Expression and purification of 15N-labeled Fra a 1, a strawberry allergen, to prepare samples for NMR measurements.

Raw strawberries contain allergens that cause oral allergic syndrome. Fra a 1 is one of the major allergens in strawberries and might decrease their allergenicity by heating, likely due to structural changes in the allergen leading to decreased recognition of the allergens in the oral cavity. In the present study, to understand the relationship between allergen structure and allergenicity, the expression and purification of 15N-labeled Fra a 1 were examined and the sample was used for NMR analysis. Two isoforms, Fra a 1.01 and Fra a 1.02, were used and expressed in E. coli BL21(DE3) in M9 minimal medium. Fra a 1.02 was purified as a single protein by using the GST tag approach, whereas histidine × 6-tag (his6-tag) Fra a 1.02 was obtained both as the full-length (∼20 kDa) and a truncated (∼18 kDa) form. On the other hand, his6-tag Fra a 1.01 was purified as a homogeneous protein. 15N-labeled HSQC NMR spectra suggested that Fra a 1.02 was thermally denatured at lower temperatures than Fra a 1.01, despite the high amino acid sequence homology (79.4%) of these isoforms. Furthermore, the samples in the present study allowed us to analyze ligand binding that probably affects structural stability. In conclusion, GST tag was effective for obtaining a homogeneous protein when his6-tag failed to give a single form, and the present study provided a sample that could be used for NMR studies of the details of the allergenicity and structure of Fra a 1.

A single amino acid substitution converts a histidine decarboxylase to an imidazole acetaldehyde synthase.

Histidine decarboxylase (HDC; EC 4.1.1.22), an enzyme that catalyzes histamine synthesis with high substrate specificity, is a member of the group II pyridoxal 5'-phosphate (PLP) -dependent decarboxylase family. Tyrosine is a conserved residue among group II PLP-dependent decarboxylases. Human HDC has a Y334 located on a catalytically important loop at the active site. In this study, we demonstrated that a HDC Y334F mutant is capable of catalyzing the decarboxylation-dependent oxidative deamination of histidine to yield imidazole acetaldehyde. Replacement of the active-site Tyr with Phe in group II PLP-dependent decarboxylases, including mammalian aromatic amino acid decarboxylase, plant tyrosine/DOPA decarboxylase, and plant tryptophan decarboxylase, is expected to result in the same functional change, given that a Y-to-F substitution at the corresponding residue (number 260) in the HDC of Morganella morganii, another group II PLP-dependent decarboxylase, yielded the same effect. Thus, it was suggested that the loss of the OH moiety from the active-site Tyr residue of decarboxylase uniquely converts the enzyme to an aldehyde synthase.

The ellagitannin trimer rugosin G inhibits recombinant human histidine decarboxylase.

Rugosin G, an ellagitannin trimer, was isolated from the water-soluble fraction of red rose petals, and its inhibitory activity against recombinant human histidine decarboxylase was investigated. Rugosin G showed potent inhibition compared to ellagitannin monomers and a dimer with macrocyclic structure (oenothein B), suggesting the potent inhibition of rugosin G was attributed to its linear oligomeric conformation. Abbreviations: HDC, histidine decarboxylase; Me2CO, acetone; EtOAc, ethyl acetate.

Analysis of major paralogs encoding the Fra a 1 allergen based on their organ-specificity in Fragaria × ananassa.

KEY MESSAGE: Fra a 1 protein in strawberry causes oral allergic syndrome. Over 39 Fra a 1 paralogs have been identified in strawberry genome. Fra a 1.01 is major accumulating protein in edible organs. Strawberry fruits contain allergenic proteins that cause oral allergic syndrome. The hypothesized major allergen is Fra a 1, an ortholog of the birch pollen allergen protein Bet v 1. We organized Fra a 1 genes and analyzed their localizations at the transcriptional and translational levels. In total, 15 new Fra a 1 proteins were identified from the genomic database, increasing the total number of Fra a 1 to 30 proteins encoded by 39 genes. Fra a 1.02 was mostly expressed in receptacles, and Fra a 1.01 in achenes, when analyzed by RNA sequencing. Immunoblotting showed that the Fra a 1.01 protein was broadly accumulated in strawberry organs, while the Fra a 1.02 protein was mostly expressed in receptacles. Recombinant Fra a 1.01 strongly reacted with human IgE. The mRNA and protein expression levels of Fra a 1 did not correlate, indicating the importance of protein levels when evaluating the abundance of allergens in strawberry. Based on the localizations, accumulation levels and reactivity to human IgE, we determined that Fra a 1.01 was the most important allergen, followed by Fra a 1.02, and then other Fra a 1 proteins. The information obtained here will be useful for selecting the target Fra a 1 paralogs when breeding hypoallergenic strawberry.

Characterization of a Monoclonal Antibody against Syringate Derivatives: Application of Immunochemical Detection of Methyl Syringate in Honey.

Syringic acid is one of the key skeletal structures of plant-derived chemicals. The derivatives of syringic acid have certain biological functions. In this study, a monoclonal antibody to syringic acid-based phytochemicals was prepared and characterized. The obtained antibody reacted with methyl syringate, syringic acid, and leonurine. Methyl syringate is a characteristic compound found in manuka honey, other honey varieties, and plants. Manuka honey was fractionated using HPLC, and the reactivity of the fractions with the antibody was examined. The antibody reacted with the fraction in which methyl syringate was eluted. The amount of methyl syringate in honeys as estimated by ELISA using the antibody had a good linearity compared with that estimated by HPLC. These results suggest that the antibody is applicable for the immunochemical detection of syringic acid derivatives in plants and foods.

Inhibition of Morganella morganii Histidine Decarboxylase Activity and Histamine Accumulation in Mackerel Muscle Derived from Filipendula ulumaria Extracts.

Filipendula ulmaria, also known as meadowsweet, is an herb; its extract was examined for the prevention of histamine production, primarily that caused by contaminated fish. The efficacy of meadowsweet was assessed using two parameters: inhibition of Morganella morganii histidine decarboxylase (HDC) and inhibition of histamine accumulation in mackerel. Ellagitannins from F. ulmaria (rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A) were previously shown to be potent inhibitors of human HDC; and in the present work, these compounds inhibited M. morganii HDC, with half maximal inhibitory concentration values of 1.5, 4.4, 6.1, and 6.8 µM, respectively. Application of the extracts (at 2 wt%) to mackerel meat yielded significantly decreased histamine accumulation compared with treatment with phosphate-buffered saline as a control. Hence, F. ulmaria exhibits inhibitory activity against bacterial HDC and might be effective for preventing food poisoning caused by histamine.

Competitive immunochromatographic assay for leptosperin as a plausible authentication marker of manuka honey.

Manuka honey is known as one of the premium honeys because of its unique property: a potent antibacterial activity. Leptosperin, methyl syringate 4-O-ß-d-gentiobioside, has been specifically identified in manuka honey. Because leptosperin is relatively stable under warmer conditions, measuring leptosperin levels may be applied to authenticate manuka honey. In this study, an immunochromatographic separation and quantification of leptosperin techniques have been developed. The concentration of leptosperin measured by immunochromatography was significantly correlated with the concentration measured by high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA). Because the immunochromatographic method is rapid and reliable, it could be applied to on-site quality control or inspection of honey samples by a beekeeper, a manufacturer, an inspector, a retailer, or a consumer.

Immunochemical authentication of manuka honey using a monoclonal antibody specific to a glycoside of methyl syringate.

Leptosperin, a novel glycoside of methyl syringate, is exclusively present in manuka honey derived from the Leptospermum species Leptospermum scoparium. Quantification of leptosperin might thus be applicable for authentication of honey. The concentration of leptosperin has high linearity with antibacterial activity. We established a monoclonal antibody to leptosperin and characterized the antibody in detail by a competitive enzyme-linked immunosorbent assay (ELISA), comparing the results with those of the high-performance liquid chromatography (HPLC) method for validation. The antigen in manuka honey was confirmed as leptosperin by HPLC fractionation with quantitation by an ELISA. Leptosperin contents of 50 honey samples were analyzed by an established ELISA, which can handle 20 samples (duplicate) on one 96-well plate. Significant coincidence with the chemical quantitation was observed. Immunochemical quantitation of leptosperin would be an economical and facile method for the possible authentication of manuka honey, allowing many honey samples to be processed and analyzed by an ELISA simultaneously.

The effect of thermal history on the elasticity of K-type gellan gels.

Elasticity of potassium type gellan gels prepared at different thermal histories was examined using dynamic viscoelastic measurements. The storage Young's modulus E' decreased with increasing cooling rate during gelation. Once gel formation occurred, thermal history at lower temperature did not influence the elastic modulus and thermal stability of the gellan gels. On the other hand, thermal history around gelation temperature influenced strongly the elastic modulus and thermal stability of resulting gels. When the gellan solution was kept for a certain time before cooling at a temperature near the gelation temperature, it was found that gels with higher elastic modulus and thermal stability were formed.

Plausible authentication of manuka honey and related products by measuring leptosperin with methyl syringate.

Manuka honey, obtained from Leptospermum scoparium flowers in New Zealand, has strong antibacterial properties. In this study, plausible authentication of the manuka honey was inspected by measuring leptosperin, methyl syringate 4-O-ß-D-gentiobiose, along with methyl syringate. Despite a gradual decrease in methyl syringate content over 30 days at 50 °C, even at moderate 37 °C, leptosperin remained stable. A considerable correlation between nonperoxide antibacterial activity and leptosperin content was observed in 20 certified manuka honey samples. Leptosperin and methyl syringate in manuka honey and related products were analyzed using HPLC connected with mass spectrometry. One noncertified brand displayed significant variations in the leptosperin and methyl syringate contents between two samples obtained from different regions. Therefore, certification is clearly required to protect consumers from disguised and/or low-quality honey. Because leptosperin is stable during storage and specific to manuka honey, its measurement may be applicable for manuka honey authentication.

Inhibitory activity of Filipendula ulmaria constituents on recombinant human histidine decarboxylase.

Histidine decarboxylase (HDC) catalyses the formation of histamine, a bioactive amine. Agents that control HDC activity are beneficial for treating histamine-mediated symptoms, such as allergies and stomach ulceration. We searched for inhibitors of HDC from the ethyl acetate extract of the petal of Filipendula ulmaria, also called meadowsweet. Rugosin D, rugosin A, rugosin A methyl ester (a novel compound), and tellimagrandin II were the main components; these 4 ellagitannins exhibited a non-competitive type of inhibition, with K(i) values of approximately 0.35-1 µM. These K(i) values are nearly equal to that of histidine methyl ester (K(i)=0.46 µM), an existing substrate analogue inhibitor. Our results show that food products contain potent HDC inhibitors and that these active food constituents might be useful for designing clinically available HDC inhibitors.

Structural study reveals that Ser-354 determines substrate specificity on human histidine decarboxylase.

Histamine is an important chemical mediator for a wide variety of physiological reactions. L-histidine decarboxylase (HDC) is the primary enzyme responsible for histamine synthesis and produces histamine from histidine in a one-step reaction. In this study, we determined the crystal structure of human HDC (hHDC) complexed with the inhibitor histidine methyl ester. This structure shows the detailed features of the pyridoxal-5'-phosphate inhibitor adduct (external aldimine) at the active site of HDC. Moreover, a comparison of the structures of hHDC and aromatic L-amino acid (L-DOPA) decarboxylase showed that Ser-354 was a key residue for substrate specificity. The S354G mutation at the active site enlarged the size of the hHDC substrate-binding pocket and resulted in a decreased affinity for histidine, but an acquired ability to bind and act on L-DOPA as a substrate. These data provide insight into the molecular basis of substrate recognition among the group II pyridoxal-5'-phosphate-dependent decarboxylases.

Purification, crystallization and preliminary X-ray analysis of human histidine decarboxylase.

The core domain of a human histidine decarboxylase mutant was purified and cocrystallized with the inhibitor L-histidine methyl ester. Using synchrotron radiation, a data set was collected from a single crystal at 100 K to 1.8 Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 215.16, b = 112.72, c = 171.39 Å, ß = 110.3°. Molecular replacement was carried out using the structure of aromatic L-amino-acid decarboxylase as a search model. The crystal contained three dimers per asymmetric unit, with a Matthews coefficient (V(M)) of 3.01 Å(3) Da(-1) and an estimated solvent content of 59.1%.

Viscoelasticity and phase separation of aqueous Na-type gellan solution.

Viscoelastic and phase behaviors of Na-type gellan were investigated. The complex shear modulus of aqueous gellan solutions was monitored as a function of concentration, temperature, and molar mass. At relatively low concentrations, the loss shear modulus, G'', increased steeply at a certain temperature upon cooling, which was attributed to helix formation. Above a certain concentration, a steep increase and then gradual decrease of G'' was observed. The decrease of G'' was significant and is considered to be due to anisotropic phase formation, judging from the macroscopic phase separation into upper isotropic and lower anisotropic phases of a gellan solution kept quiescently in a vial. Polarized light microscopy produced an image showing a nematic liquid crystalline phase. Although gel formation was not observed when gellan alone was used in the present study, the gellan formed a gel in the presence of an appropriate amount of salt.

Model study for large deformation of physical polymeric gels.

A model for large deformation of polymer gels with physical cross-linking is developed and shown to be in good agreement with experimental stress-strain curves which show strain hardening in intermediate strains followed by strain softening in large deformations near the yield strain. The model takes into account the coil-helix transition equilibrium and allows for the distribution of the end-to-end distance. The gel is considered to be formed by long flexible chains and crystalline zones acting as junctions of the chains. The number of segments contained in a flexible chain is variable due to the equilibrium between the two regions. As the end-to-end distance increases due to the deformation, more and more segments are reeled out from the junction zone. Finally, one end of the chain is librated from the junction and the chain becomes dangling. The appearance of dangling chains causes the strain softening because they cease to contribute to the elasticity. From the parameter dependence of the stress-strain relations, it was found that the yield behavior depends strongly on the distribution of end-to-end distance. The yield strain is approximately given by the ratio of the upper limit of the number of segments and the average end-to-end distance. The standard deviation of the end-to-end distance affects significantly the width of the peak in the stress-strain curve, thus determining the degree of strain softening.

Food components inhibiting recombinant human histidine decarboxylase activity.

Histidine decarboxylase (HDC) catalyzes histamine formation from histidine. Histamine is a bioactive amine acting as a neurotransmitter as well as a chemical mediator. Phenolic food components have been tested for their ability to inhibit recombinant human HDC. Epicatechin gallate (ECG) was found to be a potent inhibitor as it inhibited HDC activity in a competitive manner with Ki = 10 muM against l-histidine. Epigallocatechin gallate (EGCG) showed time-dependent inhibition which disappeared under anaerobic conditions. It is probable that time-dependent inhibition could be due to the result of autoxidation of EGCG. The initial burst observed for EGCG suggests that EGCG itself is involved in HDC inhibition as observed for ECG. Our present results have shown that the tested food components can inhibit HDC activity. This inhibition likely affects histamine biosynthesis and possibly leads to controlling the biological action induced by histamine. Therefore, those food components exhibiting HDC inhibitory activity might be potentially useful in controlling histamine-induced biological actions.

The reinforcement of gellan gel network by the immersion into salt solution.

The effect of immersion into salt solutions on rheological properties of gellan gels was investigated. The storage Young's modulus of gellan gels increased with time during the immersion into salt solutions. The increase of the storage Young's modulus can not be explained solely by change in the concentration of gellan. The ellipticity at 202 nm decreased by the immersion, suggesting the formation and aggregation of gellan helices. It was considered that during immersion cations penetrated into gellan gels to induce the formation and aggregation of gellan helices in gels, resulting in reinforcement of the gel network.

Synergistic gel formation of xyloglucan/gellan mixtures as sudied by rheology, DSC, and circular dichroism.

The gelation behavior of mixtures of tamarind seed xyloglucan (TSX) and sodium form gellan (Na-G) was investigated. The storage and loss shear moduli, G' and G'', of the mixtures showed that a thermoreversible gel was obtained although each polysaccharide alone did not form a gel at experimental conditions. The viscoelastic behavior of the mixtures showed a gel formation of TSX and Na-G induced by synergistic interaction. This synergistic interaction was also revealed by differential scanning calorimetry (DSC) and circular dichroism. Although TSX alone did not show any peak in DSC curves, mixtures with only a small amount of Na-G, which by itself did not show any peak, showed a single peak. With increasing Na-G content, another peak began to appear at the same temperature at which a peak of Na-G alone appeared. Thermally induced changes in circular dichroism of the mixtures were different from those expected from the individual behavior of TSX and Na-G.