Characterization of prokaryotic recombinant Aspergillus ribotoxin alpha-sarcin.
The Aspergillus ribonuclease alpha-sarcin is toxic to intact mammalian cells but the mechanism by which it enters the cells to reach its ribosomal RNA substrate is unclear. Here we have compared the cytotoxicity of alpha-sarcin to that of ricin, another catalytic toxin that targets the same rRNA sequence but whose mechanism of cell entry is better understood. Intact ricin binds to cell surface components and enters the cells by receptor-mediated endocytosis, whereas the catalytic polypeptide of ricin (the A chain or RTA) which, like alpha-sarcin, is unable to bind to surface components directly and enters cells by fluid phase uptake. Recombinant alpha-sarcin was produced in Escherichia coli and purified to homogeneity. The protein was soluble, stable and its ability to inhibit in vitro protein synthesis was indistinguishable from that of native alpha-sarcin. Further, recombinant alpha-sarcin had the same in vitro protein synthesis inhibition activity as ricin A chain. The cytotoxicity of alpha-sarcin and ricin A chain to HeLa cells was also the same. The cytotoxicity of alpha-sarcin was due to its RNAase activity rather than to specific membrane effects at the cell surface, since a mutant containing a single substitution at a putative key catalytic residue had reduced ribonuclease activity and an equivalent reduction in cytotoxicity. One interpretation of the data is that a-sarcin enters mammalian cells in the same way as free ricin A chain.
Sylvester
I D
ID
Department of Biological Sciences, University of Warwick, Coventry, UK.
Roberts
L M
LM
Lord
J M
JM
D001230:Aspergillus / Q000235:genetics*; D001692:Biological Transport; D003603:Cytotoxins / Q000494:pharmacology; D004722:Endoribonucleases / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D009154:Mutation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics; D012276:Ricin / Q000494:pharmacology
D003603:Cytotoxins; D005656:Fungal Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C014801:alpha-sarcin; D012276:Ricin; D004722:Endoribonucleases