Antigenic regions of tumor necrosis factor alpha and their topographic relationships with structural/functional domains.
The immunogenic regions of human Tumor Necrosis Factor alpha (huTNF) have been mapped by studying the interaction between various mouse anti-huTNF sera and synthetic huTNF fragments, spanning the entire sequence of huTNF. Three main immunogenic regions were identified within residues 1-23, 95-116 and 137-157 of huTNF and two other less immunogenic regions within residues 117-136 and 37-55. The same huTNF regions were found to contain antigenic sites by binding studies with cognate anti-peptide sera. Competitive binding experiments with shorter synthetic subfragments provided evidence for the location of strong antigenic sites within residues 1-10, 17-23, 104-112 and 137-143. In particular the immunodominant site was found to be located within residues 104-112. huTNF regions corresponding to residues 24-36, 56-75, 76-94, and 147-157 resulted to be not or poorly antigenic. However, treatment of huTNF with Triton X-100 under conditions that partially dissociate the oligomeric quaternary structure resulted in the exposition of sites recognized by sera against peptides huTNF [56-75] and huTNF [76-94], suggesting that antigenic sites not accessible in the oligomeric huTNF are exposed in the dissociated form. The principal antigenic sites in the oligomeric molecule are localized in the flexible N-terminal part and in hydrophilic segments located in the "middle/top" region of the molecule, opposite to the C-terminus. Protein segments of the "bottom" region, close to the C-terminus, were poorly immunoreactive. Neutralization assays of TNF cytolytic activity on L-M cells showed that binding of antibodies to epitopes located in the "middle/top" regions of huTNF does not affect its cytolytic activity, supporting the hypothesis of a receptor binding site location at the "bottom" of TNF trimer.
Molecular Immunology and Biochemistry Unit, Tecnogen SCpA, Milan, Italy.
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A
Corti
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Fassina
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F
Marcucci
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Cassani
D000818:Animals; D000917:Antibody Formation; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D017830:Octoxynol; D010446:Peptide Fragments; D011092:Polyethylene Glycols; D011956:Receptors, Cell Surface / Q000276:immunology; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D000939:Epitopes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols; D017830:Octoxynol