Effect Of PH Shifts And Glycation On IgE-binding Capacity And Conformational Structure Of Tropomyosin From Short-neck Clam (Ruditapes Philippinarum)

The short-neck clam(Ruditapes philippinarum) is the most popular farming species with high economic value,delicious flavour and rich nutrition in China, Southeast Asia and Japan. However, the short-neck clam has also been recognized as one of the most frequent causes of food allergies as a common kind of molluscs. Tropomyosin is considered to be a major allergen both in molluscs and crustaceans. This research focuses on the properties of short-neck clam tropomyosin, assessment of pH-induced and glycation-induced changes in conformational structures and potential allergenicity of tropomyosin.Further analysis on the epitopes mapping of tropomyosin was carried out combining with bioinformatic approaches. The main results are as follow:1. The major allergen protein tropomyosin of short-neck clam with the molecular weight 37 kDa was extracted and identified by immunoblotting using rabbit anti-shrimp tropomyosin polyclonal antibody. The results of properties analysis showed that the carbohydrates in tropomyosin were 6.34% and isoelectric point was 5.1. Short-neck clam tropomyosin was stable in the process of thermal and decomposition temperature was 109.98℃. The full-length cDNAs encoding tropomyosin was cloned by RACE technology, whose cDNA contained an open reading frame composed of 855 bp coding for 284 amino acid residues.2. As defined with circular dichroism (CD), an unfolded structure was found at pH values ranging from 2.0-5.0, followed by the loss of secondary structure at a pH of 1.0. Correspondingly, surface hydrophobicity was reduced by 97.7% when pH was reduced from 7.0 to 1.0. Further indirect ELISA and dot-blot results of pH shifted tropomyosin showed that potential allergenicity correlated well with structural changes, as well as with SGF digestibility. Allergenicity decreased significantly with unfolding of the protein and was stable when surface hydrophobicity recovered back to neutral conditions. These results showed that conformational changes in tropomyosin induced by pH shifting significantly influenced the allergenicity of tropomyosin, and that the resulting changes occurred predominately in the acidic pH range. For some short-neck clam allergic patients, a pH shifting processing may be an efficient way to reduce the hazards of clam consumption.3. The aim of the study was to assess glycation-induced changes in conformational structures and potential allergenicity of tropomyosin from short-neck clams with ribose. Changes in potential allergenicity were characterized by immunological techniques, while circular dichroism (CD) and available lysine were determined to define conformational changes. An unfolded structure was found as the time increasing during glycation. Correspondingly, the amounts of available lysine as reduced by 45.5% and surface hydrophobicity was increased by 192% when the reaction time was 12 h. Indirect ELISA and dot-blot results of glycation showed that potential allergenicity correlated well with structural changes. Allergenicity decreased significantly with unfolding of the protein. These results showed that conformational changes in tropomyosin induced by glycation significantly influenced the allergenicity of tropomyosin.4. Homology between short-clam tropomyosin and other tropomyosin of molluscs and crustacean was analyzed by bioinformatics approaches. Results showed that the sequences of different tropomyosins were conservative, which indicated that the high homology of tropomyosin. DNAStar and AntheProt prediction system with two prediction websites were used to predict the epitopes of short-calm tropomyosin. As a result,10 peptides were predicted, among which 4 peptides were compounded and confirmed according to the experimental results.The experimental results showed that any modifications that induce structural changes in proteins may affect antibody-binding capacity. Conformational changes, such as protein unfolding or exposure of buried amino acid, may explain a reduction in antigenicity and allergenicity of allergens upon treatment. Protein fragments or specific epitopes that are sufficient for IgE binding may be maintained, depending on the intensity of treatment. The results of the epitope prediction also contribute to the control of allergens, which is expected to develop hypoallergenic shellfish food and geve supports to improve the safety of shellfish food.