UBC Theses and Dissertations
Molecular dissection of Mitogillin, a fungal ribotoxin Kao, Richard Yi Tsun
Fungal ribotoxins, such as mitogillin and the related Aspergillus toxins restrictocin and α-sarcin, are small (~17 kDa) basic ribosome-inactivating proteins (RIPs) which catalytically inactivate the large ribosomal subunits of all organisms tested so far; they act as specific ribonucleases by hydrolyzing one single phosphodiester bond in the universally conserved α-sarcin/ricin loop (SRL) of 23 - 28S rRNAs and are among the most potent inhibitors of protein synthesis known. The site of cleavage occurs between G4325 and A4326 (rat ribosome numbering), which is conserved among the large subunit rRNAs of all living species. Amino acid sequence comparison of fungal ribotoxins and guanyl/purine ribonucleases has identified domains or residues likely to be involved in ribonucleolytic activity or cleavage specificity. The amino acid residues involved in the cytotoxic activities of mitogillin were investigated by introducing hydroxylamine-induced point mutations into a recombinant Metmature mitogillin gene (mitogillin with a Met codon at the N-terminus and no leader sequence) constructed from an Aspergillus fumigatus cDNA clone. These constructs were cloned into a yeast expression vector under the control of the GAL1 promoter and transformed into Saccharomyces cerevisiae. Upon induction of mitogillin expression, surviving transformants revealed that substitutions of certain amino acid residues on mitogillin abolished its cytotoxicity. Non-toxic mutant genes were cloned into an E.coli expression vector, the proteins over-expressed and purified to homogeneity, and their activities examined by in vitro ribonucleolytic assays. These studies identified the His49Tyr, Glu95Lys, Arg120Lys, and His136Tyr mutations as having profound impact on the ribonucleolytic activities of mitogillin suggesting that these residues are key components of the active site contributing to the catalytic activities of mitogillin. Fifteen deletion mutants (each 4 to 8 amino acid deletions) in motifs of mitogillin lacking amino acid sequence homology with guanyl/purine ribonucleases were constructed by site-directed mutagenesis. Analyses of the purified mutant proteins identified the lysine rich region loop 4 (L4) region and β sheet 1-loop 1-β sheet 2 (B1-L1-B2) domain in fungal ribotoxins as contributing to ribosome-targeting and modulating the catalytic activity of the toxin; these regions show strong sequence similarity to ribosomal proteins and elongation factors. Nine mutant mitogillins that were capable of digesting polyinosinic acid [poly(I)] but unable to cleave the SRL in rabbit reticulocyte ribosomal RNA were isolated after screening E. coli encoding partially degenerate oligonucleotide sequences in the lysine rich L4 region (Lys106-Phe107-Asp108-Ser 109-Lys110-Lys111-Pro112-Lys 113) of the mitogillin gene. Further investigation by site-directed mutagenesis indicated that Lys111 plays an important role in substrate recognition; a Lys111Gln change markedly reduced the ability of mitogillin to specifically cleave the rabbit ribosomal RNA or a 35-mer oligoribonucleotide mimicking the SRL. In addition, a variant mitogillin with an Asn7Ala substitution in the B1-L1-B2 domain exhibited elevated ribonucleolytic activity and reduced substrate specificity, suggesting the involvement of this domain in substrate selection. This mutational study of mitogillin taken together with the recently published X-ray structure of restrictocin (a close relative of mitogillin) supports the hypothesis that the specific cleavage properties of ribotoxins are the result of natural genetic engineering of T1- like ribonucleases in which the ribosomal targeting elements of ribosome-associated proteins were inserted into nonessential regions of the nuclease protein.
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