Receptor and antibody interactions of human interleukin-3 characterized by mutational analysis.
Human interleukin-3 (hIL-3) is a regulator of proliferation and differentiation of multipotent hemopoietic progenitor cells. Mutants of hIL-3 have been constructed by oligonucleotide-directed mutagenesis and expressed in Escherichia coli and Bacillus licheniformis. Purified muteins were assayed for induction of DNA synthesis in IL-3-dependent human cells and for binding to the IL-3 receptor. Residues at the NH2 and COOH termini together comprising one-quarter of the molecule could be removed without loss of biological function. Deletions of 6-15 residues within the central part of the molecule caused a large reduction (up to 5 logs) but no complete loss of activity. Substitution of evolutionary conserved residues resulted in a strong decrease of biological activity and demonstrated that the S-S bridge is an essential structural element in hIL-3. Interestingly, four muteins displayed a significantly higher potency of binding to the IL-3 receptor than in stimulating DNA synthesis. These results demonstrate that receptor binding may be (partly) disconnected from activation of DNA synthesis. Analysis of hIL-3 muteins demonstrated that the majority of monoclonal antibodies are directed against a small portion of the IL-3 molecule. The neutralizing potential of individual monoclonal antibodies could be increased by a combination of antibodies directed against nonoverlapping epitopes.
Dorssers
L C
LC
Department of Molecular Biology, Dr. Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.
Mostert
M C
MC
Burger
H
H
Janssen
C
C
Lemson
P J
PJ
van Lambalgen
R
R
Wagemaker
G
G
van Leen
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D004252:DNA Mutational Analysis; D000939:Epitopes; D006801:Humans; D007377:Interleukin-3 / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D016185:Receptors, Interleukin-3 / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007377:Interleukin-3; D016185:Receptors, Interleukin-3