Histidine-15: an important role in the cytotoxic activity of human tumor necrosis factor

The amino acids that are required for the cytotoxic activityof recombinant human tumor necrosis factor- (TNF) were investigatedby chemical modification and oligonucleotide-directed site-specificmutagenesis. TNF contains three histidine residues, locatedat positions 15, 73 and 78. The histidine-specific reagent diethylpyrocarbonate(DEP) was used to chemically modify TNF. The chemical inactivationof the in vitro cytotoxic activity of this lymphokine (usingmurine L929 target cells) was found to be time- and dose-dependent.Inactivated TNF failed to compete with fully bioactive [¹²⁵I]TNFfor human MCF-7 target cell receptors. Mutant polypeptides ofTNF were genetically engineered by oligonucoleotide-directedsite-specific mutagenesis. The cytotoxicity of a double histidinemutant, in which histidine-73 and histidine-78 were replacedwith glutamine, was not altered and was chemically inactivatedby DEP. Substituting glutamine for histidine-15 resulted in10–15% of the wild-type bioactivity. Replacing histidine-15with either asparagine, lysine or glycine resulted in a biologicallyinactive molecule. The data show that the histidine residueat position 15 is an amino acid that is required for the cytotoxicactivity of TNF.