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,...

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Bibliographic Details
Published in:The Journal of biological chemistry 1992-07, Vol.267 (20), p.14266-14269
Main Authors: MEROPOL, N. J, ALTMANN, S. W, SHANAFELT, A. B, KASTELEIN, R. A, JOHNSON, G. D, PRYSTOWSKY, M. B
Format: Article
Language:English
Subjects:
Alanine
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Animals
Binding, Competitive
Biological and medical sciences
Cell Division - drug effects
Cell Line
Cloning, Molecular
DNA Replication - drug effects
Fundamental and applied biological sciences. Psychology
Glycoproteins
Glycosylation
Granulocyte-Macrophage Colony-Stimulating Factor - genetics
Granulocyte-Macrophage Colony-Stimulating Factor - metabolism
Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology
Kinetics
Mice
Mutagenesis, Site-Directed
Protein Conformation
Proteins
Receptors, Granulocyte-Macrophage Colony-Stimulating Factor - metabolism
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)49707-X