Molecular Structure and Granulocyte/Macrophage Colony-Stimulating Factor Activity

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Abstract
Granulocyte/macrophage colony-stimulating factor (GM-CSF) plays a critical role in myeloid differentiation and in several immune and inflammatory processes. GM-CSF binds to specific cellular receptors (GM-CSFR) which belong to a recently described supergene family. These receptors are potential targets for pharmacologic design and such design depends on a molecular understanding of ligand–receptor interactions. We present our initial studies evaluating the potential active sites of the molecule. The sites on the GM-CSF molecule that were studied represent two α-helices predicted to be critical for GM-CSF activity, as implicated by human–murine chimeric molecule studies. These helices are predicted to be adjacent in native GM-CSF. Peptides corresponding to amino acids 17–31 and 78–99 of GM-CSF were synthesized and cross-linked to one another in two different orientations. The ability of anti-GM-CSF to bind the individual and complexed peptides was evaluated by both ELISA and radioimmunoassay. Significant binding to all peptides was demonstrated. A preferred orientation of the two peptides was apparent, and this agreed with the predicted model structures. Antibodies were developed against the coupled peptides, and these demonstrated significant cross-reactivity with recombinant human GM-CSF. Additionally, analyses of anti-peptide antisera binding studies predict these two amino acid sequences to lie in parallel planes to one another in the native human GM-CSF molecule.

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