Significance of the Putative Upstream Polybasic Nuclear Localisation Sequence for the Biological Activity of Human Interferon-Gamma

Abstract

Interferon-gamma (IFNγ) accomplishes its multiple biological effects by activating the STAT transcription factors, which are translocated to the nucleus through a specific nuclear localization sequence(s) (NLS) located in the IFNγ molecule. Two putative NLS have been pointed out in the human interferon gamma (hIFN): an upstream located in helix E (residues 83-89) and a downstream located at the C-terminal unstructured region (residues 124-132). To investigate the significance of the putative upstream NLS for the biological activity of hIFN we have introduced a point mutation in the hIFN gene to disturb the polybasic sequence typical for the conventional NLS. In the new gene a gln codon was substituted for the Lys88 codon and the mutated gene was cloned and expressed in E. coli LE392. This mutation led to a 1000-fold decrease in both hIFN antiviral and antiproliferative activities. When co-incubated with the wild-type hIFN (standard), the mutant hIFN competed for the cellular receptors that led to a 30% inhibition of the standard activity. This indicates that the mutation does not interfere with the interaction of the protein to the receptor but probably affects the intracellular signal transduction pathway. To avoid any putative compensatory effect in the function of the upstream NLS caused by the downstream C-terminal NLS, 21 C-terminal codons were deleted from the mutant hIFN gene. The latter resulted in only 10-fold additional decrease in biological activity and 50% inhibition of the standard activity in the competition assay. Our data indicate that the upstream NLS is endowed with a greater functional significance for the hIFNmediated signal transduction rather than the downstream NLS.