A human interleukin 3 analog with increased biological and binding activities.
Human interleukin 3 (IL-3) variants generated by site-directed mutagenesis were analyzed in multiple biological and binding assays to identify residues critical for IL-3 activity. Two mutants carrying substitutions in the predicted hydrophilic region within the first alpha-helix, [Ala21,Leu22]IL-3 and [Ala21,Leu22,Ala25]IL-3 showed loss of biological activity and high-affinity binding. Mutants in a second predicted hydrophilic region, [Ala44,Leu45,Ala46]IL-3 and [Ala44,Ala46]IL-3, however, showed similar biological and binding activities to wild-type IL-3. Mutations in a C-terminal hydrophilic region that overlaps the fourth predicted alpha-helix led to either loss or gain of function. IL-3 analogs [Glu104,Asp105]-, [Leu108]-, [Asn108]-, [Thr108]-, and [Ala101,Leu108]IL-3 were less active than wild-type IL-3, whereas [Ala101]IL-3 and [Val116]IL-3 were 2- to 3-fold more potent. Significantly, the double mutant [Ala101,Val116]IL-3 exhibited a 15-fold greater potency than native IL-3. Receptor binding studies showed that [Ala101,Val116]IL-3 exhibited increased binding to the high- and low-affinity receptors of monocytes. These results show the generation of an IL-3 analog with increased biological and binding activities and support a model where the C terminus of IL-3 interacts with the alpha chain of the IL-3 receptor, making this region a useful focus for the development of more potent IL-3 agonists or antagonists.
Lopez
A F
AF
Division of Human Immunology, Institute of Medical and Veterinary Science, Adelaide, South Australia.
Shannon
M F
MF
Barry
S
S
Phillips
J A
JA
Cambareri
B
B
Dottore
M
M
Simmons
P
P
Vadas
M A
MA
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007377:Interleukin-3 / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology; D016297:Mutagenesis, Site-Directed; D016185:Receptors, Interleukin-3 / Q000378:metabolism; D013329:Structure-Activity Relationship
D007377:Interleukin-3; D016185:Receptors, Interleukin-3