Identification of amino acids critical for IgE-binding to sequential epitopes of bovine kappa-casein and the similarity of these epitopes to the corresponding human kappa-casein sequence.
BACKGROUND
The delineation of allergenic (i.e. IgE-binding) epitopes in cow's milk proteins and the amino acids (AAs) critical for IgE-binding is necessary to understand better the structural properties of an allergen and to develop more efficacious immunotherapeutic reagents. Furthermore, this information may enable us to understand better cross-sensitivity between different allergens.


METHODS
Eleven peptides, 10-14 AAs in length, representing the IgE-binding epitopes of kappa-casein were synthesized on a derivatized cellulose membrane with single AA substitutions at each position. Membranes were incubated with pooled sera from 15 milk-allergic patients and individual sera from 10 of the patients included in the pool.


RESULTS
For 10/11 allergenic peptides, one to five different single AA substitutions resulted in elimination of IgE-binding of pooled patient sera. Overall at least one mutated peptide could be found for these 10 IgE-binding sites that resulted in a reduction of IgE-binding in at least 80% of the patients who recognized the native protein. Furthermore, the IgE-binding region at AA104-112 on bovine kappa-casein showed a high degree of similarity with the human kappa-casein, respectively, including the AAs critical for IgE-binding.


CONCLUSIONS
This finding suggests that critical AAs should be assessed with both pooled and individual patient sera to account for the B-cell epitope heterogeneity between patients, with cow's milk allergy. In addition, we identified two potentially cross-reactive peptides between bovine and human caseins of unknown clinical relevance.
Han
N
N
Division of Pediatric Allergy & Immunology, Jaffe Institute for Food Allergy, The Mount Sinai School of Medicine, New York, NY 10029-6574, USA.
Järvinen
K M
KM
Cocco
R R
RR
Busse
P J
PJ
Sampson
H A
HA
Beyer
K
K
D000293:Adolescent; D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000378:metabolism*; D016269:Milk Hypersensitivity / Q000209:etiology* / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism
D002364:Caseins; D000939:Epitopes; D010455:Peptides; D007073:Immunoglobulin E