Shellfish allergy is a prevalent, long-lasting disorder usually persisting throughout life. However, the allergen information is incomprehensive in crab. This study aimed to identify a novel allergen in crab, show its potential in diagnosis and reduce the allergenicity by food processing. A 21-kDa protein was purified from Scylla paramamosain and confirmed as sarcoplasmic calcium binding protein (SCP) by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Total RNA was isolated from crab muscle, and a rapid amplification of cDNA was performed to obtain an ORF of 579 bp that coded for 193 amino acid residues. According to the results of circular dichroism analysis and ELISA assay, the recombinant SCP (rSCP) expressed in Escherichia coli showed similar physicochemical and immunoreactive properties to native SCP (nSCP). Additionally, the extensive cross reactivity of SCP among different species and the bidirectional IgE cross-reactivity between nSCP and rSCP were detected by iELISA. The allergenicity of rSCP was reduced via Maillard reaction or enzymatic cross-linking reaction, which was confirmed by the results of scanning electron microscopy, dot blot, and digestion assay. A straightforward and reproducible way was developed to obtain high yields of rSCP that maintains structural integrity and full IgE reactivity, which could compensate the low specific IgE-titers of most patient sera for future diagnosis. Furthermore, the Maillard reaction and enzymatic cross-linking reaction were effective approaches for the production of hypoallergenic seafood.
The enzymatic cross-linking of proteins to form high-molecular-weight compounds may alter their sensitization potential. The IgG-/IgE-binding activity, digestibility, allergenicity, and oral tolerance of cross-linked tropomyosin with tyrosinase (CTC) or horseradish peroxidase (CHP) were investigated. ELISA results demonstrated CTC or CHP reduced its IgE-binding activity by 34.5 ± 1.8 and 63.5 ± 0.6%, respectively. Compared with native tropomyosin or CTC, CHP was more easily digested into small fragments; CHP decreased the degranulation of RBL-2H3 cells and increased endocytosis by dendritic cells. CHP can induce oral tolerance and reduce allergenicity in mice by decreasing IgE and IgG1 levels in serum, the production of T-cell cytokines, and the percentage composition of dendritic cells. These findings demonstrate CHP has more potential of reducing the allergenicity than CTC via influencing the morphology of protein, changing the original method of antigen presentation, modulating the Th1/Th2 immunobalance, and inducing the oral tolerance of the allergen tropomyosin.
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