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.
a-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 threedimensional 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 In this work, Arg121 of a-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosomeinactivating activity of a-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of a-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of a-sarcin and presumably of the other fungal ribotoxins.Keywords: Aspergillus protein; cytotoxin; protein-lipid interaction; ribotoxin; a-sarcin. a-Sarcin is the best characterized member of a family of fungal cytotoxic ribosome-inactivating proteins (ribotoxins) that cleave one single phosphodiester bond at an evolutionarily conserved sequence of the larger rRNA [1], impairing the binding of elongation factors EF-1 and EF-2 [2]. This well known exquisite ribonucleolytic action in cell-free systems has been also recently observed in intact cells, namely human rhabdomyosarcoma cells, where the toxic effect produces cell death via apoptosis [3]. a-Sarcin is internalized by the target cells via endocytosis involving acidic endosomes and the Golgi [3,4]. This traffic might be related to the known ability of a-sarcin to interact with model membranes. In fact, besides its specific ribonuclease activity, this highly polar protein binds to acidic lipid vesicles promoting their aggregation and fusion [5,6] and resulting in translocation of the protein across the model membranes [7,8].Ribotoxins of the a-sarcin family display a high degree of sequence similarity although they are secreted by a variety of different moulds [9-11], i.e. restrictocin and mitogillin, two other members of this family, display 85% sequence identity to a-sarcin, which is only one residue longer [12][13][14]. These ribotoxins belong to a larger group of microbial extracellular RNases, represented by the noncytotoxic RNases T1 and U2 [10,11,15], based on their sequence and three-dimensional structure similarities. Thus, a-sarcin [15], restrictocin [16] and RNase T1 [17] display an identical architecture and connectivity of the secondary structure elements in the three proteins. The catalytic mechanisms of a-sarcin and RNase T1 are similar [18][19][20] and related to that of bovine pancreatic RNase A [21]. They behave as cyclizing RNases, the overall reaction being composed of two steps, cyclizati...
Z O . 2000. The yield of puri®ed recombinant a-sarcin increases approximately three-to fourfold when this toxin is co-expressed in Escherichia coli with thioredoxin. This increased production is attributed to the existence, in the presence of thioredoxin, of a reducing environment which allows rearrangement of incorrect disulphide bonds to produce the soluble native conformation. The protein thus produced retains the structural, spectroscopic and enzymatic features of the natural fungal a-sarcin.
Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as ␣-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of ␣-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type ␣-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of ␣-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of ␣-sarcin on ribosomes.
Asparagine 54 of alpha-sarcin is a conserved residue within the proteins of the ribotoxin family of microbial ribonucleases. It is located in loop 2 of the protein, which lacks repetitive secondary structure elements but exhibits a well-defined conformation. Five mutant variants at this residue have been produced and characterized. The spectroscopic characterization of these proteins indicates that the overall conformation is not changed upon mutation. Activity and denaturation assays show that Asn-54 largely contributes to protein stability, and its presence is a requirement for the highly specific inhibitory activity of these ribotoxins on ribosomes.
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