Deletion of the NH2-terminal β-Hairpin of the Ribotoxin α-Sarcin Produces a Nontoxic but Active Ribonuclease

Abstract

Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA. ␣-Sarcin, the best characterized member of this family, is a potent cytotoxin that promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with nontoxic ribonucleases of the RNase T1 family. However, significant structural differences between these two groups of proteins are related to the presence of a long amino-terminal ␤-hairpin in ribotoxins and to the different length of their unstructured loops. The aminoterminal deletion mutant ⌬(7-22) of ␣-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type protein to degrade rRNA in intact ribosomes. The results indicate that ␣-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the ␤-hairpin of the protein. In addition, this latter protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human rhabdomyosarcoma cells.

Fungal extracellular ribotoxins as insecticidal agents

“…This mutant retains the structural features of the wild-type protein, as well as its ability to interact with membranes, but lacks the characteristic ribonucleolytic activity of ribotoxins (Lacadena et al, 1995(Lacadena et al, , 1999. SDS-PAGE of proteins, Western blots, protein hydrolysis, amino acid analysis, and spectroscopic characterization were performed according to standardized procedures described before (García-Ortega et al, 2000, 2002Lacadena et al, 1994;Martínez-Ruiz et al, 2001). According to all these criteria, the three proteins used in this study were purified to homogeneity and retained their structural and functional properties.…”

Lactococcus lactis as a vehicle for the heterologous expression of fungal ribotoxin variants with reduced IgE-binding affinity

“…This assay was performed at pH 7.0 in 15% (w/v) polyacrylamide gels containing 0.1% (w/v) SDS and 0.3 mg/mL of the homopolynucleotide. In these zymograms, proteins exhibiting ribonuclease activity appear as colorless bands after appropriate distaining (Lacadena et al, 1999;Kao et al, 2001;García-Ortega et al, 2002). Mice (n = 6) were inoculated daily via intragastric gaugage with 2 x 10 7 L. lactis cells suspended in 200 μl of bicarbonate solution during 14 consecutive days.…”

“…Suitable deoxyoligonucleotides were used as primers for PCR amplification, using a series of plasmids constructed before as templates (Lacadena et al, 1994(Lacadena et al, , 1995García-Ortega et al, 2002, containing the cDNA corresponding to the five proteins studied: Asp f 1, α-sarcin, Asp f 1 Δ(7-22), and α-sarcins Δ(7-22) and H137Q. These amplified DNA fragments were flanked by NgoMIV and BamHI restriction sites that were used to clone the proteins into the corresponding cloning sites of the plasmid pT1NX, in frame with the secretion signal leader of the lactococcal usp45 gene placed under the control of the constitutive promoter P1 (Steidler et al, 1995.…”

“…The second of them (residues 7-22) juts out as a solvent-exposed protuberance and is one of the protein regions with highest conformational flexibility (Pérez-Cañadillas et al, 2000, 2002. Deletion of this β-sheet results in a Δ(7-22) mutant that shows no significant conformational differences except for the deleted region (García-Mayoral et al, 2004) but has lost its ability to specifically recognize the ribosome and is much less cytotoxic (García-Ortega et al, 2002).…”

“…In this work, the extracellular 5 production by L. lactis of the above α-sarcin and Asp f 1 deletion variants is presented. Both wild-type proteins were also included in the study, as well as a properly folded, but catalytically inactive α-sarcin H137Q mutant, as a control (Lacadena et al, 1995;García-Ortega et al, 2002. The possibility of using this strategy as a potential immunomodulating therapeutic approach is discussed.…”