Requirement of hydrophilic amino-terminal residues for granulocyte- macrophage colony-stimulating factor bioactivity and receptor binding

N. J. Meropol, S. W. Altmann, A. B. Shanafelt, R. A. Kastelein, G. D. Johnson, M. B. Prystowsky

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)14266-14269
Number of pages4
JournalJournal of Biological Chemistry
Volume267
Issue number20
StatePublished - Jan 1 1992
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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