Qiu-Feng Cai

Stability of major allergen tropomyosin and other food proteins of mud crab (Scylla serrata) by in vitro gastrointestinal digestion

Stability in simulated gastric fluid is regarded as an important parameter for the estimation of food allergenicity. In this study, the digestive stability of allergenic protein tropomyosin (TM) and other food proteins from mud crab in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion assay system was investigated and compared by SDS-PAGE and Western blot. In SGF system, proteins such as actin and the original band of myosin heavy chain (MHC) were rapidly degraded within a short period of time, while TM was relatively resistant to pepsin digestion. In SIF system, MHC was easily decomposed, while TM and actin were similarly resistant to digestion. Further study by IgE-immunoblotting and inhibition ELISA using sera from crab-allergic patients indicated that allergenicity of TM was partially decreased, suggesting proteinase digestion is effective in reducing the allergenicity of crab TM.

Effects of different processing methods on digestibility of Scylla paramamosain allergen (tropomyosin).

Crustacean allergy is a significant health problem around the world, and tropomyosin (TM) represents the major allergen of crustaceans. The aim of this study was to evaluate three processing methods (boiling, CUB, HPS) and identify the one method that is most effective in the degradation of TM and reduction of its IgE-binding reactivity, and make it easier to be decomposed during gastrointestinal digestion. SDS-PAGE analysis indicated that boiling had little impact on the digestive stability of TM. In contrast, combined ultrasound and boiling (CUB), and high pressure steaming (HPS) both could accelerate the digestion of TM. Similarly, western blotting and inhibition ELISA also demonstrated that the reactivity of IgG/IgE-binding of TM that was extracted from processed crab was partially decreased after treating with CUB or HPS. Among the three processing methods, HPS was the most effective method to accelerate the digestion of TM in gastrointestinal digestion, and reduce the reactivity of IgG/IgE-binding of TM. These results suggest that proper processing of crab could promote the degradation of TM in simulated gastrointestinal digestion, reduce the reactivity of IgG/IgE-binding of TM, and decrease the incidence of crab hypersensitivity in humans.

Purification, cloning, expression and immunological analysis of Scylla serrata arginine kinase, the crab allergen

BACKGROUND Although crustaceans have been reported to be one of the most common causes of IgE-mediated allergic reactions, there are no reports about the characterization and identification of arginine kinase (AK) from the mud crab (Scylla serrata) as allergen. In the present study, the purification, molecular cloning, expression and immunological analyses of the IgE allergen AK from the mud crab were investigated. RESULTS The results showed that cloned DNA fragments of AK from the mud crab had open reading frames of 1021 bp, predicted to encode proteins with 356 amino acid residues. Sequence alignment revealed that mud crab AK shares high homology with other crustacean species. Mud crab AK gene was further recombined with the vector of pGEX-4T-3 and expressed in Escherichia coli BL 21. 2-D electrophoresis suggested that native AK (nAK) and recombinant AK (rAK) shared the same molecular weight of 40 kDa, and the pI is 6.5 and 6.3, respectively. The nAK and rAK were further confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Immunoblotting analysis and colloidal gold immunochromatographic assay (GICA) using sera from subjects with crustacean allergy confirmed that the nAK and rAK reacted positively with these sera, indicating AK is a specific allergen of mud crab. CONCLUSION Both of purified nAK and rAK reacted positively with sera from subjects with crustacean allergy in immunoblotting and GICA analysis, indicating AK is a common allergen of mud crab. In vitro expressed AK is proposed as a source of the protein for immunological or clinical studies.

Purification, Cloning, and Immunological Characterization of Arginine Kinase, a Novel Allergen of Octopus fangsiao

Arginine kinase (AK) is an important enzyme participating in energy metabolism in invertebrates, but, to date, there have been no reports that AK from octopus is an allergen. In this study, octopus AK was purified, and its molecular biological, immunological, and physicochemical characterizations were analyzed. The results showed that octopus AK was purified and confirmed by mass spectrometry for the first time, and its molecular mass was 38 kDa. The full-length gene sequence of octopus AK encompassed 1209 bp and was predicted to encode a protein with 348 amino acid residues. The homology of octopus AK and crustacean AK was about 54%, but the similarity between their three-dimensional structures was high. Octopus AK could react with mouse anti-shrimp AK and rabbit anti-crab AK polyclonal antibody singly. Octopus AK could also react with specific IgE of the sera from octopus-allergic patients effectively, whereas crab AK could inhibit the reaction between them. Finally, the IgE-binding activity of octopus AK could be reduced in the processes of thermal or acid-alkali treatment. In summary, AK was identified as a novel allergen in octopus, which had a sensitizing ability similar to that of crustacean AK. This is significant in allergy diagnosis and the treatment of octopus-allergic disorders.

Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi)

Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21kDa and 21.5kDa, respectively, as estimated by SDS-PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35°C and 40°C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45°C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca(2+) and Mg(2+) and inactivated by Fe(2+), Zn(2+), Mn(2+), Cu(2+), Al(3+), Ba(2+) and Co(2+) to different degrees. Apparent Km values of trypsin A and B were 2.18μM and 1.88μM, and Kcat values were 81.6S(-1) and 111.3S(-1) for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.

Comparative study of in vitro digestibility of major allergen, tropomyosin and other proteins between Grass prawn (Penaeus monodon) and Pacific white shrimp (Litopenaeus vannamei).

BACKGROUND Stability in simulated gastric fluid is supposed to be an important parameter for the estimation of food allergenicity. In the present study, the digestive stability of allergenic protein tropomyosin (TM) and other food proteins from Grass prawn and Pacific white shrimp in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion assay system was investigated and comparatively studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and inhibition enzyme-linked immunosorbent assay (ELISA). RESULTS In the SGF system, proteins such as actin and myosin heavy chain (MHC) were rapidly degraded within a short period of time, while TM was relatively resistant to pepsin digestion. In the SIF system, MHC was also easily decomposed, while TM and actin were resistant to digestion. Western blotting using a specific polyclonal antibody against TM indicated that the degradation pattern of shrimp TM by SGF and SIF was almost unaffected by the presence of other myofibrillar proteins. Further study by IgE immunoblotting and inhibition ELISA using sera from crustacean-allergic patients indicated that IgE binding of TM was decreased. CONCLUSION Proteinase digestion is effective in reducing IgE binding of shrimp TM. It is also of interest to notice that Pacific white shrimp TM had higher digestion stability than Grass prawn TM. However, Pacific white shrimp TM revealed enhanced IgE binding over that of TM from Grass prawn and thus it is possibly more allergenic.

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.

Purification, physicochemical and immunological characterization of arginine kinase, an allergen of crayfish (Procambarus clarkii)

Arginine kinase (AK) has attracted considerable attention because it has been identified as a shellfish allergen. However, little information is available about AK in crayfish (Procambarus clarkii). In this study, crayfish AK was purified and cloned. Its physicochemical properties, processing stability, and immunological characteristics were analyzed. Crayfish AK was purified by column chromatography, which revealed a single band with molecular mass of 40 kDa; this result was further confirmed by mass spectrometry. The full-length gene sequence of crayfish AK was 1462 bp and encoded a protein of 357 amino acid residues. The results of this study revealed that crayfish AK is a glycoprotein with an isoelectric point of approximately 6.5. Thermal stability assays revealed that crayfish AK easily forms aggregates at temperatures >44°C and was stable at pH 4.0-8.0. SDS-PAGE and dot blotting were used to assess processing stability of purified AK. The results revealed that the IgE-binding activity of crayfish AK is reduced after boiling.

Purification and characterization of parvalbumins from silver carp (Hypophthalmichthy molitrix)

BACKGROUND As the largest producer and consumer of freshwater fish in the world, many people suffer from allergy for consuming freshwater fish in China. However, the allergen profiles of freshwater fish are rarely known. RESULTS Parvalbumins (PVs) from the white muscle of silver carp (Hypophthalmichthy molitrix) were purified by ammonium sulfate fractionation and column chromatography including DEAE-Sepharose and Superdex 75. Three PV isoforms-PV-I, PV-II, and PV-III-were obtained and their molecular masses as estimated by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 12, 11, and 14 kDa, respectively. All the PVs could be detected by anti-frog PV monoclonal antibody. PV-I and PV-II were quite possibly glycoproteins, while PV-III was not glycosylated, as analyzed by periodic acid-Schiff (PAS) staining. Thermal stability revealed that PV-I and PV-II easily formed polymers, while these proteins were stable in a pH range of 4.0-10.0. A PV gene encoding 110 amino acid residues was cloned and it revealed high identity with PVs from other species of fish. CONCLUSION Three isotypes of PV were purified to homogeneity and one distinct PV gene was cloned in silver carp white muscle.

Biochemical characterization of chymotrypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus).

Two chymotrypsins (chymotrypsins A and B) have been purified to homogeneity from the hepatopancreas of Japanese sea bass ( Lateolabrax japonicus ) by ammonium sulfate fractionation and chromatographies on DEAE-Sepharose and Phenyl-Sepharose. Two-dimensional electrophoresis (2-DE) analysis revealed that the molecular masses of chymotrypsins A and B were approximately 27.0 and 27.5 kDa, respectively. Their respective isoelectric points were 8.0 and 7.0. Purified chymotrypsins also revealed a single band on native-PAGE, whereas their mobilities were quite different. Optimum temperature and pH of chymotrypsins A and B were 45 degrees C and 8.0, respectively. Both enzymes were strongly inhibited by chymostatin, phenylmethanesulfonyl fluoride (PMSF), and Pefabloc SC, but slightly inhibited by metalloproteinase inhibitor of 1,10-phenanthroline and EDTA. Using Suc-Leu-Leu-Val-Tyr-MCA as substrate, apparent K(m) values of chymotrypsins A and B were 0.8 and 1.1 microM and k(cat) values were 2.7 and 2.0 s(-1), respectively. The N-terminal amino acid sequences of chymotrypsins A and B were determined to the 21st and 18th residues, respectively, and were identical. These sequences exhibited high identities to chymotrypsins from other animals. The digestive effect of the two chymotrypsins on myofibrillar proteins indicated their effectiveness in the degradation of food proteins.