The effect of C-terminal processing on the activity of human interferon-gamma.
Homogeneous recombinant human interferon-gamma (IFN-gamma) obtained from Escherichia coli (E. coli) was treated with a protease-containing fraction prepared from mechanically lysed E. coli cells. Polyacrylamide gel electrophoresis of the resulting product revealed two major components of molecular weight less than that of intact IFN-gamma. These were purified by ion exchange chromatography in the presence of 7 M urea and shown to have intact IFN-gamma N-terminal sequences, suggesting that they resulted via C-terminal cleavages of IFN-gamma. Amino acid analysis indicated that 4 C-terminal residues of IFN-gamma were lacking in one, and 15 in the other. The species lacking 4 C-terminal residues had activities virtually indistinguishable from those of IFN-gamma in antiviral and growth inhibitory assays using Encepharomyocarditis-treated HeLa or T98G cells and in a macrophage activation assay using macrophage-like U937 cells. The species lacking 15 C-terminal residues had markedly decreased activities in each of these assays, and had decreased binding affinity for IFN-gamma cell surface receptors. These observations define the C-terminal residues important for IFN-gamma's biological activity--information which should be useful in designing analogs of IFN-gamma with enhanced or altered biological activities.
Arakawa
T
T
Amgen Inc., Thousand Oaks, CA 91320.
Narachi
M A
MA
Hsu
Y R
YR
Everett
R R
RR
Lai
P H
PH
Fish
E N
EN
D000596:Amino Acids / Q000032:analysis; D000998:Antiviral Agents / Q000494:pharmacology; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D010450:Endopeptidases / Q000378:metabolism; D006367:HeLa Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma / Q000378:metabolism / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate
D000596:Amino Acids; D000998:Antiviral Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D007371:Interferon-gamma; D010450:Endopeptidases