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

Neal J. Meropol, Scott W. Altmann, Armen B. Shanafelt, Robert A. Kastelein, G. Douglas Johnson, Michael B. Prystowsky

Research output: Contribution to journalArticle

20 Citations (Scopus)

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 - Jul 15 1992
Externally publishedYes

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Granulocyte-Macrophage Colony-Stimulating Factor Receptors
Granulocyte-Macrophage Colony-Stimulating Factor
Bioactivity
Alanine
COS Cells
Glycosylation
Granulocytes
Mutagenesis
Glycoproteins
Macrophages
Peptides
Conformations
Substitution reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Requirement of hydrophilic amino-terminal residues for granulocyte-macrophage colony-stimulating factor bioactivity and receptor binding. / Meropol, Neal J.; Altmann, Scott W.; Shanafelt, Armen B.; Kastelein, Robert A.; Johnson, G. Douglas; Prystowsky, Michael B.

In: Journal of Biological Chemistry, Vol. 267, No. 20, 15.07.1992, p. 14266-14269.

Research output: Contribution to journalArticle

Meropol, Neal J. ; Altmann, Scott W. ; Shanafelt, Armen B. ; Kastelein, Robert A. ; Johnson, G. Douglas ; Prystowsky, Michael B. / Requirement of hydrophilic amino-terminal residues for granulocyte-macrophage colony-stimulating factor bioactivity and receptor binding. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 20. pp. 14266-14269.
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AU - Altmann, Scott W.

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AU - Kastelein, Robert A.

AU - Johnson, G. Douglas

AU - Prystowsky, Michael B.

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AB - 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.

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