Mud crab ( Scylla serrata), which is widely consumed, can cause severe allergic symptoms. Eight linear epitopes and seven conformational epitopes of tropomyosin (TM) from S. serrata were identified using phage display. The conformational epitopes were formed based on the coiled-coil structure of TM. Most of the epitopes were located in the regions where primary structures were conserved among crustacean TM. Twelve synthetic peptides were designed according to the epitopes and trypsin-cutting sites of TM, among them, three synthetic peptides (including one linear epitope and two conformational epitopes) were recognized by all of the patient sera using inhibitory dot blotting. A triple-variant (R90A-E164A-Y267A) was constructed based on the critical amino acids of the TM epitope. The IgE-binding activity of the triple-variant was significantly reduced compared with that of native TM. The results of phage display and site-directed mutagenesis offered new information regarding conformational epitopes of TM.

The mud crab (Scylla paramamosain) is widely consumed but can cause a severe food allergic reaction. To reduce allergenicity to arginine kinase (AK), site-directed mutagenesis was used to destroy disulfide bonds or mutate critical amino acids of conformational epitopes. Three hypoallergenic mutant AKs (mAK1, mAK2, and mAK3) were generated, with the immunoreactivity decreasing by 54.2, 40.1, and 71.4%, respectively. In comparison to recombinant AK (rAK), the structure of mAKs was clearly changed. Additionally, antisense peptides were designed on the basis of linear epitopes and pepsin-cutting sites of AK. Five peptide aptamers were screened by molecular docking and then analyzed by the immunoglobulin E inhibition enzyme-linked immunosorbent assay and human Laboratory of Allergic Diseases 2 mast cell degranulation assay. The peptide aptamers could significantly inhibit allergenicity of rAK and mAKs, and the inhibitory effect of peptide aptamer 3 was slightly better than the others. These results provide synergistic methods to reduce allergenicity to AK, which could be applied to other shellfish allergens.

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BACKGROUND Myofibril‐bound serine proteinase (MBSP) comprises a class of trypsin‐type serine proteinases that can specifically cleave the carboxyl side of peptide bonds of arginine or lysine residues. To obtain higher specificity MBSPs, mutants were designed based on the substrate‐binding pocket and constructed through site‐directed mutagenesis. RESULTS The wild‐type (WT) and mutants were expressed in Pichia pastoris, and its enzymatic properties indicated that the mutants D170S, D170T, D170K and D170T/G203A were not biologically active. However, S171A specifically cleaved arginine residues and G203A preferably hydrolyzed lysine residues. The secondary structures of mutants were approximately similar to that of the WT according to the results of circular dichroism spectroscopy. Additionally, molecular docking showed that substrates were able to combine with S171A within the critical areas through hydrophobic interaction, hydrogen bonds and van der Waals electrostatic force. CONCLUSION The structural stability of MBSP was consistent using amino acid substitution. Due to the interaction pattern of substrates and mutant enzyme changes, only S171A was selective in cutting Arg residues, which illustrates how the catalytic triad and substrate‐binding pocket of MBSP plays an important role during enzymatic hydrolysis of substrates. © 2018 Society of Chemical Industry