Javier Lacadena

Overproduction and purification of biologically active native fungal α-sarcin in Escherichia coli

An efficient system was developed to produce, in Escherichia coli, large amounts of native alpha-sarcin (alpha Sar), a cytotoxin from the mold Aspergillus giganteus. The protein has been purified to homogeneity with a yield of 1.5 micrograms/ml of original culture. The constructed expression vector (pINPG alpha S) is based on the synthesis of a fusion protein between alpha Sar and a modified version of the OmpA signal peptide. This peptide seems to favour the postranslational processing of the fusion protein. The purified recombinant alpha-sarcin (re-alpha Sar) is structurally identical to the mature fungal protein according to the following criteria: N-terminal amino acid (aa) sequence, aa composition, electrophoretic mobility, chromatographic behaviour, immunoreactivity and spectroscopic features. Indeed, the recombinant protein recovered is completely functional, since it cleaves, in vitro, eukaryotic rRNA and it is able to interact with phospholipid vesicles with the same specificity as the native fungal alpha Sar.

Role of histidine‐50, glutamic acid‐96, and histidine‐137 in the ribonucleolytic mechanism of the ribotoxin α‐sarcin

α‐Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis—histidine‐50, glutamic acid‐96, and histidine‐137—were changed to glutamine. Three dif‐ ferent single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three‐dimensional structure of these variants of α‐sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of α‐sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild‐type α‐sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of α‐sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by α‐sarcin. Proteins 1999;37:474–484. ©1999 Wiley‐Liss, Inc.

The cytotoxin α‐sarcin behaves as a cyclizing ribonuclease

The hydrolysis of adenylyl(3′→5′)adenosine (ApA) and guanylyl(3′→5′)adenosine (GpA) dinucleotides by the cytotoxic protein α‐sarcin has been studied. Quantitative analysis of the reaction has been performed through reverse‐phase chromatographic (HPLC) separation of the resulting products. The hydrolysis of the 3′‐5′ phosphodiester bond of these substrates yields the 2′‐3′ cyclic mononucleotide; this intermediate is converted into the corresponding 3′‐monophosphate derivative as the final product of the reaction. The values of the apparent Michaelis constant (K M), k cat and k cat/K M have also been calculated. The obtained results fit into a two‐step mechanism for the enzymatic activity of α‐sarcin and allow to consider this protein as a cyclizing RNase.

Characterization of a natural larger form of the antifungal protein (AFP) from Aspergillus giganteus

Two major proteins, alpha-sarcin and an antifungal polypeptide (AFP), are secreted by the mould Aspergillus giganteus MDH 18894 when it is cultured for 70-80 h. A third major protein is also found in the extracellular medium at 48-60 h, but it disappears as the culture proceeds. This protein has been isolated and characterized in terms of apparent molecular mass, electrophoretic and chromatographic behaviour, NH2-terminal primary structure, amino acid content, spectroscopical features, reactivity against anti-AFP antibodies, and antifungal activity. Based on the obtained results it would be an extracellular inactive precursor form of AFP, designated as the large form of AFP (lf-AFP). Its amino acid composition is identical to that of AFP but containing six extra residues. NH2-terminal sequence analysis of the first eight amino acid residues of this polypeptide revealed that the extra residues can be perfectly accommodated within the DNA-deduced sequence of the precursor form of AFP. Its alignment with precursor sequences of different proteins, secreted by a variety of Aspergillus spp., reveals the existence of a common tetrapeptide at the carboxy-terminal end of their leader peptides. This sequence would be Ile/Leu-Xaa-Yaa-Arg, being mostly Xaa and Yaa an acid residue (Asp/Glu) and alanine, respectively. The presence of lf-AFP as an extracellular protein would be in perfect agreement with the existence of this tetrapeptide motif, that can be involved in the protein secretion mechanisms of filamentous fungi.

Structural basis for the catalytic mechanism and substrate specificity of the ribonuclease α-sarcin

α‐Sarcin is a ribosome‐inactivating protein which selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA. The solution structure of α‐sarcin has been determined on the basis of 1898 distance and angular experimental constraints from NMR spectroscopy. It reveals a catalytic mechanism analogous to that of the T1 family of ribonucleases while its exquisite specificity resides in the contacts provided by its distinctive loops.

MEMBRANE INTERACTION OF A BETA -STRUCTURE-FORMING SYNTHETIC PEPTIDE COMPRISING THE 116-139TH SEQUENCE REGION OF THE CYTOTOXIC PROTEIN ALPHA -SARCIN

alpha-Sarcin is a cytotoxic protein that strongly interacts with acid phospholipid vesicles. This interaction exhibits a hydrophobic component although alpha-sarcin is a highly polar protein. A peptide comprising the amino acid sequence corresponding to the 116-139th segment of the alpha-sarcin cytotoxin has been synthesized by a standard fluoren-9-yl-methoxycarbonyl-based solid phase method. Its primary structure is: (116)-NPGPARVIYTYPNKVFCGIIAHTK-(139). Two beta-strands have been predicted in this region of alpha-sarcin, where the less polar stretches of the protein are found. The synthetic peptide interacts with negatively charged large unilamellar vesicles of either natural or synthetic phospholipids. An apparent fragmentation of the vesicles is produced by the peptide based on electron microscopy studies. The peptide promotes leakage of the intravesicular aqueous contents and lipid mixing of bilayers. The packing of the phospholipid molecules is greatly perturbed by the peptide, as deduced from the drastic changes induced by the peptide in cooperative properties associated with the phase transition of the bilayers. At saturating peptide/phospholipid ratios, the phase transition of dimyristoylphosphatidylglycerol vesicles is abolished. All of these effects are saturated at about 0.3 peptide/lipid molar ratio. The peptide adopts a mostly random structure in aqueous solution. A conformation composed of a high proportion of antiparallel beta-sheet is induced as a consequence of the interaction with the phospholipid vesicles in opposition to trifluoroethanol that promotes alpha-helical peptide structures, as deduced from circular dichroism measurements. The obtained results are discussed in terms of the potential involvement of the region comprising residues 116-139 of alpha-sarcin in the hydrophobic interactions of this cytotoxic protein with membranes.

The insecticidal protein hirsutellin A from the mite fungal pathogen Hirsutella thompsonii is a ribotoxin.

The mite fungal pathogen Hirsutella thompsonii produces a single polypeptide chain, insecticidal protein named hirsutellin A (HtA) that is composed of 130 amino acid residues. This protein has been purified from its natural source and produced as a recombinant protein in Escherichia coli. Spectroscopic analysis has determined that the two protein forms are indistinguishable. HtA specifically inactivates ribosomes and produces the α‐fragment characteristic of ribotoxin activity on rRNA. Behaving as a cyclizing ribonuclease, HtA specifically cleaves oligonucleotides that mimick the sarcin/ricin loop of the ribosome, as well as selected polynucleotides and dinucleosides. HtA interacts with phospholipid membranes as do other ribotoxins. As a consequence of its ribonuclease activity and its ability to interact with cell membranes, HtA exhibits cytotoxic activity on human tumor cells. On the basis of these results, HtA is considered to be a member of the ribotoxin group of proteins, although it is significantly smaller (130 aa) than all known ribotoxins that are composed of 149/150 amino acids. Ribotoxins are members of a larger family of fungal ribonucleases whose members of smaller size (100/110 aa) are not cytotoxic. Thus, the characterization of the fungal ribotoxin HtA represents an important milestone in the study of the diversity and the function of fungal ribonucleases. Proteins 2008.

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which − due to its severity − is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149‐amino acid protein belongs to the ribotoxin family, whose best characterized member is α‐sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Δ(7–22) have been produced as recombinant proteins; the deleted region corresponds to an exposed β‐hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Δ(7–22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted β‐hairpin is involved in ribosome recognition and is a significant allergenic region.

Involvement of the amino-terminal β-hairpin of the Aspergillus ribotoxins on the interaction with membrances and nonspecific ribonuclease activity

Ribotoxins are a family of potent cytotoxic proteins from Aspergillus whose members display a high sequence identity (85% for about 150 amino acid residues). The three‐dimensional structures of two of these proteins, α‐sarcin and restrictocin, are known. They interact with phospholipid bilayers, according to their ability to enter cells, and cleave a specific phosphodiester bond in the large subunit of ribosome thus inhibiting protein biosynthesis. Two nonconservative sequence changes between these proteins are located at the amino‐terminal β‐hairpin of α‐sarcin, a characteristic structure that is absent in other nontoxic structurally related microbial RNases. These two residues of α‐sarcin, Lys 11 and Thr 20, have been substituted with the equivalent amino acids in restrictocin. The single mutants (K11L and T20D) and the corresponding K11L/T20D double mutant have been produced in Escherichia coli and purified to homogeneity. The spectroscopic characterization of the purified proteins reveals that the overall native structure is preserved. The ribonuclease and lipid‐perturbing activities of the three mutants and restrictocin have been evaluated and compared with those of α‐sarcin. These proteins exhibit the same ability to specifically inactivate ribosomes, although they show different activity against nonspecific substrate analogs such as poly(A). The mutant variant K11L and restrictocin display a lower phospholipid‐interacting ability correlated with a decreased cytotoxicity. The results obtained are interpreted in terms of the involvement of the amino‐terminal β‐hairpin in the interaction with both membranes and polyadenylic acid.

Assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin ?-sarcin

α‐Sarcin, a potent cytotoxic protein from Aspergillus giganteus, contains two tryptophan residues at positions 4 and 51. Two single, W4F and W51F, and the double mutant, W4/51F, have been produced and purified to homogeneity. These two residues are neither required for the highly specific ribonucleolytic activity of the protein on the ribosomes (production of the so called α‐fragment) nor for its interaction with lipid membranes (aggregation and fusion of vesicles), although the mutant forms involving Trp‐51 show a decreased ribonuclease activity. Proton NMR data reveal that no significant changes in the global structure of the enzyme occur upon replacement of Trp‐51 by Phe. Substitution of each Trp residue results in a 4 °C drop in the thermal denaturation midpoint, and the double mutants midpoint is 9°C lower. Trp‐51 is responsible for most of the near‐UV circular dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp‐51 does not show fluorescence emission. The membrane‐bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of the protein with phospholipid bilayers promotes a large increase of the quantum yield of Trp‐51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers. This indicates that the region around this residue is located in the hydrophobic core of the bilayer following protein–vesicle interaction. Proteins 2000;41:350–361.