Two allergens from Scylla paramamosain share common epitopes showed different allergenic potential in Balb/c mice.

Filamin C (FLN c) and triosephosphate isomerase (TIM) are novel allergens of crab (Scylla paramamosain) which are sharing common epitopes. This work aimed to assess their contributions to the induction and elicitation of allergenic responses. Balb/c mice were sensitized by intraperitoneal injections and challenged by intragastric gavage with purified proteins. Upon oral challenge, FLN c triggered more severe anaphylactic symptoms, higher levels of specific antibodies and histamine in serum than TIM, while TIM was a more active promotor of early specific antibody production and stimulated stronger Th2-biased responses. Combined with the results of in vitro assays, the data demonstrated that though with common epitopes, the two allergens showed a different allergenicity, TIM favored Th2 polarization in sensitization stage, while FLN c had a better ability to stimulate B cells and is highly immunogenic in oral challenge stage. The findings can help with the better understanding of allergenicity of crab allergens.

Coumarin alleviates ovalbumin-induced food anaphylaxis in a mouse model by affecting mast cell function.

Coumarin is an important organic heterocyclic compound with a wide range of sources in nature. It plays an important role in the drug discovery process due to its existence in diverse biologically active compounds and its broad bioactivity. In this study, the anti-allergic activity of coumarin was evaluated using an ovalbumin (OVA)-induced mouse food allergy model and an immunoglobulin (Ig)E mediated mouse bone marrow-derived mast cell (BMMC) model. Coumarin could alleviate the OVA-induced allergic symptoms, decrease the diarrhea rates, and promote the rectal temperature rise in allergic mice. Moreover, coumarin had the ability to reduce the levels of histamine and mouse mast cell proteinases, inhibit OVA-specific IgE, and significantly decrease the population of mast cells in the spleen and mesenteric lymph nodes. Coumarin could also significantly suppress mast cell-dependent passive cutaneous anaphylaxis. Additionally, the number of mature BMMCs was decreased as coumarin caused the suppression of c-KIT receptors. Furthermore, coumarin up-regulated the apoptosis of OVA-activated BMMCs in a concentration-dependent manner. In conclusion, coumarin displayed effective anti-food allergy activity via the regulation of mast cell function and numbers. Coumarin and its derivatives provide a new direction for the development of anti-food allergic drug components.

A comprehensive analysis of the allergenicity and IgE epitopes of myosinogen allergens in Scylla paramamosain.

BACKGROUND: Scylla paramamosain is one of the most common and serious food allergens in Asia. Therefore, research on its prevalence, accurate diagnosis, and IgE-binding pattern of the allergens is crucial. OBJECTIVE: To identify the IgE epitopes of the myosinogen allergens in S. paramamosain using phage peptide library. METHODS: The prevalence of allergy to crabs (AC) and of sensitization was analysed using a questionnaire and a serological assay. BAT was performed by flow cytometry, and its diagnostic performance was evaluated in relation to allergens purified from crab myosinogen. IgE-binding epitopes were identified by phage display using the IgE from patients with AC. Sequence- and structure-based bioinformatics analyses were performed to identify allergenic epitopes. RESULTS: Crab was the most common cause of food allergies in this study. Subjects with AC (n = 30) with clear clinical symptoms were identified by immunoblotting and BAT. All of the myosinogen allergens triggered basophil activation; surface expression of CD63 and CD203c was higher in patients allergic to AK and FLN c than in patients allergic to SCP and TIM. In addition to six conformational epitopes of SCP, six linear epitopes and eight conformational epitopes of AK were identified. Five linear epitopes and three conformational epitopes of TIM, nine linear and ten conformational epitopes of FLN c were also identified, and the sequence VH(I/T) L was appeared in epitopes of both TIM and FLN c. The number of epitopes showed consistency with the value of BAT. CONCLUSIONS AND CLINICAL RELEVANCE: BAT can be used for accurate diagnosis of AC. Identification of particular allergenic motifs could be a valuable tool for prevention, diagnosis, and treatment of food allergies.

Crystal structure determination of Scylla paramamosain arginine kinase, an allergen that may cause cross-reactivity among invertebrates.

Shellfish are one of the most common causes of food allergy. Arginine kinase (AK) is known as an important allergen in shellfish. In the present study, AK from crab (Scylla paramamosain) was purified and its crystal structure was determined. A comparison of AK from S. paramamosain to AKs of other species showed high amino acid sequence and secondary structure identity, while the superposition of crystal structures of AKs from different species revealed only slight differences. Similarity of the linear epitope regions among species was observed in the epitope alignment of AKs; conformational epitopes were located in the regions where secondary structure was conserved. The structure of S. paramamosain AK is an accurate template for the analysis of the IgE binding pattern, and the structure conservation and epitope similarity of AKs among species could help to inform our understanding of the cross-reactivity and contribute to the prediction of cross-reactivity related epitopes.

Deep-Sea-Derived Butyrolactone I Suppresses Ovalbumin-Induced Anaphylaxis by Regulating Mast Cell Function in a Murine Model.

Deep-sea-derived butyrolactone I (BTL-I), which was identified as a type of butanolide, was isolated from Aspergillus sp. Ovalbumin (OVA)-induced BALB/c anaphylaxis was established to explore the antifood allergic activity of BTL-I. As a result, BTL-I was able to alleviate OVA-induced allergy symptoms, reduce the levels of histamine and mouse mast cell proteinases, inhibit OVA-specific IgE, and decrease the population of mast cells in the spleen and mesenteric lymph nodes. BTL-I also significantly suppressed mast-dependent passive cutaneous anaphylaxis. Additionally, the maturation of bone marrow-derived mast cells (BMMCs) declined as BTL-I caused down-regulation of c-KIT receptors. Furthermore, molecular docking analyses revealed that BTL-I interacted with the inhibitory receptor, FcγRIIB. In conclusion, the reduction of mast cell function by deep-sea-derived BTL-I as well as its interactions with the inhibitory receptor, FcγRIIB, may contribute to BTL-I-related protection against food anaphylaxis.

In vitro and in vivo immunomodulatory activity of sulfated polysaccharide from Porphyra haitanensis.

The immunoregulatory activity of sulfated polysaccharide from Porphyra haitanensis (PHPS) was investigated in a RAW264.7 macrophages cell model and a BALB/c murine model. The subpopulation of dendritic cells (DCs) and regulatory T cells (Tregs) from PHPS-treated mice splenocytes were also measured by flow cytometry. Consistent with previous reports, we showed that PHPS increased the phagocytosis of RAW264.7 macrophages, and enhanced the secretion of interleukin (IL)-6, IL-10 and tumor necrosis factor-α (TNF-α). Meanwhile, PHPS induced the production of nitric oxide via the Jun N-terminal kinase (JNK) and the Janus kinase (JAK2) signaling pathways in RAW264.7 macrophages. Furthermore, PHPS promoted the proliferation of mice lymphocytes, inducing the generation of TNF-α and IL-10 in vivo, as well as the subpopulation of CD4+ splenic T lymphocytes, DCs, and Tregs. These results indicated that PHPS plays key roles in immunoregulation and may be apply to develop new health foods.

Triosephosphate Isomerase and Filamin C Share Common Epitopes as Novel Allergens of Procambarus clarkii.

Triosephosphate isomerase (TIM) is a key enzyme in glycolysis and has been identified as an allergen in saltwater products. In this study, TIM with a molecular mass of 28 kDa was purified from the freshwater crayfish (Procambarus clarkii) muscle. A 90-kDa protein that showed IgG/IgE cross-reactivity with TIM was purified and identified as filamin C (FLN c), which is an actin-binding protein. TIM showed similar thermal and pH stability with better digestion resistance compared with FLN c. The result of the surface plasmon resonance (SPR) experiment demonstrated the infinity of anti-TIM polyclonal antibody (pAb) to both TIM and FLN c. Five linear and 3 conformational epitopes of TIM, as well as 9 linear and 10 conformational epitopes of FLN c, were mapped by phage display. Epitopes of TIM and FLN c demonstrated the sharing of certain residues; the occurrence of common epitopes in the two allergens accounts for their cross-reactivity.

Inhibitory effects of Na7PMo11CuO40 on mushroom tyrosinase and melanin formation and its antimicrobial activities.

Keggin-type Cu-substituted phosphomolybdic acid (Na7PMo11CuO40, abbreviated as PMo11Cu) was synthesized and characterized. The inhibitory effects of PMo11Cu on mushroom tyrosinase and melanin formation in B16 melanoma cells were studied. The results showed that PMo11Cu could strongly inhibit the activity of tyrosinase, and it was reversible and competitive inhibitor. The IC50 value was estimated to be 0.48 mM for diphenolase activity. PMo11Cu also exhibited inhibitory effects on cell viability, cellular tyrosinase activity and melanin formation in B16 melanoma cells at concentrations ranging from 0 to 200 µM for 24 h. Furthermore, the antimicrobial activities of PMo11Cu against Sarcina lutea, Staphylococcus aureus, Bacillus subtilis and Escherichia coli were investigated. The results showed that PMo11Cu had an obvious antimicrobial activities, and it was more effective against two kinds of coccus than two kinds of bacillus. This study may provide theoretical basis for designing novel effective mushroom tyrosinase inhibitors and extend the use of polyoxometalates in the fields of food preservation and depigmentation.

Preparation, characterisation and use for antioxidant oligosaccharides of a cellulase from abalone (Haliotis discus hannai) viscera.

BACKGROUND: In China, abalone (Haliotis discus hannai) production is growing annually. During industrial processing, the viscera, which are abundant of cellulase, are usually discarded or processed into low-value feedstuff. Thus, it is of interest to obtain cellulase from abalone viscera and investigate its application for preparation of functional oligosaccharides. RESULTS: A cellulase was purified from the hepatopancreas of abalone by ammonium sulfate precipitation and two-steps column chromatography. The molecular weight of the cellulase was 45 kDa on SDS-PAGE. Peptide mass fingerprinting analysis yielded 103 amino acid residues, which were identical to cellulases from other species of abalone. Substrate specificity analysis indicated that the cellulase is an endo-1,4-ß-glucanase. Hydrolysis of seaweed Porphyra haitanensis polysaccharides by the enzyme produced oligosaccharides with degree of polymerisation of two to four, whose monosaccharide composition was 58% galactose, 4% glucose and 38% xylose. The oligosaccharides revealed 2,2'-diphenyl-1-picrylhydrazyl free radical as well as hydrogen peroxide scavenging activity. CONCLUSION: It is feasible and meaningful to utilise cellulase from the viscera of abalone for preparation of functional oligosaccharides. © 2015 Society of Chemical Industry.

Matrix Metalloproteinase 2 (MMP-2) Plays a Critical Role in the Softening of Common Carp Muscle during Chilled Storage by Degradation of Type I and V Collagens.

Matrix metalloproteinases (MMPs) are proposed to play important roles in the degradation of collagens, thus causing the post-mortem softening of fish muscle, although the specific mechanism remains largely unresolved. Previously, we reported the existence of gelatinase-like proteinases in common carp (Cyprinus carpio) muscle. The primary structures of these proteinases, however, have never been investigated. In the present study, two MMPs with molecular masses of 66 and 65 kDa were purified to homogeneity from common carp muscle by ammonium sulfate fractionation and a series of column chromatographies. Matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) analysis indicated that they are completely identical to MMP-2 from common carp. During chilled storage of common carp at 4 °C, the enzymatic activity of MMP-2 increased to 212% in 12 h while the texture profile increased over the first 2 h and gradually decreased. On the other hand, type V collagen was purified to homogeneity and a specific polyclonal antibody against this protein was prepared. Both type I and V collagens were effectively hydrolyzed by MMP-2 at 30 °C and even at 4 °C. Furthermore, injection of metalloproteinase proteinase inhibitor EDTA into the blood vessel of live common carp suppressed post-mortem tenderization significantly. All of these results confirmed that MMP-2 is a major proteinase responsible for the degradation of collagens, resulting in the softening of fish muscle during chilled storage.

Cathepsin L is an immune-related protein in Pacific abalone (Haliotis discus hannai)--Purification and characterization.

Cathepsin L, an immune-related protein, was purified from the hepatopancreas of Pacific abalone (Haliotis discus hannai) by ammonium sulfate precipitation and column chromatographies of SP-Sepharose and Sephacryl S-200 HR. Purified cathepsin L appeared as two bands with molecular masses of 28.0 and 28.5 kDa (namely cathepsin La and Lb) on SDS-PAGE under reducing conditions, suggesting that it is a glycoprotein. Peptide mass fingerprinting (PMF) analysis revealed that peptide fragments of 95 amino acid residues was high similarity to cathepsin L of pearl oyster (Pinctada fucata). The optimal temperature and pH of cathepsin L were 35 °C and pH 5.5. Cathepsin L was particularly inhibited by cysteine proteinase inhibitors of E-64 and leupeptin, while it was activated by metalloproteinase inhibitors EDTA and EGTA. The full-length cathepsin L cDNA was further cloned from the hepatopancreas by rapid PCR amplification of cDNA ends (RACE). The open reading frame of the enzyme was 981 bp, encoding 327 amino acid residues, with a conserved catalytic triad (Cys134, His273 and Asn293), a potential N-glycosylation site and conserved ERFNIN, GNYD, and GCGG motifs, which are characteristics of cathepsin L. Western blot and proteinase activity analysis revealed that the expression and enzyme activity of cathepsin L were significantly up-regulated in hepatopancreas at 8 h following Vibrio parahaemolyticus infection, demonstrating that cathepsin L is involved in the innate immune system of abalone. Our present study for the first time reported the purification, characterization, molecular cloning, and tissue expression of cathepsin L in abalone.

Purification, Characterization, and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkii.

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.

Effect of blend ratio and pH on the physical properties of edible composite films prepared from silver carp surimi and skin gelatin.

The effect of blend ratio and pH on the physical properties of surimi-gelatin composite films was investigated. Tensile strength (TS), film water solubility and soluble proteins of composite films increased with the increasing proportion of gelatin, while elongation at break (EAB) decreased. The TS of neutral films with the same ratio of surimi and gelatin were lowest, while increased at acidic or alkaline conditions. Similar tendency was also found in protein solubility and surface hydrophobicity of the film-forming solutions. On the other hand, the film water solubility and soluble proteins of neutral composite films were higher than those of acidic and alkaline films. Furthermore, it was revealed that the dissolved surimi and gelatin proteins could form strong composite films, which were insoluble in water. These results suggested that dissolved proteins were mainly involved in the formation of surimi-gelatin composite films.

Identification and inhibition of histamine-forming bacteria in blue scad (Decapterus maruadsi) and chub mackerel (Scomber japonicus).

In this study, we investigated the differences in histamine accumulation between blue scad and chub mackerel and methods of inhibiting histamine-forming bacteria and controlling histamine accumulation in fish. The free histidine contents in blue scad and chub mackerel were 1.45 and 2.75 mg/g, respectively. The histamine-forming bacteria isolated from them were identified as Citrobacter freundii, Citrobacter braakii, and Enterobacter aerogenes using 16S rDNA sequence analysis, the VITEK 2 Compact system, and MALDI-TOF MS. The histamine-producing capacities of C. freundii, C. braakii, and E. aerogenes were 470, 1,057, and 4,213 mg/liter, respectively, after culture at 37°C for 48 h. Among the different antimicrobials and preservatives tested, potassium sorbate and sodium diacetate effectively inhibited the histamine-forming bacteria and their histamine production. After chub mackerel was dipped into 0.5% potassium sorbate or sodium diacetate, its histamine content increased more slowly at room temperature. Therefore, a potassium sorbate or sodium diacetate dipping treatment could effectively control histamine accumulation in fish.

Purification, characterization, cDNA cloning and in vitro expression of a serine proteinase from the intestinal tract of sea cucumber (Stichopus japonicus) with collagen degradation activity.

Sea cucumber (Stichopus japonicus) autolysis during transportation and processing is a major problem and the specific proteinases responsible for autolysis have not yet been identified. In the present study, a 34 kDa serine proteinase (SP) was isolated to high purity from sea cucumber intestinal tract by a series of column chromatographies. Peptide mass fingerprinting revealed that six peptide fragments were identical to a proprotein convertase subtilisin/kexin type 9 preproprotein from sea cucumber A. japonicus. The enzyme hydrolyzed gelatin effectively at pH 6.0-9.0 and 35-40 °C, and the enzyme activity was strongly inhibited by SP inhibitors. Sea cucumber collagen was hydrolyzed significantly by purified SP at 37 °C and more gradually at 4 °C, suggesting that SP may be involved in autolysis. In addition, the SP gene that codes for 377 amino acid residues was cloned into an E. coli expression vector and expressed in vitro. A polyclonal antibody against rSP was prepared and found to react specifically against both rSP and endogenous SP, which may prove useful for future studies on the physiological functions of SP.

Expression and characterization of common carp (Cyprinus carpio) matrix metalloproteinase-2 and its activity against type I collagen.

Matrix metalloproteinases (MMPs) play essential roles in the metabolism of animal collagen while few reports are available for MMPs in aquatic animals. In this study, we report the complete sequence of matrix metalloproteinase-2 (MMP-2) gene from common carp (Cyprinus carpio) skeletal muscle. The full-length cDNA of MMP-2 was 2792bp which contains an open reading frame of 1974bp, corresponding to a protein of 657 amino acid residues. Based on the structural feature of MMP-2, the gene of the catalytic domain containing 351 amino acid residues was cloned and expressed in Escherichia coli. SDS-PAGE showed that the truncated recombinant MMP-2 (trMMP-2) with molecular mass of approximately 38kDa was in the form of inclusion body. The trMMP-2 was further purified by immobilized metal ion affinity chromatography. After renaturation, similar to native MMP-2, the trMMP-2 exhibited high hydrolyzing activity toward gelatin as appeared on gelatin zymography and optimal activity was at pH 8.0 and 40°C. The activity of the trMMP-2 was completely suppressed by metalloproteinase inhibitors, including EDTA, EGTA and 1,10-phenanthroline while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca(2+) was necessary for the gelatinolytic activity, suggesting it is a calcium-dependent metalloproteinase. Moreover, the trMMP-2 effectively hydrolyzed native type I collagen at 37°C and even at 4°C, implying its potential application value as a collagenase for preparation of biologically active oligopeptides.

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.

Structural characterization and IgE epitope analysis of arginine kinase from Scylla paramamosain.

Arginine kinase (AK) is reported to be the pan-allergen of shellfish. However, there is limited information on its IgE epitopes and structural characteristics. In this study, AK from Scylla paramamosain was purified and characterized. The purified AK is a glycoprotein with the molecular weight of 40 kDa and it demonstrates cross-reactivity with the related allergens present in other shellfish. The cDNA of S. paramamosain AK was cloned, which encodes 357 amino acid residues. Nine linear epitopes and seven conformational epitopes were predicted following bioinformatics analysis. In addition, the entire recombinant AK (rAK) and three partial recombinant AKs (rAK1, rAK2, and rAK3) were successfully expressed in Escherichia coli BL21 (DE3). The proteins of rAK1, rAK2 and rAK have strong IgE reactivity with the pooled sera from crab allergic patients, while rAK3 has significantly weaker IgE reactivity, which indicates that the IgE epitopes of AK are mainly distributed in the regions of rAK1 and rAK2. Furthermore, three experimental linear epitopes (epitope 1: AA 127-141, epitope 2: AA 141-155, and epitope 3: AA 211-225) were discovered in the region of rAK1 and rAK2 using synthetized overlapping peptides. The experimental linear epitopes were mapped onto the protein homology model of AK. Meanwhile, in the IgE-binding assays of the sera from nine crab allergic patients, only three sera reacted with the denatured, linear AK as shown by Western-blotting, eight sera reacted with the native, folded AK by both dot-blotting and ELISA, which indicates that the conformational IgE epitopes of S. paramamosain AK may be more predominant.

Tropomyosin contains IgE-binding epitopes sensitive to periodate but not to enzymatic deglycosylation.

Glycosylation has been reported to affect the epitopes of food allergens, however, there are few reports on its role in crab allergen. In the present study, the effect of glycosylation on the IgE-binding activity of tropomyosin (TM), a major allergen in Scylla paramamosain, was investigated. The results showed that TM was a glycoprotein with a 0.2% carbohydrate moiety and contained O-glycan. Moreover, enzymatic deglycosylation of TM by glycosidase had no effect on the IgE-binding activity of TM. In contrast, treatment with periodate resulted in a significant reduction in its IgE-binding activity.

Purification and characterisation of sarcoplasmic calcium-binding protein, a novel allergen of red swamp crayfish (Procambarus clarkii).

Crayfish sarcoplasmic calcium-binding protein (SCP) was purified. The physicochemical and polymorphic characterisations were also analysed. SCP was purified by column chromatography to reveal a single band with molecular mass of 22 kDa and further confirmed by mass spectrometry. The results of physicochemical characterisation showed that SCP was stable in the processes of thermal or acid/alkali treatment, and could be digested by simulate gastrointestinal fluid. Importantly, the comparison of SCP polymorphism using sera from crustacean-allergic patients demonstrated SCP-II had a weaker IgE-binding activity. The isoelectric points of SCP subunits a, b and c were 4.6, 4.7, and 4.8, respectively, as determined by two-dimensional electrophoresis and IgE immunoblotting analysis showed that patients' sera reacted to three subunits of SCP. Finally, it can be concluded that SCP is a stable polymorphic allergen in crayfish, and all of its isotypes and subunits have allergenicity.