Analysis of the three-dimensional antigenic structure of giant ragweed allergen, Amb t 5

Abstract

The ragweed allergens Amb t 5 and Amb a 5 are among the smallest inhaled protein allergens known, containing a single, immunodominant T-cell epitope. In this study we analyzed the B-cell epitope structure of Amb t 5. The three-dimensional structures of Amb t 5 and Amb a 5 have been determined by NMR spectroscopy, providing a rare opportunity to analyze three-dimensional antigenic sites. Amb t 5 residues likely to be important for antigenicity were identified by examining the surface area of Amb t 5 accessible to a probe of the size of an antibody molecule. After changing these residues to the corresponding Amb a 5 residues, recombinant proteins were purified and tested for loss of antigenic activity. Inhibition radio-immunoassays, using sera from 8 individuals who had received immunotherapy with giant ragweed extract, allowed the mutations to be divided into three groups: (1) mutations that had little or no effect on antibody binding, (2) mutations that caused a loss of antigenic activity to a different degree in different sera and (3) mutations that drastically reduced antigenic activity in all sera tested. This last set of mutations clustered in the third loop of Amb t 5, suggesting that antibody recognition of Amb t 5, like T-cell recognition, is primarily directed towards a single, immunodominant site.

Introduction

The structural elements of protein allergens are recognized at two levels by the immune system. T-cells recognize a short, linear peptide fragment bound to a major histocompatibility complex class II molecule, whereas B-cells produce IgE and IgG antibodies that can react either with linear epitopes or to antigenic sites that are determined by the tertiary structure of the protein (Benjamin et al., 1984; Germain, 1991). Immunochemical studies of allergens provide information about these immunogenic sites and have resulted in the development of experimental desensitization protocols that aim to provide a safer and more effective treatment for allergic diseases (Briner et al., 1993; Norman, 1993). To date, multiple allergens have been cloned and their primary structures probed for the presence of B- and T-cell epitopes (Joost van Neerven et al., 1993; Liebers et al., 1993; Seiberler et al., 1994; van Kampen et al., 1994; Zhu et al., 1995). These mapping studies have mostly been performed with synthetic peptides, a method that is sufficient for delineating T-cell epitopes and B-cell epitopes that are comprised of linear amino acid sequences. Synthetic peptides, however, can not be used to identify B-cell epitopes of many important allergens such as the major ragweed, cat and mite allergens, because antibody recognition of these molecules is primarily directed towards conformational determinants (Olson and Klapper, 1986; Nilsen et al., 1991; OBrien and Thomas, 1994; Seiberler et al., 1994; Vailes et al., 1994). Identification of conformational epitopes on structurally complex allergens can provide insight into which structural elements are important in antigenicity⧹allergenicity and may help to explain the clinically relevant allergenic crossreactivity that occurs between allergens from distantly related species (Ebner et al., 1995; Frankland, 1995).

To examine how a group of structurally complex allergens is recognized by human B-cells, we chose to study the homologous ragweed allergens, the Amb 5s. Amb t 5 (from giant ragweed, Ambrosia trifida) and Amb a 5 (from short ragweed, Ambrosiaartemisiifolia) are among the smallest protein allergens defined to date, consisting of a single polypeptide chain of 40 and 45 amino acids respectively (Mole et al., 1975; Goodfriend et al., 1985). Specific immune responsiveness to both proteins is strongly associated with DR(α,β1∗1501) (Marsh et al., 1982a, Marsh et al., 1982b; Huang et al., 1991) and cellular and molecular evidence suggest that the molecules contain a single, immunodominant T-cell epitope (Rafnar et al., 1993; Zhu et al., 1995). Amb a 5 and Amb t 5 have amino acid sequence identity of 45%, with each protein containing 8 conserved cysteine residues, all of which participate in disulfide-bond pairing. The immunological and physical properties of Amb t 5 and Amb a 5 have been studied extensively; both proteins have been cloned (Ghosh et al., 1991, Ghosh et al., 1993) and sequenced and their three-dimensional structures have been derived by NMR spectroscopy (Metzler et al., 1992a, Metzler et al., 1992b). The Amb 5 allergens thus provide an exceptional opportunity to study the relationship between structure and immunological activity. Topologically, the structures of Amb t 5 and Amb a 5 are very similar; both molecules contain a triple-stranded β-sheet, three loops and a C-terminal α-helix. Despite their overall structural homology, Amb t 5 and Amb a 5 are essentially non-crossreactive antigenically (Goodfriend et al., 1985; Roebber et al., 1985), a feature that was used in the design of this study.

Here,we examined the B-cell epitope structure of Amb t 5, using mutagenesis of recombinant proteins. Amino acids of Amb t 5 predicted to be parts of antigenic sites (Novotny et al., 1986) were mutated into the corresponding Amb a 5 amino acids. The resulting molecules were tested for loss of Amb t 5 activity and gain of Amb a 5 activity. Our results show that while different individuals have distinct profiles of epitope recognition, loop 3 of Amb t 5 contains an immunodominant B-cell epitope that is responsible for a large portion of the IgG antibody response to Amb t 5.