title: Identification of an antigenic domain near the C terminus of human granulocyte-macrophage colony-stimulating factor and its spatial localization.
abstract: 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 alpha-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 alpha-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 beta-sheet 2 and half of the alpha-helix D; peptide-[C121A]-(110-127) included the second half of the alpha-helix D and the C terminus of hGMCSF; peptide-(13-31)-Gly-Pro-Gly-(103-116) included both the alpha-helices A and D connected by the tripeptide Gly-Pro-Gly, which allows the original antiparallel orientation of the two alpha-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 alpha-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.
authors.affiliation: Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, Via G. Moruzzi, 1-56100 Pisa, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Chiarini
authors.forename: Oscar
authors.initials: O
authors.lastname: Moran
authors.forename: Roberto P
authors.initials: RP
authors.lastname: Revoltella
mesh_terms: D000941:Antigens / Q000378:metabolism*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006433:Hemocyanins / Q000276:immunology; D006801:Humans; D007106:Immune Sera; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D000941:Antigens; D007106:Immune Sera; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin