Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin α‐sarcin

Abstract

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 In this work, Arg121 of a-sarcin has been replaced by Gln or Lys.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...

The Therapeutic Potential of Fungal Ribotoxins

“…These mutational studies have revealed the involvement of several residues, which are conserved among the different microbial extracellular RNases, in catalysis. Thus, it is well known that His137 and Glu96 are the only residues that are essential for the cleavage reaction performed by α-sarcin [35,45,[70][71][72][73][74][75], whereas His-50, Tyr-48, Arg-121, and Leu-145 mostly contribute to the stabilization of the transition state [45][46][47][48], as stated above. Most of these residues are located in the central β-sheet and their side-chains point towards the concave face of the protein structure where the substrate is supposed to dock [29].…”

“…The study of the mentioned mutants suggested that it would be located within the hydrophobic core of the phospholipid bilayer once the protein-lipid complexes were formed [46,78]. Within this same idea, mutants affecting α-sarcin active site residue Arg121 (R121K and R121Q),…”

“…Mutational studies have also revealed that three other active site residues, Tyr48, Arg121, and Leu145, although not essential, appear to be determinants of the ribotoxin activity of α-sarcin [46][47][48]. Studies on the crystal structures of complexes of restrictocin with inhibitors led to the proposal that these ribotoxins may use base flipping to enable cleavage at the correct site of the SRL substrates [49].…”

“…showed that the loss of the positive charge at that position produced a dramatic impairment of the protein's ability to interact with phospholipid membranes [46].…”

Role of the basic character of α-sarcin’s NH2-terminal β-hairpin in ribosome recognition and phospholipid interaction

“…This high content of positively charged residues, mostly located at the loops and the NH 2 -terminal β-hairpin, is probably required for recognizing and binding not only to its highly negatively charged target, the SRL rRNA, but also to ribosomal proteins and cellular membranes [18,36,37]. In this context, the role of the positively charged residues located at the NH 2 -terminal β-hairpin (Table 1) in these different but closely related events has been studied in the work herein presented.…”