title: Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin.
abstract: Protein synthesis elongation factor 2 (EF-2) from eukaryotes contains a conserved post-translationally modified histidine residue known as diphthamide. Diphthamide is a unique site of ADP-ribosylation by diphtheria toxin (DT), which is responsible for cell killing. In this report, we describe the construction of DT-resistant HeLa cell lines by engineering the toxin-resistant form of its specific substrate, protein elongation factor-2. Using site-specific mutagenesis of the histidine precursor of diphthamide, the histidine residue of codon 715 in human EF-2 cDNA was substituted with one of four amino acid residue codons: leucine, methionine, asparagine or glutamine. Mutant EF-2s were subcloned into a pCMVexSVneo expression vector, transfected into HeLa cells, and DT-resistant cell clones were isolated. The protective effect of mutant EF-2s against cell killing by DT, after exposing all four mutant strains derived from HeLa cells to different concentrations of the toxin (5-20 ng/mL) was demonstrated by: (1) the normal morphological appearance of the cells; (2) their unaffected or slightly slower growth rates; (3) their undisturbed electrophoretic DNA profiles whose integrity was virtually preserved. Mutant cell strains showed also considerable levels of resistance to very high concentrations of DT, in that they maintained slower but consistent rates of cell growth. It was hence concluded that despite its strict conservation and unique modification, the diphthamide histidine appears not to be essential to the function of human EF-2 in protein synthesis. In addition, DT-resistant HeLa cell clones should prove valuable hosts for various DT gene-containing vectors that express the toxin intracellularly.
authors.affiliation: Department of Molecular Biology, Ruder Bosković Institute, Bijenicka 54, 10000 Zagreb, Croatia.
authors.forename: Milena
authors.initials: M
authors.lastname: Ivankovic
authors.forename: Ivica
authors.initials: I
authors.lastname: Rubelj
authors.forename: Maja
authors.initials: M
authors.lastname: Matulic
authors.forename: Edward
authors.initials: E
authors.lastname: Reich
authors.forename: Branko
authors.initials: B
authors.lastname: Brdar
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004167:Diphtheria Toxin / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D006367:HeLa Cells; D006639:Histidine / Q000031:analogs & derivatives* / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020652:Peptide Elongation Factor 2 / Q000235:genetics*; D014162:Transfection
chemical_list: D004167:Diphtheria Toxin; D020652:Peptide Elongation Factor 2; D006639:Histidine; C027527:diphthamide