The Structural Requirements of Epitopes with Ige Binding Capacity Demonstrated by Three Major Allergens from Fish, Egg and Tree Pollen

Abstract

Three major allergens from cod fish, egg white and tree pollen, were characterized by studies on their allergenic and antigenic structures.

The major allergen of cod fish, Allergen M “paralbumins pi 4.75”, is composed of 113 amino acid residues with a molecular weight of 12,328 daltons. It comprised three domains, AB, CD and EF, consisting of 3 helices interspaced by one loop. Each of the loops of the CD and EF domains each coordinates one Ca²⁺. The antigenicity and allergenicity of Allergen M was deduced from studying the modified protein and some particular synthetic peptides. Three sites were encompassing IgE binding epitopes namely peptides 33-44, 65-74 and 88-96.

A novel peptide (49-64), of the CD-domain, was demonstrated to be allergenically/antigenically active and cross reactive with birch pollen allergen, which incidentally was used as a negative control. This site encompassed two repetitive sequences (D-E-D-K) and (D-E-L-K), suggested to be mutually critical for the specificity of antibody binding. This hypothesis was reconfirmed by SPPS of several analogous peptides of region 39-64.

Furthermore, peptide 88-103 of the EF-domain was similarly synthesized; it functioned as a monovalent hapten, blocking and not eliciting allergic reaction.

Moreover, peptide 13-32 of domain AB, the non-calcium binding domain, was thoroughly tested. The results of PK inhibition showed clear activity and the peptide was found to function at the level of a divalent determinant.

Ovalbumin (OA) is the most dominant of five major allergens of egg white and universally used as model protein. OA allergenic epitopes were shown to be mainly determined by the primary structure and depend on certain peptide chain length.

The N-terminal decapeptide (OA 1-10) was shown to react with reaginic IgE. Direct skin test on egg allergic patients, showed no activity and the site was therefore concluded to encompasses one single Ig binding haptenic epitope.

Peptide OA 323-339, was demonstrated to be valuable in studies of T-cell recognition of protein antigens. Three analogous peptides of this region were prepared and clearly shown to be immunogenic in rabbits and to bind specific IgE from patients allergic to egg. OA 323-339 was concluded to encompass an allergenic and antigenic epitope which was recognized by human and rabbit B-lymphocytes.

Eight peptides in the region 11-122 were similarly synthesized. A test battery was performed to study this region using rabbit polyclonal antibodies and human specific IgE. Some of these sites were involved in binding of particular Ig paratopes.

Five immunogenic peptides from the major allergens of tree pollen extracts (segment 23-38), were synthesized. The selection of those peptides was setteled using two algorithms for providing the optimal hydrophobicity. All the synthetic peptides and analogues from region 23-38, could inhibit the binding of specific IgE to the intact molecules.

A minimal requirements of an allergenic epitope was clearly demonstrated to be the repetitive four amino acids peptides interspaced by 6 unrelated spacer arm. A certain minimal molecular size of 12-15 amino acids seemed necessary in all the epitopes synthesized for studying the biological activity and for a successful predictive algorithms of the helicity.

Key Words:

• Allergic diseases
• parvalbumin
• Allergen M
• immunoglobulin E
• ovalbumin
• antigenic structure
• primary structure
• solid phase peptide synthesis
• birch pollen allergens
• allergenic epitopes
• Ig paratopes