Structure-activity relationship study of human interleukin-3. Identification of residues required for biological activity by site-directed mutagenesis.
Recombinant human interleukin-3 (rhuIL-3) variants were generated by site-directed mutagenesis and expression in Escherichia coli. Amino acid deletions and substitutions were made in the previously identified epitopes of two huIL-3-specific neutralizing monoclonal antibodies (mAbs). The rhuIL-3 variants were analyzed for their ability to bind to the IL-3 receptor and to induce the proliferation of the human IL-3-dependent cell line M-O7. Several deletion mutants spanning the epitopes of these neutralizing mAbs indicated the importance of residues Pro33 and Leu34 for biological activity. Further, substitution of Pro33 with Asn (Asn33) showed an enhanced proliferative activity (4-fold) and a moderate increase in receptor binding (2-fold) compared to wild-type (wt) rhuIL-3. The most remarkable change, however, was seen with variant Gly33, which showed a 14-fold increase in promoting the growth of M-O7 cells without a significant modification in its receptor binding capacity. In contrast, substitution of Leu34 with Gly (Gly34) yielded an IL-3 variant that had a 25-fold decreased receptor binding capacity and proliferative activity, while Glu34 had properties similar to wild-type rhuIL-3. Analysis of the binding of these variants to different rhuIL-3-specific monoclonal antibodies suggested that no major modification had occurred in their conformations. These results indicate that both residues, Pro33 and Leu34, play a critical role in modulating the activity of rhuIL-3.
Lokker
N A
NA
Preclinical Research, Sandoz-Pharma Ltd., Basel, Switzerland.
Movva
N R
NR
Strittmatter
U
U
Fagg
B
B
Zenke
G
G
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D016185:Receptors, Interleukin-3 / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007377:Interleukin-3; D016185:Receptors, Interleukin-3; D011994:Recombinant Proteins; D004247:DNA