Title: Structural characterization and IgE epitope analysis of arginine kinase from Scylla paramamosain Authors
|Mao, Hai-Yan -|
|Cao, Min-Jie -|
|Cai, Qiu-Feng -|
|Su, Wen-Jin -|
|Yang, Yang -|
|Liu, Guang-Ming -|
Submitted to: Molecular Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 29, 2013
Publication Date: August 4, 2013
Citation: Mao, H., Cao, M., Maleki, S.J., Cai, Q., Su, W., Yang, Y., Liu, G. 2013. Structural characterization and IgE epitope analysis of arginine kinase from Scylla paramamosain. Molecular Immunology. 56:463-470. Interpretive Summary: Arginine kinase (AK) has been reported as an allergen in shellfish. There is limited information about the allergenic regions structural characteristics of this protein. In this study, AK from mud crab was purified and characterized. The purified AK was found to be very similar to the related allergens in other shellfish in amino acid sequence and allergenic regions. Because of this similarity, the DNA was able to be found, which was then used to produce this protein or smaller fragments, thereof, in bacteria (referred to as recombinant AK or rAK). Nine allergenic and seven structural regions were predicted by the bioinformatics analysis. The results showed rAK and two of the fragments had strong IgE reactivity with the antibodies of shellfish allergic patients, a third fragment had weak IgE reactivity, which helped us identify the regions more important for allergenicity. These allergenic regions were mapped onto the structure of the AK protein to identify the structural regions involved in allergenicity. Importantly, it was determined that if the protein was unfolded, it became less allergenic, which means any type of processing or genetic engineering to unfold this major allergen may reduce the allergenic potential of shellfish for allergic individuals.
Technical Abstract: Arginine kinase (AK) has been reported as the pan-allergen of shellfish, however, there is limited information about its IgE epitopes and structural characteristics. In this study, AK from Scylla paramamosain was purified and characterized. The purified AK was a glycoprotein with the molecular weight of 40 kDa, and it demonstrates cross-reactivity with the related allergens in other shellfish. The Scylla paramamosain AK cDNA was cloned, which encodes 357 amino acid residues. Nine linear epitopes and seven conformational epitopes were predicted by the bioinformatics analysis. In addition, the whole recombinant AK (rAK) and three partial recombinant AKs (rAK1, rAK2, and rAK3) were expressed in Escherichia coli BL21 (DE3). The results showed rAK1, rAK2, and rAK had strong IgE reactivity with the pooled sera of shellfish allergic patients, however, rAK3 had weak IgE reactivity, which indicated the IgE epitopes of AK are distributed mainly in the region of rAK1 and rAK2. Furthermore, three linear epitopes (epitope 1: AA 127-141, epitope 2: AA 141-155, and epitope 3: AA 211-225) were found in the region of rAK1 and rAK2 using synthetized peptides. The linear epitopes were mapped onto the protein homology model of AK protein. Meanwhile, in the IgE-binding assays of nine shellfish allergic patients’ sera, only three sera reacted with the denatured linear AK by Western blotting, eight sera reacted with the native folded AK by both dot-blotting and ELISA, which indicated that the conformational IgE epitopes of Scylla paramamosain AK may be more predominant.