Document Detail

Mutational analysis of the IgE epitopes in the latex allergen Hev b 5.
MedLine Citation:
PMID:  11398087     Owner:  NLM     Status:  MEDLINE    
BACKGROUND: Hev b 5 is a major latex allergen and potential candidate for an immunotherapy reagent. OBJECTIVE: The purpose of this study was to produce a hypoallergenic form of Hev b 5. METHODS: We used SPOTs analysis with alanine substitution to identify amino acids (AAs) critical for IgE binding and used site-directed mutagenesis to produce recombinant proteins with altered IgE-binding activity. RESULTS: Eleven epitopes were identified (5.1-5.11) in Hev b 5. Individual patients demonstrated variable epitope recognition, with the most intense reactivity to epitopes 5.4 and 5.7. IgE inhibition assays with synthetic peptides indicated that mutating a single epitope would not reduce IgE binding, but rather a combination of epitopes was required. After alanine substitutions to identify the important AAs, site-directed mutagenesis was used to replace the crucial AAs with alanine. Twenty clones with different combinations of altered epitopes were evaluated by means of IgE inhibition assays. Clones with mutations in single epitopes failed to reduce IgE binding, but changes to 8 epitopes (14 AAs) resulted in a 4500-fold reduction in IgE binding. Epitopes 5.7 and 5.9 were found to be cross-reactive, making Hev b 5 a multivalent allergen. CONCLUSIONS: We produced a recombinant Hev b 5 protein with significantly reduced IgE-binding activity. Changing a minimum of 3 immunodominant epitopes was required to cause a 100-fold reduction in IgE binding. Changes in 8 epitopes, particularly the cross-reactive epitopes 5.7 and 5.9, were needed to maximize the reduction in IgE binding. Mutants with reduced IgE-binding activity may prove to be valuable reagents for immunotherapy.
D H Beezhold; V L Hickey; G L Sussman
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Journal of allergy and clinical immunology     Volume:  107     ISSN:  0091-6749     ISO Abbreviation:  J. Allergy Clin. Immunol.     Publication Date:  2001 Jun 
Date Detail:
Created Date:  2001-06-08     Completed Date:  2001-07-05     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  1275002     Medline TA:  J Allergy Clin Immunol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1069-76     Citation Subset:  AIM; IM    
Laboratory of Immunobiology, Guthrie Research Institute, 1 Guthrie Square, Sayre, PA 18840, USA.
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MeSH Terms
Alanine / genetics
Allergens / chemistry,  genetics*,  immunology,  metabolism
Amino Acid Sequence
Amino Acid Substitution*
Immunodominant Epitopes / chemistry,  genetics*,  immunology
Immunoglobulin E / immunology*,  metabolism
Latex Hypersensitivity / immunology*
Molecular Sequence Data
Mutagenesis, Site-Directed
Peptides / chemical synthesis,  chemistry,  immunology
Plant Proteins
Recombinant Proteins / genetics,  immunology,  metabolism
Reg. No./Substance:
0/Allergens; 0/Immunodominant Epitopes; 0/Peptides; 0/Plant Proteins; 0/Recombinant Proteins; 0/allergen Hev b 5; 37341-29-0/Immunoglobulin E; 56-41-7/Alanine

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