The carboxyl-terminal region of human interferon γ is important for biological activity: mutagenic and NMR analysis

Abstract

Deletion of nine amino acids from the carboxyl terminus of human IFN gamma (residues 138--146; LFRGRRASQ) resulted in a 7-fold increase in specific antiviral activity. Similar increases in receptor binding affinity were seen. Deletion of residues 136 and 137 (QM) had little additional effect, but removal of Ser135 resulted in a sharp drop in antiviral activity. Further removal of residues 133 and 134 (KR) lowered antiviral activity to 1% of the peak value. Comparison of the proton NMR spectra of selected deletions down to residue 132 showed that there was no significant change in the core protein structure. Deletions down to residue 125 had the same antiviral activity as those to 132, but changes could now be seen in the aromatic proton NMR spectrum of this shorter derivative. Substitution of the homologous murine sequence between residues 124 and 130 (human SPAAKTG; murine LPESSLR) resulted in only a small decrease in antiviral activity, further suggesting that the precise sequence in this region was not critical for activity. Ser135 was substituted with a number of other amino acids with little or no change in activity. The importance of the residues between 131 and 134 for biological activity was corroborated by mutagenesis, although some substitutions in this region were tolerated.

Molecular Clonings and Sequences of Djungarian (Phodopus sungorus) and Chinese (Cricetulus griseus) Hamster Interferon-Gammas

“…The unique KRKR polycationic tail at the 3' end of the C-terminal α-helix is required for biological activity [9]. Removal of this region leads to a complete loss of activity of the respective mouse protein [7]. These hamster and gerbil sequences or woodchuck and human ones have an additional 17 or 9 amino acids at the C-terminus, respectively, compared with the mouse and rat sequences [7].…”

“…Removal of this region leads to a complete loss of activity of the respective mouse protein [7]. These hamster and gerbil sequences or woodchuck and human ones have an additional 17 or 9 amino acids at the C-terminus, respectively, compared with the mouse and rat sequences [7]. Removal of the C-terminal 9 amino acids from the human IFN-γ protein was found to significantly enhance its antiviral activity [7], so it is thought that these additional residues sterically block the proximal residues from a strong interaction with the IFN-γ receptor.…”

“…These hamster and gerbil sequences or woodchuck and human ones have an additional 17 or 9 amino acids at the C-terminus, respectively, compared with the mouse and rat sequences [7]. Removal of the C-terminal 9 amino acids from the human IFN-γ protein was found to significantly enhance its antiviral activity [7], so it is thought that these additional residues sterically block the proximal residues from a strong interaction with the IFN-γ receptor. The biological activity may increase when the 17 amino acid additional residues in 3 hamster and gerbil IFNγs are removed from the C-terminal.…”

Signaling by Covalent Heterodimers of Interferon-γ

“…␥ m -␥ and ␥-␥ m were purified as described (12). Briefly, purification involved sonication of a bacterial suspension expressing the proteins, dialysis against 20 mM Tris, 1 M NaCl, pH 7.5, adsorption onto a nickel-Sepharose 6B column, and elution with 25 mM imidazole, 20 mM Tris, pH 7.5, 1 M NaCl, 5 mM benzamidine.…”

“…It was previously demonstrated that wild type IFN-␥ (␥⅐␥) (33-34 kDa) can bind to two IFN-␥R1 EC s (40 kDa) or two cell surface receptors simultaneously (3,12,13). Thus, a soluble ternary complex would have a molecular mass of 114 kDa, and a binary complex would have a molecular mass of 74 kDa.…”