. DNA encoding saporin L3 was cloned into a pPICZαB expression vector and expressed in P. pastoris under the alcohol dehydrogenase AOX1 promoter. A fusion protein of saporin L3 containing the pre-pro-sequence of the α-mating factor, the c-myc epitope, and the His tag was excreted from the P. pastoris cells and isolated from the culture medium. Autoprocessing of the α-mating factor yielded truncated saporin L3 (amino acids 22–280), the c-myc epitope, and the His tag expressed optimally as a 32 kDa construct following methanol induction. Saporin L3 was also expressed with specific alanines and/or serines mutated to cysteine. Native and Cys mutant saporins are kinetically similar. The recombinant expression of saporin L3 and its mutants permits the production and investigation of this high-activity ribosome-inactivating protein.

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Polynucleotide:Adenosine Glycosidase Is the Sole Activity of Ribosome-Inactivating Proteins on DNA

Cited by 52 publications

References 2 publications

Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

Comparison of the Presence of Shiga Toxin 1 in Food Matrices as Determined by an Enzyme-Linked Immunosorbent Assay and a Biological Activity Assay

Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity

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