Naslov
Site-specific mutagenesis of the histidine precursor of diphthamide in the human elongation factor-2 gene confers resistance to diphtheria toxin

Autori
Ivanković, Milena ; Rubelj, Ivica ; Matulić, Maja ; Reich, Edward ; Brdar, Branko

Izvornik
Mutation research. Genetic toxicology and environmental mutagenesis (1383-5718) 609 (2006), 1; 34-42

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
EF-2; diphtheria toxin; site-specific mutagenesis

Sažetak
Protein synthesis elongation factor-2 (EF-2) from eukaryots 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 the site-specific mutagenesis of the histidine precursor of diphthamide, histidine 715 in human EF-2 was substituted with one of four amino acids: leucine, methionine, asparagine or glutamine. Mutant EF-2s were cloned into pCMVexSVneo expression vector, transfected into HeLa cells and DT-resistant cell clones 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 was demonstrated by their: (1) normal morphological appearance ; (2) unaffected or slightly slower growth rates ; (3) undisturbed electrophoretic DNA profiles whose integrity was almost totally preserved. 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 may be useful as a host for various DT gene-containing vectors that express the toxin intracellularly.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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