title: Structural characterization and IgE epitope analysis of arginine kinase from Scylla paramamosain.
abstract: 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.
authors.affiliation: College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, 43 Yindou Road, Xiamen, Fujian 361021, China.
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Mao
authors.forename: Min-Jie
authors.initials: MJ
authors.lastname: Cao
authors.forename: Soheila J
authors.initials: SJ
authors.lastname: Maleki
authors.forename: Qiu-Feng
authors.initials: QF
authors.lastname: Cai
authors.forename: Wen-Jin
authors.initials: WJ
authors.lastname: Su
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Guang-Ming
authors.initials: GM
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001122:Arginine Kinase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D003386:Brachyura / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D005512:Food Hypersensitivity / Q000097:blood / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology* / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D010802:Phylogeny; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000939:Epitopes; D007527:Isoenzymes; D010455:Peptides; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D001122:Arginine Kinase
keywords: AA; Arginine kinase; ELISA; Gene expression; IgE epitope; MW; PDB; Protein homology modeling; RCSB protein data bank; SDS-PAGE; Shellfish allergy; Synthesized peptides; amino acid number; enzyme-linked immunosorbent assay; isoelectric point; molecular weight; pI; sodium dodecylsulfate polyacrylamide gel electrophoresis