Identification of an antigenic region near the C-terminus of human granulocyte-macrophage colony-stimulating factor and its spatial relationship with functional domains.

The goal of this study was to map an epitope on the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) at its C terminus, a region whose integrity is fundamental in maintaining the normal function of this molecule. Residues including the fourth -helix (D, 103-116) were analyzed for their role in the interaction with antibodies (Abs) raised against the protein. Five peptides homologous to different segments of the C terminus of hGM-CSF were synthesized. Peptide-(102-121) included the same residues of the -helix D and the next five amino acids toward the C terminus; peptide-[E108A]-(102-121) introduced the mutation E108A in order to verify the role of acidic residues; peptide-[C96A](93-110) encompassed the -sheet 2 and half of the -helix D; peptide-[C121A]-(110-127) included the second half of the -helix D and the C terminus of hGMCSF; peptide-(13-31)-Gly-Pro-Gly-(103-116) included both the -helices A and D connected by the tripeptide Gly-Pro-Gly, which allows the original antiparallel orientation of the two -helices to be maintained. Both anti-protein and anti-peptide-(102-121) antibodies, capable of neutralizing the stimulatory activity of hGMCSF in the bone marrow colony-forming assays, recognized a specific epitope in the C terminus of hGM-CSF. Molecular modeling estimated the surface accessibility of hGM-CSF and the stability of the synthetic peptides in aqueous solution. Altogether, our results showed that the immunogenic region includes part of the -helix D and the residues 116-120, which are external to this helix and particularly exposed on the protein surface, confirming the feasible participation of this region in antibody binding.