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1.
Sci Rep ; 9(1): 11680, 2019 08 12.
Article in English | MEDLINE | ID: mdl-31406218

ABSTRACT

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.


Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Carcinoembryonic Antigen/genetics , Colorectal Neoplasms/therapy , Endoribonucleases/chemistry , Fungal Proteins/chemistry , Immunotoxins/pharmacology , Recombinant Fusion Proteins/pharmacology , Animals , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/immunology , Biomarkers, Tumor/genetics , Biomarkers, Tumor/immunology , Carcinoembryonic Antigen/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/genetics , Colorectal Neoplasms/immunology , Colorectal Neoplasms/mortality , Endoribonucleases/genetics , Endoribonucleases/immunology , Fungal Proteins/genetics , Fungal Proteins/immunology , Gene Expression , Humans , Immunotoxins/chemistry , Immunotoxins/genetics , Male , Mice , Mice, Nude , Pichia/genetics , Pichia/metabolism , Plasmids/chemistry , Plasmids/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Survival Analysis , Tumor Burden/drug effects , Xenograft Model Antitumor Assays
2.
Article in English | MEDLINE | ID: mdl-20232776

ABSTRACT

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.


Subject(s)
Allergens/immunology , Aspergillus fumigatus/immunology , Fungal Proteins/immunology , Hypersensitivity/therapy , Allergens/genetics , Animals , Antigens, Plant , Female , Fungal Proteins/genetics , Gene Deletion , Hypersensitivity/immunology , Hypersensitivity/pathology , Immunoglobulin E/blood , Lung/pathology , Mice , Mice, Inbred BALB C , Nasal Mucosa/pathology
3.
Curr Protein Pept Sci ; 8(6): 558-72, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18220843

ABSTRACT

Actinoporins are a family of 20-kDa, basic proteins isolated from sea anemones, whose activity is inhibited by preincubation with sphingomyelin. They are produced in monomeric soluble form but, when binding to the plasma membrane, they oligomerize to produce functional pores which result in cell lysis. Equinatoxin II (EqtII) from Actinia equina and Sticholysin II (StnII) from Stichodactyla helianthus are the actinoporins that have been studied in more detail. Both proteins display a beta-sandwich fold composed of 10 beta-strands flanked on each side by two short alpha-helices. Two-dimensional crystallization on lipid monolayers has allowed the determination of low-resolution models of tetrameric structures distinct from the pore. However, the actual structure of the pore is not known yet. Wild-type EqtII and StnII, as well as a nice collection of natural and artificially made variants of both proteins, have been produced in Escherichia coli and purified. Their characterization has allowed the proposal of a model for the mechanism of pore formation. Four regions of the actinoporins structure seem to play an important role. First, a phosphocholine-binding site and a cluster of exposed aromatic residues, together with a basic region, would be involved in the initial interaction with the membrane, whereas the amphipathic N-terminal region would be essential for oligomerization and pore formation. Accordingly, the model states that pore formation would proceed in at least four steps: Monomer binding to the membrane interface, assembly of four monomers, and at least two distinct conformational changes driving to the final formation of the functional pore.


Subject(s)
Cell Membrane/metabolism , Pore Forming Cytotoxic Proteins/chemistry , Pore Forming Cytotoxic Proteins/metabolism , Protein Folding , Sea Anemones/cytology , Sea Anemones/metabolism , Amino Acid Sequence , Animals , Molecular Sequence Data , Pore Forming Cytotoxic Proteins/genetics , Solubility
4.
Eur J Biochem ; 268(23): 6190-6, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11733014

ABSTRACT

Alpha-sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three-dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of alpha-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome-inactivating activity of alpha-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of alpha-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of alpha-sarcin and presumably of the other fungal ribotoxins.


Subject(s)
Cytotoxins/chemistry , Cytotoxins/toxicity , Endoribonucleases/chemistry , Endoribonucleases/toxicity , Fungal Proteins , Mycotoxins/chemistry , Mycotoxins/toxicity , Amino Acid Substitution , Apoptosis/drug effects , Arginine/chemistry , Aspergillus/enzymology , Aspergillus/genetics , Cytotoxins/genetics , Endoribonucleases/genetics , Humans , Liposomes , Models, Molecular , Mutagenesis, Site-Directed , Mycotoxins/genetics , Phospholipids , Protein Conformation , Tumor Cells, Cultured
6.
Protein Sci ; 10(8): 1658-68, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11468362

ABSTRACT

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, alpha-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 beta-hairpin of alpha-sarcin, a characteristic structure that is absent in other nontoxic structurally related microbial RNases. These two residues of alpha-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 alpha-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 beta-hairpin in the interaction with both membranes and polyadenylic acid.


Subject(s)
Allergens , Aspergillus/chemistry , Cytotoxins/metabolism , Endoribonucleases/metabolism , Fungal Proteins/metabolism , Ribonucleases/metabolism , Amino Acid Substitution , Antigens, Plant , Aspergillus/genetics , Aspergillus/metabolism , Cytotoxins/chemistry , Cytotoxins/genetics , Endoribonucleases/chemistry , Endoribonucleases/genetics , Endoribonucleases/toxicity , Escherichia coli/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/toxicity , Humans , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Molecular Structure , Mutagenesis, Site-Directed , Mycotoxins/chemistry , Mycotoxins/genetics , Mycotoxins/metabolism , Mycotoxins/toxicity , Protein Denaturation , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Synthesis Inhibitors/chemistry , Protein Synthesis Inhibitors/metabolism , Ribonucleases/chemistry , Ribonucleases/genetics , Ribonucleases/toxicity , Ribosomes/metabolism , Spectrometry, Fluorescence , Temperature , Tumor Cells, Cultured
7.
Biochim Biophys Acta ; 1545(1-2): 122-31, 2001 Feb 09.
Article in English | MEDLINE | ID: mdl-11342038

ABSTRACT

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.


Subject(s)
Cnidarian Venoms/chemistry , Hemolysin Proteins/chemistry , Anilino Naphthalenesulfonates/metabolism , Circular Dichroism , Hydrogen-Ion Concentration , Osmolar Concentration , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Sodium Chloride/pharmacology , Spectrometry, Fluorescence , Structure-Activity Relationship , Temperature
8.
Eur J Biochem ; 268(7): 2113-23, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11277935

ABSTRACT

Alpha-sarcin is a ribosome-inactivating protein that has been well characterized in vitro, but little is known about its toxicity in living cells. We have analyzed the mechanism of internalization of alpha-sarcin into human rhabdomyosarcoma cells and the cellular events that result in the induction of cell death. No specific cell surface receptor for alpha-sarcin has been found. The toxin is internalized via endocytosis involving acidic endosomes and the Golgi, as deduced from the ATP requirement and the effects of NH4Cl, monensin and nigericin on its cytotoxicity. Specific cleavage of 28S rRNA in cultured rhabdomyosarcoma cells, associated with protein biosynthesis inhibition, has been detected. alpha-Sarcin kills rhabdomyosarcoma cells via apoptosis: incubation of cells with alpha-sarcin at a concentration below its IC50 induces internucleosomal genomic DNA fragmentation, reversion of membrane asymmetry, activation of caspase-3-like activity and cleavage of poly(ADP-ribose)polymerase. Apoptosis is not a general direct consequence of protein biosynthesis inhibition, as deduced from the comparative analysis of the effects of alpha-sarcin and cycloheximide; the latter does not induce apoptosis even at concentrations far beyond its IC50, where protein biosynthesis is null. Experiments with a catalytically inactive alpha-sarcin mutant, neither toxic nor apoptotic, reveal that induced apoptosis is directly related to the effects of catalytic activity of the toxin on the ribosomes. The caspase inhibitor z-VAD-fmk does not suppress protein synthesis inhibition by alpha-sarcin. Together, these data suggest that alpha-sarcin-induced caspase activation is a pathway downstream of the 28S rRNA catalytic cleavage and consequent protein biosynthesis inhibition.


Subject(s)
Apoptosis/drug effects , Endoribonucleases/pharmacology , Fungal Proteins , Protein Synthesis Inhibitors/pharmacology , Caspase 3 , Caspases/metabolism , Cycloheximide/pharmacology , DNA Fragmentation , Electrophoresis, Polyacrylamide Gel , Endocytosis , Flow Cytometry , Humans , Molecular Weight , Phosphatidylserines/metabolism , RNA, Ribosomal/metabolism , Rhabdomyosarcoma/metabolism , Tumor Cells, Cultured
9.
Proteins ; 41(3): 350-61, 2000 Nov 15.
Article in English | MEDLINE | ID: mdl-11025546

ABSTRACT

alpha-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 alpha-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 degrees C drop in the thermal denaturation midpoint, and the double mutant's midpoint is 9 degrees 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.


Subject(s)
Cytotoxins/chemistry , Endoribonucleases/chemistry , Fungal Proteins , Protein Synthesis Inhibitors/chemistry , Tryptophan/chemistry , Anthracenes/pharmacology , Circular Dichroism , Cytotoxins/genetics , Endoribonucleases/drug effects , Endoribonucleases/genetics , Fluorescence Polarization , Hot Temperature , Models, Molecular , Molecular Probes , Mutation , Mycotoxins/chemistry , Mycotoxins/genetics , Nuclear Magnetic Resonance, Biomolecular , Phosphatidylglycerols , Protein Conformation , Protein Denaturation , Recombinant Proteins/chemistry , Spectrophotometry, Ultraviolet
10.
FEMS Microbiol Lett ; 189(2): 165-9, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10930732

ABSTRACT

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.


Subject(s)
Endoribonucleases/genetics , Pichia/genetics , Amino Acid Sequence , Base Sequence , Chromatography, Affinity , Cloning, Molecular , Endoribonucleases/isolation & purification , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
11.
J Mol Biol ; 299(4): 1061-73, 2000 Jun 16.
Article in English | MEDLINE | ID: mdl-10843858

ABSTRACT

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.


Subject(s)
Allergens , Aspergillus/chemistry , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Animals , Antigens, Plant , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Hydrogen Bonding , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Secondary , Rats , Ribonucleases/chemistry , Solutions , Static Electricity , Structure-Activity Relationship , Substrate Specificity , Surface Properties
12.
Biophys J ; 78(6): 3186-94, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10827995

ABSTRACT

Sticholysin II (Stn II), a potent cytolytic protein isolated from the sea anemone Stichodactyla helianthus, has been crystallized on lipid monolayers. With Fourier-based methods, a three-dimensional (3D) model of Stn II, up to a resolution of 15 A, has been determined. The two-sided plane group is p22(1)2, with dimensions a = 98 A, b = 196 A. The 3D model of Stn II displays a Y-shaped structure, slightly flattened, with a small curvature along its longest dimension (51 A). This protein, with a molecular mass of 19. 2 kDa, is one of the smallest structures reconstructed with this methodology. Two-dimensional (2D) crystals of Stn II on phosphatidylcholine monolayers present a unit cell with two tetrameric motifs, with the monomers in two different orientations: one with its longest dimension lying on the crystal plane and the other with this same axis leaning at an angle of approximately 60 degrees with the crystal plane.


Subject(s)
Cnidarian Venoms/chemistry , Hemolysin Proteins/chemistry , Liposomes , Animals , Cholesterol , Crystallization , Fourier Analysis , Image Processing, Computer-Assisted , Microscopy, Electron , Models, Molecular , Phosphatidylcholines , Sea Anemones , Software , Sphingomyelins
13.
Protein Expr Purif ; 18(1): 71-6, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10648171

ABSTRACT

The cDNA coding for the cytolytic toxins sticholysin I and sticholysin II from the sea anemone Stichodactyla helianthus has been isolated, cloned in pUC18, and sequenced. A 6His-tagged version of sticholysin II has been overproduced in Escherichia coli and purified to homogeneity in milligram amounts. Conformational and functional analyses of recombinant sticholysin II do not reveal any significant difference when compared to the natural cytolysin.


Subject(s)
Cnidarian Venoms/biosynthesis , Cnidarian Venoms/genetics , Hemolysin Proteins/biosynthesis , Hemolysin Proteins/genetics , Sea Anemones/genetics , Sea Anemones/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Cnidarian Venoms/isolation & purification , DNA Primers/genetics , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Escherichia coli/genetics , Hemolysin Proteins/isolation & purification , Molecular Sequence Data , Organic Chemicals , Protein Conformation , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid
14.
Proteins ; 37(3): 474-84, 1999 Nov 15.
Article in English | MEDLINE | ID: mdl-10591106

ABSTRACT

alpha-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 different 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 alpha-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 alpha-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 alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.


Subject(s)
Aspergillus/chemistry , Cytotoxins/chemistry , Endoribonucleases/chemistry , Fungal Proteins/chemistry , Glutamic Acid/chemistry , Histidine/chemistry , Catalytic Domain , Circular Dichroism , Cytotoxins/biosynthesis , Cytotoxins/genetics , Endoribonucleases/biosynthesis , Endoribonucleases/genetics , Escherichia coli/metabolism , Fungal Proteins/biosynthesis , Fungal Proteins/genetics , Magnetic Resonance Spectroscopy , Molecular Conformation , Mutagenesis, Site-Directed , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spectrometry, Fluorescence
15.
FEBS Lett ; 455(1-2): 27-30, 1999 Jul 16.
Article in English | MEDLINE | ID: mdl-10428465

ABSTRACT

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.


Subject(s)
Cytotoxins/chemistry , Sea Anemones/chemistry , Animals , Biopolymers , Chromatography, Gel , Circular Dichroism , Cytotoxins/isolation & purification , Cytotoxins/pharmacology , Hydrogen-Ion Concentration , Molecular Weight , Spectrophotometry, Ultraviolet
17.
Mol Membr Biol ; 15(3): 141-4, 1998.
Article in English | MEDLINE | ID: mdl-9859111

ABSTRACT

alpha-Sarcin is a cytotoxic protein that specifically inactivates ribosomes. The protein translocates across phospholipid membranes. Oligomerization of the protein occurs upon interaction with membranes. Chemically cross-linked protein oligomers have been obtained by treatment of protein-vesicle complexes with the membrane impermeant reagent bis-(sulfosuccinimidyl) suberate. These structures are only obtained in the presence of acidic lipid vesicles composed of either natural or synthetic phospholipids. Such oligomers are not produced in concentrated protein solutions in the absence of vesicles. The formation of the chemically stabilized oligomers is saturated at the same lipid to protein molar ratio as all the perturbations caused by alpha-sarcin on lipid vesicles. Results are discussed in terms of the involvement of oligomer formation on protein translocation across membranes.


Subject(s)
Cytotoxins/metabolism , Endoribonucleases/metabolism , Fungal Proteins/metabolism , Lipid Bilayers/metabolism , Oligopeptides/metabolism , Phospholipids/metabolism , Animals , Aspergillus , Cattle
18.
Biochemistry ; 37(45): 15865-76, 1998 Nov 10.
Article in English | MEDLINE | ID: mdl-9843392

ABSTRACT

The electrostatic behavior of titrating groups in alpha-sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0-8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the alpha-sarcin-2'-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, the pKa values for active site residues His50, Glu96, and His137 were 7.7, 5.2, and 5.8 in the free enzyme and 7.6, approximately 4.8, and 6.8 in the alpha-sarcin-2'-GMP complex, respectively. The pKa values and the activity profile against ApA, as a function of pH, are in agreement with the proposed enzymatic mechanism (in common with RNase T1 and the family of the microbial ribonucleases), in which Glu96 and His137 act as a general base and general acid, respectively. In almost all microbial ribonucleases, a Phe-His interaction is present, which affects the pKa of one of the His residues at the active site (His137). The absence of this interaction in alpha-sarcin would explain the lower pKa value of this His residue, and provides an explanation for the decreased RNase activity of this protein as compared to those of other microbial ribonucleases.


Subject(s)
Cytotoxins/chemistry , Endoribonucleases/chemistry , Fungal Proteins/chemistry , Aspergillus , Catalysis , Cytotoxins/metabolism , Endoribonucleases/metabolism , Enzyme Activation , Fungal Proteins/metabolism , Guanine Nucleotides/chemistry , Histidine/chemistry , Hydrogen-Ion Concentration , Macromolecular Substances , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protons , Ribonuclease T1/chemistry , Static Electricity , Titrimetry
19.
Protein Expr Purif ; 12(3): 315-22, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9535698

ABSTRACT

alpha-Sarcin is a ribosome-inactivating protein from the mold Aspergillus giganteus. The methylotrophic yeast Pichia pastoris has been transformed with two plasmids (pHILD2prealphaS and pHILS1prealphaS), which contain the complete alpha-sarcin cDNA, including its original fungal leader peptide, under the control of yeast alcohol oxidase promoter. The second one is indeed fused to the signal sequence of P. pastoris acid phosphatase. The transformed yeasts secreted both mature and pro-alpha-sarcin. The presence of this pro-alpha-sarcin in the yeast extracellular medium is due to an inefficient recognition of the pro-sequence by a putative Kex2p-like endopeptidase. A third plasmid accounting for a single mutation of the alpha-sarcin leader peptide was designed to produce a more efficient Kex2p recognition motif. This approach resulted in the extracellular production of only the mature protein, suggesting the existence of a two-step mechanism for processing its leader peptide. This recombinant alpha-sarcin is identical to the original fungal protein, according to activity and spectroscopic criteria. In addition, pro-alpha-sarcin, which has been characterized for the first time, also exhibits ribonucleolytic activity as the mature protein does. Therefore, protection of the producing cells against this kind of ribotoxins may depend on an efficient recognition of the signal sequence followed by translocation of the nascent polypeptide to the endoplasmic reticulum.


Subject(s)
Cytotoxins/genetics , Endoribonucleases/genetics , Fungal Proteins , Gene Expression Regulation, Fungal/genetics , Mycotoxins/genetics , Pichia/genetics , Protein Precursors/genetics , Amino Acid Sequence , Aspergillus/genetics , Aspergillus/metabolism , Base Sequence , Circular Dichroism , Cytotoxins/chemistry , Cytotoxins/metabolism , DNA, Fungal/chemistry , DNA, Fungal/genetics , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Molecular Sequence Data , Mycotoxins/chemistry , Mycotoxins/metabolism , Pichia/metabolism , Plasmids/chemistry , Plasmids/genetics , Protein Precursors/chemistry , Protein Precursors/metabolism , Protein Sorting Signals/chemistry , Protein Sorting Signals/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
20.
Eur J Biochem ; 252(2): 284-9, 1998 Mar 01.
Article in English | MEDLINE | ID: mdl-9580155

ABSTRACT

A potent hemolytic polypeptide, sticholysin II, has been purified to homogeneity from the sea anemone Stichodactyla helianthus. The protein produces leakage of aqueous contents of model lipid vesicles composed of either phosphatidylcholine or sphingomyelin if cholesterol is present in these membranes. The leakage has been analyzed by measuring the dequenching of the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonic acid, coencapsulated with its quencher N,N'-p-xylenebispyridinium bromide, upon dilution of the vesicle contents into the external medium. The protein displays a maximum effect on vesicles containing 20-25% cholesterol. Leakage is also produced in vesicles composed of mixtures of phosphatidylcholine and sphingomyelin, the maximum effect being observed for 20-30% sphingomyelin molar content. The extent of the leakage is dependent on the molecular mass of the vesicle entrapped solutes in the range 445-960 Da. This suggests the involvement of a pore of about 1 nm in diameter based on the limiting size observed for the leakage of the different solutes. Oligomerization of the protein is apparently involved in the membrane permeabilization, based on the kinetic analysis of the leakage process which is shown to proceed through an all-or-none mechanism.


Subject(s)
Hemolysin Proteins/pharmacology , Lipid Bilayers/metabolism , Peptides/pharmacology , Permeability/drug effects , Sea Anemones/chemistry , Amino Acid Sequence , Animals , Cholesterol/metabolism , Drug Compounding , Fluorescent Dyes/metabolism , Kinetics , Liposomes/metabolism , Molecular Sequence Data , Naphthalenes/metabolism , Particle Size , Phosphatidylcholines/metabolism , Protein Conformation , Sphingomyelins/metabolism
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