DNA‐nuclease activity of the single‐chain ribosome‐inactivating proteins dianthin 30, saporin 6 and gelonin

Abstract

The single-chain ribosome-inactivating proteins (scRIPs) The so-RIPs asparin 1, dianthin 30, gelonin, momordin, PAP-S identified and mapped in pBR322.(pokeweed antiviral protein from Phytolacca americana seeds) and saporin 6 were highly purified as described previously [15]. HPLCpurified saporin 6, whose specific N-glycosidase activity had been Agarose and acrylamide/bis-acrylamide for gel electrophoresis were to the A-chains of ricin and related toxins [2], which are purchased from Clontech Laboratories, Palo Alto, CA (USA). All known to depurinate the ribosomal RNA by cleaving the Nother chemicals were of analytical grade.glycosidic bond in the A4324 position of the 28S RNA fracRestriction nucleases employed were: PvuII (from Proteus vulgaris) tion, so rendering it incapable of binding to the elongation incubation buffer: Tris-HCl 10 raM, NaC1 100 mM, MgCI2 10 mM, factors, with the consequent arrest of protein synthesis [3,4]. dithioerythritol 1 mM, pH 7.5 at 37°C; PstI (from Providencia stuartii) and HinfI (from Haemophylus influenzae Rf) incubation buffer: These proteins have attracted interest in the construction of Tris-HC1 50 mM, NaCI 10 raM, MgCI2 10 mM, dithioerythritol 1 immunotoxins after conjugation with monoclonal antibodies raM, pH 7.5 at 37°C; BamHI (from Bacillus amyloliquefacens H) for the selective killing of neoplastic cells [5 7].and HindIII (from Haemophylus influenzae Rd com-10) incubation To better understand perspectives and limits of the sc-RIPs buffer: Tris-HC1 10 mM, NaC1 100 raM, MgCI2 5 mM, 2-mercaptoethanol 1 mM, pH 8 at 37°C. in clinical trials, we previously focused our attention on the antiproliferative effect of RIPs as native molecules or in corn-2.2. DNase activity assay bination with other drugs. We have demonstrated that the pBR322 and OX174 plasmid DNA (250 ng), single-stranded M13 native form of the sc-RIP saporin 6 exerts an antiproliferative phage, lambda phage DNA, linearized plasmid DNA (250 ng) were incubated with or without various amounts (250-0.25 ng) of the dieffect in vitro in human breast cancer, leukemia and melanoverse proteins in a final volume of 10 ~tl at 37°C for various times. The ma cells to a very different extent and with diverse kinetics, reactions were carried out in a range of 5 60 mM NaC1 (or MgC12), l dependent on the specific cell target analyzed [8][9][10]. The efmM Tris-HC1, 0.1 mM EDTA. The range of pH was 6.5-8. At the fect may be potentiated by other drugs: a strong synergism end of the incubations, all the samples were extracted with phenol has been obtained by combination of saporin 6 with lonidfollowed by ether and precipitated [17]. Digestion with restriction endonucleases was carried out as specified above. The samples were amine in breast cancer cells in vitro [11]. More recent results analyzed in 0.8% agarose and/or in 6% polyacrylamide gel electroobtained by others also suggest a possible penetration of the phoresis. DNA bands were visualized by staining with ethidium brosc-RIPs into cells [12,13]. In the light of our resu...

The Cytotoxic Activity of Ribosome-inactivating Protein Saporin-6 Is Attributed to Its rRNA N-Glycosidase and Internucleosomal DNA Fragmentation Activities

“…These include Tyr 72 , Tyr 120 , Glu 176 , Arg 179 , and Trp 208 , which have been proposed to be present in the active site and play an important role in the catalysis (21). The role of two other invariant residues, Tyr 16 and Arg 24 , proposed to be important for structural stability of ricin (33), has also been studied. These residues were mutated to alanine to generate seven single mutants, Y16A, R24A, Y72A, Y120A, E176A, R179A, and W208A.…”

“…Similarly, the codons for the invariant residues Tyr 16 and Arg 24 were also mutated to that for alanine. All of the mutations were carried out by oligonucleotide-mediated site-directed mutagenesis (25).…”

“…Alterations of these residues could therefore affect the folding rate or thermodynamic stability of the protein. In order to ascertain the role of corresponding residues, Tyr 16 and Arg 24 , in saporin-6, they were mutated to alanine, and the mutants were analyzed for various functional activities. Saporin-6 has been reported to contain DNA nuclease activity, and recently saporin-L1 has been shown to act on various forms of mammalian DNA (10,24).…”

Saporin as a novel suicide gene in anticancer gene therapy

“…Moreover, a number of studies have demonstrated specific DNAse-like activity of RIPs, including SAP, as evidenced by DNA fragmentation. 13,14 Such DNA damage, characterized by micronuclei formation and the appearance of cytosolic oligonucleosomes, might play a role in the induction of apoptosis upon cell exposure to the toxin and contribute to target cell killing.…”

Isolation and Characterization of a Novel Ribosome-Inactivating Protein from Root Cultures of Pokeweed and Its Mechanism of Secretion from Roots

“…A type II RIP consists of an A chain, functionally equivalent to a type I RIP, which is attached to a sugarbinding B chain (for review, see Mehta and Boston, 1998;Tumer et al, 1999;Nielsen and Boston, 2001). Besides RNA N-glycosidase activity, some RIPs have ribonuclease, DNase, DNA glycosylase, and apurinic/apyrimidic lyase activities (Li et al, 1991;Roncuzzi and Gasperi-Campani, 1996;Nicolas et al, 1997Nicolas et al, , 1998Nicolas et al, , 2000Hudak et al, 2000). In addition, RIPs from Trichosanthes kirilowii cell cultures have been demonstrated to possess chitinase activity (Remi Shih et al, 1997).…”