Liangtao Lv

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.

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.

Allergenicity of acrolein-treated shrimp tropomyosin evaluated using RBL-2H3 cell and mouse model: Acrolein modulates allergenicity of tropomyosin by RBL-2H3 cell and mouse model

BACKGROUND Food processing effects can modify protein functional properties. However, protein was oxidized inevitably by lipid peroxidation during food processing. Acrolein, a primary by-product of lipid peroxidation, can modify the structural and functional properties of protein. The aim of the research was to analyze the effect of acrolein on allergenicity of TM, a major allergen in shrimp. RESULTS The overall allergenic effects of acrolein-treated TM were evaluated using female BALB/c mice and a mediator-releasing RBL-2H3 cell line. Acrolein-treated TM significantly decreased TM-specific immunoglobulin E/G1 levels, and histamine and mMCP-1 release in mouse serum. Release of inflammatory mediators such as β-hexosaminidase, histamine, cysteinyl leukotriene and prostaglandin D2 was clearly suppressed after acrolein treatment. CONCLUSION These results indicate that acrolein-induced tropomyosin modification can decrease the allergenicity of TM. This reduction contributes to allergenic potential changes in shrimp during processing and preservation.

Advanced glycation endproducts in 35 types of seafood products consumed in eastern China

Advanced glycation endproducts (AGEs) have been recognized as hazards in processed foods that can induce chronic diseases such as cardiovascular disease, diabetes, and diabetic nephropathy. In this study, we investigated the AGEs contents of 35 types of industrial seafood products that are consumed frequently in eastern China. Total fluorescent AGEs level and Nε-carboxymethyl-lysine (CML) content were evaluated by fluorescence spectrophotometry and gas chromatography-mass spectrometry (GC-MS), respectively. The level of total fluorescent AGEs in seafood samples ranged from 39.37 to 1178.3 AU, and was higher in canned and packaged instant aquatic products that were processed at high temperatures. The CML content in seafood samples ranged from 44.8 to 439.1 mg per kg dried sample, and was higher in roasted seafood samples. The total fluorescent AGEs and CML content increased when seafood underwent high-temperature processing, but did not show an obvious correlation. The present study suggested that commonly consumed seafood contains different levels of AGEs, and the seafood processed at high temperatures always displays a high level of either AGEs or CML.

Structural changes of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) treated shrimp tropomyosin decrease allergenicity

The aim of this study was to analyze the effect of AAPH on the conformational structure and allergenicity of shrimp tropomyosin (TM). The structure of AAPH-TM was evaluated by SDS-PAGE, fluorescence, circular dichroism (CD) and ultraviolet light (UV), and the allergenicity was evaluated by in vivo and in vitro methods. Results showed that AAPH can induce structural changes through TM aggregations. These aggregations can decrease the IgG/IgE binding capacity on immunoblot analysis. Further competitive inhibition ELISA (ciELISA) results showed the IC50 of AAPH-TM (AAPH 0-25 mmol/l) changed from 0.086 to 46.2 μg/ml, which correlated with TM structural changes. An RBL-2H3 cell assay showed that release rate of β-hexosaminidase and histamine decreased by 51.6% and 68.1% with AAPH (5 mmol/l) treatment, respectively. A mouse model showed AAPH-TM could decrease levels of IgE/IgG1, release of histamine and mMCP-1 in sera. In conclusion, AAPH induced TM aggregation can cause structural changes and decrease the allergenicity.