Structural characterization and IgE epitope analysis of arginine kinase from Scylla paramamosain

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• 作者：Hai-Yan Mao ; Min-Jie Cao ; Soheila J. Maleki ; Qiu-Feng Cai ; Wen-Jin Su ; Yang Yang ; Guang-Ming Liu
• 关键词：AA ; amino acid number ; ELISA ; enzyme-linked immunosorbent assay ; SDS-PAGE ; sodium dodecylsulfate polyacrylamide gel electrophoresis ; PDB ; RCSB protein data bank ; pI ; isoelectric point ; MW ; molecular weight
• 刊名：Molecular Immunology
• 出版年：2013
• 出版时间：31 December, 2013
• 年：2013
• 卷：56
• 期：4
• 页码：463-470
• 全文大小：1970 K

文摘

Arginine kinase (AK) is reported to be the pan-allergen of shellfish. However, there is limited information on its IgE epitopes and structural characteristics. In this study, AK from Scylla paramamosain was purified and characterized. The purified AK is a glycoprotein with the molecular weight of 40 kDa and it demonstrates cross-reactivity with the related allergens present in other shellfish. The cDNA of S. paramamosain AK was cloned, which encodes 357 amino acid residues. Nine linear epitopes and seven conformational epitopes were predicted following bioinformatics analysis. In addition, the entire recombinant AK (rAK) and three partial recombinant AKs (rAK1, rAK2, and rAK3) were successfully expressed in Escherichia coli BL21 (DE3). The proteins of rAK1, rAK2 and rAK have strong IgE reactivity with the pooled sera from crab allergic patients, while rAK3 has significantly weaker IgE reactivity, which indicates that the IgE epitopes of AK are mainly distributed in the regions of rAK1 and rAK2. Furthermore, three experimental linear epitopes (epitope 1: AA 127-141, epitope 2: AA 141-155, and epitope 3: AA 211-225) were discovered in the region of rAK1 and rAK2 using synthetized overlapping peptides. The experimental linear epitopes were mapped onto the protein homology model of AK. Meanwhile, in the IgE-binding assays of the sera from nine crab allergic patients, only three sera reacted with the denatured, linear AK as shown by Western-blotting, eight sera reacted with the native, folded AK by both dot-blotting and ELISA, which indicates that the conformational IgE epitopes of S. paramamosain AK may be more predominant.