Preliminary Study On The Allergenicity And Structure-activity Relationship Of Arginine Kinase From Scylla Paramamosain

The safety of seafood has obtained more attention for it could cause allergic diseases. Crabis one of the eight major allergic foods proposed by FAO. To date, two clinically relevantallergens of crab, Tropomyosin (TM) and Arginine kinase (AK), were identified as the panallergens in shellfish. Due to the epitope was the key factor in the antigen-antibody bindingreaction, the study of food allergen epitope was essential to the development of hypoallergenicfood. The researach on TM was sufficiently, its8IgE epitopes were found, however, there islimited information on its IgE epitopes and structural characteristics.In this study, Scylla paramamosain was selected as the material. AK from it was purified byammonium sulfate fractionation and anion exchange column chromatography. The result of2-DPAGE and PAS staining indicated that AK is a glycoprotein with the molecular weight of40kDaand isoelectric point of6.5. The thermal and pH stability of AK was determined, the resultsshowed that AK was stable in acid or alkali but not stable to heat (>44°C), AK was becamedimer and polymer. Digestive stability of AK was analyzed by simulated gastrointestinaldigestion assay, the results showed that AK was digested by pepsin but not digested by trypsin,and the pepsin hydrolysate of AK had IgE bingding activity. The inhibition Western-blottingassay demonstrated that AK had cross-reactivity with the related allergens present in othershellfish. In the IgE-binding assays of the sera from nine crab allergic patients, only three serareacted with the denatured, linear AK as shown by Western-blotting, eight sera reacted with thenative, folded AK by both dot-blotting and ELISA, which indicates that the conformational IgEepitopes of Scylla paramamosain AK may be more predominant.The cDNA of Scylla paramamosain AK was cloned by the method of RT-PCR and RACE,which was1457bp with an open reading frame of1074bp. The ORF encodes357amino acidresidues, its predicted molecular was40.29kDa and isoelectric point was6.44. Both thenucleotide and amino acide sequence were avilable in GenBank/EBI databases, the accessionnumber were JN828652and AFA45340, respectively. The bioinformatics analysis of Scyllaparamamosain AK showed it belongs to phosphagen_kinase super-family and had two structuraldomains: ATP: guanidophosphotransferase N-terminal domain and ATP: guanidophosphotransferase C-terminal domain. Its putative enzymatic active site is located at271-277and N-glycosylation site at the position of215-217. A homology model of Scylla paramamosain AK was constructed by SWISS-MODEL server. The Nine linear epitopes and sevenconformational epitopes were predicted by the DNA STAR Protean and DiscoTope2.0softwear.In addition, the entire recombinant AK (rAK) and three partial recombinant AKs (rAK1,rAK2, and rAK3) were successfully expressed in Escherichia coli BL21(DE3). rAK1, rAK2andrAK have strong IgE reactivity with the pooled sera from crab allergic patients, while rAK3hassignificantly weaker IgE reactivity, which indicates that the IgE epitopes of AK are mainlydistribute in the regions of rAK1and rAK2. Furthermore, three linear epitopes (epitope1: AA127-141, epitope2: AA141-155, and epitope3: AA211-225) were found in the region of rAK1and rAK2using synthetized peptides. The linear epitopes were mapped onto the proteinhomology model of AK. It is worth noting that the three linear epitopes were included in theregion of rAK1and rAK2.In summary, this paper gives a systematic analysis on the allergenicity and structure-activityrelationship of arginine kinase from Scylla paramamosain. The type and advantage area ofepitope were primarily confirmed. The results will lay the foundation for understannding of craballergens and provide the theoretical basis for development of hypoallergenic food products andsafer immunodetection/immunotherapy.