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IgE structure-function relationships defined by sequence directed antibodies induced by synthetic peptides
1Division of Molecular Biology, Medical Biology Institute, La Jolla, CA 92037.
Six peptides, representing contiguous amino acid sequences within the C epsilon 2, C epsilon 3 and C epsilon 4 domains of murine IgE, were selected for synthesis on the basis of overall hydropathy, degree of homology to both human and rat IgE and, where possible, inclusion of a native cysteine residue. Antibodies were produced against each peptide by immunizing rabbits with peptide-KLH conjugates. Each anti-peptide antiserum exhibited good reactivity with the corresponding immunizing peptide (titer: 10(-4) to 10(-5] and four of the six antisera exhibited a distinct preferential murine IgE reactivity compared to four other murine immunoglobulin classes (IgG1, IgG2b, IgM and IgA). In addition, one antiserum (anti-epsilon peptide 5), raised against a peptide with 80% homology to human IgE, reacted comparably with both human and murine IgE. Each IgE-reactive antiserum was screened for the ability to stimulate mediator release from IgE-sensitized rat basophilic leukemia (RBL) cells. Two of the four IgE-reactive antisera strongly stimulated 3H-serotonin release (anti-epsilon peptides 4 and 5), one antiserum showed weak activity (anti-epsilon peptide 3) and the remaining anti-peptide serum (anti-epsilon peptide 6), which exhibited the highest anti-IgE reactivity, exhibited no detectable stimulatory activity. Individual anti-peptide antibodies were subsequently tested for the potential to bind to receptor-bound IgE. Anti-epsilon peptide 3 was shown to exhibit the least binding, anti-epsilon peptide 6 showed the highest magnitude of binding while anti-epsilon peptides 4 and 5 exhibited intermediate values. We conclude from this study that sequences defined by epsilon-peptides 4 and 5 are not significantly involved in the receptor binding mechanism whereas epsilon-peptide 3 is likely to be most proximal to the IgE-receptor recognition site of those sequences studied. Finally, we suggest that the epsilon-peptide 6 sequence is in such an orientation in cell-bound IgE that, while it is accessible to external antibody, effective cross-linking of the IgE-receptor complex cannot be achieved through this determinant.
Six peptides, representing contiguous amino acid sequences within the C epsilon 2, C epsilon 3 and C epsilon 4 domains of murine IgE, were selected for synthesis on the basis of overall hydropathy, degree of homology to both human and rat IgE and, where possible, inclusion of a native cysteine residue. Antibodies were produced against each peptide by immunizing rabbits with peptide-KLH conjugates. Each anti-peptide antiserum exhibited good reactivity with the corresponding immunizing peptide (titer: 10(-4) to 10(-5] and four of the six antisera exhibited a distinct preferential murine IgE reactivity compared to four other murine immunoglobulin classes (IgG1, IgG2b, IgM and IgA). In addition, one antiserum (anti-epsilon peptide 5), raised against a peptide with 80% homology to human IgE, reacted comparably with both human and murine IgE. Each IgE-reactive antiserum was screened for the ability to stimulate mediator release from IgE-sensitized rat basophilic leukemia (RBL) cells. Two of the four IgE-reactive antisera strongly stimulated 3H-serotonin release (anti-epsilon peptides 4 and 5), one antiserum showed weak activity (anti-epsilon peptide 3) and the remaining anti-peptide serum (anti-epsilon peptide 6), which exhibited the highest anti-IgE reactivity, exhibited no detectable stimulatory activity. Individual anti-peptide antibodies were subsequently tested for the potential to bind to receptor-bound IgE. Anti-epsilon peptide 3 was shown to exhibit the least binding, anti-epsilon peptide 6 showed the highest magnitude of binding while anti-epsilon peptides 4 and 5 exhibited intermediate values. We conclude from this study that sequences defined by epsilon-peptides 4 and 5 are not significantly involved in the receptor binding mechanism whereas epsilon-peptide 3 is likely to be most proximal to the IgE-receptor recognition site of those sequences studied. Finally, we suggest that the epsilon-peptide 6 sequence is in such an orientation in cell-bound IgE that, while it is accessible to external antibody, effective cross-linking of the IgE-receptor complex cannot be achieved through this determinant.