Mapping and characterization of antigenic epitopes of arginine kinase of Scylla paramamosain.
Arginine kinase (AK) is a panallergen present in crustaceans, which can induce an immunoglobulin (Ig) E-mediated immune response in humans. The aim of this work was to map and characterize the antigenic epitopes of Scylla paramamosain AK. Specific-protein-A-enriched IgG raised in rabbits against purified S. paramamosain AK was used to screen a phage display random peptide library. Five AK mimotope clones were identified among 20 random clones after biopanning. Four conformational epitopes D3A4K43M1A5T49T44I7, L31K33V35T32E11E18F14S34D37, V177G172M173D176Q178T174L181K175L187, and R202L170Y203E190P205W204L187T206Y145 were identified with the program LocaPep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively. On the basis of biopanning, six IgE-specific peptides were mapped with synthetic overlapping peptides using the sera from crab-allergic patients, and four seropositive peptides (amino acids 113-127, 127-141, 141-155, and 204-218) were confirmed as linear epitopes in a degranulation assay in RBL-2H3 cells. Stability experiments showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability.
College of Food and Biological Engineering, Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Jimei University, 43 Yindou Road, Xiamen 361021, Fujian, China.
Yang
Y
Yang
College of Food and Biological Engineering, Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Jimei University, 43 Yindou Road, Xiamen 361021, Fujian, China.
Min-Jie
MJ
Cao
School of Biosciences, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Marcos
M
Alcocer
College of Food and Biological Engineering, Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Jimei University, 43 Yindou Road, Xiamen 361021, Fujian, China.
Qing-Mei
QM
Liu
College of Food and Biological Engineering, Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Jimei University, 43 Yindou Road, Xiamen 361021, Fujian, China.
Dan-Xia
DX
Fei
College of Food and Biological Engineering, Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Jimei University, 43 Yindou Road, Xiamen 361021, Fujian, China.
Hai-Yan
HY
Mao
College of Food and Biological Engineering, Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Jimei University, 43 Yindou Road, Xiamen 361021, Fujian, China. Electronic address: gmliu@jmu.edu.cn.
Guang-Ming
GM
Liu
D000485:Allergens / Q000235:genetics / Q000276:immunology; D000818:Animals; D001122:Arginine Kinase / Q000235:genetics / Q000276:immunology; D060829:Arthropod Proteins / Q000235:genetics / Q000276:immunology; D003386:Brachyura / Q000235:genetics* / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005260:Female; D005512:Food Hypersensitivity / Q000235:genetics / Q000276:immunology; D006801:Humans; D008297:Male; D019151:Peptide Library; D011817:Rabbits; D017747:Seafood / Q000009:adverse effects*
D000485:Allergens; D060829:Arthropod Proteins; D000939:Epitopes; D019151:Peptide Library; D001122:Arginine Kinase
Arginine kinase; Denaturating processing; Epitope; Mast cells; Phage display; Synthetic peptides