Relationships between T cell and IgE/IgG4 epitopes of the Anisakis simplex major allergen Ani s 1.

BACKGROUND: Anisakiasis is a global disease caused by the consumption of raw or lightly cooked fish parasitized with third-stage Anisakis larvae. Anisakis simplex allergens may cause severe allergic reactions including angio-oedema, urticaria and anaphylaxis. Approximately 80% of allergic patients have allergen-specific IgE against Ani s 1, and the diagnostic value of testing for antibodies to Ani s 1 has been extensively demonstrated. However, no previous studies have investigated the molecular aspects of the allergic response to Ani s 1. Knowledge of allergen-specific T cell and B cell (IgE and IgG4) epitopes is important for elucidating the immunological mechanisms underlying allergic responses, and for understanding why particular proteins behave as allergens. OBJECTIVE: To elucidate the main T cell- and B cell (IgE and IgG4)- binding regions of Ani s 1. METHODS: T cell epitopes were identified by peptide proliferation assays using T cell lines derived from peripheral blood mononuclear cells of 11 patients with Anisakis allergy, and IgE and IgG4 epitopes were identified by microarray immunoassay using sera from a different group of 11 patients with Anisakis allergy. RESULTS: Several T cell epitopes of Ani s 1 were identified, of which Ani s 1145-156 , Ani s 1151-162 and Ani s 1163-171 located at the C-terminal end of the protein were the most relevant. IgE and IgG4 recognized largely the same peptides, including Ani s 122-41 , Ani s 125-44 , Ani s 127-47 , Ani s 137-56 and Ani s 194-113 . CONCLUSIONS AND CLINICAL RELEVANCE: This is the first report describing the T cell epitopes of an important allergen of A. simplex, and the first B cell epitope study of this allergen in the Spanish population. This information can help to elucidate the mechanisms underlying the allergic response to Ani s 1, potentially leading to therapeutic and diagnostic advances.

Evolutionary transition pathways for changing peptide ligand specificity and structure.

We identified evolutionary pathways for the inter- conversion of three sequentially and structurally unrelated peptides, GATPEDLNQKL, GLYEWGGARI and FDKEWNLIEQN, binding to the same site of the hypervariable region of the anti-p24 (HIV-1) monoclonal antibody CB4-1. Conversion of these peptides into each other could be achieved in nine or 10 single amino acid substitution steps without loss of antibody binding. Such pathways were identified by analyzing all 7 620 480 pathways connecting 2560 different peptides, and testing them for CB4-1 binding. The binding modes of intermediate peptides of selected optimal pathways were characterized using complete sets of substitution analogs, revealing that a number of sequential substitutions accumulated without changing the pattern of key interacting residues. At a distinct step, however, one single amino acid exchange induces a sudden change in the binding mode, indicating a flip in specificity and conformation. Our data represent a model of how different specificities, structures and functions might evolve in protein-protein recognition.

X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: basis of substrate specificity.

The homodimeric enzyme form of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa ATCC 17933 crystallizes readily with the space group R3. The X-ray structure was solved at 2.6 A resolution by molecular replacement. Aside from differences in some loops, the folding of the enzyme is very similar to the large subunit of the quinoprotein methanol dehydrogenases from Methylobacterium extorquens or Methylophilus W3A1. Eight W-shaped beta-sheet motifs are arranged circularly in a propeller-like fashion forming a disk-shaped superbarrel. No electron density for a small subunit like that in methanol dehydrogenase could be found. The prosthetic group is located in the centre of the superbarrel and is coordinated to a calcium ion. Most amino acid residues found in close contact with the prosthetic group pyrroloquinoline quinone and the Ca(2+) are conserved between the quinoprotein ethanol dehydrogenase structure and that of the methanol dehydrogenases. The main differences in the active-site region are a bulky tryptophan residue in the active-site cavity of methanol dehydrogenase, which is replaced by a phenylalanine and a leucine side-chain in the ethanol dehydrogenase structure and a leucine residue right above the pyrrolquinoline quinone group in methanol dehydrogenase which is replaced by a tryptophan side-chain. Both amino acid exchanges appear to have an important influence, causing different substrate specificities of these otherwise very similar enzymes. In addition to the Ca(2+) in the active-site cavity found also in methanol dehydrogenase, ethanol dehydrogenase contains a second Ca(2+)-binding site at the N terminus, which contributes to the stability of the native enzyme.

Stepwise transformation of a cholera toxin and a p24 (HIV-1) epitope into D-peptide analogs.

We have transformed two peptide epitopes into D-peptide analogs: VPGSQHIDS derived from cholera toxin recognized by the antibody TE33, and GATPQDLNTML from the HIV-1 capsid protein p24 recognized by the antibody CB4-1. The transformation process was performed by stepwise substitution of each single epitope position by all 19 D-amino acids and glycine followed by antibody binding studies and selection of one D-analog for further transformation. Thus, each transformation step introduced one novel D-position into the peptide. For both epitopes complete D-analogs were obtained. The cholera toxin-derived variant dwGsqhydp binds to the antibody TE33 with higher affinity than its original epitope, whereas in the case of the p24-derived analog saGdwwGkssl lower affinity was detected. Both D-peptides are completely stable in serum for several days. Antibody interaction models for both D-molecules were generated by computer-assisted modelling based on the crystal structures of the starting complexes. Compared with the L-peptides, the binding conformation of dwGsqhydp is very similar, whereas saGdwwGkssl displays a completely different interaction mode.