Importance of disulfide linkage for constructing the biologically active human interleukin-2.
Recombinant human interleukin-2 (rIL-2) produced in Escherichia coli possesses a free thiol group at Cys-125 and a disulfide linkage between Cys-58 and Cys-105, as in the case for natural human interleukin-2. Treatment of rIL-2 with 200 mM dithiothreitol resulted in the cleavage of the Cys-58-Cys-105 disulfide bond. The reduced form of rIL-2 thus obtained retained only 10% of the in vitro biological activity of the native form, as measured by the ability to stimulate the growth of an IL-2-dependent mouse natural killer cell line, NKC3. Far-uv circular dichroism studies indicated that the cleavage of the disulfide bond results in a decrease of alpha-helix content. Near-uv circular dichroism studies suggested that the native molecule is folded into a rigid tertiary structure, while the reduced form showed a spectrum similar to that of rIL-2 denatured in the presence of 6 M guanidine.HCl. The once-reduced molecule was readily reoxidized in the presence of 10 microM Cu2+ to form the native molecule with full biological activity. These results strongly demonstrate that the Cys-58-Cys-105 disulfide linkage in the IL-2 molecule is essential for constructing a rigid and biologically active form of IL-2.
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis*; D006801:Humans; D007376:Interleukin-2 / Q000032:analysis*; D010084:Oxidation-Reduction; D010434:Pepsin A / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis
D000596:Amino Acids; D004220:Disulfides; D007376:Interleukin-2; D011994:Recombinant Proteins; D010434:Pepsin A