Fasciola hepatica and F. gigantica. Such parasites cause serious illness in numerous domestic animals and also in humans. Following infection, the parasite secretes a variety of molecules that immediately interact with the host immunity to establish successful infection. These molecules include cathepsin L peptidase 1 (CatL1); the highly investigated diagnostic and vaccine antigens using various animal models. However, a few studies have analyzed the potentials of FhCatL1 as a diagnostic or vaccine antigen using bioinformatic tools and much less for FgCatL1. The present study provides inclusive and exclusive information on the physico-chemical, antigenic and immunogenic properties of F. hepatica cathepsin L1 (FhCatL1) protein using multiple bioinformatic analysis tools and several online web servers. Also, the validation of our employed available online servers was conducted against a huge collection of previously published studies focusing on the properties of FhCatL1as a diagnostic and vaccine antigen.Fascioliasis is a parasitic foodborne disease caused by the liver flukes, For this purpose, the secondary, tertiary, and quaternary structure of FhCatL1 protein were also predicted and analyzed using the SWISS-MODEL server. Validation of the modeled structures was performed by Ramachandran plots. The antigenic epitopes of the protein were predicted by IEDB server.Our findings revealed the low similarity of FhCatL1 with mammalian CatL1, lacking signal peptides or transmembrane domain, and the presence of 33 phosphorylation sites. Also, the containment of FhCatL1 for many topological, physico-chemical, immunological properties that favored its function of solubility and interaction with the immune components were reported. In addition, the earlier worldwide reports documented the high efficacy of FhCatL1 as a diagnostic and vaccine antigen in different animals. Altogether, FhCatL1 is considered an excellent candidate for using in commercialized diagnostic assays or vaccine products against fascioliasis in different animal species. Our assessment also included FgCatL1 and reported very similar findings and outputs to those of FhCatL1. Fasciola hepatica and F. gigantia are common foodborne trematode parasites widespread worldwide. They affect a wide variety of host species causing fascioliasis. Fasciola parasites are major pathogens of ruminants and have a substantial impact on the health and wellbeing of animals. Severe infections can be fatal as a result of widespread liver damage caused by the migration of large numbers of young flukes. Chronic infections lead to anemia, weight loss and poor condition. However, low-grade infections are common and asymptomatic and are associated with slower growth and lower milk production  proteins that possess strong antigenic characteristics. These tests demonstrated a high sensitivity (86.1–100 %) and specificity (70–99.3 %)  and CL2 are the most abundant of proteases in ES antigen and the two proteins accounting for 67 % and 28% of total cathepsin L proteins, respectively . HoweverBioinformatics is one of the most current study fields to better address and monitor biological challenges . It is fEpitopes are typically identified by showing that certain peptide fragments and certain proteins have antigenic reactivity. Since the accessibility, hydrophilicity, and mobility of polypeptide chain segments have been linked to the position of epitopes in proteins, it is conceivable to infer from the primary structure, in which linear peptides are most likely to correspond to those epitopes . In ordeF. hepatica cathepsin L1 (FhCatL1) protein using multiple bioinformatic analysis tools and several online web servers. In addition, data of bioinformatic analyses have been compared with the previous heritage of available studies investigated the potentials of FhCatL1and FgCatL1in immnunodiagnostic and vaccine researches.Few studies have analyzed the potentials of FhCatL1 as diagnostic or vaccine antigens using bioinformatic tools and much less for FgCatL1 , 20. NevF. hepatica cathepsin L1 (FhCatL1) was obtained in FASTA format from the GenBank (AJ279092.1) database at https://www.ncbi.nlm.nih.gov. Then, using the corresponding cathepsin L1 proteins from FhCatL1, as well as those from F. gigantica (JQ342985.1), humans (BC142983.1), and cattle, multiple sequence alignment was performed using Clustal Omega  (BC134741.1). For full matching with FhCatL1, first 15 amino acid (MRLFILAVLTVGVLG) was deleted from our analyzed F. gigantica (JQ342985.1).The amino acid sequence for https://services.healthtech.dtu.dk/service.php?SignalP-5.0) was utilized. A Sec signal peptide (Sec/SPI), a Lipoprotein signal peptide (Sec/SPII), a Tat signal peptide (Tat/SPI), or no signal peptide at all can be found in the protein (Other). Additionally, using the TMHMM 2.0 online server , putative transmembrane domains of FhCatL1 and FgCatL1 were predicted  and molecular weight of FhCatL1 and FgCatL1 was calculated. The molecular weight and diameter of the selected amino acids from FhCatL1 and FgCatL1of number 311 were determined.To identify the signal peptide in the sequence, the SignalP online tool  used ensembles of neural networks to show the locations of phosphorylation sites for serine, threonine, and tyrosine. Two web servers were used to clarify the FhCatL1 and FgCatL1 secondary structure, including the Garnier-Osguthorpe-Robson (GOR IV) servers (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_gor4.html) and position specific iterated prediction (PSIPRED) (http://bioinf.cs.ucl.ac.uk/psipred). Then, three-dimensional (3D) protein models were created using SWISS-MODEL, a fully automated online server for protein structure homology modeling that may be found at (https://swissmodel.expasy.org/) , graphical representations of linear epitopes were created based on average flexibility, beta-turn, alpha-helix, hydrophobicity, and the percentage of accessible residues and molecular weight (The NetPhos 3.1 server (PSIPRED) , 23, bassy.org/) . Using tr weight .http://tools.immuneepitope.org/bcell/) was employed by the IEDB server to predict the B-cell epitopes. ElliPro predictor was used to analyze linear and conformational (discontinuous) epitopes based on 3D structures prediction of FhCatL1. Crystal Structure of ProCathepsin L1 from Fasciola hepatica (206X) that showed high similarity with our target sequence of FhCatL1, as it is available as PDB file, the required input data for ElliPro searches was used for further analysis of FhCatL1 (http://tools.iedb.org/ellipro/). Similarly, Disco Tope tool that can predict the conformational antibody epitope via 3D structures was used employing the same the protein model 206X (http://tools.iedb.org/discotope/). A summary for the predictor servers used in the current study for analyses of different properties of FhCatL1 was illustrated in Based on the physicochemical characteristics of amino acids including hydrophilicity , and antF. gigantica, as well as human and cattle amino acids as examples for highly susceptible host species for fascioliasis. Similarity was 94.2, 44.4, and 40.2% for cathepsin L1 of F. gigantica, human and cattle, respectively . Similarly, FgCatL1 (JQ342985.1) consists of 311 amino acids with estimated MW of 35.2 kDa with similar diameter to FhCatL1 , six threonine (T), and eleven tyrosine (Y) as shown in The FhCatL1 protein contained three main secondary structure components, consisting of 53.38 % random coil, 24.44 % extended strand, and 22.19 % alpha helix, as shown by PSIPRED and GOR IV servers. For FgCatL1 amino acid sequences encompasses 52.41 % random coil, 25.08 % extended strand, and 22.51 % α-helix in secondary structure , S6. ProZ-score was −1.12 which is close to zero suggesting high accuracy and reliability of protein modeling  protein models were constructed for FhCatL1and FgCatL1using a fully automated protein structure homology-modeling web server called SWISS-MODEL. In total, 50 SWISS-MODEL templates were made for FhCatL1 protein sequence, among which a single model highly matching our target was built. The model showed highest coverage and 92.26% sequence identity. Global model evaluation GMQE and QMEANDisCo of any analyzed model gives an overall model quality measurement between 0 and 1, with higher numbers indicating higher expected quality. GMQE and QMEANDisCo in our model was 0.96 and 0.93, respectively, suggesting high quality of expected model measurement and assessment –D. The s protein modeling . In addimodeling . Red col FgCatL1 .via Ramachandran plot from SWISS-MODEL server. The model demonstrated MolProbity (0.92), Clash score (0.21), Poor rotamers (1.0) and Ramachandran favored (95.78%), no Ramachandran outliers, C-Beta deviations or bad bonds, Rotamer outliers (1.16%), bad angels (30 / 6856) and twisted proline (2/18)  at SWISS-MODEL repository for all tested characters of oligomerization, stoichiometry, topology, and the interface similarity based on quaternary structure analysis  . These re (2/18) . As suche (2/18) , 37. More (2/18) . Furthermilarity . Our taranalysis –E. The danalysis . As 206Xructures . Furtherructures . These rructures . Thus, oFasciola hepatica (206X.1A or 206X.1B) was assessed followed by visualization of 2D charts and Jmol visualization of the predicted epitopes. As shown in ProtScale online tool was applied to predict linear B-cell epitopes for FhCatL1 and FgCatL1 according to hydrophobicity, alpha-helix, beta-turn, average flexibility, percent of accessible residue, and molecular weight . Strong in silico methods an appealing complementary approach -based indirect ELISAs demonstrated high efficacy in detection of specific antibodies in infected animals including sheep  antigen has proven its potential in discriminating between infected and non-infected sera collected from susceptible animals. In this regard, various forms of FgCatL1 could detect fascioliasis in buffaloes, sheep, or cattle sera either using rFgCatL1  showed a high efficacy in diagnosis of fascioliasis using indirect ELISAs against sera obtained from Sika deer  in eliciting an immune response and conferring protection against challenge infection in both rabbit , and sheol group , 83.Fasciola progressively emerge with wide resistance to available anti-parasitic drugs and the burden of parasites. Different preparations of FhCatL1 including recombinant protein or mimotope and either used solitary or in combination with other antigens succeeded to confer efficient protective potentials in different animal models. However, further studies are required to improve the protective potentials, eg. by mixing with potent adjuvants, testing the underestimated F. gigantica, and finding out novel protective indices. As the vaccine preparation and testing are costly procedures, initial assessment of the efficacy will be highly advantageous in many aspects particularly from economic viewpoint. Bioinformatic analysis has been already providing this benefit for vaccine researches including those conducted for Fasciola assessments  or molecules   , 85. CurFasciola- or other parasites-derived proteins via computational and online software programs will reduce the time, effort and cost before application of practical assays and experiments. Useful relevant information was also presented for FgCatL1 as a diagnostic and vaccine candidates, although it was much less than those of FhCatL1.We revealed the first description of multiple sequence alignment, isoelectric point, phosphorylation sites, and morpho-physical properties from protein database of FhCatL1. Moreover, first assessment of FhCatL1 was applied for secondary structure using PSIPRED and GOR IV servers, tertiary, and quaternary structure using SWISS-MODEL, and linear B- cell epitopes using ProtScale online available tool. The bioinformatic analyses in the present study revealed the high antigenicity, immunogenicity, hydrophilicity, surface accessibility, and flexibility indexes for FhCatL1 protein. Thus, application of on-line available bioinformatic analysis tools can be used for evaluation and validation of basic structures and properties and to predict antigenicity and immunogenicity of protein based on amino acid sequence. Such data are additionally supported with numerous previous literatures that corroborating the high efficiency of FhCatL1 as diagnostic and vaccine antigen. Hence, we recommend FhCatL1 as a suitable antigen for developing diagnostic tests and vaccines against fascioliasis in different animal models. Prediction of the antigenic or immunogenic properties of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Conceptualization and investigation: RF and AA. Formal analysis, project administration, and writing—original draft: RF. Methodology: RF, SM, E-SE-A, HA, and MO. Resources and writing—review and editing: RF, SM, E-SE-A, HA, OS, MO, and AA. All authors contributed to the article and approved the submitted version.


Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominantly inherited genetic disease characterized by recurrent numbness and limb weakness. HNPP can be easily missed or misdiagnosed because of electrophysiological heterogeneity and atypical clinical symptoms. To date, diagnosis of HNPP remains a challenge for clinicians.Here, we report the case of a 12-year-old woman diagnosed with HNPP, which was initially diagnosed with Guillain–Barré Syndrome (GBS) and treated with intravenous immunoglobulin (IVIG).Repeat electrodiagnostic studies and genetic testing confirmed the diagnosis of HNPP.The patient was treated with neurotrophic drugs and health education, including avoiding maintenance of a certain posture for extended periods, which could damage the peripheral nerves.The patient was discharged 5 days later. The patient was free from recurrence after 6 months of follow-up.This case highlights the complexity of HNPP diagnosis and emphasizes the importance of early identification. The clinical presentation of hereditary neuropathy with liability to pressure palsies (HNPP), such as limb numbness, fatigue, and/or pain, is variable. And electromyography lacks specificity. There is a need to distinguish HNPP from other diseases, such as Guillain–Barré Syndrome (GBS) or diabetic peripheral neuropathy, and it can be easily missed or misdiagnosed. In turn, this may increase patients’ financial burden and lead to doctor–patient contradictions. Here, we present a case of HNPP misdiagnosed as GBS. We also reviewed the available literature on the pathogenesis, clinical manifestations, and examination and treatment of HNPP. This study aimed to improve the awareness of HNPP and reduce the rate of misdiagnoses and missed diagnoses.The patient was 12 years old, junior school student with no medical history. She was referred to our hospital in November 2021 because of recurrent right upper limb weakness that had persisted for 4 years. Symptoms started to appear at the age of 8 years with the right upper limb having predominantly proximal muscle weakness, which improved after 1 to 2 weeks. The patient’s quality of life was unaffected during the interictal period. She first sought psychiatric help before 6 months, and then a full diagnostic work-up, including serum tests, magnetic resonance imaging , and cerebrospinal fluid cytology, was negative. Electrophysiological examination suggested a demyelinating neuropathy. As a result, she was initially diagnosed with GBS, treated with intravenous immunoglobulin (IVIG), and discharged in a stable condition after 5 days. She presented with recurrence of right upper limb weakness two weeks prior to presentation. Additionally, the patient’s father had a previous diagnosis of GBS.Neurological examination showed weakness in the right upper extremity  and functional limitation of the upper limb. Her grip was vigorous. The muscular strength of the remainder of the limb was normal, and the tendon reflex was absent. In addition, signs of meningeal irritation and the Babinski reflex were also negative.Electrophysiological examinations were repeated. Data from nerve conduction velocities suggested multiple neurological abnormalities. Prolonged distal motor latencies and slow conduction velocities were also observed Table . The F-wIn contrast, genetic testing revealed deletion of the peripheral myelin protein 22 (PMP22) gene region on chromosome 17, confirming the diagnosis of HNPP  of the head and cervical spine. This finding was consistent with previous literature. However, nerve MRI might not be essential and can be used to further shorten the differential diagnosis.Although data on imaging descriptions of the HNPP are limited, they have attracted more research attention. Treatment is based on neurotrophic drugs as well as symptomatic and supportive therapies. The key to HNPP lies in prevention, mainly to avoid nerve compression caused by exercise or heavy physical work.Currently, no specific therapy is available for HNPP.In summary, our analysis showed that the clinical manifestations of HNPP included limb weakness and/or numbness. Patients may have subclinical symptoms, and the scope of electrophysiological examination should be expanded in suspicious cases. The gold standard for the diagnosis of HNPP is identification of PMP22 deletions or point mutations. In addition, HNPP should be diagnosed as early as possible to avoid unnecessary loss due to misdiagnosis.Thanks to the patient, all the participants and researchers involved in this study.Data curation: Xueqing Tong.Writing – original draft: Yandeng Li, Guangqin Li.Writing – review & editing: Jianming Zhu, Zhendong Pi.


Tea is a non-alcoholic drink containing various active ingredients, including tea polysaccharides (TPSs). TPSs have various biological activities, such as antioxidant, anti-tumor, hypoglycemic, and anti-cancer activities. However, TPSs have a complex composition, which significantly limits the extraction and isolation methods, thus limiting their application. This paper provides insight into the composition, methodological techniques for isolation and extraction of the components, biological activities, and functions of TPSs, as well as their application prospects. Tea is an important cash crop with a long history of medicinal and dietary benefits, especially in Asian countries, such as China, Thailand, Japan, and India ,2. It haTea leaves contain about 1.5–13% of TPS ,16. TPSsTPSs are non-starch protein-bound acidic polysaccharides containing 44.2% neutral sugar, 43.1% glyoxylate, and 3.5% protein. TPSs mainly contain monosaccharides, such as glucose (Glc), galactose , arabinose (Ara), rhamnose (Rha), xylose (Xyl), galacturonic acid , mannose (Man), ribose (Rib), and glucuronide (GulA)  Figure .TPSs are also bioactive components of tea with excellent activity, and their crude forms are extracted via simple processing methods. Various processing methods alter the fractions of crude TPSs . Two cruTPSs can be divided into neutral polysaccharides (NTPSs) and acidic polysaccharides (ATPSs). NTPSs contain 82.7% of total sugars, of which 12.9% consists of glyoxylates, while ATPSs contain 85.5% of total sugars, of which 39.8% consists of glyoxylates. Gal (67.6%) is the main sugar composition in NTPSs, while Ara, Rha, GalA, and Gal are the main sugar compositions in ATPSs . HoweverThe variation in chemical composition and biological activity of TPSs may be due to the preparation methods and raw materials . VariousSeveral functional experiments have shown that TPSs have biological activities, including immunomodulatory, anti-tumor, antioxidant, and anti-diabetic activities.Ganoderma lucidum polysaccharide (LLPs) from samples of L. lucidus Turcz herbal tea and evaluated the systemic immunomodulatory activity of LLPs in mice. They found that LLPs can exhibit an overall synergistic stimulatory effect on specific and non-specific immune functions in mice. Moreover, medium and high doses of LLPs significantly increased thymic and splenic organ indices (p < 0.05). This finding suggests that polysaccharides extracted from Ganoderma lucidum tea can enhance the immune system and may be considered a biological response regulator [Intracellular polysaccharides (IPSs) isolated from fermented Fuzhuan brick tea (FBT) have immunomodulatory activity in vitro. Specifically, IPSs enhance the immune function of cyclophosphamide (Cy)-induced immunosuppressed mice in vivo by improving immune organ index and immunoglobulins. IPSs also attenuate Cy-induced intestinal barrier damage and promote the expression of tight junction protein and mucin, thus enhancing intestinal barrier function . Cao et egulator .p < 0.01). Higher concentrations  of TSPSs had a stronger proliferative effect on lymphocytes [4 Da) and showed that GTP could inhibit the growth of prostate cancer (PC)-3 cells by inducing apoptosis [Wei et al. investigated the biological activity of tea seed polysaccharides (TSPSs) and showed that TSPSs can significantly inhibit the growth of K562 cells. TSPSs at 50 μg/mL showed the highest inhibitory activity with an inhibition ratio of more than 38.44 ± 2.22%  and turkey polysaccharide (HJP) can significantly enhance glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities and reduce malondialdehyde (MDA) levels in mice . Han et t manner . Fan et t manner . Liu et t manner . Wang ett manner . Chen ett manner . Shu et t manner .Camellia sinensis) via an aqueous extraction method and evaluated their anti-diabetic activity. The TPSs exhibited significant antioxidant and anti-diabetic (α-glucosidase inhibition and anticoagulant) activities. Particularly, Pu’er TPS had good anti-diabetic activity [Camellia sinensis L.) and showed that black TPSs have stronger α-glucosidase inhibitory and anti-diabetic activities than the other selected tea polysaccharides [Nelumbo nucifera polysaccharide (NNP-2) from lotus seed tea and investigated its anti-diabetic activity and the mechanism of regulating glucose metabolism in HepG2 cells. They found that NNP-2 has biological activity against α-glucosidase (IC50 = 97.32 µg/mL) and could improve intracellular glucose levels by regulating the IRS1/PI3 K/Akt pathway in HepG2 cells [TPSs in coarse old tea leaves are widely used to treat diabetes in Chinese and Japanese folklore . The hypactivity . Wang etactivity . Wu et aactivity . Chung eactivity . Mao et activity . Deng etactivity . Guo et charides . Luo et charides . Le et aG2 cells .Bai et al. investigated the effects of crude polysaccharides from Fuzhuan brick tea (CFBTPS) and purified fractions (FBTPS-3) extracted from porcupine tea on the regulation of immune function and intestinal microbiota in mice, and found the following results: CFBTPS and FBTPS-3 could restore the levels of various physiological indicators, such as body weight, diet, immunoglobulins, and immune organ indices in mice. CFBTPS and FBTPS-3 could also repair intestinal damage and regulate the intestinal microbiota. Moreover, FBTPS could regulate immune function in mice .TPSs have a hypoglycemic effect. Numerous studies have demonstrated that TPSs have significant therapeutic effects on diabetes. Tang et al. investigated the biological activities of two fractions (JSP-1 and JSP-2) of water-soluble polysaccharides (JSPs) extracted from Eton tea and showed that JSP-1 and JSP-2 could scavenge DPPH and hydroxyl radicals and protect pancreatic islet cells. These results indicate the antioxidant and hypoglycemic activities of JSP-1 and JSP. However, JSP-1 had stronger antioxidant and hypoglycemic activities than JSP-2 .Obesity has become a global health problem due to changes in diet and living habits. Several studies have shown that tea can regulate lipid metabolism. Wu et al. found that polysaccharide extract can reduce the weight of rats with hfD-induced obesity, improve body composition, and increase the content of fatty acids in feces, thus preventing hfD-induced obesity. They also explored the mechanism of polysaccharide extracts in regulating lipid metabolism via gene chip array and found that polysaccharide extracts mainly affect gene expression of the lipid metabolism pathway, thus causing biochemical changes, such as increasing fecal fatty acid content and decreasing body fatty acid content . Xu et aChen et al. investigated the therapeutic effect of TPSs on colitis in mice. They showed that TPSs could alleviate colitis by restoring various physical indices  and promoting intestinal barrier function .Polysaccharides have natural advantages in biomedical applications due to the unique multifunctional groups on their structure. Polysaccharides can be modified to polysaccharide-based nanoparticles with various structures via simple chemical or biochemical methods. Moreover, polysaccharides can be used as a carrier of different drugs since their structures have good biocompatibility and degradability and cannot easily leak into the blood, thus achieving longer blood circulation ,79,80,81For example, polysaccharide-gel-coated drugs can be orally taken to specific sites to deliver their effects .Wu et al. designed and synthesized polysaccharide-based porous microspheres (PPMs) via the inverse emulsion polymerization method and evaluated their drug delivery capabilities and functionalities. The PPMs loaded with mitomycin-C (MMC) could promote the sustained release of MMC at the target site, thus reducing the toxic effects on normal tissues and exhibiting stronger tumor-suppressive effects .Common plastic products are environmental pollutants. As a result, polysaccharides have attracted much attention because they can be developed into biodegradable materials and coatings ,85. FruiThe variation in chemical composition and biological activity of TPSs may be due to the different preparation methods and raw tea samples. Some common TPS preparation methods have low yields and alter the active components during extraction. Therefore, an efficient, standardized, and simple method should be established for purifying TPSs from various tea leaves to obtain chemically stable TPS products.Various types of polysaccharide drugs have been introduced into clinical practice in the past 20 years for the treatment of various diseases in China and worldwide. About 30 types of polysaccharide drugs with anti-tumor, anti-infection, anti-rheumatism, anti-peptic ulcer, and immune functions are currently used in clinics. The drugs may be in the form of polysaccharide drugs, anti-radiation drinks, chewing gums, and various health products. TPSs can be added to coffee, beer, and other foods to make unique functional foods. TPSs can also act as food additives in beverages, pastries, and oral liquids. However, in-depth research on TPSs is necessary to discover more valuable aspects, including the auxiliary treatment of diabetes.


Kinetic studies of the inhibition processes indicated the tannins provided inhibition through competitive–uncompetitive mixed reactions. In silico molecule docking showed that tannins were bound to the active site of tyrosinase via hydrogen and electrovalent bonds. Circular dichroism (CD) observed the structural variation in the tyrosinase after reacting with the tannins. Fluorescence quenching analysis and free radical scavenging assays indicated that the tannins had copper ion chelating and antioxidant activities, which may also contribute to inhibition. The intracellular inhibition assay revealed that the melanin was reduced by 34.50% in B16F10 cells. These results indicate that these tannins can be applied as whitening agents in the cosmetics industry.The application of hops could be extended to obtain higher commercial values. Tannins from hops were assessed for their tyrosinase inhibition ability, and the associated mechanisms were explored. Nuclear magnetic resonance (NMR) and high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS) revealed that the hop tannins were characterized as condensed tannins with (epi)catechin and (epi)gallocatechin as subunits and an average polymerization degree of 10.32. Tyrosinase inhibition assay indicated that hop tannins had an IC Melanin is responsible for providing protection in human skin against ultraviolet radiation, stress from environmental pollutants, and toxic drugs and chemicals. However, abnormal melanin production could result in melasma, solar lentigo, freckles, acne scars, age spots, and other dermatological problems . It is rConsequently, many natural compounds, discovered from plants and especially food materials, have been evaluated for their tyrosinase inhibition ability. These compounds can be classified as resveratrol derivates, which were mainly found in grapes and wines, and inhibited tyrosinase through suicide substrate inhibition with limited cytotoxicity effects ; diphenyHumulus lupulus L.) are conventionally used in beer brewing to impart an attractive aroma and bitter taste. Hops are mainly produced in the U.S., Germany, and the Czech Republic, and their annual production has almost reached 100K tons [w/w) of the hops, can be easily extracted using hot water [Hops  was dissolved in a 10 mL 50% methanol solution and then loaded onto a Sephadex LH-20 column . The column was washed with a 50% methanol solution  to remove simple phenolics, proteins, and sugars. The tannin fraction was then collected by elution with a 70% acetone–water solution (1.0 L eluent). After removing organic solvents and water from the tannin fraction by rotary evaporation (35 °C) and lyophilization, purified tannins were obtained and used as samples for the following experiments.13CNMR spectra were obtained with a Bruker Ascend 400 MHz NMR Spectrometer . Purified tannins (30 mg) were dissolved in a 750 µL CD4O–D2O solution  and then transferred into a nuclear magnetic tube for 13CNMR analysis [analysis . SignalsTo obtain subunits for the HPLC–ESI–MS/MS study, the tannins were treated with an acid–cleavage derivatization process prior to analysis. The method was referenced in our previous report  and sligThe HPLC–ESI–MS/MS analysis was carried out based on previous reports ,16. The 50), and (2) an inhibition kinetic assay. All the reactions were undertaken in a phosphate buffer solution , while tyrosinase  and L-DOPA  were chosen as the model enzyme and substrate. The mean molecular weight of the tannins was determined as 3581 Da  of the tannins was calculated using the inhibition rate–inhibitor concentration plot (Hop tannin solutions (50 µL) of different concentrations  were each mixed with 1 mL of L-DOPA solution (0.5 mM) and preheated at 30 °C for 10 min. Afterwards, 50 μL of tyrosinase solution (0.1 mg/mL) was added to start the reaction; then, 300 μL of the reaction solution was immediately transferred into a 96-well microtiter plate, and the absorbances (475 nm) were recorded at the beginning (0 min) and at the end (10 min) of the reaction by a microplate reader . The inhibition rate was calculated using Equation (1), and the absorbance of the tyrosinase-L-DOPA solution without tannin addition was used as a control.ion plot .475/min) of DOPA–quinone formation was monitored (475 nm) throughout the reaction, but under different tannin concentrations , different enzyme concentrations , as well as different DOPA concentrations . The inhibition type was evaluated according to the Lineweaver–Burk plot and the adjusted Michaelis–Menten Equation (2):0 is the corresponding initial velocity, S is the substrate concentration, and KM and Vmax are the Michaelis’s constant and maximum reaction speed, respectively, which were obtained from the vertical and horizontal intercepts from the Lineweaver–Burk plot.The initial velocity , slope, and intercept of the Lineweaver–Burk plot were then fitted to a secondary plot through Equations (3) and (4) to obtain the inhibition constant (KI) and the inhibition constant in the enzyme−substrate complex (KIS).The KThe tyrosinase and tannins were dissolved in PBS (50 mM) to obtain sample solutions with a constant tyrosinase concentration (0.4 mg/mL) but different tannin concentrations . After a 30 min (37 °C) reaction, the sample solutions were transferred into a quartz sample cell (0.1 cm path length) mounted onto a MOS-450 CD spectropolarimeter  . The spe2 force field to obtain their preferential conformations.Molecule dockings were performed using an open-source AutoDock Vina software package . The crystallographic structure of the tyrosinase  was taken from RCSB Protein Data Bank and used as the initial protein model. The model was then subjected to water and tropolone removal, followed by polar hydrogen addition, a missing atom correction, and a Gasteiger charge assignment. Subunits of the tannins, including catechin, epicatechin, gallocatechin, and epigallocatechin, were constructed with Chem Draw 17.0 software  and geometrically optimized with an MMReferencing Heitz’s research , moleculThe antioxidant activity of the tannins was assessed through 3-ethylbenzthiazolin-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging analyses. The ABTS assay was conducted following the method described previously ,28 and m50).For the DPPH assay, the procedure was carried out following a previously described method . In shorThe copper ion chelating ability of the tannins was measured by fluorescence quenching analysis. The protocol was conducted in accordance with published research  and adju2 atmosphere. After incubation, the cell pellet was washed with PBS (10 mM) and dispersed by pipetting with 0.25% trypsin (1 mL) and harvested for cytotoxicity, cellular tyrosinase inhibition, and cellular melanin production analysis.B16F10 melanoma cells  were seeded and incubated in a cell culture bottle filled with Dulbecco’s modified eagle medium (DMEM) , penicillin (100 U/mL), and streptomycin (100 µg/mL)) for 3 days in a 37 °C incubator filled with 5% CO6 cell intensity) were seeded into a 6-well plate and incubated for 12 h. Then, the supernatant was replaced with 2 mL DMEM media with different hop tannin concentrations  and then incubated for 48 h. After incubation, the medium was removed and the cell pellet was washed three times with PBS solution; the pellet was then dispersed with 500 μL of PBS buffer  and treated with a freeze/thaw  process following a centrifuge  to obtain the dissolved intercellular substance. The dissolved intercellular substance (20 μL) was reacted with 180 μL of L-DOPA solution (1 mM) in a 96-well plate. After a 30 min reaction (at 37 °C), its absorption was recorded at 475 nm and the cellular tyrosinase inhibition rates were calculated using Equation (5), while cells cultured without tannin addition (hop tannin concentration = 0 μM) were used as a control.n is the absorbance obtained from samples and D0 is the absorption of the sample prepared without tannin addition.The cellular tyrosinase inhibition was measured as described in the report from Si et al. . B16F10 6 cell intensity) were transferred into a 6-well plate and incubated for 12 h [n is the absorbance obtained from the samples and E0 is the absorption of the sample prepared without tannin addition. The schematic diagram of the study is presented in B16F10 cells , while 271 u and 287 u were attributed to the loss of both water and cysteamine moieties (77 + 18 u). These fragment ions demonstrated that the related subunits were released from the extension position of the tannins .In general, the subunits and their derivates, including (epi)catechin (291 u), (epi)catechin–cysteamine (366 u), and (epi)gallocatechin–cysteamine (382 u), were identified. The relative contents of these subunits were 8.83%, 78.76%, and 12.40%, respectively. Furthermore, hop tannins were found to have (epi)catechin as a terminal unit, and (epi)catechin and (epi)gallocatechin as extension units. This structural character is in agreement with the findings of Kennedy et al. . Based o50. Kojic acid, a famous skin-whitening agent [The tyrosinase inhibition ability of hop tannins was evaluated and expressed as ICng agent , was als50 value of 76.52 ± 6.56 μM, which is comparable to kojic acid (49.54 ± 2.08 μM). Kojic acid has previously been proven to have stronger tyrosinase inhibition ability than many other commercial inhibitors, such as arbutin, ascorbic acid, and phloroglucinol [The results  implied glucinol . These rAfter fitting the initial velocities with their corresponding tyrosinase concentrations, the tyrosinase concentration–reaction rate plots were obtained and are presented in M) were both affected by changing inhibitor concentrations, but more importantly, they imply that tannins inhibited tyrosinase activity in a competitive–uncompetitive mixed way. In other words, hop tannins showed inhibition ability through binding on either active or nonactive sites of tyrosinase.The Lineweaver–Burk plots B, which I) and of the inhibitor–tyrosinase–substrate complex (KIS) were obtained through slopes of the secondary fitted lines . This indicates that the hops tannin–tyrosinase complex showed similar binding forces to the hops tannin–L-DOPA–tyrosinase complex [One of the most important mechanisms for competitive inhibition is the structural similarity between inhibitors and substrates . L-DOPA,ed lines C,D. As e complex , which aThe secondary structures of tyrosinase, before and after binding with tannins, were observed through CD spectra, and are detailed in To provide insight into the tyrosinase inhibition, molecular docking was performed. The subunits, including catechin, epicatechin, gallocatechin, and epigallocatechin, were chosen as ligand models and were applied for the docking. The complexes with the lowest energies are shown in The subunits were all observed as embedding into the active site and binding with tyrosinase by forming hydrogen bonds with HIS-244, SER-282, and MET-280 . These rMany compounds with copper chelating ability and antioxidant activities were identified to have tyrosinase inhibition activity, not only because the oxidation state of copper ions embedded into the active site plays a key role in the tyrosinase-catalyzed reactions, but also because the L-DOPA to DOPA–quinone transition is basically an oxidation reaction .2+ chelating ability of hop tannins was assessed through fluorescence analysis and is shown in 2+ concentrations in the solution. This phenomenon can be attributed to the fluorescence quenching induced by the Cu2+ chelation. The fluorescence emissions of the tannins are believed to be from benzene rings with hydroxyl groups [The Cul groups . The abul groups . The hopl groups . In the · and ABTS·+ radical scavenging abilities in comparison with a known antioxidant (ascorbic acid) and a tyrosinase inhibitor (hydroquinone)  . Hop tanbic acid . CompareIn general, hop tannins presented outstanding tyrosinase inhibition ability, not only via binding on the active or nonactive sites of tyrosinase, but also by affecting the tyrosinase structure, and by its copper ion chelating ability and antioxidant activity.The intracellular tyrosinase and the corresponding melanin inhibition ability of hop tannins are evaluated and presented in Hop tannins are composed of (epi)catechin as a terminal subunit and (epi)catechin and (epi)gallocatechin as extension subunits, and have an average polymerization degree of 10.32. They present the multifunctional tyrosinase inhibition effects of binding onto the active site and forming a hydrogen bond with tyrosinase; chelating with copper ions, thereby influencing the valence state of the ions; affecting the structure of tyrosinase; and presenting antioxidant abilities during an enzyme catalysis reaction. As a consequence, the tannins show a competitive–uncompetitive mixed inhibition to tyrosinase. Hop tannins also presented intracellular tyrosinase and melanin inhibition abilities, a 66.67% tyrosinase inhibition rate, and a 34.65% melanin inhibition rate when 10 μM of tannins was used. These results demonstrate that hop tannins may have the potential to be applied as a whitening agent in the cosmetics industry.


Lipid transfer proteins (LTPs) were identified as allergens in a large variety of pollens and foods, including cereals. LTPs belong to the prolamin superfamily and display an α-helical fold, with a bundle of four α-helices held together by four disulfide bonds. Wheat LTP1 is involved in allergic reactions to food. To identify critical structural elements of antibody binding to wheat LTP1, we used site-directed mutagenesis on wheat recombinant LTP1 to target: (i) sequence conservation and/or structure flexibility or (ii) each disulfide bond. We evaluated the modifications induced by these mutations on LTP1 secondary structure by synchrotron radiation circular dichroism and on its antigenicity with patient’s sera and with mouse monoclonal antibodies. Disruption of the C28–C73 disulfide bond significantly affected IgE-binding and caused protein denaturation, while removing C13–C27 bond decreased LTP1 antigenicity and slightly modified LTP1 overall folding. In addition, we showed Lys72 to be a key residue; the K72A mutation did not affect global folding but modified the local 3D structure of LTP1 and strongly reduced IgE-binding. This work revealed a cluster of residues , four of which embedded in disulfide bonds, which play a critical role in LTP1 antigenicity. In plants, LTPs are associated with different events and signalling pathways related to development and defence against pathogens and environmental stress2. Although their precise function(s) is(are) still largely unknown, recent findings indicate that they are at least involved in lipid transfer between organelles4. The presence of lipid ligands is also a frequent characteristic of plant allergens and appears relevant to allergic sensitization events5. Plant LTPs belong to the prolamin protein superfamily, which includes several important types of allergens related to legumes, tree nuts, cereals, fruits and vegetables7. The LTP family comprises two structurally related types, LTP1 and LTP28. Both are small globular proteins with Mr ≈ 9–13 kDa, and a basic pI (9–10). LTP1 and LTP2 display low sequence identities but a similar α-helical fold, with a bundle of four α-helices numbered H1–H4 held together by four disulfide bonds, allowing formation of a hydrophobic cavity9. The 3D structure of wheat LTP1 has been resolved by nuclear magnetic resonance (NMR) (PDB ID 1GH1)10 and X-ray crystallography (PDB ID 1BWO)11. The protein contains a skeleton of eight cysteine residues forming four disulfide bonds that are located at positions C3–C50, C13–C27, C28–C73 and C48–C87.Lipid transfer proteins (LTPs), also known as non-specific lipid transfer proteins (ns-LTPs), form a widespread family of plant proteins characterized by a hydrophobic cavity that allows binding with a large variety of lipids12, as well as plant-food allergens in a large variety of foods, particularly in Rosaceae fruits and in their Prunoideae subfamily16.LTPs, especially LTP1, have been identified as pollen respiratory allergens, such as in mugwort (Art v 3)19). Although less prevalent than fruits, cereals—notably maize and wheat21—may also be involved in the cross-reaction between different LTP sources. In this context, wheat LTP1 Tri a 14 was shown to be involved in severe reactions such as anaphylaxis and wheat-dependent exercise-induced anaphylaxis22.LTP allergy syndrome, initially described for older children and adults in Spain and Italy, is characterized by multiple plant-food sensitizations and potentially severe clinical reactions in patients sensitized to LTP1. Peach is the most frequent offending food for these patients, and immunoglobulin E (IgE) specific for peach LTP1 Pru p 3 is detected in a very high percentage of patients  and sera from children allergic to wheat in foods. We found that only a few sequential epitopes are recognized by IgE from only a few patients34, suggesting that most LTP1 B-cell epitopes are conformational. This is probably a consequence of the high thermal stability of this protein during food processing and its resistance to gastrointestinal digestion, allowing sensitization37. Identifying conformational epitopes requires time-consuming and extensive strategies. The most common technique for locating and mapping conformational epitopes is directed mutagenesis of the protein allergen. A complementary approach, known as mimotope mapping, uses random peptide libraries that mimic epitopes. Both techniques have been applied successfully to identify IgE-binding and T-cell epitopes of peach LTP Pru p 340. The mimotope approach has also revealed a wheat LTP1 conformational epitope for IgE from patients with baker’s asthma35 and its probable contribution to cross-reactivity between Pru p 3 and Tri a 14. Recently, conformational epitopes were mapped on the mugwort pollen allergen Art v 3 using directed mutagenesis in combination with a novel approach, H/D (hydrogen/deuterium) exchange memory NMR. This method measures differences in signals between the allergen–antibody complex and free allergen41.Identification of epitopes on plant LTPs has focused on understanding the molecular mechanisms of sensitization to these pan-allergens frequently involved in cross-reactions. Findings would be useful in the context of allergen immunotherapy that aims at developing safe and effective treatment of food allergies using peptides, variants or modified proteins34. A large set of protein variants was produced to evaluate the role of each disulfide bond and how some amino acid residues contribute to IgE/LTP1 interactions. Mutations were notably designed to evaluate the importance of the conservation of primary amino acid sequences among several plant-food LTPs and of their location in the 3D structure as key elements for IgE-binding. Indeed, despite mild conservation in primary structure  directed against natural and denatured LTP1. Modification of LTP1 triggering capacity was also assessed by rat basophilic leukaemia (RBL) cell degranulation assay. Finally, structures of wheat recombinant LTP1 and mutant LTP1s (Mut-LTPs) were analysed by SRCD (synchrotron radiation circular dichroism) and by computational analysis for one hypoallergenic variant.37QARSQSDRQS46, which was previously recognized by both patients with allergy to food and wheat LTP1-sensitized mice34. Some of these residues are also present in the conformational epitope (H35N36R39S40S42D43) recognized by a pool of sera from Spanish patients with baker’s asthma, co-sensitized with peach and wheat LTPs35  Fig. a, or bec74V75L77P78Y79T80 from the conformational epitope described for patients with baker’s asthma in Tordesillas et al.35. The introduction of three alanine between the N76 and L77 or R39 and S40 residues was done to increase the loop size between H2-H3 helices and H3-H4 helices, respectively.Three other residues  located at the end of α-helix 4 and in the flexible C-terminal domain of LTP1 were targeted as well to design three additional variants within the first mutation set. N76 and K72 have relatively high accessibility in the LTP1 3D structure, with K72 being a highly conserved residue among plant LTP1s Fig. , Table 1Accordingly, to study the contribution of each disulfide bond, we designed a second set of four variants  by removing one bond at a time  in order to produce them in a soluble and well-folded form42. Only proteins produced in the soluble fraction after bacterial lysis were submitted to purification by Ni2+-affinity chromatography followed by anion-exchange chromatography. The purity of recombinant proteins was checked by 15% SDS-PAGE and western blot (not shown). A few hundred micrograms of each mutant were obtained and used for subsequent experiments.Recombinant wild-type wheat LTP1 (Wt-LTP) and the series of variants were expressed in 2 = 0.9491, p < 0.0001, n = 27; Fig. p > 0.9999, n = 27)  to measure concentrations in specific IgE for Wt-LTP, reduced/alkylated LTP1 (Ra-LTP) and 13 Mut-LTPs. For better clarity, we separately analysed the variants affecting sequence conservation and structure flexibility with either single or double mutations Fig. .Figure 3–1, whereas the K72A mutant showed a dramatic and systematic decrease in IgE-binding .The introduced mutations had a significant effect on IgE-binding capacity compared with Wt-LTP, except for the R39A-D43N and D43N variants, which showed no difference as regards to specific IgE concentration median values  in the range < 0.1–10 ng mL–1 and 0.1–30 ng mL–1 for Nat-LTP and Wt-LTP, respectively (not shown). For variants, EC50 values were compared with those obtained with Wt-LTP. For the K72A mutant, a 30-fold increase in EC50 was observed for three sera, while for three other sera the EC50 increase was in the range of 103–104, which indicates markedly reduced eliciting potency. The two other mutations  did not change, increased or decreased (increase in EC50 by a factor of 50–500) the ability of proteins to induce degranulation, depending on the sera. Even when performed with a limited number of sera, these results confirmed the ELISA results and identified the K72A mutant as the one with the greatest effect on LTP1 antigenicity.To confirm our results at the cellular level, in a system that mimics in vivo triggering, we assayed variants inducing a significant reduction in IgE-binding (K72A) or no effect (D43N and R39A-D43N) for their ability to induce basophil degranulation (RBL cells), with three to six sera . All sera tested showed very low or no binding at all to these two variants (83–100% reduction in specific IgE concentrations compared with Wt-LTP).To better understand the contribution of disulfide bonds to wheat LTP1 IgE reactivity, we measured IgE-binding to Nat-LTP, to the same protein after reduction and alkylation (Ra-LTP) and to recombinant disulfide bond variants of LTP1s by ELISA. The results presented in Fig. 50 measured for Ra-LTP was between 100 and 104 times higher than that measured for Nat-LTP. Significant increases in EC50 were also observed with the C13S-C27S and C28S-C73S variants compared with Wt-LTP . For the two other variants (C3S-C50S and C48S-C87S), mutations increased, let unchanged or decreased (increase up to 50 or 100 the EC50 ratios) the ability to induce degranulation, depending on the sera. These results confirm the ELISA results and show the important role played by the C13-C27 and C28-C73 disulfide bonds in wheat LTP1 antigenicity and triggering activity.We compared the RBL degranulation capacity of Wt-LTP and of the different disulfide bond variants, using three to six sera Fig. b. We alsTo evaluate the effect of 3D structure on LTP1 antigenicity, mice were immunized either with Nat-LTP or Ra-LTP. Monoclonal antibodies were obtained and characterized in ELISA for their reactivity towards the immunogens and Wt-LTP, as well as towards variants selected for their reduced IgE-binding capacity. The following variants (C13S-C27S and K72A) and  were used for their strong or slight effect on IgE binding respectively. The C28S-C73S LTP1 variant was not available for this analysis. From mice immunized with Nat-LTP, 14 clones secreting mAbs that bound to this LTP were obtained; none of them recognized Ra-LTP . Six mAbs also reacted with Wt-LTP and Nat-LTP, two of them with equal intensity; four other mAbs showed less reactivity compared with Ra-LTP. Four mAbs from this immunization did not bind to Nat-LTP. Nonetheless, three of them displayed a limited but significant response to Wt-LTP.To relate the effect of introduced mutations on antibody binding (human IgE and mouse mAbs) and degranulation events to changes in protein folding, we analysed the selected variants using SRCD Fig. . Wt-LTP,The CD spectra of LTP1 variants targeting sequence conservation and/or flexibility presented in Fig. Estimated changes to secondary structures are reported in Fig. The S40G-S42G double mutation induced a different effect, with a decrease in α-helices (− 6%) and antiparallel β-sheets (− 14%) and an increase in parallel (+ 10%) β-sheet structures and other structures (+ 7%). The turn structures remained stable. R39A, D43E-R44K and K72A variants did not show large variations in their percent of secondary structures.As shown in Fig. Some small variations in the C13S-C27S mutant particularly at the shoulder around 185–190 nm could be observed.Secondary structure estimation for the C28S-C73S mutant showed a drop in α-helix content (− 20%), a marked increase in turn (+ 8%) and antiparallel strands (+ 8%), and a moderate increase in unordered structures (+ 4%) Fig. d. The C4Given the high number of proteins studied, we computed the Pearson correlation between the Wt-LTP CD signal and those of the other proteins Fig. e. The va43 and VLDP44 web servers, employing the 1GH1 PDB file or the K72A 3D structure model generated from DynaMut as templates  of 0.665 kcal mol–1 K–1  and volume (− 75.55 Å3) upon K72A mutation compared with the wild-type protein  may fold independently, in accordance with the presence of a rather long spacer. In addition, IgE-binding to Wt-LTP was comparable to that of Nat-LTP, indicating that Trx does not interfere with IgE interactions. This strategy has already been validated with Ara h 2, a major peanut allergen expressed in fusion with the carrier protein MBPThirteen substitution variants of wheat LTP1 were produced in fusion with Trx and evaluated for IgE-binding with sera from patients allergic to wheat. Four of these variants  showed reduced binding by a limited number of sera, and two of them (C48S-C87S and R39A-D43N) also displayed loss of recognition by two anti-Nat-LTP mAbs. Three other variants  exhibited very significant reduction in IgE-binding for all or most sera. These mutations also markedly affected the protein’s triggering activity in the RBL assay. Similarly, variants C13S-C27S and K72A  lost their antigenicity for a set of mAbs obtained by immunization with Nat-LTP, in contrast to Wt-LTP.47. In the case of wheat LTP1, it has been shown that the presence of lipid within the protein cavity increased its flexibility and accessibility to digestive enzymes, properties that may also influence its allergenic potential37.All other point or double mutations preserved IgE-binding. Surprisingly, several mutations  caused a small but significant increase in LTP1 binding by IgE compared with Wt-LTP. The explanation for this phenomenon remains to be determined. Nevertheless, mutation may induce discrete changes, such as more favourable surface electrostatics and topology that may enhance interaction with IgE. The mutations might increase area sizes on mobile epitope residues. This seems to be the case for some LTP1 variants . These mutations may also induce discrete changes elsewhere in protein structure, enabling easier accessibility. As an example, the presence of lipids could increase the accessibility of some flexible protein regions and was recently shown to impact the IgE-binding capacity of several plant LTPs34 and has also been observed to be important for other LTPs48. Among the four LTP1 disulfide bonds that are characteristic of the prolamin superfamily, we identified two (C13-C27 and C28-C73) as critical elements for wheat LTP1 antigenicity. C28-C73 is also essential for protein self-assembly. Indeed, SRCD analysis showed that disruption of C28-C73, which links H2 and H4 helices, causes denaturation of the protein, with almost total disappearance of α-helix structures. A similar effect was previously observed after reduction/alkylation of all disulfide bonds34. Conversely, disruption of C13-C27, which links H1 and H2 helices, did not lead to protein denaturation. Rather, C13-C27 disruption induced only a small change in the SRCD spectra compared with Wt-LTP, indicating slight modification of overall folding, whereas IgE-binding and triggering capacity were notably reduced. These two disulfide bonds include the double cysteine motif (C27-C28) and hold H1, H2 and H4 helices together. By doing so, they probably serve as scaffolding and help in gathering the epitope residues. Yet some of these cysteine residues may also be directly involved in interaction of LTP1 with IgE. Our previous study on wheat LTP1 localized a sequential B-cell epitope at position #24–33, comprising the cysteine doublet C27-C28. This epitope was detected with exceptionally high intensity compared with other bound LTP1 peptides34. In the case of Art v 3 (from mugwort pollen)41, C74 was evidenced as an epitope residue in direct interaction with antibodies.The structural integrity and preservation of disulfide bonds is crucial for IgE-binding to wheat LTP1Two other disulfide bonds, C3-C50 and C48-C87, link the H3 helix to the N- and C-terminal domains of the protein, respectively. Mutation of the C3-C50 bond did not change the secondary structure of wheat LTP1 and had almost no effect on IgE-binding or basophil triggering activity. Mutation of the C48-C87 bond only slightly modified LPT1 protein folding and antigenicity.Parietaria judaica pollen)48, disruption of Cys14–Cys29 and Cys30–Cys75 bridging caused the loss of specific IgE-binding and of triggering activity for most patients, whereas a mutant lacking the other bridges retained its activity. The importance of the cysteine doublet in preserving LTP1 structure and allergenicity was also described for Pru p 333. Apart from these two bridges, holding α-helices H1, H2 and H4 together might be an important structural element of LTP1 antigenicity.This difference in disulfide bond involvement and the essential role of C13-C27 and C28-C73 in LTP allergenicity has also been reported for other LTPs. Indeed, in Par j 1 41. Its position between a proline that reduces the trypsin proteolytic activity and a cysteine forming a disulfide bridge may assure its protection from enzymatic digestion.In addition to residues C13, C27, C28 and C73, our data point to K72 as a key residue in IgE-binding and basophil triggering for all sera tested. SRCD results show that the K72A variant does not impact structure folding. This result points out that K72A may be directly involved in the interaction with the antibody via the relative protrusion of its lateral chain. Substitution of K72A by a small alanine residue induced changes in electrostatic properties, decreased protein volume and solvent accessible surface, and modified non-covalent interactions with residues from the H1 and H4 helices and from the loop between those helices Fig. a. This sWith respect to the 3D structure of LTP1, the five residues whose mutation greatly affected IgE-binding  cluster on the protein surface . As expected, we observed a visible influence of these single and double mutations in the SRCD spectra of the protein or variations in the content of the secondary structure. Despite these structural modifications, weak reductions in LTP1 antigenicity were detected in only two of these variants  and only for a limited number of sera. Other studies on Pru p 3 also observed a large effect of similar mutations only when they were in combination (e.g. simultaneous mutations of 39–40–43–44–76–77–78–79)38. Similarly, N77 appeared in a conformational epitope area of Art v 3 in combination with other residues 41.Other mutations aimed to change electrostatic potential, volume or distance on LTP1 portions 39–44 or N76-L77 that belong to sequential epitopes (37–46 and 66–80) previously identified in food allergy to wheat or baker’s asthma41. This sub-group of anti-Nat-LTP mAbs could enable generation of recombinant monoclonal IgE as substitutes mimicking patients reactivity49.Additional characterization of the modifications induced by some mutations  on LTP1 structure and antigenicity was carried out with mouse mAbs directed against natural and denatured LTP1. Like patient sera, none of the anti-Nat-LTP mAbs recognized Ra-LTP. About half (8/14) of them equally bound to Nat-LTP and Wt-LTP. Very interestingly, these mAbs react similarly to patient sera towards the set of tested LTP1 variants, indicating that they surely bind the dominant IgE epitope regions containing the cluster C13, C27, C28, C73 and K72. This was also the case of mAbs generated to Art v 3, which showed good overlap with patient IgEIn contrast to epitopes bound by anti-Nat-LTP, immunization with Ra-LTP mainly generated mAbs directed to new epitope sites that were moderately or not accessible in Nat-LTP or Wt-LTP but more accessible in the cysteine variants. Even with no or little modification observed in SRCD, binding of cysteine variants  by anti-Ra-LTP mAbs indicated some change in the 3D structure of wheat LTP1 and in accessibility to some of these mAb epitopes. However, high-resolution 3D studies would be needed to provide more information about structural differences.The reactivity of the panel of mAbs generated by immunization with purified Nat-LTP or Ra-LTP indicated small differences in the antigenicity of Nat-LTP and Wt-LTP. However, because most patient sera reacted equally to Nat-LTP and Wt-LTP in ELISA, the human polyclonal IgE repertoire appeared mostly restricted to regions with similar structural features or that may be less sensitive than mAbs to structural variations between the two proteins.In conclusion, the similar reactivity of some mAbs and of IgE from allergic patients highlights the significant involvement of some residues in wheat LTP1 antigenicity.52 and proposed as a factor in facilitating allergenic activity. However, this cluster might contain several overlapping or non-overlapping epitopes53. Our work also highlights the hierarchy of disulfide bridges and the special participation of C13-C27 and C28-C73 in maintaining protein structure and allergenic potency. This result, also observed for LTPs in peach and P. judaica pollen, could eventually prove generic to numerous allergenic LTPs. Aside from these critical elements, we observed a moderate role of residues D43, R39, C48 and C87 in LTP1 antigenicity.We observed for the first time a cluster of residues  that appear essential for IgE-binding on wheat LTP1 or for IgE cross-linking on the FcεRI receptor. Among other properties, these residues play a definite role in the 3D structure of the antigenic site for its maintenance (disulfide bonds) or in terms of protrusion (K72). Spatial clustering and steric orientation of epitopes have been reported in food and respiratory allergensThe three variants C13S-C27S, C28S-C73S and K72A appear to be promising hypoallergenic components. The efficacy of such protein variants as candidates usable in allergy immunotherapy remains to be investigated. In particular, their reduced triggering capacity as well as their immunogenicity should be evaluated on a larger sample of patient sera. In particular, the C13S-C27S and K72A variants, which folded quite similarly to Wt-LTP, should be tested for their capacity to modulate an allergic response to wheat LTP1.n = 27) from patients diagnosed with food allergy to wheat were selected based on their sensitization to wheat LTP1 established by ELISA . All methods were performed in accordance with the relevant guidelines and regulations.Serum samples (SA Table . These sltp expression vector enabling production of recombinant wild-type wheat LTP1 (UniProt P24296) as a fusion protein (Wt-LTP) with Trx was kindly provided by Dr K. Elmorjani  and used as a template to perform site-directed mutagenesis by a polymerase chain reaction (PCR)-based strategy. The wheat LTP1 coding sequence was inserted into the pET32-b expression vector  between NcoI and HindIII restriction sites. cDNA was inserted as translational fusion protein with N-terminal Trx separated by a 50-residue-long spacer as previously reported42. The spacer contained six His-tag peptides for purification on a Ni2+ affinity column.The pETDP54. Ra-LTP was obtained as follows: Nat-LTP was incubated with 0.1 M dithiothreitol  for 2 h at 37 °C, then with 0.5 M iodoacetamide (Merck) in 0.5 M NaOH for 30 min in the dark. Afterwards, the protein solutions were dialysed overnight at room temperature, with change of dialysis bath every 8 h. Recombinant expression was done in Escherichia coli Origami strain (Novagen) as described in Elmorjani et al.42, except that recombinant LTP1 was not cleaved from Trx. After cell lysis by sonication, the soluble fraction  was applied on a HisTrap FF column  for Ni2+ affinity chromatography. Elution was performed with a 50–300 mM imidazole gradient. This first purification step was followed by anion exchange chromatography with Resource Q resin  on an Äkta Avant system . The column was equilibrated with Tris 20 mM pH 8 before injection of the protein solution, and elution was performed by a NaCl gradient (up to 1 M) in Tris–HCl 20 mM pH 8. The proteins were extensively dialysed against water then lyophilized. The purity was assessed by SDS-PAGE, and the protein concentration was assayed with the BCA reagent kit  protein assay using BSA as standard. Protein identity was checked by mass spectrometry analysis . Recombinant Trx from E. coli (GenBank accession no. M26133) used as a control for IgE reactivity was purchased from Merck (#T0910 reference).Nat-LTP was purified as described by Douliez et al.https://pymol.org). Solvent accessibility area (or surface) (SASA) was calculated using the web server55. Sequence conservation was explored by multiple sequence alignment performed by Jalview56 and Clustal Omega . Sequences were collected from the UniProt database (https://www.uniprot.org/) and queried on the keyword “NLTP1”. The DynaMut web tool (http://biosig.unimelb.edu.au/dynamut/)43 was used to predict the mutation effect on protein thermodynamic properties using the 1GH1 PDB file as a template. The impact of mutation on volume and surface properties was computed using the VLDP and protein volume web servers (VLDP: https://www.dsimb.inserm.fr/dsimb_tools/vldp/)44 using the wild-type or mutated protein generated by DynaMut as templates. Finally, wild-type or mutated protein 3D models were used as templates to predict protein electrostatic properties using PyMol.Visualization and manipulation of LTP1 3D structure were done using PyMOL software  or Ra-LTP (group 2) emulsified in TiterMax Gold Adjuvant . Three days after the second immunization, the mice were sacrificed. For each group, the popliteal lymph nodes were removed, and their lymphocytes were suspended by perfusion, pooled and and fused with the SP2/0 myeloma cell line using standard protocol for hybridoma preparation. Hybridoma supernatants from the fusion were screened for the presence of anti-immunogen antibodies using an indirect ELISA. Nat-LTP or Ra-LTP were coated overnight at 5 µg/mL in PBS on MaxiSorp ELISA plates . After three washes in PBS-0.05% Tween 20, wells were saturated with 4% skimmed milk powder in PBS for 2 h at room temperature. After three washes in PBS-0.05% Tween 20, hybridoma supernatants were incubated for 1 h at room temperature. After three washes in PBS-Tween 0.05%, mAbs bound to antigen were detected with peroxidase-conjugated goat anti-mouse IgG (H + L)  and revealed with ortho-phenylenediamine (Merck). Selected hybridomas were cloned by dilution. Animal experiments were carried out at the INRAE facilities, which are authorized by the local veterinary department (authorization no. 44502). All animal experiments were approved by the Ethics Committee of the Pays de la Loire (registration no. CEEA-2011.26). The study was carried out, where appropriate, in compliance with the ARRIVE guidelines.The reactivity of mAbs directed to either Nat-LTP or Ra-LTP to Mut-LTP, Nat-LTP and Ra-LTP was assessed by indirect ELISA as described above, using four dilutions of hybridoma supernatant . For each mAb, titration curves were built and the highest dilution of the supernatant giving a signal at the plateau of the curve was determined for each Mut-LTP, Nat-LTP or Ra-LTP. When no reactivity was observed, the reported dilution was set at zero for the next calculation. For Nat-LTP mAbs, we calculated the ratio of the dilution determined for either Mut-LTP or for Ra-LTP to the dilution determined for Nat-LTP. For Ra-LTP mAbs, we calculated the ratio of the dilution determined for each Mut-LTP or for Nat-LTP to the dilution determined for Ra-LTP. Reactivity of a mAb to each LTP1 variant form was considered equivalent to that observed on the immunogen (Nat-LTP or Ra-LTP) if the ratio was superior to 0.1, reduced if the ratio was between 0.01 and 0.1, and absent if the ratio was inferior to 0.01..27. Nat-LTP, Ra-LTP, or Trx (Merck) solubilized in PBS buffer were coated at 5 µg mL–1 in carbonate buffer, and sera were diluted 1:10 or 1:40. All determinations were carried out in triplicate. A range of standard IgEs (human serum immunoglobulin E: NIBSC ref. 75/502) from 0.8 ng mL–1 up to 160 ng mL–1 were processed by sandwich ELISA, using rabbit anti-human IgE  as capture antibody. The specific IgE concentrations in the sera were calculated from the fluorescent signal and the standard curve after adjusting a four-parameter logistic regression curve (GraphPad Software) to the data.LTP- and Trx- specific IgE concentrations were measured by F-ELISA as described in Mameri et al57. Proteins  were tested at six concentrations  with six sera diluted to 1:10 . Sera were selected based on availability and IgE concentrations specific for Nat-LTP and Wt-LTP. We calculated EC50 for each serum and protein, as the protein concentration allowing 50% of the degranulation maximum.Activation of rat basophil leukaemia RBL cells was carried out with the SX38 P4B7 clone expressing the human FcεRI α-, β- and γ-chains as described.58 and Gohon et al.59 on the DISCO beamline at the SOLEIL synchrotron facility . Briefly, proteins were buffer exchanged with 10 mM phosphate buffer, pH 8, containing 100 mM Na2SO4. CD spectra recorded at 25 °C at wavelengths between 170 and 280 nm with a data pitch of 1 nm were measured at least three times for each protein sample and its buffer counterparts. After subtraction of averaged buffer spectra from those of samples, the 263–270 nm region was set to zero. The resulting spectra were calibrated with d-10-camphorsulfonic acid using CDtoolX software60. Spectra were normalized to protein concentrations and optical path length and presented in Δε units. Spectral deconvolution, fitting and secondary structure percentages were calculated using the publicly available BeStSel web server61. Spectra were analysed according to wavelength range quality and accuracy validity: this corresponds to the photomultiplier high tension remaining below half of its total variation. Normalized root-mean-square deviations provided the most accurate fit for each spectrum. Value ranged from 0.01 to 0.08.SRCD spectra were recorded as previously described in Vindigni et alStatistical analyses were performed using GraphPad Prism, version 9.1.2 for Windows  with the significance level set at 0.05. IgE reactivity towards Nat- and Wt-LTP and Trx was compared using ANOVA (Dunn’s multiple comparisons test), while a Wilcoxon test was applied to compare IgE reactivity towards Nat-LTP and variants, as these data often failed to demonstrate a Gaussian distribution .Supplementary Legends.Supplementary Figure S1.Supplementary Figure S2.Supplementary Table S1.