A deletion variant of the Aspergillus fumigatus ribotoxin Asp f 1 induces an attenuated airway inflammatory response in a mouse model of sensitization.

BACKGROUND: Aspergillus fumigatus is the most prevalent airborne fungal pathogen, and the ribotoxin Asp f 1 is one of its major allergens. Alpha-Sarcin is a natural variant of Asp f 1 produced by the nonpathogenic fungus Aspergillus giganteus. Both proteins show a sequence identity of 87% and almost identical 3-dimensional structures. Alpha-Sarcin delta(7-22) is a deletion mutant that displays reduced immunoglobulin (Ig) E reactivity and is much less cytotoxic than wild-type proteins against human transformed cells. OBJECTIVE: A murine model of sensitization to Asp f 1 was established to test the response elicited by this alpha-sarcin delta(7-22) deletion mutant. METHODS: BALB/c mice were treated intraperitoneally with different mixtures of recombinant wild-type Asp f 1 and/or a suspension of a commercially available A. fumigatus standard extract. Mice were then intranasally challenged with Asp f 1 or alpha-sarcin delta(7-22). Sera were collected for subsequent measurement of Ig levels and histological analysis of the nostrils and lungs. RESULTS: Sensitization to Asp f 1 was successful only when the purified protein was first administered together with the A fumigatus suspension. The model was characterized by elevated levels of total IgE in serum and histological lesions in the lungs and nostrils. These symptoms were less severe when the deletion variant was the protein administered, thus confirming in vivo its lower toxic character. CONCLUSIONS: An easily reproducible mouse model of A fumigatus Asp f 1 sensitization was established. This model revealed alpha-sarcin delta(7-22) to be a potential candidate for immunotherapy.

A novel Carcinoembryonic Antigen (CEA)-Targeted Trimeric Immunotoxin shows significantly enhanced Antitumor Activity in Human Colorectal Cancer Xenografts.

Immunotoxins are chimeric molecules, which combine antibody specificity to recognize and bind with high-affinity tumor-associated antigens (TAA) with the potency of the enzymatic activity of a toxin, in order to induce the death of target cells. Current immunotoxins present some limitations for cancer therapy, driving the need to develop new prototypes with optimized properties. Herein we describe the production, purification and characterization of two new immunotoxins based on the gene fusion of the anti-carcinoembryonic antigen (CEA) single-chain variable fragment (scFv) antibody MFE23 to α-sarcin, a potent fungal ribotoxin. One construct corresponds to a conventional monomeric single-chain immunotoxin design (IMTXCEAαS), while the other one takes advantage of the trimerbody technology and exhibits a novel trimeric format (IMTXTRICEAαS) with enhanced properties compared with their monomeric counterparts, including size, functional affinity and biodistribution, which endow them with an improved tumor targeting capacity. Our results show the highly specific cytotoxic activity of both immunotoxins in vitro, which was enhanced in the trimeric format compared to the monomeric version. Moreover, the trimeric immunotoxin also exhibited superior antitumor activity in vivo in mice bearing human colorectal cancer xenografts. Therefore, trimeric immunotoxins represent a further step in the development of next-generation therapeutic immunotoxins.

Conformational study of the antitumor protein alpha-sarcin.

The antitumor protein alpha-sarcin is a single polypeptide chain produced by the mold Aspergillus giganteus. It inhibits protein synthesis in some tumor cells by inactivating the larger ribosomal subunit. The secondary structure of the molecule has been studied by circular dichroism and predictive methods. The protein contains about 40% of periodic structures, mainly located at both extremes of the polypeptide chain. beta-Turns and aperiodic conformation appear at the central part of the molecule. Two different tyrosine populations have been observed in alpha-sarcin. Attempts to correlate solvent accessibility and particular protein regions have been carried out by using CD in the near-ultraviolet region, fluorescence and absorbance spectroscopies as well as acrylamide quenching and hydropathy profiles. Five different pH-induced conformational transitions are detected. Two of them, at pH 2.5 and 10.2, are denaturing transitions. These results are explained in terms of the structural features of this molecule, and related to its ribonucleolytic activity and ability to cross cell membranes.

Spectroscopic characterization of the alkylated alpha-sarcin cytotoxin: analysis of the structural requirements for the protein-lipid bilayer hydrophobic interaction.

alpha-Sarcin is a ribosome-inactivating protein that translocates across lipid bilayers, these two abilities explaining its cytotoxic character. This protein is composed of a single polypeptide chain with two disulfide bridges. Reduction and carboxyamidomethylation of alpha-sarcin results in protein unfolding, based on the results of the spectroscopic characterization of the chemically modified protein. The absorption and fluorescence emission bands of the tryptophan residues of the modified protein appear blue- and red-shifted, respectively. Far-UV circular dichroism analysis reveals the presence of residual secondary structure (beta-strands and turns) in the alkylated protein. This retains its ability to interact with lipid bilayers. It promotes vesicle aggregation, lipid-mixing between bilayers and leakage of the intravesicular aqueous contents. The modified protein tends to abolish the phase transition of acid phospholipids as detected by differential scanning calorimetry and depolarization measurements of fluorescence-labelled vesicles. The protein gain access to vesicle-entrapped trypsin. The fluorescence emission of the tryptophan residues is blue-shifted upon interaction of the protein with the bilayers, and anthracene incorporated into the hydrophobic core of the membranes quenches the tryptophan fluorescence emission of the protein. The secondary structure of the alkylated protein interacting with lipid vesicles has been studied by infrared spectroscopy. An increase in the alpha-helix and turn contents and a concomitant decrease in the beta-structure content are observed upon interaction with the bilayers. The results obtained are discussed in terms of the structural requirements for the interaction of alpha-sarcin with lipid membranes.

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.

Modulation by the ratio S-adenosylmethionine/S-adenosylhomocysteine of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase.

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. S-Adenosylmethionine/S-adenosylhomocysteine ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio S-adenosylmethionine/S-adenosylhomocysteine can modulate phosphorylation of a specific protein.

Effect of the antitumour protein alpha-sarcin on the thermotropic behaviour of acid phospholipid vesicles.

The antitumour protein alpha-sarcin modifies the thermotropic behaviour of phospholipid vesicles. This has been studied by fluorescence depolarization measurements and differential scanning calorimetry. A surface protein-phospholipid interaction is detected by measuring the polarization degree of TMA-DPH-labelled vesicles. At the higher protein/lipid molar ratios studied, the alpha-sarcin-vesicles complexes exhibit different thermotropic behaviour depending on whether they are prepared above or below the Tm of the corresponding phospholipid. Labelling of the protein with photoactive phospholipids has also been considered. alpha-Sarcin penetrates the bilayer deep enough to be labelled with the photoactive group located at the C-12 of the fatty acid acyl chain of phospholipids forming vesicles.

Thermal unfolding of the cytotoxin alpha-sarcin: phospholipid binding induces destabilization of the protein structure.

The effect of membrane binding on the structure and stability of the cytotoxin alpha-sarcin has been studied by differential scanning calorimetry, Fourier-transform infrared and fluorescence spectroscopic techniques. The thermal unfolding of alpha-sarcin in aqueous solution fits into a two-state transition characterized by a transition temperature (Tm) of 52.6 degrees C and a calorimetric enthalpy (delta Hcal) of 136 kcal/mol. Upon interaction with phosphatidylglycerol vesicles, alpha-sarcin undergoes conformational changes, as deduced from the FTIR and fluorescence emission spectra. These changes result in a decreased Tm and delta Hcal values for the thermal unfolding of phospholipid-bound alpha-sarcin. The lower Tm value for lipid-bound alpha-sarcin is also observed at the level of secondary and tertiary structures, based on analyses of both the amide I' infrared spectrum and the tryptophan emission of the protein as a function of temperature, respectively. The results obtained indicate a protein destabilization promoted by the phospholipid interaction.

Partially folded states of the cytolytic protein sticholysin II.

Sticholysin II (Stn II) is a cytolytic protein produced by the sea anemone Stichodactyla helianthus, its effect being related to pore formation. The conformation of the protein and its temperature-induced transitions, in the 1.5-12.0 pH range and in the 0-0.5 M NaCl concentration interval, have been studied by circular dichroism and fluorescence spectroscopy. At temperature < 35 degrees C, the protein maintains the same, high beta-structure content, folded conformation in the 1.5-11.0 pH range and ionic strength up to 0.5 M. In the 1.5-3.5 pH range and ionic strength > or = 0.1 M, Stn II shows a thermal transition, resulting in a partially folded state characterized by: (i) a native-like content of regular secondary structure, as detected by far-UV CD; (ii) a largely disordered tertiary structure, as detected by near-UV CD, with partially exposed tryptophan residues according to their fluorescence emission; and (iii) ability to bind the hydrophobic probe 2-anilinonaphthalene-6-sulfonic acid. In the pH range 4.0-10.5, thermally-induced protein aggregation occurs. The obtained results demonstrate the existence of partially folded state of Stn II, which may contribute to the pore formation ability of this cytolysin.

The highly refined solution structure of the cytotoxic ribonuclease alpha-sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity.

alpha-Sarcin selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA, that inactivates the ribosome. The elucidation of the three-dimensional solution structure of this 150 residue enzyme is a crucial step towards understanding alpha-sarcin's conformational stability, ribonucleolytic activity, and its exceptionally high level of specificity. Here, the solution structure has been determined on the basis of 2658 conformationally relevant distances restraints (including stereoespecific assignments) and 119 torsional angular restraints, by nuclear magnetic resonance spectroscopy methods. A total of 60 converged structures have been computed using the program DYANA. The 47 best DYANA structures, following restrained energy minimization by GROMOS, represent the solution structure of alpha-sarcin. The resulting average pairwise root-mean-square-deviation is 0.86 A for backbone atoms and 1.47 A for all heavy atoms. When the more variable regions are excluded from the analysis, the pairwise root-mean-square deviation drops to 0.50 A and 1.00 A, for backbone and heavy atoms, respectively. The alpha-sarcin structure is similar to that reported for restrictocin, although some differences are clearly evident, especially in the loop regions. The average rmsd between the structurally aligned backbones of the 47 final alpha-sarcin structures and the crystal structure of restrictocin is 1.46 A. On the basis of a docking model constructed with alpha-sarcin solution structure and the crystal structure of a 29-nt RNA containing the sarcin/ricin domain, the regions in the protein that could interact specifically with the substrate have been identified. The structural elements that account for the specificity of RNA recognition are located in two separate regions of the protein. One is composed by residues 51 to 55 and loop 5, and the other region, located more than 11 A away in the structure, is the positively charged segment formed by residues 110 to 114.

1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease alpha-Sarcin.

The ribosome-inactivating protein alpha-Sarcin (alpha S) is a 150-residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure-dynamics-function relationships in this system, we have carried out the assignment of the 1H and 15N NMR spectrum of alpha S on the basis of homonuclear (1H-1H) and heteronuclear (1H-15N) two-dimensional correlation spectra of a uniformly 15N-labeled sample, and two selectively 15N-labeled (Tyr and Phe) samples, as well as a single three-dimensional experiment. The secondary structure of alpha S, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N-terminal beta-hairpin, a short alpha-helical segment, and a C-terminal beta-sheet of five short strands arranged in a + 1, + 1, + 1, + 1 topology, connected by long loops in which the 13 Pro residues are located.

A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens.

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.

Overproduction and purification of biologically active native fungal alpha-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.

The cytotoxin alpha-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 alpha-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 (KM), kcat and kcat/KM have also been calculated. The obtained results fit into a two-step mechanism for the enzymatic activity of alpha-sarcin and allow to consider this protein as a cyclizing RNase.

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

alpha-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 a-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.

Sticholysin II, a cytolysin from the sea anemone Stichodactyla helianthus, is a monomer-tetramer associating protein.

Sticholysin II (Stn-II) is a pore-forming cytolysin. Stn-II interacts with several supports for size exclusion chromatography, which results in an abnormal retardation precluding molecular mass calculations. Sedimentation equilibrium analysis has revealed that the protein is an associating system at neutral pH. The obtained data fit a monomer-tetramer equilibrium with an association constant K4c of 10(9) M(-3). The electrophoretic pattern of Stn-II treated with different cross-linking reagents, in a wide range of protein concentrations, corroborates the existence of tetrameric forms in solution. A planar configuration of the four monomers, C4 or D2 symmetry, is proposed from modelling of the cross-linking data.

gamma-Acetylenic-gamma-aminobutyrate as an enzyme-activated inhibitor of D-amino acid transaminase.

Bacterial D-amino acid transaminase undergoes complete inactivation by gamma-acetylenic GABA. This inactivation is completely prevented by D-alanine and partially prevented by L-alanine. During inactivation the coenzyme portion of the enzyme undergoes significant spectral changes.

Ribonuclease U2: cloning, production in Pichia pastoris and affinity chromatography purification of the active recombinant protein.

RNase U2 is an endoribonuclease secreted by the fungus Ustilago sphaerogena. Its genomic DNA (rnu2), containing an intron of 116 bp, has been isolated and cloned. The corresponding cDNA has also been synthesized. The recombinant RNase U2 was successfully produced in Pichia pastoris, fused to the yeast alkaline phosphatase signal peptide. The recombinant RNase U2, purified by affinity chromatography, contains three extra amino acids at its amino-terminal end and retains the enzymatic and spectroscopic properties of the natural fungal protein.

Ribotoxins are a more widespread group of proteins within the filamentous fungi than previously believed.

Alpha-sarcin, restrictocin and mitogillin are the best known members of the family of fungal ribotoxins. In recent years, new members of this family have been discovered and characterised. In this work, we study the occurrence of ribotoxins among different species of fungi. The presence of ribotoxins has been identified in some new species by means of genetic studies, as well as expression and activity assays. The ribotoxin genes have been partially sequenced, and demonstrate a high degree of similarity. These studies demonstrate that these toxins are more widespread than previously considered. This is surprising, considering the ribotoxins are such specific and potent toxins, of unknown biological function. These studies confirm the hypothesis that these proteins are naturally engineered toxins derived from ribonucleases of broad substrate specificity.