Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies.
The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown.
The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG4 binding regions.
The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg2+, but not Ca2+, binding was determined by band shift using SDS-PAGE. IgE and IgG4 epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients.
The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG4. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera.
This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG4 binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.
Knowledge of potential pathogens in seafood is of major significance for human health. The high rates of parasitation of fish all over the world make Anisakis a serious health hazard. In fact, Anisakiasis is a growing zoonotic disease in countries where consumption of raw/marinated fish is high. Moreover, Anisakiasis could be under diagnosed in countries where the consumption of these dishes is less common, since it could be easily misdiagnosed as appendicitis, gastric ulcer or other food allergies. Allergen structural studies are essential for the development of specific diagnostic tests and novel immunotherapy strategies. In the present study, we have elucidated for the first time the tertiary structure of Ani s 5 Anisakis allergen and its IgE and IgG4 regions implicated in allergic response. Ani s 5 belongs to the SXP/RAL-2 protein family. Several members of this family have been detected in animal and plant parasitic nematodes. As no homologs have been identified outside the Nematoda, these proteins may be suitable targets for controlling the damage caused by these parasites. Our work reveals that the structure of Ani s 5 resembles that of Calmodulin but binds Mg2+ instead of Ca2+, which suggests some putative functions for SXP/RAL-2 proteins.