Reduction in Allergenicity and Induction of Oral Tolerance of Glycated Tropomyosin from Crab.

Tropomyosin (TM) is an important crustacean (Scylla paramamosain) allergen. This study aimed to assess Maillard-reacted TM (TM-G) induction of allergenic responses with cell and mouse models. We analyzed the difference of sensitization and the ability to induce immune tolerance between TM and TM-G by in vitro and in vivo models, then we compared the relationship between glycation sites of TM-G and epitopes of TM. In the in vitro assay, we discovered that the sensitization of TM-G was lower than TM, and the ability to stimulate mast cell degranulation decreased from 55.07 ± 4.23% to 27.86 ± 3.21%. In the serum of sensitized Balb/c mice, the level of specific IgE produced by TM-G sensitized mice was significantly lower than TM, and the levels of interleukins 4 and interleukins 13 produced by Th2 cells in spleen lymphocytes decreased by 82.35 ± 5.88% and 83.64 ± 9.09%, respectively. In the oral tolerance model, the ratio of Th2/Th1 decreased from 4.05 ± 0.38 to 1.69 ± 0.19. Maillard reaction masked the B cell epitopes of TM and retained some T cell epitopes. Potentially, Maillard reaction products (MRPs) can be used as tolerance inducers for allergen-specific immunotherapy.

Effects of the Maillard reaction on the epitopes and immunoreactivity of tropomyosin, a major allergen in Chlamys nobilis.

Scallop (Chlamys nobilis) causes an IgE-mediated food allergy; however, studies of the allergens in its musculus are not sufficiently comprehensive. In this context, the target protein was purified from scallops and confirmed to be the major allergen tropomyosin (TM) using proteomic technology and serological testing. Subsequently, seven potential IgE epitopes of TM were obtained using phage display technology with IgE enrichment from the serum of scallop-sensitized patients and identified via inhibition enzyme-linked immunosorbent assays. A method for the Maillard reaction of TM and xylose was established, and Maillard-reacted TM (MR-TM) showed significantly decreased immunobinding activity and CD63 and CD203c expression in basophils compared with TM. Furthermore, shotgun proteomics analysis showed that eleven specific amino acids (K12, R15, K28, K76, R125, R127, K128, R133, R140, K146, and K189) of the six IgE epitopes of TM were modified after the Maillard reaction. Overall, the immunoactivity of MR-TM was reduced, which provides a theoretical reference for the development of hypoallergenic foods.

Cloning, Expression, and Epitope Identification of Myosin Light Chain 1: An Allergen in Mud Crab.

Mud crab (Scylla paramamosain) is a commonly consumed seafood as a result of its high nutritional value; however, it is associated with food allergy. The current understanding of crab allergens remains insufficient. In the present study, an 18 kDa protein was purified from crab muscle and confirmed to be myosin light chain 1 (MLC1) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. Total RNA was isolated and amplified to obtain a MLC1 open reading frame of 462 bp, encoding 154 amino acids. A structural analysis revealed that recombinant MLC1 (rMLC1) expressed in Escherichia coli contained α-helix and random coil. Moreover, rMLC1 displayed strong immunoactivity by dot blot and a basophil activation test. Furthermore, seven allergenic epitopes of MLC1 were predicted, and five critical epitope regions were identified by an inhibition enzyme-linked immunosorbent assay and human mast cell degranulation assay. This comprehensive research of an allergen helps to conduct component-resolved diagnoses and immunotherapies related to crab allergies.