Paper Citing NAMD - Abstract
Chiarini, R.; Moran, O.; Revoltella, R.P.
Identification of an antigenic domain near the C terminus of human granulocyte-macrophage colony-stimulating factor and its spatial localization
JOURNAL OF BIOLOGICAL CHEMISTRY, 279:37908-37917, SEP 3 2004
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 hGM-CSF; 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 hGM-CSF 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.
