Albumins Research Articles

Imaging Interorganelle Phospholipid Transport by Extended Synaptotagmins Using Bioorthogonally Tagged Lipids.

The proper distribution of lipids within organelle membranes requires rapid interorganelle lipid transport, much of which occurs at membrane contact sites and is mediated by lipid transfer proteins (LTPs). Our current understanding of LTP mechanism and function is based largely on structural studies and in vitro reconstitution. Existing cellular assays for LTP function use indirect readouts, and it remains an open question as to whether substrate specificity and transport kinetics established in vitro are similar in cellular settings. Here, we harness bioorthogonal chemistry to develop tools for direct visualization of interorganelle transport of phospholipids between the plasma membrane (PM) and the endoplasmic reticulum (ER). Unnatural fluorescent phospholipid analogs generated by the transphosphatidylation activity of phospholipase D (PLD) at the PM are rapidly transported to the ER dependent in part upon extended synaptotagmins (E-Syts), a family of LTPs at ER-PM contact sites. Ectopic expression of an artificial E-Syt-based tether at ER-mitochondria contact sites results in fluorescent phospholipid accumulation in mitochondria. Finally, in vitro reconstitution assays demonstrate that the fluorescent lipids are bona fide E-Syt substrates. Thus, fluorescent lipids generated in situ via PLD activity and bioorthogonal chemical tagging can enable direct visualization of the activity of LTPs that mediate bulk phospholipid transport at ER-PM contact sites.

PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with the occurrence of pear fruit russeting

Non-specific lipid-transfer proteins (nsLTPs) are a group of small, cysteine-rich proteins that are involved in the transport of cuticular wax and other lipid compounds. Accumulating evidence suggests that dynamic changes in cuticular waxes are strongly associated with fruit russeting, an undesirable visual quality that negatively affects consumer appeal in pears. Currently, the regulatory role of nsLTPs in cuticular wax deposition and pear fruit skin russeting remains unclear. Here, we characterized the variations of cuticular waxes in non-treated (russeted) and preharvest bagging treated (non-russeted) pear fruits throughout fruit development and confirmed that the contents of cuticular waxes were significantly negatively correlated with the occurrence of pear fruit russeting. Based on RNA-Sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) analyses, two nsLTP genes (PpyLTP36 and PpyLTP39) were identified, which exhibited high expression levels in non-russeted pear fruit skins and were significantly repressed during fruit skin russeting. Subcellular localization analysis demonstrated that PpyLTP36 and PpyLTP39 were localized to the plasma membrane (PM). Further, transient Virus-Induced Gene Silencing (VIGS) analyses of PpyLTP36 and PpyLTP39 in pear fruits significantly reduced cuticular wax deposition. In conclusion, PpyLTP36 and PpyLTP39 are involved in the transmembrane transport of cuticular wax and are associated with pear fruit skin russeting.

Apolipoprotein E knockout, but not cholesteryl ester transfer protein (CETP)-associated high-density lipoprotein cholesterol (HDL-C) lowering, exacerbates muscle wasting in dysferlin-null mice

BackgroundDysferlin-deficient limb-girdle muscular dystrophy type 2B (Dysf) mice are notorious for their mild phenotype. Raising plasma total cholesterol (CHOL) via apolipoprotein E (ApoE) knockout (KO) drastically exacerbates muscle wasting in Dysf mice. However, dysferlinopathic patients have abnormally reduced plasma high-density lipoprotein cholesterol (HDL-C) levels. The current study aimed to determine whether HDL-C lowering can exacerbate the mild phenotype of dysferlin-null mice.MethodsHuman cholesteryl ester transfer protein (CETP), a plasma lipid transfer protein not found in mice that reduces HDL-C, and/or its optimal adapter protein human apolipoprotein B (ApoB), were overexpressed in Dysf mice. Mice received a 2% cholesterol diet from 2 months of age and characterized through ambulatory and hanging functional tests, plasma analyses, and muscle histology.ResultsCETP/ApoB expression in Dysf mice caused reduced HDL-C (54.5%) and elevated ratio of CHOL/HDL-C (181.3%) compared to control Dysf mice in plasma, but without raising CHOL. Compared to the severe muscle pathology found in high CHOL Dysf/ApoE double knockout mice, Dysf/CETP/ApoB mice did not show significant changes in ambulation, hanging capacity, increases in damaged area, collagen deposition, or decreases in cross-sectional area and healthy myofibre coverage.ConclusionsCETP/ApoB over-expression in Dysf mice decreases HDL-C without increasing CHOL or exacerbating muscle pathology. High CHOL or nonHDL-C caused by ApoE KO, rather than low HDL-C, likely lead to rodent muscular dystrophy phenotype humanization.

Innate lymphoid cells in immunoglobulin E-mediated food allergy.

Recognition of the importance of innate lymphoid cells (ILCs) in the immune mechanisms of food allergy has grown in recent years. This review summarizes recent findings of ILCs in immunoglobulin E (IgE)-mediated food allergy. New research on ILCs in the context of the microbiome and other atopic diseases are also considered with respect to how they can inform understanding of the role of ILCs in food allergy. ILCs can mediate allergic and tolerogenic responses through multiple pathways. A novel subset of interleukin (IL)-10 producing ILC2s are associated with tolerance following immunotherapy to grass pollen, house dust mite allergy and lipid transfer protein allergy. ILC2s can drive food allergen-specific T cell responses in an antigen-specific manner. A memory subset of ILC2s has been identified through studies of other atopic diseases and is associated with effectiveness of response to therapy. The role of ILCs in food allergy and oral tolerance is relatively understudied compared to other diseases. ILCs can modulate immune responses through several mechanisms, and it is likely that these are of importance in the context of food allergy. Better understanding of theses pathways may help to answer fundamental questions regarding the development of food allergy and lead to novel therapeutic targets and treatment.

Soybean transcription factor GmNF-YB20 confers resistance to stripe rust in transgenic wheat by regulating nonspecific lipid transfer protein genes.

Worldwide food security is severely threatened by the devastating wheat stripe rust disease. The utilization of resistant wheat cultivars represents the most cost-effective and efficient strategy for combating this disease. However, the lack of resistant resources has been a major bottleneck in breeding for wheat disease resistance. Therefore, revealing novel gene resources for combating stripe rust and elucidating the underlying resistance mechanism is of utmost urgency. In this study, we identified that the soybean NF-YB transcription factor GmNF-YB20 in wheat provides resistance to the stripe rust fungus (Puccinia striiformis f. sp. tritici, Pst). Wheat lines with stable overexpression of the GmNF-YB20 enhanced resistance against multiple Pst races. Transcriptome profiling of GmNF-YB20 transgenic wheat under Pst infection unveiled its involvement in the lipid signaling pathway. RT-qPCR assays suggested that GmNF-YB20 increased transcript levels of multiple nonspecific lipid transfer protein (LTP) genes during wheat-Pst interaction, luciferase reporter analysis illustrates that it activates the transcription of TaLTP1.50 in wheat protoplast, and GmNF-YB20 overexpressed wheat plants had higher total LTP content in vivo during Pst infection. Overexpression of TaLTP1.50 in wheat significantly increased resistance to Pst, whereas knockdown of TaLTP1.50 exhibited the opposite trends, indicating that TaLTP1.50 plays a positive role in wheat resistance. Taken together, our findings provide perspective regarding the molecular mechanism of GmNF-YB20 in wheat and highlight the potential use for wheat breeding.

Proviral role of ATG2 autophagy related protein in tomato bushy stunt virus replication through bulk phospholipid transfer into the viral replication organelle.

Subversion of cellular membranes and membrane proliferation are used by positive-strand RNA viruses to build viral replication organelles (VROs) that support virus replication. The biogenesis of the membranous VROs requires major changes in lipid metabolism and lipid transfer in infected cells. In this work, we show that tomato bushy stunt virus (TBSV) hijacks Atg2 autophagy related protein with bulk lipid transfer activity into VROs via interaction with TBSV p33 replication protein. Deletion of Atg2 in yeast and knockdown of Atg2 in Nicotiana benthamiana resulted in decreased TBSV replication. We found that subversion of Atg2 by TBSV was important to enrich VRO membranes with phosphatidylethanolamine (PE), phosphatidylserine (PS) and PI(3)P phosphoinositide. Interestingly, inhibition of autophagy did not affect the efficient recruitment of Atg2 into VROs, and overexpression of Atg2 enhanced TBSV replication, indicating autophagy-independent subversion of Atg2 by TBSV. These findings suggest that the proviral function of Atg2 lipid transfer protein is in VRO membrane proliferation. In addition, we find that Atg2 interacting partner Atg9 with membrane lipid-scramblase activity is also coopted for tombusvirus replication. Altogether, the subversion of Atg2 bridge-type lipid transfer protein provides a new mechanism for tombusviruses to greatly expand VRO membranes to support robust viral replication.

Unbiased MD simulations identify lipid binding sites in lipid transfer proteins.

The characterization of lipid binding to lipid transfer proteins (LTPs) is fundamental to understand their molecular mechanism. However, several structures of LTPs, and notably those proposed to act as bridges between membranes, do not provide the precise location of their endogenous lipid ligands. To address this limitation, computational approaches are a powerful alternative methodology, but they are often limited by the high flexibility of lipid substrates. Here, we develop a protocol based on unbiased coarse-grain molecular dynamics simulations in which lipids placed away from the protein can spontaneously bind to LTPs. This approach accurately determines binding pockets in LTPs and provides a working hypothesis for the lipid entry pathway. We apply this approach to characterize lipid binding to bridge LTPs of the Vps13-Atg2 family, for which the lipid localization inside the protein is currently unknown. Overall, our work paves the way to determine binding pockets and entry pathways for several LTPs in an inexpensive, fast, and accurate manner.

The ORP9-ORP11 dimer promotes sphingomyelin synthesis

Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.

The ORP9-ORP11 dimer promotes sphingomyelin synthesis.

Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.

Model Mechanism for Lipid Uptake by the Human STARD2/PC-TP Phosphatidylcholine Transfer Protein.

The human StAR-related lipid transfer domain protein 2 (STARD2), also known as phosphatidylcholine (PC) transfer protein, is a single-domain lipid transfer protein thought to transfer PC lipids between intracellular membranes. We performed extensive μs-long molecular dynamics simulations of STARD2 of its apo and holo forms in the presence or absence of complex lipid bilayers. The simulations in water reveal ligand-dependent conformational changes. In the 2 μs-long simulations of apo STARD2 in the presence of a lipid bilayer, we observed spontaneous reproducible PC lipid uptake into the protein hydrophobic cavity. We propose that the lipid extraction mechanism involves one to two metastable states stabilized by choline-tyrosine or choline-tryptophane cation-π interactions. Using free energy perturbation, we evaluate that PC-tyrosine cation-π interactions contribute 1.8 and 2.5 kcal/mol to the affinity of a PC-STARD2 metastable state, thus potentially providing a significant decrease of the energy barrier required for lipid desorption.

Walnut Jug r 1 is Responsible for Primary Sensitization among Patients Suffering Walnut-Hazelnut 2S Albumin Cross-Reactivity.

Walnut and hazelnut coallergy is a frequent manifestation in clinical practice whose molecular basis remains unclear. For this purpose, walnut-hazelnut cross-reactivity was evaluated in 20 patients allergic to one or both tree nuts and sensitized to their 2S albumins. Immunoblotting assays showed that 85% of patients recognized Jug r 1, walnut 2S albumin, which was associated with the development of severe symptoms; 50% of them corecognized hazelnut 2S albumin, Cor a 14. Both allergens were isolated using chromatographic techniques. Inhibition ELISAs revealed that Jug r 1 strongly inhibited the binding of Cor a 14-specific IgE, but Cor a 14 only partially inhibited Jug r 1-specific IgE binding. Our results showed that patients sensitized to walnut/hazelnut 2S albumins were not a homogeneous population. There were patients sensitized to specific epitopes of walnut 2S albumins and patients sensitized to cross-reactive epitopes between walnut and hazelnut, with Jug r 1 being the primary sensitizer.

Gastrointestinal Symptoms: Under the Tip of the Iceberg in Lipid Transfer Protein Food Allergy.

Gastrointestinal Symptoms: Under the Tip of the Iceberg in Lipid Transfer Protein Food Allergy.

Allergenic Biomarkers in the Molecular Diagnosis of IgE-Mediated Wheat Allergy.

IgE-mediated wheat allergy can take on various forms, including childhood food allergy to wheat, wheat-dependent exercise-induced anaphylaxis in young adults, baker's respiratory allergy/asthma in workers exposed to wheat flour inhalation, and contact urticaria that is caused by hydrolyzed wheat proteins in some cosmetics, and that is sometimes associated with a food allergy. Singleplex and multiplex immunoassays detect specific IgE antibodies to wheat allergenic molecular biomarkers such as omega-5 gliadin Tri a 19, lipid transfer protein Tri a 14, and alpha-amylase inhibitors. The fluorescence enzyme immunoassay with capsulated cellulose polymer solid-phase coupled allergens is a commonly used singleplex assay. Multiplex methods include the ELISA-based macroarray immunoassay using nano-bead technology and a microarray immunoassay on polymer-coated slides. Another promising diagnostic tool is the basophil activation test performed with omega-5 gliadin and other wheat protein types. Detailed comprehension of the structural and immunological features of the numerous wheat allergens significant in clinical settings is imperative for advancing diagnostic biomarkers for IgE-mediated wheat allergies.

Food-triggered anaphylaxis in adults.

Adult food allergy, either unresolved from childhood, or new-onset in adult-life, is known to be increasingly prevalent. Although much of the reported anaphylaxis in adults is due to drug reactions, foods are becoming an increasingly important trigger, affecting adults of all ages, with a wide variation in food triggers which are often quite different to those reported in children. Peanuts are well known to cause anaphylaxis in some adult populations, but other legumes such as soy may be more relevant in others. Reactions to natto, fermented soybeans, are currently mainly reported in Japan, but changing dietary practices and an increase in plant-based eating mean natto, other forms of soy and other legumes are increasingly linked to anaphylaxis in Western countries. Anaphylaxis to red meat, caused by sensitization to galactose-α-1,3-galactose and first reported in North America, is now a more world-wide concern. Co-factor induced anaphylaxis is increasingly associated with both wheat allergy and lipid transfer protein allergy. More research is urgently needed to characterize adult food allergy, its triggers and symptom severity. Unusual food triggers and potential co-factors should be considered, so that anaphylaxis in adults can be correctly managed, not merely labelled as idiopathic.

Xylem Sap Proteome Analysis Provides Insight into Root-Shoot Communication in Response to flg22.

Xylem sap proteomics provides crucial insights into plant defense and root-to-shoot communication. This study highlights the sensitivity and reproducibility of xylem sap proteome analyses, using a single plant per sample to track over 3000 proteins in two model crop plants, Solanum tuberosum and Hordeum vulgare. By analyzing the flg22 response, we identified immune response components not detectable through root or shoot analyses. Notably, we discovered previously unknown elements of the plant immune system, including calcium/calmodulin-dependent kinases and G-type lectin receptor kinases. Despite similarities in the metabolic pathways identified in the xylem sap of both plants, the flg22 response differed significantly: S. tuberosum exhibited 78 differentially abundant proteins, whereas H. vulgare had over 450. However, an evolutionarily conserved overlap in the flg22 response proteins was evident, particularly in the CAZymes and lipid metabolism pathways, where lipid transfer proteins and lipases showed a similar response to flg22. Additionally, many proteins without conserved signal sequences for extracellular targeting were found, such as members of the HSP70 family. Interestingly, the HSP70 response to flg22 was specific to the xylem sap proteome, suggesting a unique regulatory role in the extracellular space similar to that reported in mammalians.

Analysis of Brain, Blood, and Testis Phenotypes Lacking the Vps13a Gene in C57BL/6N Mice.

The Vps13a gene encodes a lipid transfer protein called VPS13A, or chorein, associated with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), mitochondria-endosomes, and lipid droplets. This protein plays a crucial role in inter-organelle communication and lipid transport. Mutations in the VPS13A gene are implicated in the pathogenesis of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder characterized by chorea, orofacial dyskinesias, hyperkinetic movements, seizures, cognitive impairment, and acanthocytosis. Previous mouse models of ChAc have shown variable disease phenotypes depending on the genetic background. In this study, we report the generation of a Vps13a flox allele in a pure C57BL/6N mouse background and the subsequent creation of Vps13a knockout (KO) mice via Cre-recombination. Our Vps13a KO mice exhibited increased reticulocytes but not acanthocytes in peripheral blood smears. Additionally, there were no significant differences in the GFAP- and Iba1-positive cells in the striatum, the basal ganglia of the central nervous system. Interestingly, we observed abnormal spermatogenesis leading to male infertility. These findings indicate that Vps13a KO mice are valuable models for studying male infertility and some hematological aspects of ChAc.

Leave no stone unturned: Exploring the metaproteome of beerstone for the identification of archaeological beer production

In archaeological contexts, identifying processes of beer production and consumption has contributed to our understanding of agriculture, labor mobilization, economic surplus, feasting, gender dynamics, social structure, tribute, community, identity and politics. Nevertheless, in the absence of pictorial representations and characteristic objects, beer brewing is difficult to identify in the archaeological record, and molecular methods are often limited by constraints of preservation and specificity. A potential target for studies of ancient beer production are residues formed during brewing activity, including beerstone, a calcium oxalate residue. Here, we apply shotgun proteomics analyses to a sample of modern beerstone to explore this residue's capacity as a marker for beer in archaeological contexts. The beerstone proteome was compared to the protein profiles of ungerminated and germinated barley to identify key proteins indicative of malted grains which may be encased by the residue. Proteins matching to barley grain (Hordeum vulgare) and Baker's/Brewer's yeast (Saccharomyces cerevisiae) were successfully identified in the beerstone. In particular, we identified hordeins, lipid transfer proteins, trypsin/α-amylase inhibitors, and protein Z, which are barley proteins abundant in proteomic characterisations of beer. In comparison to ungerminated and germinated barley grains, we find that beerstone preserves only a subset of the barley proteome, with the residue being more reflective of the final brewing product than of earlier brewing steps such as malting. Overall, we demonstrate that beerstone has potential to entrap and preserve proteins reflective of the beer-making process and identify proteins that we might anticipate in future archaeological analyses.

Comprehensive study of chiral herbicide flusulfinam uptake, translocation, degradation, and subcellular distribution in rice (Oryza sativa L.)

The biological behavior of flusulfinam, a potential commercial chiral herbicide for rice, has not been well explored. Herein, the uptake of chiral flusulfinam by rice and its transport, degradation, and subcellular distribution in rice (Oryza sativa L.) were investigated. The enantiomeric fraction (EF) in roots was 0.54 during 0 d to 7 d in hydroponic laboratory conditions. The bioconcentration factor of flusulfinam enantiomers was 2.1, suggesting an absence of observed enantioselectivity in the absorption process. Notably, the EF in the shoots decreased to 0.35 on the 7th day. The translocation factors of R- and S-flusulfinam were 0.12 and 0.27, respectively, indicating a preferential transfer of the S-flusulfinam from the root to the shoot. Flusulfinam was identified in the root after spraying. The translocation factors of R- and S-flusulfinam were consistently similar, signifying the capacity for downward movement without enantioselectivity. Interestingly, the degradation half-lives of R- and S-flusulfinam in the total plant were 5.50 and 5.06 d (p < 0.05), respectively, supporting the preferential degradation of S-flusulfinam throughout the total plant. Flusulfinam primarily entered the roots via the apoplastic pathway and was subsequently transported within the plant through aquaporins and ion channels. The subcellular distribution experiment revealed the predominant accumulation of flusulfinam enantiomers in soluble components (84%) with no enantioselectivity in these processes. There was upregulation lipid transfer protein-2 and carboxylesterases15 genes, which could explain the preferential transport and degradation of S-flusulfinam. This study is important in assessing the environmental risk associated with flusulfinam and ensuring food safety.

The Long-Distance Transport of Jasmonates in Salt-Treated Pea Plants and Involvement of Lipid Transfer Proteins in the Process.

The adaption of plants to stressful environments depends on long-distance responses in plant organs, which themselves are remote from sites of perception of external stimuli. Jasmonic acid (JA) and its derivatives are known to be involved in plants' adaptation to salinity. However, to our knowledge, the transport of JAs from roots to shoots has not been studied in relation to the responses of shoots to root salt treatment. We detected a salt-induced increase in the content of JAs in the roots, xylem sap, and leaves of pea plants related to changes in transpiration. Similarities between the localization of JA and lipid transfer proteins (LTPs) around vascular tissues were detected with immunohistochemistry, while immunoblotting revealed the presence of LTPs in the xylem sap of pea plants and its increase with salinity. Furthermore, we compared the effects of exogenous MeJA and salt treatment on the accumulation of JAs in leaves and their impact on transpiration. Our results indicate that salt-induced changes in JA concentrations in roots and xylem sap are the source of accumulation of these hormones in leaves leading to associated changes in transpiration. Furthermore, they suggest the possible involvement of LTPs in the loading/unloading of JAs into/from the xylem and its xylem transport.

Recombinant production of Morus alba lipid transfer protein and prediction of its allergenic epitopes

BackgroundWhite mulberry (Morus alba) pollen grains trigger allergic diseases including asthma, allergic rhinitis, and allergic conjunctivitis. Non-specific lipid transfer proteins (nsLTPs) are robust allergens found in higher plants. Although it was isolated and immunologically characterized in the fruits of Morus nigra, it has not yet been determined whether nsLTP is one of the M. alba pollen allergens. ObjectiveThis study aims to examine the possible allergenicity of M. alba pollen nsLTP by producing it recombinantly, followed by immunoblotting. MethodsTotal RNA was isolated from M. alba pollen grains, and the nsLTP gene was obtained by reverse transcriptase polymerase reaction. It was cloned into Escherichia coli using pQE2 plasmid and purified by immobilized metal affinity chromatography following the expression. To confirm the immunoreactivity of the purified r-nsLTP, immunoblotting was performed with the serum of patients allergic to M. alba pollen. B-cell epitopes of nsLTP were also predicted using the bioinformatics tools, namely Bioinformatics Predicted Antigenic Peptides, BepiPred-2.0, and the Immune Epitope Database, whereas T-cell epitopes were estimated by using NetMHCIIpan-4.0 and NetMHCII-2.3 servers. ResultsThe r-nsLTP showed positive results in immunoblotting with sera of 7/7 (100%) of M. alba allergic patients. Five B-cell epitopes (26–31, 43–50, 65–71, 92–100, 105–115) and three T-cell epitopes (57–65, 78–86, 105–113) were predicted by in silico analyses. ConclusionData obtained in this study are expected to support the use of r-nsLTP in solutions for diagnosis and treatment of M. alba allergy and cross-reactivities between nsLTPs, as well as recombinant allergen production for vaccination.