Role of disulfide bridges in determining the biological activity of interleukin 3.

Abstract

Total chemical synthesis of analog proteins was used to examine the requirement for specific disulfide bridges for the biological activity of interleukin 3 (IL-3), a growth factor that stimulates multiple lineages of hemopoietic cells. Four structural analogs of the mature, 140 amino acid murine IL-3 molecule were synthesized in which specific cysteine residues were replaced by alanines. In a quantitative IL-3 assay, based on [3H~thymidine incorporation into factordependent cells, the IL-3 analog with alanines substituted for all four cysteines-i.e., [Alal7,7980, 4l41IL-3-had 1/500th as much activity as the molecule synthesized according to the native sequence. The two analogs [Cys17,79, [Cys17,140,Ala79 8]IL-3 had similarly low activity, whereas the [Cysl780 A.la79,141IL-3 analog had 2000-fold higher activity than these three analogs, and 3-fold higher than the molecule with the native sequence. This shows that in IL-3 a single disuflide bridge, between cysteines 17 and 80, is required for biological activity that approximates physiological levels. This disulfide probably stabilizes the tertiary structure of the protein to give a conformation that is optimal for function.The growth and differentiation of cells of the hemopoietic lineage are regulated by several glycoprotein growth factors (1). One of these, which has been best characterized in the mouse, is now widely termed interleukin 3 (IL-3) but has also been termed P-cell-stimulating factor, mast cell growth factor, burst-promoting activity, and multi-colony-stimulating factor (2-7). IL-3 stimulates multiple hemopoietic cell types, including pluripotential stem cells, committed progenitors, and some mature cells (1,7,8). Murine IL-3 has been purified to homogeneity (4, 9), and cDNA and genomic DNA clones that encode the protein have been isolated (10-12). The protein sequence deduced from the DNA sequences (10, 11) and N-terminal amino acid sequence analysis (9, 13) indicated that mature IL-3 contains 140 amino acid residues. However, a shorter form, lacking the first 6 amino acids, has also been identified (4). Recent experiments with antibodies to synthetic peptide fragments indicate that the IL-3 produced by activated T cells is the full-length, 140-residue form (14). Neither the location of disulfide bridges (if any) nor the three-dimensional structure of the molecule is known.We have investigated the structural basis for IL-3 function by a total chemical synthesis approach (15). The ability to reproducibly synthesize IL-3 with high activity has allowed us to systematically vary its structure and compare the biological activities of the analogs with those of the molecule with the native sequence. In our previous study we demonstrated that the cysteine at position 17 was essential for detectable activity. A fragment corresponding to residues 18-140, and therefore lacking this cysteine, had barely detectable activity, whereas a fragment (residues 17-140) that included the cysteine at position 17 had high activity. The most likely explanation of t...

Structure-function relationships of interleukin-3. An analysis based on the function and binding characteristics of a series of interspecies chimera of gibbon and murine interleukin-3.

“…(48). In addition to 26% amino acid identity and 40% homology based on conservative substitutions, the biologically critical disulfide bond of IL-3 is conserved throughout evolution ( 18,25 ). Furthermore, Pakula and Sauer have recently shown that surface side chain changes have little impact on the tertiary structure ofpolypeptides (49).…”

“…Initial studies using truncated synthetic polypeptides of murine IL-3 suggested that the first disulfide bond and the amino-terminal half ofthe molecule are responsible for biological activity (17,18). Recently, two groups have reported on the binding sites ofneutralizing mAbs of human IL-3 (19,20).…”

Enhanced hematopoietic activity of a human granulocyte/macrophage colony-stimulating factor-interleukin 3 fusion protein.

“…Helices were then packed by using the combinatorial packing algorithm of Cohen et al (20). The resulting structures were screened for compactness and consistency by using known disulfide constraints (21,22) and a preference for antiparallel helices.…”

The Receptor Binding Site of Human Interleukin-3 Defined by Mutagenesis and Molecular Modeling

“…A distance of 2.5 Å between the side chain nitrogen of Lys 28 and one of the carboxyl oxygen atoms of Glu 366 in this model suggests a salt bridge between Lys 28 of IL-3 and Glu 366 of the ␤ subunit. Although this interaction may facilitate the proper docking of the ligand, its contribution to the overall binding energy is relatively small considering the 2-4-fold decrease in binding affinity when this potential interaction was disrupted by the E366A mutant of the ␤ subunit (29) or by the K28E mutant of IL-3 (20).…”