Specific human granulocyte-macrophage colony-stimulating factor antagonists.
Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hemopoietic growth factor and activator of mature myeloid cell function. We have previously shown that residue 21 in the first helix of GM-CSF plays a critical role in both biological activity and high-affinity receptor binding. We have now generated analogues of GM-CSF mutated at residue 21, expressed them in Escherichia coli, and examined them for binding, agonistic, and antagonistic activities. Binding experiments showed that GM E21A, E21Q, E21F, E21H, E21R, and E21K bound to the GM-CSF receptor alpha chain with a similar affinity to wild-type GM-CSF and had lost high-affinity binding to the GM-CSF receptor alpha-chain-common beta-chain complex. From these mutants, only the charge reversal mutants E21R and E21K were completely devoid of agonistic activity. Significantly we found that E21R and E21K antagonized the proliferative effect of GM-CSF on the erythroleukemic cell line TF-1 and primary acute myeloid leukemias, as well as GM-CSF-mediated stimulation of neutrophil superoxide production. This antagonism was specific for GM-CSF in that no antagonism of interleukin 3-mediated TF-1 cell proliferation or tumor necrosis factor alpha-mediated stimulation of neutrophil superoxide production was observed. E. coli-derived GM E21R and E21K were effective antagonists of both nonglycosylated and glycosylated wild-type GM-CSF. These results show that low-affinity GM-CSF binding can be dissociated from receptor activation and have potential clinical significance for the management of inflammatory diseases and certain leukemias where GM-CSF plays a pathogenic role.
Division of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, South Australia.
T R
TR
Hercus
C J
CJ
Bagley
B
B
Cambareri
M
M
Dottore
J M
JM
Woodcock
M A
MA
Vadas
M F
MF
Shannon
A F
AF
Lopez
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects*; D003001:Cloning, Molecular; D006224:Cricetinae; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000494:pharmacology*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D007951:Leukemia, Myeloid; D016297:Mutagenesis, Site-Directed; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C101659:granulocyte-macrophage colony-stimulating factor, Arg(21)-; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013936:Thymidine