Zhenxing Li

Mapping IgE binding epitopes of major shrimp (Penaeus monodon) allergen with immunoinformatics tools

Tropomyosin, a myofibrillar protein, was described as the major allergen in crustaceans. The objective of this study was to screen and identify the linear IgE epitopes of tropomyosin in Penaeus monodon. Three immunoinformatics tools were used to predict the potential epitopes and the resultant epitopes were confirmed by dot-blot inhibition with sera from shrimp allergic subjects. As the result, 10 peptides were predicted and assembled by solid-phase synthesis. Eight epitopes were identified by dot-blot inhibition test, among which peptide 4 and 8 had not been reported by previous researches. Moreover, peptide 6 and 10 had IgE binding capability with all sera, while peptide 5, 8 and 9 could bind with 80% of the sera. Amino acid analysis demonstrated that Y, E, R, F and S presented more frequently in epitopes. Although further research should be done, it seems that immunoinformatics-driven epitope prediction can benefit epitope identification with high accuracy and efficiency.

Effect of malondialdehyde treatment on the IgE binding capacity and conformational structure of shrimp tropomyosin

The aim of this work was to determine the effect of malondialdehyde (MDA) treatment of shrimp tropomyosin (TM) with respect to IgE binding capacity and conformational structure. Following treatment with MDA, changes in TM molecular weight were characterized by SDS-PAGE and TM cross-linking was observed, then the aggregates were recognized by IgG/IgE in immunoblot analysis. Meanwhile, TM allergenicity slowly decreased following MDA treatment. These data show a correlation between the loss of TM structure and allergenic potential. TM secondary structure became more disordered following treatment with increasing concentrations of MDA. Moreover, the enhancement of surface hydrophobicity was also in accordance with the effect caused by MDA. Additionally, MDA treatment resulted in an increase in carbonyl content and a decrease in free amine groups and available lysine. The results suggest that MDA-induced conformational changes in TM can significantly influence the antigenicity and allergenicity of TM.

Effect of 4-hydroxy-2-nonenal treatment on the IgE binding capacity and structure of shrimp (Metapenaeus ensis) tropomyosin

Lipid peroxidation can react with free amines of proteins and induce modification of structural and functional properties. This study presents the IgE binding capacity and structural changes of shrimp tropomyosin (TM) under oxidative stress with 4-hydroxy-2-nonenal (HNE). IgE binding capacity was evaluated with the dot-blot assay and inhibition enzyme-linked immunosorbent assay. A decrease in IgE binding capacity of TM was found with 0.01mM HNE treatment, which was more significant when the HNE concentration was increased to 0.5mM. The conformational changes of TM, as characterized by fluorescence spectroscopy, circular dichroism spectroscopy and ultraviolet absorption spectroscopy, correlated well with IgE binding capacity changes. Further LC-ESI-MS/MS analyses showed that the side-chain groups of alanine, leucine, lysine and histidine had been modified by HNE. These results suggested that the HNE-induced conformational changes of TM significantly influenced its allergenicity and that these changes were caused by the modification of specific amino acids residues.

Effect of pH shifts on IgE-binding capacity and conformational structure of tropomyosin from short-neck clam (Ruditapes philippinarum)

The aim of the present study was to assess pH-induced changes in conformational structures and potential allergenicity of tropomyosin from short-neck clams. As defined with circular dichroism (CD), an unfolded structure was found at pH values ranging from 2.0 to 5.0, followed by the loss of secondary structure at pH of 1.0. Correspondingly, surface hydrophobicity was reduced by 97.7% when pH was reduced from 7.0 to 1.0. Further indirect ELISA and dot-blot results of pH shifted tropomyosin showed that potential allergenicity correlated well with structural changes, as well as with SGF digestibility. Allergenicity decreased significantly with unfolding of the protein and was stable when surface hydrophobicity recovered back to neutral conditions. These results showed that conformational changes in tropomyosin induced by pH shifting significantly influenced the allergenicity of tropomyosin, and that the resulting changes occurred predominately in the acidic pH range.

Protein carbonylation during electron beam irradiation may be responsible for changes in IgE binding to turbot parvalbumin.

The present study aimed to investigate the relationship between protein carbonylation and changes of the IgE reactivity of turbot parvalbumin (PV) following electron beam (EB) irradiation. The concentration of protein carbonyls, specific IgE binding, and IgE binding inhibition between irradiated and oxidized PV were assessed. Irradiation resulted in a 3-fold enhancement in the protein carbonyl content. In purified PV irradiated with a 10-kGy dose, specific IgE binding was reduced by 91.2±6.2%. When raw PV was treated with reactive oxygen species (ROS), the protein carbonyl content increased 17.6-fold, with the specific IgE binding being reduced by 87.9±6.5% at an ROS concentration of 10 nmol/mL. The IgE binding inhibition between irradiated and oxidized PV was investigated using an inhibition ELISA. Results showed that oxidized PV can inhibit the binding between irradiated PV and specific IgE with an IC50 of 8.2-58 ng according to different doses of irradiation. These findings suggest that EB irradiation reduces specific IgE binding, probably by the induction of protein carbonylation.

Effect of transglutaminase-catalyzed glycosylation on the allergenicity and conformational structure of shrimp (Metapenaeus ensis) tropomyosin

Tropomyosin (TM), a myofibrillar protein, is a major allergen in shrimp. The aim of this study was to evaluate the effect of transglutaminase (TGase)-catalyzed glycosylation on the potential allergenicity and conformational structure of TM in Metapenaeus ensis. Results showed that glycosylation of TM induced unfolding of the primary protein structure followed by loss of the secondary structure. Cleavage of certain free amino groups was observed during TGase-catalyzed glycosylation. The glycosylation rate correlated with reaction temperature. Western blotting and indirect ELISA with TM-specific polyclonal antibodies from rabbit and sera from patients allergic to shrimp demonstrated that antigenicity and potential allergenicity of TM decreased, which correlated well with the conformational changes in its structure. Considering TGase is widely utilized in the food industry, these results indicate that TGase-catalyzed glycosylation has the potential to serve as a mild method for reducing the allergenicity of shrimp products.

Identification of oxidative modification of shrimp (Metapenaeus ensis) tropomyosin induced by malonaldehyde

Abstract In this study, shrimp tropomyosin was subjected to malonaldehyde (MDA)-induced oxidative stress in aqueous situation. The in vivo cross-linking of tropomyosin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the structural changes were investigated by differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy (FTIR). The location of the resulting protein carbonyls was determined by mass spectrometry. The SDS-PAGE of the cross-linked tropomyosin showed four new bands corresponding to two, three, four, and five-time molecular weights of tropomyosin. The conformational structures were partly destroyed because of the thermal denaturation at higher temperatures. The α-helix content increased, and new chemical bonds were formed by the MDA modification. These physiochemical properties were confirmed by the identification of amino acid side-chain modifications by using liquid chromatography–tandem mass spectrometry. Lysine (Lys), glutamine (Gln), and asparagine (Asn) residues in tropomyosin were modified at four lysine (Lys-76, 168, 189, and 233), five glutamine (Gln-61, 70, 118, 147, and 247), and four asparagine (Asn-17, 107, 203, and 215) sites. Apart from these MDA modification sites, oxidized methionine was observed by data search. These results indicate that some active food ingredients, such as MDA, can react with the side chains of amino acids resulting in structural changes in the protein.

Effect of tyrosinase-aided crosslinking on the IgE binding potential and conformational structure of shrimp (Metapenaeus ensis) tropomyosin

The present study was performed to determine crosslinking and oxidative reactions catalyzed by tyrosinase (Tyr), caffeic acid (CA) and their combination with respect to IgE binding potential and conformational structure of shrimp tropomyosin (TM). Cross-links and IgE binding potentials were analyzed by SDS-PAGE, western blot and indirect ELISA. While structural changes were characterized using surface hydrophobicity, ultraviolet (UV), fluorescence and circular dichroism (CD) spectroscopies. Maximum reduction in the IgG (37.19%) and IgE binding potentials (49.41%) were observed when treated with 2000 nkat/g Tyr + CA, as indicated by ELISA analyses. These findings correlated well with the denaturation of protein, as evident by slight blue shift and alterations in the ellipticities observed via structural analyses. The results demonstrated that addition of CA mediator with Tyr pronouncedly enhanced crosslinking, and altered the conformational structure, thereby mitigated allergenicity of TM, thus showing promise in developing novel food structures with reduced allergenic potential.

Determining the effect of malondialdehyde on the IgE-binding capacity of shrimp tropomyosin upon in vitro digestion

BACKGROUND Stability in simulated gastric fluids is considered an important parameter for the estimation of food allergenicity. Moreover, proteins in food are highly susceptible to lipid oxidation during processing and preservation. In this study, the change in the IgE-binding capacity of malondialdehyde (MDA)-treated shrimp tropomyosin (TM) following in vitro digestion was investigated by SDS-PAGE and western blot. RESULTS Shrimp TM treated with different concentrations of MDA was slightly degraded and became increasingly resistant to pepsin digestion over time. While untreated TM was rapidly degraded, MDA-treated TM showed some resistance and was degraded by trypsin only after increasing the digestion time. Results of immunoblotting studies on IgE using sera from patients allergic to shrimp indicated that the IgE-binding capacity of TM and MDA (50 mmol L-1 )-treated TM decreased slightly after pepsin digestion and significantly decreased after trypsin digestion. CONCLUSION The study indicated that the resistance of TM to degradation increased after oxidation. The treatment with proteases, especially trypsin, is quite effective in decreasing the IgG/IgE-binding capacity of shrimp TM.

Effects of brown seaweed polyphenols, α-tocopherol, and ascorbic acid on protein oxidation and textural properties of fish mince (Pagrosomus major) during frozen storage.

BACKGROUND Frozen storage of minced fish is currently one of the most important techniques to maintain its functional properties. However, some deterioration does occur during frozen storage and cause quality loss. The effects of brown seaweed polyphenols, α-tocopherol, and ascorbic acid on lipid and protein oxidation and textural properties of red sea bream (Pagrosomus major) during 90 days of frozen storage at -18 °C were investigated. RESULTS All added antioxidants at 1 g kg-1 resulted in significantly lower thiobarbituric acid-reactive substances (TBARS) compared to the control during the 45 days of frozen storage. The antioxidants were also effective in retarding protein oxidation concerning to total sulfhydryl content and protein carbonyl content. Brown seaweed polyphenols and α-tocopherol significantly retarded the inactivation of Ca2+ -ATPase activity during the first 45 days, whereas ascorbic acid had no such effect. The antioxidant activity showed either an invariable or decrease trend after 45 days storage. Adding antioxidants had a significant effect on the breaking force of the gels during the frozen storage period. CONCLUSION These results indicate that brown seaweed polyphenols and α-tocopherol can be used to prevent oxidative reactions and thus maintain the structure of the gel formed by fish mince during frozen storage.