Purification and characterization of parvalbumins, the major allergens in red stingray (Dasyatis akajei).
Fish has received increasing attention because it induces IgE-mediated food allergy. Parvalbumin (PV) represents the major allergen of fish, and IgE cross-reactivity to PV in various teleost fish species has been shown, while little information is available about allergens in elasmobranch fish. In this study, two PV isoforms (named as PV-I and PV-II) from red stingray (Dasyatis akajei) were purified to homogeneity by a series of procedures including ammonium sulfate precipitation and column chromatographies of DEAE-Sepharose and Sephacryl S-200. Purified PVs revealed a single band on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of PV-I and PV-II were 12.29 and 11.95 kDa, respectively, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot using antifrog PV monoclonal antibody (PARV-19) showed positive reactions to the two proteins, confirming that they were PVs, although their immunological reactivities were weaker than those of PV from silver carp. The N-terminal amino acid sequence of PV-I was determined, and comparison with PVs from other fish species showed low homology between teleost and elasmobranch fish. The isoelectric points of PV-I and PV-II were 5.4 and 5.0, respectively, as determined by two-dimensional electrophoresis (2-DE), suggesting that both isoforms belong to the α-group. IgE immunoblotting analysis showed that sera from fish-allergic patients reacted to both PV-I and PV-II from red stingray. Thermal stability revealed that PV-I easily formed oligomers than PV-II, which might contribute to the maintenance of its allerginicity during heat processing.
College of Biological Engineering, Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Jimei, Xiamen, China, 361021.
Qiu-Feng
QF
Cai
Guang-Ming
GM
Liu
Teng
T
Li
Kenji
K
Hara
Xi-Chang
XC
Wang
Wen-Jin
WJ
Su
Min-Jie
MJ
Cao
D000485:Allergens / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D029941:Fish Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010320:Parvalbumins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D055550:Protein Stability; D016415:Sequence Alignment; D012860:Skates, Fish / Q000276:immunology*
D000485:Allergens; D029941:Fish Proteins; D010320:Parvalbumins