Characterization of recombinant shrimp allergen Pen a 1 (tropomyosin).

Abstract

Tropomyosin (Pen a 1) from brown shrimp, Penaeus aztecus, has been identified as the only major shrimp allergen. Since beef, pork and chicken are other tropomyosin-containing foods that are not very allergenic, tropomyosins can serve to investigate the contribution of the structural properties of a protein to its allergenicity. The aim of this study was to determine the primary structure of Pen a 1 and to identify IgE-binding epitopes. The screening of a unidirectional expression cDNA library from shrimp tail muscle with the Pen-a-1-specific monoclonal antibody 4.9.5 resulted in 4 positive Escherichia coli clones. Immunoblot analysis with human sera from shrimp-allergic subjects demonstrated IgE binding of all 4 recombinant shrimp proteins. Three of 4 expressed recombinant proteins have a molecular weight of approximately 36 kD, consistent with the molecular weight of natural Pen a 1. The DNA sequence analysis identified these recombinant shrimp proteins as tropomyosin and could be aligned with the sequence of greasyback shrimp (Metapenaeus ensis) tropomyosin (Met e 1). In order to characterize contiguous IgE-binding epitopes of Pen a 1, a peptide library (Novagen epitope mapping system) expressing 10-30 amino-acid-residue-long recombinant Pen a 1 peptides was constructed and screened with human IgE. Four recombinant, IgE-reactive Pen a 1 peptides were selected and sequenced. They show various degrees of sequence identity with tropomyosins of other arthropods, such as fruitfly and house dust mite, helminths and vertebrates.