A neutralizing monoclonal antibody binds to an epitope near the amino terminus of murine granulocyte-macrophage colony-stimulating factor.
A rat anti-murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) monoclonal antibody, A2, that neutralizes bioactivity in vitro was isolated. The binding epitope recognized by this antibody was identified using human-murine hybrid GM-CSF proteins. A2 was unable to immunoprecipitate a hybrid (hm7) protein containing the human GM-CSF sequence for the first 11 amino terminal amino acids, and the mGM-CSF sequence for amino acids 12-124. In contrast, A2 did recognize a hybrid which substitutes human GM-CSF amino acids 23-36 in the murine sequence. These data suggest that this neutralizing antibody recognizes an epitope at the amino terminus of mGM-CSF. Because hm7 did maintain in vitro bioactivity, it is probable that the epitope recognized by the neutralizing antibody is not itself part of the receptor-binding domain of mGM-CSF; rather, it is likely that neutralization occurs as a result of antibody binding near the receptor-binding site, with steric inhibition of mGM-CSF binding to its receptor. Interestingly, monoclonal antibody A2 does not recognize mGM-CSF glycosylation species corresponding to predicted maximal O-glycosylation variants. The presence of O-glycosylation sites within the antibody-binding epitope was confirmed using site-directed mutagenesis. Potential O-glycosylation sites in native mGM-CSF were removed by introducing conservative amino acid substitutions, and expected molecular weight reductions were obtained. These findings are consistent with previous reports that suggest the importance of the integrity of residues near the amino terminus to GM-CSF bioactivity.
Meropol
N J
NJ
Hematology-Oncology Section, Hospital of the University of Pennsylvania, Philadelphia 19104.
Kreider
B L
BL
Lee
V M
VM
Kaushansky
K
K
Prystowsky
M B
MB
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D000939:Epitopes / Q000032:analysis / Q000737:chemistry*; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000737:chemistry; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D011233:Precipitin Tests; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016178:Granulocyte-Macrophage Colony-Stimulating Factor