Effect of acid and in vitro digestion on conformation and IgE-binding capacity of major oyster allergen Cra g 1 (tropomyosin)

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Lane M, molecular mass marker (kDa)；Lane 1, no pepsin control; Lane 2–6, acid-treated Cra g 1 was digested by SGF for 0, 5, 15, 30 and 60min, respectively." ] ] ] "textoCompleto" => "IntroductionAllergic diseases have long been considered an important health issue in the world. Food allergy is a typical disease that belongs to a type-I allergic reaction mediated by IgE.<a class="elsevierStyleCrossRef" href="#bib0005">1</a> It is estimated that about 3–8% of children and up to 3% of adults are affected by food allergies.<a class="elsevierStyleCrossRef" href="#bib0010">2</a> Among all food allergies, shellfish allergy is one of the most common types with a prevalence of 0.3% in the world population. Oyster, one of the typical representatives of shellfish, provides essential nutrients and benefits for human health. With the increase in production and consumption of oyster, the allergic diseases caused by it have also increased. Sensitized individuals exposed to oyster may suffer a series of immediate allergic reactions, which are characterized by gastrointestinal diseases, vascular plaques, itchy throat and even life-threatening anaphylaxis.<a class="elsevierStyleCrossRefs" href="#bib0015">3,4</a> To date, six shrimp allergens have been identified, including tropomyosin, myosin light chain, arginine kinase, troponin C, sarcoplasmic calcium binding protein and triose phosphate isomerase,<a class="elsevierStyleCrossRefs" href="#bib0025">5–7</a> whereas tropomyosin is the only well-identified allergen in oyster.<a class="elsevierStyleCrossRef" href="#bib0020">4</a> Ishikawa et al.<a class="elsevierStyleCrossRef" href="#bib0040">8</a> isolated the oyster major allergen, tropomyosin, from Crassostrea gigas for the first time and named it Cra g 1. Experiments have demonstrated that tropomyosin, a coil-coiled dimeric protein with a molecular weight of about 34–38kDa, is a highly conserved actin-binding protein in muscle.<a class="elsevierStyleCrossRefs" href="#bib0045">9–13</a>Some high-acid commercial products containing oyster residues, such as salad dressings, are increasingly consumed. In these products, organic acids, like vinegar, may affect the physicochemical properties of protein.<a class="elsevierStyleCrossRef" href="#bib0070">14</a> The treatment of acids may produce some chemical modifications to allergenic proteins, which would eventually lead to changes in the conformation and allergenicity of them.<a class="elsevierStyleCrossRefs" href="#bib0075">15–17</a> In addition, simulated gastrointestinal fluid (SGF) and simulated intestinal fluid (SIF) digestion assay system are considered to be an important method to estimate food allergy.<a class="elsevierStyleCrossRef" href="#bib0090">18</a> Astwood et al.<a class="elsevierStyleCrossRef" href="#bib0095">19</a> performed simulated digestion experiments on common allergens and non-allergenic proteins, and found that allergenic proteins were highly resistant to digestion while non-allergenic proteins were quickly degraded. But some opposite results have been achieved in other literature, showing that allergenic and non-allergenic proteins have similar resistance to digestion.<a class="elsevierStyleCrossRefs" href="#bib0100">20,21</a> So far, there have been few reports on the stability, conformation and IgE-binding capacity of acid-treated oyster tropomyosin and its digests by protease. Hence, studies on the alterations of structure and immunoreactivity of tropomyosin during such processing are needed to ascertain the effects of acid and protease on its allergenic potential.Therefore, the aim of our present study was to express recombinant Cra g 1 in Escherichia coli strain BL21 (DE3) and investigate the effects of acid and protease treatment on the conformation and IgE-binding capacity of this protein. These results will be useful to reduce the risk of foods containing oyster ingredients for oyster-allergic subjects.Materials and methodsHuman seraSerum samples from seven oyster-allergic subjects were obtained from PlasmaLab International (Everett, WA, USA). All seven subjects had a clinical history of at least two or more symptoms within 1h of oyster intake, including pruritus, allergic rhinitis, gastrointestinal disease and diarrhea. A serum pool from these patients was made by mixing equal aliquots of IgE. Negative control sera were obtained from three subjects without adverse reactions after ingestion of shellfish. All sera were stored at −80°C until used. All experiments were approved by China National Research Institute of Food and Fermentation Industries (Approval number: 2017–0012).Cloning, expression and purification of recombinant Cra g 1According to the Cra g 1 gene sequence (NCBI accession number <a href="ncbi-n:AB444943.1">AB444943.1</a>), one pair of primers was designed: P1: 5′-CGGAATTCTGGACAGCATCAAGAAGAA-3′, P2: 5′-TATTGCGGCCGCTTCTTATTATTTTTCCATGGG-3′. The underlined portions are the restriction enzyme sites for EcoRⅠand NotⅠ. Then the Cra g 1 gene was amplified and inserted into the pET-21a vector. The resulting plasmid was verified by restriction digestion and sequencing from both ends of the inserted segment.The recombinant plasmid was transformed into BL21 (DE3), plated on ampicillin-containing plates, and cultured overnight at 37°C. Single colonies were inoculated into 20mL of ampicillin-containing LB medium overnight at 37°C with shaking. 10mL of overnight culture was transferred to 1L fresh LB medium and grown at 37°C. When the OD600nm reached 0.6–0.8, 0.5mM IPTG was added to induce expression. After culturing at 18°C for 18h, the cells were harvested by centrifugation, resuspended in solution (20mmol L−1 Tris-HCl pH 8.0) and ultrasonicated (on for 3s, off for 3s, 99 cycles). Then, it was filtered through a 0.22μm filter after centrifugation at 15000rpm for 30min. The filtered protein solution was loaded onto a Ni2+ charged HiTrap chelating HP column of an ÄKTA-fast protein liquid chromatography (FPLC) system. After washing the column, the bound Cra g 1 was eluted with a linear 20–500mmolL−1 gradient of imidazole in the same buffer. To further purify the protein, the concentrated elution was applied onto a gel-filtration Hi-load Superdex-75 column equilibrated with a buffer containing 20mmolL−1 Tris-HCl, pH 7.5, 150mmolL−1 NaCl and 1mmol L−1 DTT. All purification procedures described here were performed at 4°C. The purified Cra g 1 was analyzed on 15% SDS-PAGE and stored at −80°C until used.Protein quantificationThe total protein content of the purified extract was determined using the Quick Start Bradford Assay kit (Bio-Rad, Hercules, CA, USA) in accordance with the manufacturer’s instructions. Bovine serum albumin was used as the protein standard.Acid treatment of Cra g 1The purified recombinant tropomyosin was treated under acidic condition to study the effects of different pH values and induction time on Cra g 1. The specific procedure was as follows: 0.5mgmL−1 of Cra g 1 was treated at three pH gradients at pH 5.0, 3.5 and 2.0 for 1 and 3h, respectively. Cra g 1 without acid treatment was used as control.SGF digestion assaySGF was prepared as described in the United States Pharmacopoeia.<a class="elsevierStyleCrossRef" href="#bib0110">22</a> SGF consists of porcine pepsin (275 U/mg) in 25mmolL−1 NaCl with pH 2.0. For digestion assays, a centrifuge tube (10mL) containing SGF was preheated at 37°C prior to addition of test protein to adequately simulate human gastric fluid temperature. A ratio was determined (1:100 pepsin to protein, w:w) for tests at five different time points (0, 5, 15, 30, and 60min). The digestion was performed at 37°C and terminated by adding 200mmolL−1 Na2CO3. The protein sample for 0-minute time point was added to SGF and immediately stopped with Na2CO3. For the control, the protein sample was mixed with SGF without pepsin. All the experiments were performed in triplicate.SIF digestion assaySIF was prepared as described in the United States Pharmacopoeia and was composed of trypsin in 0.05molL−1 KH2PO4 with pH 7.5.<a class="elsevierStyleCrossRef" href="#bib0110">22</a> For digestion assays, a centrifuge tube (10mL) containing SIF was preheated at 37°C prior to the addition of test protein. For final digestion, a ratio (1:100 trypsin to protein, w:w) was selected for all tests at six different time points (0, 15, 30, 60, 120, and 240min). The tests were carried out at 37°C and heated at 95°C for 5min to terminate reaction. At the 0-minute time point, the protein sample was added to SIF and immediately heated at 95°C for 5min. For the control, the protein sample was mixed with SIF without trypsin. All the experiments were performed in triplicate.Electrophoresis analysis (SDS-PAGE)The sample solution (20μL) was mixed with 5μL of the loading buffer, heat-denatured, and loaded into each well of the SDS-PAGE gel (5% stacking gel, 15% running gel). The molecular weight was determined by comparing the electrophoretic mobility of molecular weight marker proteins (14.4–116kDa) purchased from Takara Biotechnology Company (Dalian, China).Circular dichroism (CD) spectroscopyThe CD spectrum of the protein sample (0.3mgmL−1) was measured with a Jasco J-810 Spectropolarimeter (Jasco Inc., Easton, MD, USA) and the average of three spectra was taken as the final data. For wavelength analysis, samples were scanned with step increments of 0.2nm and a band width of 2.0nm. The spectral range was 190–250nm and the scanning speed was 200nmmin−1. The baseline spectrum of buffer was subtracted from each spectrum and the resulting value was converted to molar ellipticity (θ) using the analysis function built into the Jasco software.ANS-binding fluorescence measurementsThe hydrophobic surface was monitored by ANS-conjugated fluorescence using an F-4500 fluorescence spectrophotometer (Hitachi Inc., Tokyo, Japan). 1.0mL of different samples were mixed with 15μL of 5.0mM ANS solution and reacted for 30min at 25°C. Both the excitation and emission slit widths were 5nm and the scan speed was 1200nmmin−1. 300μL aliquots of each sample were added to a quartz cuvette with 1cm path length. Experiments were performed in triplicate and their average values were taken as the final results.IgE enzyme-linked immunosorbent assay (ELISA)The samples were diluted to 5μgmL−1 with CBS solution, 100μL of each sample was taken to a 96-well plate (Costar Inc., Cambridge, MA, USA) and coated at 4°C overnight. After washing four times with Tween 20/PBS (PBS-T), 350μL of 5% skimmed milk powder was added and incubated at 37°C for 1h. It was washed and 100μL of pool of sera from allergic patients diluted (1:50) in 2% skimmed milk powder was added and incubated for 3h at room temperature. After washing, 100μL of peroxidase-labeled goat anti-human IgE antibody (1:1000；KPL Inc., Gaithersburg, MD, USA) was added to wells and then plates were incubated at room temperature for 1h. After washing five times with PBS-T and three times with PBS, 200μL of TMB solution was added to wells for 30min, 50μL of 2M H2SO4 was used to stop the reaction. Finally, the O.D. values were measured with a spectrophotometer (Dynex TechnologiesInc., Chantilly, VA, USA) at 450nm. The same procedure was performed with sera from non-allergic subjects to determine the extent of non-specific binding, which was subtracted from test sera data. Three parallel experiments were performed for each sample and the average values were taken as the final data.Statistical analysisData were processed using Origin 8.0 software (OriginLab Corp., Northampton, MA, USA). All quantitative data for the experiments were mean±standard deviation. Significant difference in means between the samples was determined at a 5% confidence level (P<0.05).Results and discussionExpression and purification of Cra g 1The DNA fragment encoding Cra g 1 was cloned into expression vector pET-21a and confirmed by restriction enzyme digestion and DNA sequencing. The protein expression was carried out in E. coli BL21 (DE3). Cells were collected and analyzed by SDS-PAGE. After two-step purification by Ni2+ affinity chromatography and S-75 gel-filtration chromatography, the recombinant protein was purified to near homogenous (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>, lane 1). The final yield of this protein is 1mgmL−1 and the purified protein was stable after storage at −80°C.<elsevierMultimedia ident="fig0005"></elsevierMultimedia>Effect of acid treatment on conformation and allergenicity of Cra g 1The SDS-PAGE of Cra g 1 treated with different pH and time was shown in <a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>. After acid treatment at pH 5.0, 3.5 and 2.0 for 1h and 3h, an apparent protein band of approximately 37kDa was still observed (<a class="elsevierStyleCrossRef" href="#fig0005">Fig. 1</a>, lanes 2–7), and did not change under acidic conditions. Almost no degradation produced low molecular weight protein bands.To analyze acid-induced changes in the structure of Cra g 1, CD spectra measurements were allowed to monitor the folding changes. The CD spectrum has a peptide bond absorbing signal in the far-UV region and reflects the contents of the regular secondary structure feature of protein. From our CD spectra (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>A and B), we found that the original Cra g 1has a positive peak (maximum) at nearly 198nm and two negative peaks (minimum) at nearly 208nm and 222nm. Moreover, the molar ellipticity 222/208 ratio is greater than 1. These results indicated that it is a typical α-helical protein and forms a double-stranded coil-coiled structure. On the other hand, significant changes in the intensity of signal could be observed after acid treatment at pH 5.0, 3.5 and 2.0 for 3h. When the pH ranged from 5.0 to 2.0, the increase in the intensity, coupled with the minimum, was shifted to a lower wavelength, indicating that α-helix content of tropomyosin was further reduced and coil-coiled structure was partially damaged. The results of acid induction for 1h were not as obvious as those of 3h. Next, ANS-binding fluorescence was used to determine the hydrophobicity of Cra g 1 after acid processing. The surface hydrophobicity of acid-treated Cra g 1 was increased with pH environments ranging from 5.0 to 2.0 for 1h or 3h. Surprisingly, after 3h of acid treatment at pH 2.0, the surface hydrophobicity of Cra g 1 was almost three times compared to that of the original form (<a class="elsevierStyleCrossRef" href="#fig0010">Fig. 2</a>C). The IgE-binding capacity of acid-induced tropomyosin was evaluated by indirect ELISA (<a class="elsevierStyleCrossRef" href="#fig0015">Fig. 3</a>). As we speculated, the untreated Cra g 1 showed higher IgE reactivity, however, acid-treated tropomyosin reduced IgE-binding capacity when exposed to low pH environments, particularly at pH 2.0 and 3.5 for 1h and 3h (P<0.05).<elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="fig0015"></elsevierMultimedia>Our results illustrated that Cra g 1 did not undergo degradation under acidic conditions, indicating that it was resistant to acid, which agreed well with previous reports.<a class="elsevierStyleCrossRef" href="#bib0085">17</a> In addition, the conformation of the protein altered greatly during processing, which suggested that the secondary structure was partially destructed and more hydrophobic regions were exposed on the surface of Cra g 1. Generally, ionic interactions and hydrophobic interactions are considered to be the most important factors in forming the three-dimensional structure of protein.<a class="elsevierStyleCrossRef" href="#bib0115">23</a> Acids could affect the protonation state of the charged amino acid, alpha-carboxyl, alpha-amino terminal groups as well as hydrophobic surface of protein, which may ultimately alter the possible IgE-binding epitopes and potential allergenicity. Thus, we might deduce that steric structure of Cra g 1 was changed under acidic conditions, leading to destroying or modifying several antigenic epitopes on molecular surface. These changes markedly reduced IgE reactivity compared to the original protein, which is consistent with previous experimental results.<a class="elsevierStyleCrossRef" href="#bib0120">24</a> In order to further explore the conformational and allergic alterations of acid-treated Cra g 1 during human digestion, we finally selected the protein that was to deal with acid for 3h at pH 2.0 to use in simulated gastrointestinal fluid digestion experiments and the digestion time in SGF and SIF was designated as 1h and 4h, respectively, based on the detention time of foods in digestive system.Digestibility of acid-treated Cra g 1 by SGFIn the present study, acid-treated Cra g 1 was subjected to SDS-PAGE analysis after SGF digestion (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>). Cra g 1 was relatively resistant in SGF and was only slightly degraded even at higher digestion periods. Many digestion-resistant fragments (about 36, 22 and 15kDa) progressively intensified with prolongation of digestion time, while intact protein did not completely degrade at 60min by pepsin.<elsevierMultimedia ident="fig0020"></elsevierMultimedia>From the CD spectra, the signal obviously changed after SGF digestion (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>A). With increasing digestion time, the intensity of CD signals was increased and the minimum gradually shifted to lower wavelengths, implying the secondary structures were varied drastically. Hydrophobicity of Cra g 1 after pepsin treatment was determined by ANS-binding fluorescence measurements. Interestingly, compared to the control, the surface hydrophobicity of the protein was decreased by 4%, 27%, 34%, and 40% when digestion time was adjusted from 5 to 60min (P<0.05) (<a class="elsevierStyleCrossRef" href="#fig0025">Fig. 5</a>B). To further analyze whether SGF digestion affects the allergenicity of acid-treated Cra g 1, we performed an indirect ELISA experiment using pooled oyster-allergic subjects’ sera. Compared with the undigested sample, IgE-binding ability showed a decreasing trend from 5 to 60min, especially at 30 and 60min (P<0.05) (<a class="elsevierStyleCrossRef" href="#fig0030">Fig. 6</a>).<elsevierMultimedia ident="fig0025"></elsevierMultimedia><elsevierMultimedia ident="fig0030"></elsevierMultimedia>Pepsin is the only proteolytic enzyme in the stomach, which prefers to cleave peptide bonds following Phe or Tyr residues, as well as other hydrophobic amino acids.<a class="elsevierStyleCrossRef" href="#bib0125">25</a> Pepsin could only slightly hydrolyze oyster tropomyosin, demonstrating that it has relative resistance to pepsin digestion (<a class="elsevierStyleCrossRef" href="#fig0020">Fig. 4</a>). A possible reason is that the structural changes of Cra g 1 caused by acid produced some modifications to the enzyme cleavage sites and fewer Phe and Tyr residues were exposed on the surface of the protein, thus diminishing the degree of hydrolysis. Additionally, we might infer that pepsin, at least in part, altered the conformation of the protein during digestive processing, resulting in hydrophobic residues being masked and several antigenic epitopes that bound serum IgE were buried inside the protein (<a class="elsevierStyleCrossRefs" href="#fig0025">Figs. 5 and 6</a>). This phenomenon was also observed by Huang YY et al.;<a class="elsevierStyleCrossRef" href="#bib0130">26</a> they demonstrated that mud crab (Scylla serrata) tropomyosin reduced IgE-binding ability after pepsin digestion.Digestibility of acid-treated Cra g 1 by SIFThe degradative patterns of acid-treated Cra g 1 by SGF and SIF were quite different. Under SIF conditions, more obvious degraded fragments appeared (<a class="elsevierStyleCrossRef" href="#fig0035">Fig. 7</a>). Cra g 1 was digested into three main proteolytic fragments with sizes of approximately 36, 22 and 20kDa. All these fragments and the intact original protein remained even after 4h by trypsin digestion.<elsevierMultimedia ident="fig0035"></elsevierMultimedia>The CD far ultraviolet signal of acid-treated Cra g 1 after SIF digestion is shown in <a class="elsevierStyleCrossRef" href="#fig0040">Fig. 8</a>A. The spectra gradually showed a slight blue shift in the course of digestion, and the curve was nearly flat after 240min. These results demonstrated that the regular α-helices that dominate in tropomyosin structure diminished when the protein environment varied. Besides, compared with the undigested Cra g 1, the surface hydrophobicity increased by 28%, 43%, 46%, 53% and 58% after 15, 30, 60, 120 and 240min, respectively (P<0.05) (<a class="elsevierStyleCrossRef" href="#fig0040">Fig. 8</a> B). The IgE-binding ability of Cra g 1 after SIF digestion was further analyzed by ELISA (<a class="elsevierStyleCrossRef" href="#fig0045">Fig. 9</a>). Surprisingly, contrary to the SGF results, all digested samples have significantly higher IgE reactivity than the control (P<0.05). Possibly, more IgE-binding epitopes in degraded fragments or original protein were generated by trypsin digestion. Therefore, this treatment may not be effective in decreasing the allergenic ability of the protein.<elsevierMultimedia ident="fig0040"></elsevierMultimedia><elsevierMultimedia ident="fig0045"></elsevierMultimedia>The sensitization of potential allergens is routinely evaluated using simulated digestion studies. Our results showed that digestive patterns by trypsin and pepsin were very different because of cleavage specificities of the two proteases. Trypsin mainly cleaves peptide bonds following Lys and Arg residues at the P1 position with higher specificity.<a class="elsevierStyleCrossRef" href="#bib0135">27</a> Although there are several cleavage sites for trypsin and pepsin in Cra g 1 primary structure,<a class="elsevierStyleCrossRef" href="#bib0140">28</a> acid-treated protein and its degraded fragments were resistant to digestion (<a class="elsevierStyleCrossRefs" href="#fig0020">Figs. 4 and 7</a>). One reason for this might be that cleavage sites on the molecular surface were eliminated due to acid treatment. Hence oyster tropomyosin, as well as its digestion-resistant fragments, could survive and reach the intestinal immune system. Furthermore, antigenicity of acid-induced Cra g 1 decreased in SGF, while it increased significantly in SIF (<a class="elsevierStyleCrossRefs" href="#fig0030">Figs. 6 and 9</a>). This phenomenon may result from conformational alterations of protein (<a class="elsevierStyleCrossRef" href="#fig0040">Fig. 8</a>), which exposed more hydrophobic groups and additional immunogenic epitopes by trypsin digestion. It is also possible that new IgE-binding epitopes were produced in proteolytic fragments by SIF, which contributed to the IgE-binding reactivity. To our knowledge, this is the first report that antigenicity of acid-treated oyster tropomyosin increased under SIF conditions. This explains why sensitized individuals still suffer severe allergic reactions even if they intake high-acid oyster products such as salad dressings. Besides, the ELISA data are a relative result because IgE-binding capacity does not equate to allergenicity completely.<a class="elsevierStyleCrossRef" href="#bib0145">29</a> Some degraded fragments may bind IgE but not enable basophils or mast cells to release mediators. Consequently, the next steps are needed to assess the allergic changes of tropomyosin through release of histamine from basophils and skin prick test following acid and protease treatment. Identification of immunogenic epitopes in intact Cra g 1 and its proteolytic fragments during the same processing should be carried out to further expound the molecular mechanism of interactions between the protein and oyster-specific IgE.In conclusion, alterations in secondary structure and exposure or burial of hydrophobic amino acid residues were responsible for conformational changes, resulting in the disruption or formation of IgE-binding epitopes, and ultimately affected immunogenic activity. For oyster tropomyosin, treatment with acid and pepsin, rather than trypsin, is an effective way of reducing IgE-binding capacity.Conflict of interestThe authors have no conflict of interest to declare." "textoCompletoSecciones" => array:1 [ "secciones" => array:8 [ 0 => array:3 [ "identificador" => "xres1427779" "titulo" => "Abstract" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Introduction and Objectives" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Results" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Conclusion" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec1304200" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "sec0005" "titulo" => "Introduction" ] 3 => array:3 [ "identificador" => "sec0010" "titulo" => "Materials and methods" "secciones" => array:11 [ 0 => array:2 [ "identificador" => "sec0015" "titulo" => "Human sera" ] 1 => array:2 [ "identificador" => "sec0020" "titulo" => "Cloning, expression and purification of recombinant Cra g 1" ] 2 => array:2 [ "identificador" => "sec0025" "titulo" => "Protein quantification" ] 3 => array:2 [ "identificador" => "sec0030" "titulo" => "Acid treatment of Cra g 1" ] 4 => array:2 [ "identificador" => "sec0035" "titulo" => "SGF digestion assay" ] 5 => array:2 [ "identificador" => "sec0040" "titulo" => "SIF digestion assay" ] 6 => array:2 [ "identificador" => "sec0045" "titulo" => "Electrophoresis analysis (SDS-PAGE)" ] 7 => array:2 [ "identificador" => "sec0050" "titulo" => "Circular dichroism (CD) spectroscopy" ] 8 => array:2 [ "identificador" => "sec0055" "titulo" => "ANS-binding fluorescence measurements" ] 9 => array:2 [ "identificador" => "sec0060" "titulo" => "IgE enzyme-linked immunosorbent assay (ELISA)" ] 10 => array:2 [ "identificador" => "sec0065" "titulo" => "Statistical analysis" ] ] ] 4 => array:3 [ "identificador" => "sec0070" "titulo" => "Results and discussion" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "sec0075" "titulo" => "Expression and purification of Cra g 1" ] 1 => array:2 [ "identificador" => "sec0080" "titulo" => "Effect of acid treatment on conformation and allergenicity of Cra g 1" ] 2 => array:2 [ "identificador" => "sec0085" "titulo" => "Digestibility of acid-treated Cra g 1 by SGF" ] 3 => array:2 [ "identificador" => "sec0090" "titulo" => "Digestibility of acid-treated Cra g 1 by SIF" ] ] ] 5 => array:2 [ "identificador" => "sec0095" "titulo" => "Conflict of interest" ] 6 => array:2 [ "identificador" => "xack498286" "titulo" => "Acknowledgements" ] 7 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2019-07-05" "fechaAceptado" => "2019-08-28" "PalabrasClave" => array:1 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1304200" "palabras" => array:6 [ 0 => "Cra g1" 1 => "Acid" 2 => "SGF" 3 => "SIF" 4 => "Conformation" 5 => "IgE-binding" ] ] ] ] "tieneResumen" => true "resumen" => array:1 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "Introduction and ObjectivesThe production and consumption of oysters is increasing annually because it can provide essential nutrients and benefit for human health, leading to frequent occurrence of severe allergic reactions observed in sensitized individuals. The aim of the present study was to investigate the effects of acid and protease treatment on the conformation and IgE-binding capacity of recombinant Crassostrea gigas tropomyosin (Cra g 1). ResultsUnder acidic conditions, Cra g 1 did not undergo degradation, however, the changes obvious in the intensity of CD signal and ANS-binding fluorescence were observed, which was associated with a decrease in antibody reactivity. In simulated gastrointestinal fluid (SGF) and simulated intestinal fluid (SIF) digestion system, acid-treated Cra g 1 was relatively resistant to digestion, but the degradative patterns were very different. Moreover, owing to alterations of secondary structure and hydrophobic surface of the protein during digestive processing, antigenicity of acid-induced Cra g 1 reduced in SGF while it increased significantly in SIF. ConclusionTo our knowledge, this is the first study reporting that antigenicity of acid-treated oyster tropomyosin increased after SIF digestion. These results revealed that treatment with acid and pepsin, rather than trypsin, was an effective way of reducing IgE-binding capacity of tropomyosin from oyster." "secciones" => array:3 [ 0 => array:2 [ "identificador" => "abst0005" "titulo" => "Introduction and Objectives" ] 1 => array:2 [ "identificador" => "abst0010" "titulo" => "Results" ] 2 => array:2 [ "identificador" => "abst0015" "titulo" => "Conclusion" ] ] ] ] "NotaPie" => array:1 [ 0 => array:3 [ "etiqueta" => "1" "nota" => "These authors contributed equally to this work." "identificador" => "fn0005" ] ] "multimedia" => array:9 [ 0 => array:8 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 783 "Ancho" => 1250 "Tamanyo" => 75832 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0005" "detalle" => "Figure " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "SDS-PAGE analysis of effect of acid on recombinant Cra g 1. Lane M, molecular mass marker (kDa)；Lane 1, purified Cra g 1; Lanes 2–4, Cra g 1 was treated by acid for 1h at pH 5.0, 3.5 and 2.0, respectively; Lanes 5–7, Cra g 1 was treated by acid for 3h at pH 5.0, 3.5 and 2.0, respectively." ] ] 1 => array:8 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 2511 "Ancho" => 2915 "Tamanyo" => 359933 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0010" "detalle" => "Figure " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "Conformational changes of Cra g 1 before and after acid treatment. (A) CD spectra of Cra g 1 treated with acid for 1h at different pH; (B) CD spectra of Cra g 1 treated with acid for 3h at different pH; (C) ANS-binding fluorescence of Cra g 1 treated at different pH and time. *P<0.05 vs. untreated protein. All data are presented as the mean±SD (n=3)." ] ] 2 => array:8 [ "identificador" => "fig0015" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 1119 "Ancho" => 1628 "Tamanyo" => 86573 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0015" "detalle" => "Figure " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "ELISA for IgE reactivity to Cra g 1 after acid treatment for 1 and 3h. *P<0.05 vs. untreated protein. Data are presented as the mean±SD (n=3)." ] ] 3 => array:8 [ "identificador" => "fig0020" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 833 "Ancho" => 1250 "Tamanyo" => 95267 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0020" "detalle" => "Figure " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "SDS-PAGE analysis of effect of SGF digestion on acid-treated Cra g 1. Lane M, molecular mass marker (kDa)；Lane 1, no pepsin control; Lane 2–6, acid-treated Cra g 1 was digested by SGF for 0, 5, 15, 30 and 60min, respectively." ] ] 4 => array:8 [ "identificador" => "fig0025" "etiqueta" => "Figure 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 1062 "Ancho" => 2504 "Tamanyo" => 289465 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0025" "detalle" => "Figure " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "Conformational changes of acid-treated Cra g 1 during SGF digestion. (A) CD spectra of acid-treated Cra g 1 after different digestion time; (B) ANS-binding fluorescence of acid-treated Cra g 1 after different digestion time. *P<0.05 vs. control. Data represent the mean±SD (n=3)." ] ] 5 => array:8 [ "identificador" => "fig0030" "etiqueta" => "Figure 6" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr6.jpeg" "Alto" => 1193 "Ancho" => 1648 "Tamanyo" => 90879 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0030" "detalle" => "Figure " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "ELISA for IgE reactivity to acid-treated Cra g 1 after different SGF digestion time. *P<0.05 vs. control. Data represent the mean±SD (n=3)." ] ] 6 => array:8 [ "identificador" => "fig0035" "etiqueta" => "Figure 7" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr7.jpeg" "Alto" => 769 "Ancho" => 1250 "Tamanyo" => 60076 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0035" "detalle" => "Figure " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "SDS-PAGE analysis of effect of SIF digestion on acid-treated Cra g 1. Lane M, molecular mass marker (kDa); Lane 1, no trypsin control; Lane 2–7, acid-treated Cra g 1 was digested by SIF for 0, 15, 30, 60, 120 and 240min, respectively." ] ] 7 => array:8 [ "identificador" => "fig0040" "etiqueta" => "Figure 8" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr8.jpeg" "Alto" => 1053 "Ancho" => 2501 "Tamanyo" => 257195 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0040" "detalle" => "Figure " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "Conformational changes of acid-treated Cra g 1 during SIF digestion. (A) CD spectra of acid-treated Cra g 1 after different digestion time; (B) ANS-binding fluorescence of acid-treated Cra g 1 after different digestion time. *P<0.05 vs. control. Data represent the mean±SD (n=3)." ] ] 8 => array:8 [ "identificador" => "fig0045" "etiqueta" => "Figure 9" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr9.jpeg" "Alto" => 1127 "Ancho" => 1660 "Tamanyo" => 90058 ] ] "detalles" => array:1 [ 0 => array:3 [ "identificador" => "at0045" "detalle" => "Figure " "rol" => "short" ] ] "descripcion" => array:1 [ "en" => "ELISA for IgE reactivity to acid-treated Cra g 1 after different SIF digestion time. *P<0.05 vs. control. 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Jiangtao Zhang1, Wenying Liu1, Lei Fang, Ruizeng Gu, Jun Lu

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Johnljsmith@163.com

Corresponding authors at: China National Research Institute of Food & Fermentation Industries, No.24, Jiuxianqiao Middle Road, Chaoyang District, Beijing, China.

, Guoming Li

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297756628@qq.com

Corresponding authors at: China National Research Institute of Food & Fermentation Industries, No.24, Jiuxianqiao Middle Road, Chaoyang District, Beijing, China.

Beijing Engineering Research Center of Protein & Functional Peptides, China National Research Institute of Food and Fermentation Industries, Beijing 100015, PR China

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          "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">SDS-PAGE analysis of effect of SGF digestion on acid-treated <span class="elsevierStyleItalic">Cra g</span> 1&#46; Lane M&#44; molecular mass marker &#40;kDa&#41;&#65307;Lane 1&#44; no pepsin control&#59; Lane 2&#8211;6&#44; acid-treated <span class="elsevierStyleItalic">Cra g</span> 1 was digested by SGF for 0&#44; 5&#44; 15&#44; 30 and 60<span class="elsevierStyleHsp" style=""></span>min&#44; respectively&#46;</p>"
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Allergic diseases have long been considered an important health issue in the world&#46; Food allergy is a typical disease that belongs to a type-I allergic reaction mediated by IgE&#46;<a class="elsevierStyleCrossRef" href="#bib0005"><span class="elsevierStyleSup">1</span></a> It is estimated that about 3&#8211;8&#37; of children and up to 3&#37; of adults are affected by food allergies&#46;<a class="elsevierStyleCrossRef" href="#bib0010"><span class="elsevierStyleSup">2</span></a> Among all food allergies&#44; shellfish allergy is one of the most common types with a prevalence of 0&#46;3&#37; in the world population&#46; Oyster&#44; one of the typical representatives of shellfish&#44; provides essential nutrients and benefits for human health&#46; With the increase in production and consumption of oyster&#44; the allergic diseases caused by it have also increased&#46; Sensitized individuals exposed to oyster may suffer a series of immediate allergic reactions&#44; which are characterized by gastrointestinal diseases&#44; vascular plaques&#44; itchy throat and even life-threatening anaphylaxis&#46;<a class="elsevierStyleCrossRefs" href="#bib0015"><span class="elsevierStyleSup">3&#44;4</span></a> To date&#44; six shrimp allergens have been identified&#44; including tropomyosin&#44; myosin light chain&#44; arginine kinase&#44; troponin C&#44; sarcoplasmic calcium binding protein and triose phosphate isomerase&#44;<a class="elsevierStyleCrossRefs" href="#bib0025"><span class="elsevierStyleSup">5&#8211;7</span></a> whereas tropomyosin is the only well-identified allergen in oyster&#46;<a class="elsevierStyleCrossRef" href="#bib0020"><span class="elsevierStyleSup">4</span></a> Ishikawa et al&#46;<a class="elsevierStyleCrossRef" href="#bib0040"><span class="elsevierStyleSup">8</span></a> isolated the oyster major allergen&#44; tropomyosin&#44; from <span class="elsevierStyleItalic">Crassostrea gigas</span> for the first time and named it <span class="elsevierStyleItalic">Cra g</span> 1&#46; Experiments have demonstrated that tropomyosin&#44; a coil-coiled dimeric protein with a molecular weight of about 34&#8211;38<span class="elsevierStyleHsp" style=""></span>kDa&#44; is a highly conserved actin-binding protein in muscle&#46;<a class="elsevierStyleCrossRefs" href="#bib0045"><span class="elsevierStyleSup">9&#8211;13</span></a></p><p id="par0010" class="elsevierStylePara elsevierViewall">Some high-acid commercial products containing oyster residues&#44; such as salad dressings&#44; are increasingly consumed&#46; In these products&#44; organic acids&#44; like vinegar&#44; may affect the physicochemical properties of protein&#46;<a class="elsevierStyleCrossRef" href="#bib0070"><span class="elsevierStyleSup">14</span></a> The treatment of acids may produce some chemical modifications to allergenic proteins&#44; which would eventually lead to changes in the conformation and allergenicity of them&#46;<a class="elsevierStyleCrossRefs" href="#bib0075"><span class="elsevierStyleSup">15&#8211;17</span></a> In addition&#44; simulated gastrointestinal fluid &#40;SGF&#41; and simulated intestinal fluid &#40;SIF&#41; digestion assay system are considered to be an important method to estimate food allergy&#46;<a class="elsevierStyleCrossRef" href="#bib0090"><span class="elsevierStyleSup">18</span></a> Astwood et al&#46;<a class="elsevierStyleCrossRef" href="#bib0095"><span class="elsevierStyleSup">19</span></a> performed simulated digestion experiments on common allergens and non-allergenic proteins&#44; and found that allergenic proteins were highly resistant to digestion while non-allergenic proteins were quickly degraded&#46; But some opposite results have been achieved in other literature&#44; showing that allergenic and non-allergenic proteins have similar resistance to digestion&#46;<a class="elsevierStyleCrossRefs" href="#bib0100"><span class="elsevierStyleSup">20&#44;21</span></a> So far&#44; there have been few reports on the stability&#44; conformation and IgE-binding capacity of acid-treated oyster tropomyosin and its digests by protease&#46; Hence&#44; studies on the alterations of structure and immunoreactivity of tropomyosin during such processing are needed to ascertain the effects of acid and protease on its allergenic potential&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">Therefore&#44; the aim of our present study was to express recombinant <span class="elsevierStyleItalic">Cra g</span> 1 in <span class="elsevierStyleItalic">Escherichia coli</span> strain BL21 &#40;DE3&#41; and investigate the effects of acid and protease treatment on the conformation and IgE-binding capacity of this protein&#46; These results will be useful to reduce the risk of foods containing oyster ingredients for oyster-allergic subjects&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Human sera</span><p id="par0020" class="elsevierStylePara elsevierViewall">Serum samples from seven oyster-allergic subjects were obtained from PlasmaLab International &#40;Everett&#44; WA&#44; USA&#41;&#46; All seven subjects had a clinical history of at least two or more symptoms within 1<span class="elsevierStyleHsp" style=""></span>h of oyster intake&#44; including pruritus&#44; allergic rhinitis&#44; gastrointestinal disease and diarrhea&#46; A serum pool from these patients was made by mixing equal aliquots of IgE&#46; Negative control sera were obtained from three subjects without adverse reactions after ingestion of shellfish&#46; All sera were stored at &#8722;80<span class="elsevierStyleHsp" style=""></span>&#176;C until used&#46; All experiments were approved by China National Research Institute of Food and Fermentation Industries &#40;Approval number&#58; 2017&#8211;0012&#41;&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Cloning&#44; expression and purification of recombinant <span class="elsevierStyleItalic">Cra g</span> 1</span><p id="par0025" class="elsevierStylePara elsevierViewall">According to the <span class="elsevierStyleItalic">Cra g</span> 1 gene sequence &#40;NCBI accession number <a href="ncbi-n:AB444943.1">AB444943&#46;1</a>&#41;&#44; one pair of primers was designed&#58; P1&#58; 5&#8242;-CG<span class="elsevierStyleUnderline">GAATTC</span>TGGACAGCATCAAGAAGAA-3&#8242;&#44; P2&#58; 5&#8242;-TATT<span class="elsevierStyleUnderline">GCGGCCGC</span>TTCTTATTATTTTTCCATGGG-3&#8242;&#46; The underlined portions are the restriction enzyme sites for EcoR&#8544;and Not&#8544;&#46; Then the <span class="elsevierStyleItalic">Cra g</span> 1 gene was amplified and inserted into the pET-21a vector&#46; The resulting plasmid was verified by restriction digestion and sequencing from both ends of the inserted segment&#46;</p><p id="par0030" class="elsevierStylePara elsevierViewall">The recombinant plasmid was transformed into BL21 &#40;DE3&#41;&#44; plated on ampicillin-containing plates&#44; and cultured overnight at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Single colonies were inoculated into 20<span class="elsevierStyleHsp" style=""></span>mL of ampicillin-containing LB medium overnight at 37<span class="elsevierStyleHsp" style=""></span>&#176;C with shaking&#46; 10<span class="elsevierStyleHsp" style=""></span>mL of overnight culture was transferred to 1<span class="elsevierStyleHsp" style=""></span>L fresh LB medium and grown at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; When the OD<span class="elsevierStyleInf">600</span><span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleInf">nm</span> reached 0&#46;6&#8211;0&#46;8&#44; 0&#46;5<span class="elsevierStyleHsp" style=""></span>mM IPTG was added to induce expression&#46; After culturing at 18<span class="elsevierStyleHsp" style=""></span>&#176;C for 18<span class="elsevierStyleHsp" style=""></span>h&#44; the cells were harvested by centrifugation&#44; resuspended in solution &#40;20<span class="elsevierStyleHsp" style=""></span>mmol L<span class="elsevierStyleSup">&#8722;1</span> Tris-HCl pH 8&#46;0&#41; and ultrasonicated &#40;on for 3<span class="elsevierStyleHsp" style=""></span>s&#44; off for 3<span class="elsevierStyleHsp" style=""></span>s&#44; 99 cycles&#41;&#46; Then&#44; it was filtered through a 0&#46;22<span class="elsevierStyleHsp" style=""></span>&#956;m filter after centrifugation at 15000<span class="elsevierStyleHsp" style=""></span>rpm for 30<span class="elsevierStyleHsp" style=""></span>min&#46; The filtered protein solution was loaded onto a Ni<span class="elsevierStyleSup">2&#43;</span> charged HiTrap chelating HP column of an &#196;KTA-fast protein liquid chromatography &#40;FPLC&#41; system&#46; After washing the column&#44; the bound <span class="elsevierStyleItalic">Cra g</span> 1 was eluted with a linear 20&#8211;500<span class="elsevierStyleHsp" style=""></span>mmol<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleSmallCaps">L</span><span class="elsevierStyleSup">&#8722;1</span> gradient of imidazole in the same buffer&#46; To further purify the protein&#44; the concentrated elution was applied onto a gel-filtration Hi-load Superdex-75 column equilibrated with a buffer containing 20<span class="elsevierStyleHsp" style=""></span>mmol<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> Tris-HCl&#44; pH 7&#46;5&#44; 150<span class="elsevierStyleHsp" style=""></span>mmol<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> NaCl and 1<span class="elsevierStyleHsp" style=""></span>mmol L<span class="elsevierStyleSup">&#8722;1</span> DTT&#46; All purification procedures described here were performed at 4<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; The purified <span class="elsevierStyleItalic">Cra g</span> 1 was analyzed on 15&#37; SDS-PAGE and stored at &#8722;80<span class="elsevierStyleHsp" style=""></span>&#176;C until used&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Protein quantification</span><p id="par0035" class="elsevierStylePara elsevierViewall">The total protein content of the purified extract was determined using the Quick Start Bradford Assay kit &#40;Bio-Rad&#44; Hercules&#44; CA&#44; USA&#41; in accordance with the manufacturer&#8217;s instructions&#46; Bovine serum albumin was used as the protein standard&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Acid treatment of <span class="elsevierStyleItalic">Cra g</span> 1</span><p id="par0040" class="elsevierStylePara elsevierViewall">The purified recombinant tropomyosin was treated under acidic condition to study the effects of different pH values and induction time on <span class="elsevierStyleItalic">Cra g</span> 1&#46; The specific procedure was as follows&#58; 0&#46;5<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>mL<span class="elsevierStyleSup">&#8722;1</span> of <span class="elsevierStyleItalic">Cra g</span> 1 was treated at three pH gradients at pH 5&#46;0&#44; 3&#46;5 and 2&#46;0 for 1 and 3<span class="elsevierStyleHsp" style=""></span>h&#44; respectively&#46; <span class="elsevierStyleItalic">Cra g</span> 1 without acid treatment was used as control&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">SGF digestion assay</span><p id="par0045" class="elsevierStylePara elsevierViewall">SGF was prepared as described in the United States Pharmacopoeia&#46;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> SGF consists of porcine pepsin &#40;275 U&#47;mg&#41; in 25<span class="elsevierStyleHsp" style=""></span>mmol<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> NaCl with pH 2&#46;0&#46; For digestion assays&#44; a centrifuge tube &#40;10<span class="elsevierStyleHsp" style=""></span>mL&#41; containing SGF was preheated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C prior to addition of test protein to adequately simulate human gastric fluid temperature&#46; A ratio was determined &#40;1&#58;100 pepsin to protein&#44; w&#58;w&#41; for tests at five different time points &#40;0&#44; 5&#44; 15&#44; 30&#44; and 60<span class="elsevierStyleHsp" style=""></span>min&#41;&#46; The digestion was performed at 37<span class="elsevierStyleHsp" style=""></span>&#176;C and terminated by adding 200<span class="elsevierStyleHsp" style=""></span>mmol<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> Na<span class="elsevierStyleInf">2</span>CO<span class="elsevierStyleInf">3</span>&#46; The protein sample for 0-minute time point was added to SGF and immediately stopped with Na<span class="elsevierStyleInf">2</span>CO<span class="elsevierStyleInf">3</span>&#46; For the control&#44; the protein sample was mixed with SGF without pepsin&#46; All the experiments were performed in triplicate&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">SIF digestion assay</span><p id="par0050" class="elsevierStylePara elsevierViewall">SIF was prepared as described in the United States Pharmacopoeia and was composed of trypsin in 0&#46;05<span class="elsevierStyleHsp" style=""></span>mol<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> KH<span class="elsevierStyleInf">2</span>PO<span class="elsevierStyleInf">4</span> with pH 7&#46;5&#46;<a class="elsevierStyleCrossRef" href="#bib0110"><span class="elsevierStyleSup">22</span></a> For digestion assays&#44; a centrifuge tube &#40;10<span class="elsevierStyleHsp" style=""></span>mL&#41; containing SIF was preheated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C prior to the addition of test protein&#46; For final digestion&#44; a ratio &#40;1&#58;100 trypsin to protein&#44; w&#58;w&#41; was selected for all tests at six different time points &#40;0&#44; 15&#44; 30&#44; 60&#44; 120&#44; and 240<span class="elsevierStyleHsp" style=""></span>min&#41;&#46; The tests were carried out at 37<span class="elsevierStyleHsp" style=""></span>&#176;C and heated at 95<span class="elsevierStyleHsp" style=""></span>&#176;C for 5<span class="elsevierStyleHsp" style=""></span>min to terminate reaction&#46; At the 0-minute time point&#44; the protein sample was added to SIF and immediately heated at 95<span class="elsevierStyleHsp" style=""></span>&#176;C for 5<span class="elsevierStyleHsp" style=""></span>min&#46; For the control&#44; the protein sample was mixed with SIF without trypsin&#46; All the experiments were performed in triplicate&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Electrophoresis analysis &#40;SDS-PAGE&#41;</span><p id="par0055" class="elsevierStylePara elsevierViewall">The sample solution &#40;20<span class="elsevierStyleHsp" style=""></span>&#956;L&#41; was mixed with 5<span class="elsevierStyleHsp" style=""></span>&#956;L of the loading buffer&#44; heat-denatured&#44; and loaded into each well of the SDS-PAGE gel &#40;5&#37; stacking gel&#44; 15&#37; running gel&#41;&#46; The molecular weight was determined by comparing the electrophoretic mobility of molecular weight marker proteins &#40;14&#46;4&#8211;116<span class="elsevierStyleHsp" style=""></span>kDa&#41; purchased from Takara Biotechnology Company &#40;Dalian&#44; China&#41;&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Circular dichroism &#40;CD&#41; spectroscopy</span><p id="par0060" class="elsevierStylePara elsevierViewall">The CD spectrum of the protein sample &#40;0&#46;3<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>mL<span class="elsevierStyleSup">&#8722;1</span>&#41; was measured with a Jasco J-810 Spectropolarimeter &#40;Jasco Inc&#46;&#44; Easton&#44; MD&#44; USA&#41; and the average of three spectra was taken as the final data&#46; For wavelength analysis&#44; samples were scanned with step increments of 0&#46;2<span class="elsevierStyleHsp" style=""></span>nm and a band width of 2&#46;0<span class="elsevierStyleHsp" style=""></span>nm&#46; The spectral range was 190&#8211;250<span class="elsevierStyleHsp" style=""></span>nm and the scanning speed was 200<span class="elsevierStyleHsp" style=""></span>nm<span class="elsevierStyleHsp" style=""></span>min<span class="elsevierStyleSup">&#8722;1</span>&#46; The baseline spectrum of buffer was subtracted from each spectrum and the resulting value was converted to molar ellipticity &#40;&#952;&#41; using the analysis function built into the Jasco software&#46;</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">ANS-binding fluorescence measurements</span><p id="par0065" class="elsevierStylePara elsevierViewall">The hydrophobic surface was monitored by ANS-conjugated fluorescence using an F-4500 fluorescence spectrophotometer &#40;Hitachi Inc&#46;&#44; Tokyo&#44; Japan&#41;&#46; 1&#46;0<span class="elsevierStyleHsp" style=""></span>mL of different samples were mixed with 15<span class="elsevierStyleHsp" style=""></span>&#956;L of 5&#46;0<span class="elsevierStyleHsp" style=""></span>mM ANS solution and reacted for 30<span class="elsevierStyleHsp" style=""></span>min at 25<span class="elsevierStyleHsp" style=""></span>&#176;C&#46; Both the excitation and emission slit widths were 5<span class="elsevierStyleHsp" style=""></span>nm and the scan speed was 1200<span class="elsevierStyleHsp" style=""></span>nm<span class="elsevierStyleHsp" style=""></span>min<span class="elsevierStyleSup">&#8722;1</span>&#46; 300<span class="elsevierStyleHsp" style=""></span>&#956;L aliquots of each sample were added to a quartz cuvette with 1<span class="elsevierStyleHsp" style=""></span>cm path length&#46; Experiments were performed in triplicate and their average values were taken as the final results&#46;</p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">IgE enzyme-linked immunosorbent assay &#40;ELISA&#41;</span><p id="par0070" class="elsevierStylePara elsevierViewall">The samples were diluted to 5<span class="elsevierStyleHsp" style=""></span>&#956;g<span class="elsevierStyleHsp" style=""></span>mL<span class="elsevierStyleSup">&#8722;1</span> with CBS solution&#44; 100<span class="elsevierStyleHsp" style=""></span>&#956;L of each sample was taken to a 96-well plate &#40;Costar Inc&#46;&#44; Cambridge&#44; MA&#44; USA&#41; and coated at 4<span class="elsevierStyleHsp" style=""></span>&#176;C overnight&#46; After washing four times with Tween 20&#47;PBS &#40;PBS-T&#41;&#44; 350<span class="elsevierStyleHsp" style=""></span>&#956;L of 5&#37; skimmed milk powder was added and incubated at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 1<span class="elsevierStyleHsp" style=""></span>h&#46; It was washed and 100<span class="elsevierStyleHsp" style=""></span>&#956;L of pool of sera from allergic patients diluted &#40;1&#58;50&#41; in 2&#37; skimmed milk powder was added and incubated for 3<span class="elsevierStyleHsp" style=""></span>h at room temperature&#46; After washing&#44; 100<span class="elsevierStyleHsp" style=""></span>&#956;L of peroxidase-labeled goat anti-human IgE antibody &#40;1&#58;1000&#65307;KPL Inc&#46;&#44; Gaithersburg&#44; MD&#44; USA&#41; was added to wells and then plates were incubated at room temperature for 1<span class="elsevierStyleHsp" style=""></span>h&#46; After washing five times with PBS-T and three times with PBS&#44; 200<span class="elsevierStyleHsp" style=""></span>&#956;L of TMB solution was added to wells for 30<span class="elsevierStyleHsp" style=""></span>min&#44; 50<span class="elsevierStyleHsp" style=""></span>&#956;L of 2<span class="elsevierStyleHsp" style=""></span>M H<span class="elsevierStyleInf">2</span>SO<span class="elsevierStyleInf">4</span> was used to stop the reaction&#46; Finally&#44; the O&#46;D&#46; values were measured with a spectrophotometer &#40;Dynex TechnologiesInc&#46;&#44; Chantilly&#44; VA&#44; USA&#41; at 450<span class="elsevierStyleHsp" style=""></span>nm&#46; The same procedure was performed with sera from non-allergic subjects to determine the extent of non-specific binding&#44; which was subtracted from test sera data&#46; Three parallel experiments were performed for each sample and the average values were taken as the final data&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Statistical analysis</span><p id="par0075" class="elsevierStylePara elsevierViewall">Data were processed using Origin 8&#46;0 software &#40;OriginLab Corp&#46;&#44; Northampton&#44; MA&#44; USA&#41;&#46; All quantitative data for the experiments were mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46; Significant difference in means between the samples was determined at a 5&#37; confidence level &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p></span></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Results and discussion</span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Expression and purification of <span class="elsevierStyleItalic">Cra g</span> 1</span><p id="par0080" class="elsevierStylePara elsevierViewall">The DNA fragment encoding <span class="elsevierStyleItalic">Cra g</span> 1 was cloned into expression vector pET-21a and confirmed by restriction enzyme digestion and DNA sequencing&#46; The protein expression was carried out in <span class="elsevierStyleItalic">E&#46; coli</span> BL21 &#40;DE3&#41;&#46; Cells were collected and analyzed by SDS-PAGE&#46; After two-step purification by Ni<span class="elsevierStyleSup">2&#43;</span> affinity chromatography and S-75 gel-filtration chromatography&#44; the recombinant protein was purified to near homogenous &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#44; lane 1&#41;&#46; The final yield of this protein is 1<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>mL<span class="elsevierStyleSup">&#8722;1</span> and the purified protein was stable after storage at &#8722;80<span class="elsevierStyleHsp" style=""></span>&#176;C&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Effect of acid treatment on conformation and allergenicity of <span class="elsevierStyleItalic">Cra g</span> 1</span><p id="par0085" class="elsevierStylePara elsevierViewall">The SDS-PAGE of <span class="elsevierStyleItalic">Cra g</span> 1 treated with different pH and time was shown in <a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#46; After acid treatment at pH 5&#46;0&#44; 3&#46;5 and 2&#46;0 for 1<span class="elsevierStyleHsp" style=""></span>h and 3<span class="elsevierStyleHsp" style=""></span>h&#44; an apparent protein band of approximately 37<span class="elsevierStyleHsp" style=""></span>kDa was still observed &#40;<a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#44; lanes 2&#8211;7&#41;&#44; and did not change under acidic conditions&#46; Almost no degradation produced low molecular weight protein bands&#46;</p><p id="par0090" class="elsevierStylePara elsevierViewall">To analyze acid-induced changes in the structure of <span class="elsevierStyleItalic">Cra g</span> 1&#44; CD spectra measurements were allowed to monitor the folding changes&#46; The CD spectrum has a peptide bond absorbing signal in the far-UV region and reflects the contents of the regular secondary structure feature of protein&#46; From our CD spectra &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>A and B&#41;&#44; we found that the original <span class="elsevierStyleItalic">Cra g</span> 1<span class="elsevierStyleHsp" style=""></span>has a positive peak &#40;maximum&#41; at nearly 198<span class="elsevierStyleHsp" style=""></span>nm and two negative peaks &#40;minimum&#41; at nearly 208<span class="elsevierStyleHsp" style=""></span>nm and 222<span class="elsevierStyleHsp" style=""></span>nm&#46; Moreover&#44; the molar ellipticity 222&#47;208 ratio is greater than 1&#46; These results indicated that it is a typical &#945;-helical protein and forms a double-stranded coil-coiled structure&#46; On the other hand&#44; significant changes in the intensity of signal could be observed after acid treatment at pH 5&#46;0&#44; 3&#46;5 and 2&#46;0 for 3<span class="elsevierStyleHsp" style=""></span>h&#46; When the pH ranged from 5&#46;0 to 2&#46;0&#44; the increase in the intensity&#44; coupled with the minimum&#44; was shifted to a lower wavelength&#44; indicating that &#945;-helix content of tropomyosin was further reduced and coil-coiled structure was partially damaged&#46; The results of acid induction for 1<span class="elsevierStyleHsp" style=""></span>h were not as obvious as those of 3<span class="elsevierStyleHsp" style=""></span>h&#46; Next&#44; ANS-binding fluorescence was used to determine the hydrophobicity of <span class="elsevierStyleItalic">Cra g</span> 1 after acid processing&#46; The surface hydrophobicity of acid-treated <span class="elsevierStyleItalic">Cra g</span> 1 was increased with pH environments ranging from 5&#46;0 to 2&#46;0 for 1<span class="elsevierStyleHsp" style=""></span>h or 3<span class="elsevierStyleHsp" style=""></span>h&#46; Surprisingly&#44; after 3<span class="elsevierStyleHsp" style=""></span>h of acid treatment at pH 2&#46;0&#44; the surface hydrophobicity of <span class="elsevierStyleItalic">Cra g</span> 1 was almost three times compared to that of the original form &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a>C&#41;&#46; The IgE-binding capacity of acid-induced tropomyosin was evaluated by indirect ELISA &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41;&#46; As we speculated&#44; the untreated <span class="elsevierStyleItalic">Cra g</span> 1 showed higher IgE reactivity&#44; however&#44; acid-treated tropomyosin reduced IgE-binding capacity when exposed to low pH environments&#44; particularly at pH 2&#46;0 and 3&#46;5 for 1<span class="elsevierStyleHsp" style=""></span>h and 3<span class="elsevierStyleHsp" style=""></span>h &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0095" class="elsevierStylePara elsevierViewall">Our results illustrated that <span class="elsevierStyleItalic">Cra g</span> 1 did not undergo degradation under acidic conditions&#44; indicating that it was resistant to acid&#44; which agreed well with previous reports&#46;<a class="elsevierStyleCrossRef" href="#bib0085"><span class="elsevierStyleSup">17</span></a> In addition&#44; the conformation of the protein altered greatly during processing&#44; which suggested that the secondary structure was partially destructed and more hydrophobic regions were exposed on the surface of <span class="elsevierStyleItalic">Cra g</span> 1&#46; Generally&#44; ionic interactions and hydrophobic interactions are considered to be the most important factors in forming the three-dimensional structure of protein&#46;<a class="elsevierStyleCrossRef" href="#bib0115"><span class="elsevierStyleSup">23</span></a> Acids could affect the protonation state of the charged amino acid&#44; alpha-carboxyl&#44; alpha-amino terminal groups as well as hydrophobic surface of protein&#44; which may ultimately alter the possible IgE-binding epitopes and potential allergenicity&#46; Thus&#44; we might deduce that steric structure of <span class="elsevierStyleItalic">Cra g</span> 1 was changed under acidic conditions&#44; leading to destroying or modifying several antigenic epitopes on molecular surface&#46; These changes markedly reduced IgE reactivity compared to the original protein&#44; which is consistent with previous experimental results&#46;<a class="elsevierStyleCrossRef" href="#bib0120"><span class="elsevierStyleSup">24</span></a> In order to further explore the conformational and allergic alterations of acid-treated <span class="elsevierStyleItalic">Cra g</span> 1 during human digestion&#44; we finally selected the protein that was to deal with acid for 3<span class="elsevierStyleHsp" style=""></span>h at pH 2&#46;0 to use in simulated gastrointestinal fluid digestion experiments and the digestion time in SGF and SIF was designated as 1<span class="elsevierStyleHsp" style=""></span>h and 4<span class="elsevierStyleHsp" style=""></span>h&#44; respectively&#44; based on the detention time of foods in digestive system&#46;</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Digestibility of acid-treated <span class="elsevierStyleItalic">Cra g</span> 1 by SGF</span><p id="par0100" class="elsevierStylePara elsevierViewall">In the present study&#44; acid-treated <span class="elsevierStyleItalic">Cra g</span> 1 was subjected to SDS-PAGE analysis after SGF digestion &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>&#41;&#46; <span class="elsevierStyleItalic">Cra g</span> 1 was relatively resistant in SGF and was only slightly degraded even at higher digestion periods&#46; Many digestion-resistant fragments &#40;about 36&#44; 22 and 15<span class="elsevierStyleHsp" style=""></span>kDa&#41; progressively intensified with prolongation of digestion time&#44; while intact protein did not completely degrade at 60<span class="elsevierStyleHsp" style=""></span>min by pepsin&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia><p id="par0105" class="elsevierStylePara elsevierViewall">From the CD spectra&#44; the signal obviously changed after SGF digestion &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>A&#41;&#46; With increasing digestion time&#44; the intensity of CD signals was increased and the minimum gradually shifted to lower wavelengths&#44; implying the secondary structures were varied drastically&#46; Hydrophobicity of <span class="elsevierStyleItalic">Cra g</span> 1 after pepsin treatment was determined by ANS-binding fluorescence measurements&#46; Interestingly&#44; compared to the control&#44; the surface hydrophobicity of the protein was decreased by 4&#37;&#44; 27&#37;&#44; 34&#37;&#44; and 40&#37; when digestion time was adjusted from 5 to 60<span class="elsevierStyleHsp" style=""></span>min &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>B&#41;&#46; To further analyze whether SGF digestion affects the allergenicity of acid-treated <span class="elsevierStyleItalic">Cra g</span> 1&#44; we performed an indirect ELISA experiment using pooled oyster-allergic subjects&#8217; sera&#46; Compared with the undigested sample&#44; IgE-binding ability showed a decreasing trend from 5 to 60<span class="elsevierStyleHsp" style=""></span>min&#44; especially at 30 and 60<span class="elsevierStyleHsp" style=""></span>min &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>&#41;&#46;</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia><elsevierMultimedia ident="fig0030"></elsevierMultimedia><p id="par0110" class="elsevierStylePara elsevierViewall">Pepsin is the only proteolytic enzyme in the stomach&#44; which prefers to cleave peptide bonds following Phe or Tyr residues&#44; as well as other hydrophobic amino acids&#46;<a class="elsevierStyleCrossRef" href="#bib0125"><span class="elsevierStyleSup">25</span></a> Pepsin could only slightly hydrolyze oyster tropomyosin&#44; demonstrating that it has relative resistance to pepsin digestion &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a>&#41;&#46; A possible reason is that the structural changes of <span class="elsevierStyleItalic">Cra g</span> 1 caused by acid produced some modifications to the enzyme cleavage sites and fewer Phe and Tyr residues were exposed on the surface of the protein&#44; thus diminishing the degree of hydrolysis&#46; Additionally&#44; we might infer that pepsin&#44; at least in part&#44; altered the conformation of the protein during digestive processing&#44; resulting in hydrophobic residues being masked and several antigenic epitopes that bound serum IgE were buried inside the protein &#40;<a class="elsevierStyleCrossRefs" href="#fig0025">Figs&#46; 5 and 6</a>&#41;&#46; This phenomenon was also observed by Huang YY et al&#46;&#59;<a class="elsevierStyleCrossRef" href="#bib0130"><span class="elsevierStyleSup">26</span></a> they demonstrated that mud crab &#40;<span class="elsevierStyleItalic">Scylla serrata</span>&#41; tropomyosin reduced IgE-binding ability after pepsin digestion&#46;</p></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Digestibility of acid-treated <span class="elsevierStyleItalic">Cra g</span> 1 by SIF</span><p id="par0115" class="elsevierStylePara elsevierViewall">The degradative patterns of acid-treated <span class="elsevierStyleItalic">Cra g</span> 1 by SGF and SIF were quite different&#46; Under SIF conditions&#44; more obvious degraded fragments appeared &#40;<a class="elsevierStyleCrossRef" href="#fig0035">Fig&#46; 7</a>&#41;&#46; <span class="elsevierStyleItalic">Cra g</span> 1 was digested into three main proteolytic fragments with sizes of approximately 36&#44; 22 and 20<span class="elsevierStyleHsp" style=""></span>kDa&#46; All these fragments and the intact original protein remained even after 4<span class="elsevierStyleHsp" style=""></span>h by trypsin digestion&#46;</p><elsevierMultimedia ident="fig0035"></elsevierMultimedia><p id="par0120" class="elsevierStylePara elsevierViewall">The CD far ultraviolet signal of acid-treated <span class="elsevierStyleItalic">Cra g</span> 1 after SIF digestion is shown in <a class="elsevierStyleCrossRef" href="#fig0040">Fig&#46; 8</a>A&#46; The spectra gradually showed a slight blue shift in the course of digestion&#44; and the curve was nearly flat after 240<span class="elsevierStyleHsp" style=""></span>min&#46; These results demonstrated that the regular &#945;-helices that dominate in tropomyosin structure diminished when the protein environment varied&#46; Besides&#44; compared with the undigested <span class="elsevierStyleItalic">Cra g</span> 1&#44; the surface hydrophobicity increased by 28&#37;&#44; 43&#37;&#44; 46&#37;&#44; 53&#37; and 58&#37; after 15&#44; 30&#44; 60&#44; 120 and 240<span class="elsevierStyleHsp" style=""></span>min&#44; respectively &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0040">Fig&#46; 8</a> B&#41;&#46; The IgE-binding ability of <span class="elsevierStyleItalic">Cra g</span> 1 after SIF digestion was further analyzed by ELISA &#40;<a class="elsevierStyleCrossRef" href="#fig0045">Fig&#46; 9</a>&#41;&#46; Surprisingly&#44; contrary to the SGF results&#44; all digested samples have significantly higher IgE reactivity than the control &#40;<span class="elsevierStyleItalic">P</span><span class="elsevierStyleHsp" style=""></span>&#60;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41;&#46; Possibly&#44; more IgE-binding epitopes in degraded fragments or original protein were generated by trypsin digestion&#46; Therefore&#44; this treatment may not be effective in decreasing the allergenic ability of the protein&#46;</p><elsevierMultimedia ident="fig0040"></elsevierMultimedia><elsevierMultimedia ident="fig0045"></elsevierMultimedia><p id="par0125" class="elsevierStylePara elsevierViewall">The sensitization of potential allergens is routinely evaluated using simulated digestion studies&#46; Our results showed that digestive patterns by trypsin and pepsin were very different because of cleavage specificities of the two proteases&#46; Trypsin mainly cleaves peptide bonds following Lys and Arg residues at the P<span class="elsevierStyleInf">1</span> position with higher specificity&#46;<a class="elsevierStyleCrossRef" href="#bib0135"><span class="elsevierStyleSup">27</span></a> Although there are several cleavage sites for trypsin and pepsin in <span class="elsevierStyleItalic">Cra g</span> 1 primary structure&#44;<a class="elsevierStyleCrossRef" href="#bib0140"><span class="elsevierStyleSup">28</span></a> acid-treated protein and its degraded fragments were resistant to digestion &#40;<a class="elsevierStyleCrossRefs" href="#fig0020">Figs&#46; 4 and 7</a>&#41;&#46; One reason for this might be that cleavage sites on the molecular surface were eliminated due to acid treatment&#46; Hence oyster tropomyosin&#44; as well as its digestion-resistant fragments&#44; could survive and reach the intestinal immune system&#46; Furthermore&#44; antigenicity of acid-induced <span class="elsevierStyleItalic">Cra g</span> 1 decreased in SGF&#44; while it increased significantly in SIF &#40;<a class="elsevierStyleCrossRefs" href="#fig0030">Figs&#46; 6 and 9</a>&#41;&#46; This phenomenon may result from conformational alterations of protein &#40;<a class="elsevierStyleCrossRef" href="#fig0040">Fig&#46; 8</a>&#41;&#44; which exposed more hydrophobic groups and additional immunogenic epitopes by trypsin digestion&#46; It is also possible that new IgE-binding epitopes were produced in proteolytic fragments by SIF&#44; which contributed to the IgE-binding reactivity&#46; To our knowledge&#44; this is the first report that antigenicity of acid-treated oyster tropomyosin increased under SIF conditions&#46; This explains why sensitized individuals still suffer severe allergic reactions even if they intake high-acid oyster products such as salad dressings&#46; Besides&#44; the ELISA data are a relative result because IgE-binding capacity does not equate to allergenicity completely&#46;<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">29</span></a> Some degraded fragments may bind IgE but not enable basophils or mast cells to release mediators&#46; Consequently&#44; the next steps are needed to assess the allergic changes of tropomyosin through release of histamine from basophils and skin prick test following acid and protease treatment&#46; Identification of immunogenic epitopes in intact <span class="elsevierStyleItalic">Cra g</span> 1 and its proteolytic fragments during the same processing should be carried out to further expound the molecular mechanism of interactions between the protein and oyster-specific IgE&#46;</p><p id="par0130" class="elsevierStylePara elsevierViewall">In conclusion&#44; alterations in secondary structure and exposure or burial of hydrophobic amino acid residues were responsible for conformational changes&#44; resulting in the disruption or formation of IgE-binding epitopes&#44; and ultimately affected immunogenic activity&#46; For oyster tropomyosin&#44; treatment with acid and pepsin&#44; rather than trypsin&#44; is an effective way of reducing IgE-binding capacity&#46;</p></span></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Conflict of interest</span><p id="par0135" class="elsevierStylePara elsevierViewall">The authors have no conflict of interest to declare&#46;</p></span></span>"
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          "identificador" => "xres1427779"
          "titulo" => "Abstract"
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            0 => array:2 [
              "identificador" => "abst0005"
              "titulo" => "Introduction and Objectives"
            ]
            1 => array:2 [
              "identificador" => "abst0010"
              "titulo" => "Results"
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              "titulo" => "Conclusion"
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          "titulo" => "Keywords"
        ]
        2 => array:2 [
          "identificador" => "sec0005"
          "titulo" => "Introduction"
        ]
        3 => array:3 [
          "identificador" => "sec0010"
          "titulo" => "Materials and methods"
          "secciones" => array:11 [
            0 => array:2 [
              "identificador" => "sec0015"
              "titulo" => "Human sera"
            ]
            1 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "Cloning&#44; expression and purification of recombinant Cra g 1"
            ]
            2 => array:2 [
              "identificador" => "sec0025"
              "titulo" => "Protein quantification"
            ]
            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "Acid treatment of Cra g 1"
            ]
            4 => array:2 [
              "identificador" => "sec0035"
              "titulo" => "SGF digestion assay"
            ]
            5 => array:2 [
              "identificador" => "sec0040"
              "titulo" => "SIF digestion assay"
            ]
            6 => array:2 [
              "identificador" => "sec0045"
              "titulo" => "Electrophoresis analysis &#40;SDS-PAGE&#41;"
            ]
            7 => array:2 [
              "identificador" => "sec0050"
              "titulo" => "Circular dichroism &#40;CD&#41; spectroscopy"
            ]
            8 => array:2 [
              "identificador" => "sec0055"
              "titulo" => "ANS-binding fluorescence measurements"
            ]
            9 => array:2 [
              "identificador" => "sec0060"
              "titulo" => "IgE enzyme-linked immunosorbent assay &#40;ELISA&#41;"
            ]
            10 => array:2 [
              "identificador" => "sec0065"
              "titulo" => "Statistical analysis"
            ]
          ]
        ]
        4 => array:3 [
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          "titulo" => "Results and discussion"
          "secciones" => array:4 [
            0 => array:2 [
              "identificador" => "sec0075"
              "titulo" => "Expression and purification of Cra g 1"
            ]
            1 => array:2 [
              "identificador" => "sec0080"
              "titulo" => "Effect of acid treatment on conformation and allergenicity of Cra g 1"
            ]
            2 => array:2 [
              "identificador" => "sec0085"
              "titulo" => "Digestibility of acid-treated Cra g 1 by SGF"
            ]
            3 => array:2 [
              "identificador" => "sec0090"
              "titulo" => "Digestibility of acid-treated Cra g 1 by SIF"
            ]
          ]
        ]
        5 => array:2 [
          "identificador" => "sec0095"
          "titulo" => "Conflict of interest"
        ]
        6 => array:2 [
          "identificador" => "xack498286"
          "titulo" => "Acknowledgements"
        ]
        7 => array:1 [
          "titulo" => "References"
        ]
      ]
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    "fechaRecibido" => "2019-07-05"
    "fechaAceptado" => "2019-08-28"
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          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec1304200"
          "palabras" => array:6 [
            0 => "<span class="elsevierStyleItalic">Cra g</span>1"
            1 => "Acid"
            2 => "SGF"
            3 => "SIF"
            4 => "Conformation"
            5 => "IgE-binding"
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    "resumen" => array:1 [
      "en" => array:3 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Introduction and Objectives</span><p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">The production and consumption of oysters is increasing annually because it can provide essential nutrients and benefit for human health&#44; leading to frequent occurrence of severe allergic reactions observed in sensitized individuals&#46; The aim of the present study was to investigate the effects of acid and protease treatment on the conformation and IgE-binding capacity of recombinant <span class="elsevierStyleItalic">Crassostrea gigas</span> tropomyosin &#40;<span class="elsevierStyleItalic">Cra g</span> 1&#41;&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Results</span><p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Under acidic conditions&#44; <span class="elsevierStyleItalic">Cra g</span> 1 did not undergo degradation&#44; however&#44; the changes obvious in the intensity of CD signal and ANS-binding fluorescence were observed&#44; which was associated with a decrease in antibody reactivity&#46; In simulated gastrointestinal fluid &#40;SGF&#41; and simulated intestinal fluid &#40;SIF&#41; digestion system&#44; acid-treated <span class="elsevierStyleItalic">Cra g</span> 1 was relatively resistant to digestion&#44; but the degradative patterns were very different&#46; Moreover&#44; owing to alterations of secondary structure and hydrophobic surface of the protein during digestive processing&#44; antigenicity of acid-induced <span class="elsevierStyleItalic">Cra g</span> 1 reduced in SGF while it increased significantly in SIF&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Conclusion</span><p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">To our knowledge&#44; this is the first study reporting that antigenicity of acid-treated oyster tropomyosin increased after SIF digestion&#46; These results revealed that treatment with acid and pepsin&#44; rather than trypsin&#44; was an effective way of reducing IgE-binding capacity of tropomyosin from oyster&#46;</p></span>"
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          "en" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">SDS-PAGE analysis of effect of acid on recombinant <span class="elsevierStyleItalic">Cra g</span> 1&#46; Lane M&#44; molecular mass marker &#40;kDa&#41;&#65307;Lane 1&#44; purified <span class="elsevierStyleItalic">Cra g</span> 1&#59; Lanes 2&#8211;4&#44; <span class="elsevierStyleItalic">Cra g</span> 1 was treated by acid for 1<span class="elsevierStyleHsp" style=""></span>h at pH 5&#46;0&#44; 3&#46;5 and 2&#46;0&#44; respectively&#59; Lanes 5&#8211;7&#44; <span class="elsevierStyleItalic">Cra g</span> 1 was treated by acid for 3<span class="elsevierStyleHsp" style=""></span>h at pH 5&#46;0&#44; 3&#46;5 and 2&#46;0&#44; respectively&#46;</p>"
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Article information

ISSN: 03010546
Original language: English

DOI:10.1016/j.aller.2019.08.001

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2024 January	27	33	60
2023 December	17	18	35
2023 November	16	27	43
2023 October	31	28	59
2023 September	19	18	37
2023 August	14	15	29
2023 July	21	32	53
2023 June	20	14	34
2023 May	18	5	23
2023 April	15	0	15
2023 March	13	4	17
2023 February	16	3	19
2023 January	28	3	31
2022 December	29	9	38
2022 November	15	9	24
2022 October	33	14	47
2022 September	21	8	29
2022 August	18	8	26
2022 July	10	7	17
2022 June	33	5	38
2022 May	23	14	37
2022 April	27	11	38
2022 March	25	24	49
2022 February	25	18	43
2022 January	53	12	65
2021 December	23	12	35
2021 November	23	8	31
2021 October	26	17	43
2021 September	32	11	43
2021 August	57	8	65
2021 July	13	10	23
2021 June	18	6	24
2021 May	17	9	26
2021 April	25	11	36
2021 March	13	9	22
2021 February	8	6	14
2021 January	11	3	14
2020 December	1	0	1
2020 July	0	1	1
2020 March	6	0	6
2020 January	2	0	2
2019 October	0	4	4

Show all

Allergologia et Immunopathologia is no longer published on Elsevier since the 2021 year.Transferred to Codon Publications

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Allergologia et Immunopathologia is no longer published on Elsevier since the 2021 year.Transferred to Codon Publications

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