Mutagenesis of murine granulocyte/macrophage-colony-stimulating factor reveals critical residues near the N terminus

Abstract

A number of cDNAs encoding mutant forms of the murine haemopoietic growth factor, granulocyte/macrophage-colony-stimulating factor (GM-CSF), have been derived by in vitro mutagenesis and expressed in simian COS cells. Determination of the biological activity of the mutant factors revealed that residues within the regions 11 -15, 24-37, 47-49 and 81 -89 are required for generating a functional GM-CSF molecule. In particular, truncation of either of two strongly predicted a helices near the N terminus of the molecule severely depresses the activity of the factor.A number of glycoprotein growth factors, the colonystimulating factors (CSFs), are involved in regulating the proliferation and differentiation of haemopoietic cells [l , 21. In the mouse four CSFs that stimulate the production and activation of granulocytes and macrophages have been characterized, purified and cloned (for reviews, see [l -41). Each has a distinct structure and a unique spectrum of biological activities, the latter being used for classification : G-CSF and M-CSF stimulate the formation in vitro of predominantly neutrophilic granulocyte and macrophage colonies respectively; GM-CSF and multi-CSF (interleukin-3) stimulate the formation of both macrophage and neutrophilic and eosinophilic granulocyte colonies and, in addition, multi-CSF stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies [l, 21. There is currently considerable interest in these molecules, owing in part to their therapeutic potential, and in order to provide a rational basis for creating potentially useful agonists and antagonists of the CSFs it is important to define the regions of these molecules which are essential for biological activity. We are interested in systematically investigating the structure of the murine GM-CSF molecule and to this end have chosen a 'linker-scanning' approach [5] to generate clustered point mutations and internal deletions of this molecule. These mutants were expressed in simian COS cells and their biological activities determined. By this process we have defined a series of small regions within the N-terminal third of the GM-CSF molecule that are obligatory for function. MATERIALS AND METHODS Generation ofBal3I deletion mutantsResection of the parental pGM3.2 cDNA clone with the exonuclease Bu131, ligation of BamHI linkers (5'-CGGATCCG) and recloning of truncated cDNAs were performed essentially as described previously [6]. Truncated cDNA molecules were fractionated by agarose gel electrophoresis and three fractions of different sizes recovered and recloned into pUC8 (for 5' deletions) and pUC9 (for 3' deletions) [7]. To determine the end-point of the deletion in each clone, nucleotide sequence analysis was performed on the double-stranded plasmid DNA, by the dideoxy-chaintermination method [8]. Generation of linker-scanning ( L S ) mutantsSelected 5' and 3'-deletion mutants (in plasmids pUC8 and pUC9, respectively) were cleaved with BarnHI and HindIII, the small intervening segment of polylinker removed by pr...

Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis.

“…The present work and that of Zurawski and Zurawski (22) demonstrate the importance of correlating expression levels of mutant proteins with their corresponding activities. The inability of Gough et al (8) to determine the mutant protein concentrations may explain why none of the critical regions identified in the present work coincide with their results; it is possible that insufficient expression of mutant proteins accounts for their negative assay results. Additionally, their use of large deletions to identify critical regions introduces the potential of gross structural changes that may have resulted in nonspecific inactivation of the mGM-CSF protein.…”

“…Gough et al (8) have identified residues 11-15, 24-37, 47-49, and 81-89 as required for generating a functional mGM-CSF. The present work and that of Zurawski and Zurawski (22) demonstrate the importance of correlating expression levels of mutant proteins with their corresponding activities.…”

“…Using chemically synthesized hGM-CSF protein fragments and analogs, Clark-Lewis et al (7) identified residues 1-13 and residues 122-127 as being not critical and residues [14][15][16][17][18][19][20][21][22][23][24][25] as being critical for biological activity. Mutagenesis of mGM-CSF by Gough et al (8) revealed that residues 11-15, 24-37, 47-49, and 81-89 are obligatory for function. Physical and biochemical studies indicate that both the murine and human proteins have similar secondary structures containing ahelical and ,8-sheet segments (9).…”

Interferon-resistant Human Melanoma Cells Are Deficient in ISGF3 Components, STAT1, STAT2, and p48-ISGF3γ

“…RNA Preparation and Northern Blotting-Cytoplasmic RNA was isolated by the procedure of Gough (35) and subjected to Northern blot analysis (36). The blot was probed with an ␣-32 P-labeled full-length STAT1␣/␤ cDNA (a gift from Dr. Chris Schindler, Dept.…”

Rational Design of Granulocyte-Macrophage Colony-stimulating Factor Antagonist Peptides

“…These studies indicated a critical role for amino acids 21-31 (A helix) and 77-94 (including the C helix) in mediating the activity of human GM-CSF, suggesting that the second site may be involved in binding to the GM-CSFR␣. Additional mutagenesis studies (42)(43)(44)(45), mapping of neutralizing monoclonal antibodies (46 -50), and synthetic peptide studies (47, 51, 52) suggest other potential interaction sites. Thus, in spite of considerable study, the GM-CSFR␣ interaction site(s) on GM-CSF remain incompletely characterized.…”