Requirement of hydrophilic amino-terminal residues for granulocyte-macrophage colony-stimulating factor bioactivity and receptor binding.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein required for the proliferation and differentiation of granulocyte and macrophage precursors. Previous investigations have identified regions in human and murine GM-CSF that are required for bioactivity. In the present study, alanine substitution mutagenesis was undertaken to define more precisely specific amino-terminal residues in murine GM-CSF that are involved in bioactivity and receptor binding. Five double alanine mutants were identified that showed at least 10-fold reductions in bioactivity (K14AK20A, K14AE21A, H15AK20A, H15AE21A, K20AE21A). Each of these mutants maintained a normal N-linked glycosylation pattern when expressed in COS-1 cells, suggesting that native polypeptide backbone conformation was preserved. The purified prokaryotic expression products of two mutants (K14AE21A and H15AE21A) had a 100-fold decrease in bioactivity and a decrease in receptor binding, indicating that the side chains of K14, H15, and E21 are required for optimal receptor binding and maximal bioactivity.
Meropol
N J
NJ
Hematology-Oncology Section, University of Pennsylvania, Philadelphia 19104.
Altmann
S W
SW
Shanafelt
A B
AB
Kastelein
R A
RA
Johnson
G D
GD
Prystowsky
M B
MB
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D003001:Cloning, Molecular; D004261:DNA Replication / Q000187:drug effects; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*
D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000409:Alanine