Mutational analysis of major, sequential IgE-binding epitopes in alpha s1-casein, a major cow's milk allergen.
BACKGROUND
Allergy to cow's milk is common in early childhood, and no therapy other than avoidance exists. In murine models of peanut allergy, immunotherapy with mutated, engineered, proteins appears promising.


OBJECTIVE
We sought to identify the critical amino acids (AAs) for immunoglobulin E (IgE) binding within the major B-cell epitopes of alpha(s1)-casein, a major cow's milk allergen. This will provide the necessary information to alter the cDNA to encode a protein capable of activating milk-specific T cells, but with reduced IgE-binding capacity.


METHODS
For mutational analysis of the IgE-binding epitopes, peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single or multiple AA substitutions. Membranes were immunolabeled with pooled sera from 15 cow's-milk-allergic patients and with 8 individual sera.


RESULTS
With the pooled sera, substitution of a single AA led to complete abrogation of IgE binding to 2 of 8 peptides and diminished binding in the remainder. Substitution of multiple AAs led to an abrogation of binding in the remaining peptides. In 4 of the 8 peptides, the critical AA identified with pooled sera did not result in significant reduction of IgE binding with 1 or more individual patients. For these patients, other critical AAs were identified, indicating a more heterogeneous pattern in IgE recognition.


CONCLUSIONS
This study indicates that single or multiple AA substitutions within IgE-binding epitopes result in reduced binding of milk-specific IgE antibodies by patients' sera. However, for future immunotherapeutic interventions with mutated peptides, critical AAs should be evaluated with individual patient sera to determine B-cell-epitope heterogeneity.
Cocco
Renata R
RR
Division of Pediatric Allergy and Immunology and the Jaffe Institute for Food Allergy, Mount Sinai School of Medicine, New York, NY, USA.
Järvinen
Kirsi-Marjut
KM
Sampson
Hugh A
HA
Beyer
Kirsten
K
D000293:Adolescent; D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D002364:Caseins / Q000276:immunology* / Q000378:metabolism*; D002648:Child; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D000939:Epitopes / Q000235:genetics*; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D008969:Molecular Sequence Data
D002364:Caseins; D000939:Epitopes; D007073:Immunoglobulin E