A murine model of wheat versus potato allergy: Patatin and 53kDa protein are the potential allergen from potato.

Wheat allergy is the most common around the world as gluten is the potential allergen. People diagnosed with wheat allergy were mainly substitute with other novel food such as potato though it is also being reported for allergenic manifestations. Thus there is an increasing demand for developing a BALB/c mice model to empathize the allergic properties of potato protein and its fractions. Purified potato protein showed lower IgE-binding capacity (474.39 ±â€¯0.6 ng/mL) even in higher concentration (30 mg/mL) compared to wheat gluten (1418.28 ±â€¯0.17 ng/mL, 5mg/mL). Immediate active cutaneous hypersensitivity reaction, vascular leakage, intestinal permeability and lung's inflammatory cell infiltration was also ascertained comparatively lower in potato protein than wheat gluten. Furthermore, patatin (43 kDa) and protease inhibitors (∼21 kDa) were purified and separated, and patatin exhibited higher hypersensitivity score than that of protease inhibitors. Immuno-detection assays indicated that patatin and 53 kDa protein in potato protein showed specific Ig-E binding capacity, and 53 kDa was adenosyl homocysteinase identified by LC-MS/MS.

Allergenicity of potato proteins and of their conjugates with galactose, galactooligosaccharides, and galactan in native, heated, and digested forms.

The effect of glycation of potato proteins on their immunoreactivity was studied by using a pool of human sera with specific IgE to potato proteins. Patatin conjugates were more immunoreactive than protease inhibitors ones. To better understand this behavior, the changes in patatin structure upon glycation and heat treatment were investigated. Patatin demonstrated an increase in total immunoreactivity when glycated with galactose and galactooligosaccharides. However, galactan conjugation to patatin resulted in a decrease in immunoreactivity by restricting IgE's access to the epitopes. Although the heat treatment resulted in a decrease in patatin's immunoreactivity through aggregation, it was less effective when patatin conjugates were used due to the decrease in aggregation and the secondary structural changes. Upon digestion, native patatin exhibited the largest decrease in immunoreactivity resulting from the disruption of both conformational and sequential epitopes. Patatin conjugates were less digested and had higher IgE-immunoreactivity as compared to the digested patatin.

Immunoproteomic and two-dimensional difference gel electrophoresis analysis of Arabidopsis dehydration response element-binding protein 1A (DREB1A)-transgenic potato.

To produce crops that are more tolerant to stresses such as heat, cold, and salt, transgenic plants have been produced those express stress-associated proteins. In this study, we used immunoproteomic and two-dimensional difference gel electrophoresis (2D-DIGE) methods to investigate the allergenicity of transgenic potatoes expressing Arabidopsis DREB1A (dehydration responsive element-binding protein 1A), driven by the rd29A promoter or the 35S promoter. Immunoproteomic analysis using sera from potato-allergic patients revealed several immunoglobulin E (IgE)-binding protein spots. The patterns of protein binding were almost the same between transgenic and non-transgenic potatoes. The IgE-binding proteins in potato were identified as patatin precursors, a segment of serine protease inhibitor 2, and proteinase inhibitor II by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) MS/MS. 2D-DIGE analysis revealed several differences in protein expression between non-transgenic potato and transgenic potato; those showing increased expression in transgenic potatoes were identified as precursors of patatin, a major potato allergen, and those showing decreased expression in transgenic potatoes were identified as lipoxygenase and glycogen (starch) synthase. These results suggested that transgenic potatoes may express slightly higher levels of allergens, but their IgE-binding patterns were almost the same as those of control potatoes. Further research on changes in protein expressions in response to environmental factors is required to confirm whether the differences observed in this study are due to gene transfection, rather than environmental factors.

Pectin methylesterases of pollen tissue, a major allergen in olive tree.

Olive tree (Olea europaea) pollen is a main cause of allergy in Mediterranean areas and North America. A novel allergen, Ole e 11, has been detected by proteomic techniques. Protein bands binding IgE from allergic sera were excised from a 2D electrophoresis gel and analysed by Edman degradation and MALDI-TOF MS. Four peptides were sequenced and used for designing primers to clone the cDNA codifying the protein. Ole e 11 consists of a 342 amino acid length polypeptide with a molecular mass of 37.4 kDa and a pI of 7.8. The allergen was identified as a pectin methylesterase and showed low identity with other members of this family from foods such as those from carrot (23%), orange (25%) and tomato (24%), and higher identity with those from Arabidopsis thaliana (57%) and Salsola kali (54%) pollen. The protein was overproduced in Pichia pastoris, purified, and characterized as an active enzyme. CD analysis rendered 3%alpha-helix, 50%beta-sheet and 27%beta-turns for its secondary structure, which is in agreement with other pectin methylesterase structures. The recombinant protein was demonstrated to be immunologically equivalent to the natural form by immunoblotting, indirect ELISA and inhibition experiments, using polyclonal antiserum and sera from olive pollen allergic patients. The prevalence fluctuated between 55.9% and 75.6% in three different allergic populations. The availability of this new olive pollen allergen could improve the component-resolved diagnosis. Its allergenic relevance is stepped up by the biotechnological use of these enzymes to improve organoleptic properties in processing foods and further confirms the need to include it in an accurate diagnosis.

A pectin methylesterase as an allergenic marker for the sensitization to Russian thistle (Salsola kali) pollen.

BACKGROUND: Chenopodiaceae pollen is considered the main cause of pollen allergy in desert countries and its incidence is world-wide increasing by the desertization of extensive zones. Although the correlation between the sensitization to Chenopodium album and Salsola kali pollens of patients suffering from allergy to Chenopodiaceae pollens is high, a significant number of patients exhibited IgE sensitivity exclusively towards S. kali. OBJECTIVE: To analyse this differential reactivity and to purify, clone and characterize the putative responsible allergen. METHODS: Immunoblotting was used to analyse the IgE binding to pollen extract for S. kali and C. album. The protein was isolated by two chromatographic steps and characterized by Edman degradation, mass spectrometry, finger print analysis and Concanavalin A lectin staining. Specific cDNA was amplified by polymerase chain reaction, cloned in Escherichia coli and sequenced. Immunologic characterization was performed by immunoblotting, enzyme-linked immunoassay detection and inhibition experiments using sera from 11 patients allergic to S. kali pollen. RESULTS: cDNA codifies for a mature protein of 339 amino acids plus a putative signal peptide of 23 residues and it belongs to the plant pectin methylesterase (PME) family. It is a mildly basic and polymorphic protein and was recognized by the IgE from all the patients allergic to S. kali included in the study, and was called Sal k 1. The protein was not recognized in the C. album pollen extract using the sera of these patients. CONCLUSION: Sal k 1 is a protein from the PME family with a high allergenic relevance. Considering this allergen as responsible for the different sensitization between S. kali and C. album pollen, it may be a useful marker to classify patients allergic to Chenopodiaceae allowing a safer and more specific immunotherapy.

Evaluation of patatin as a major cross-reactive allergen in latex-induced potato allergy.

BACKGROUND: Potential cross-reactions between natural rubber latex and fruit/vegetable specific immunoglobulin (Ig)E antibodies have been reported for many years. This study was designed to investigate the molecular basis of acquired food sensitization focusing on the storage protein patatin and the patatin-like latex protein Hev b 7. OBJECTIVE: The amount of potato-specific IgE in the serum of latex-allergic health care workers and children with atopic dermatitis was determined to evaluate cross-reactivity between Hev b 7 and patatin. Additionally, the stability of potato patatin to digestion was investigated. METHODS: Human serum was tested on its reactivity to latex and potato proteins by IgE immunoblotting after one-dimensional (1-D) and 2-D electrophoresis. Latex- and potato-specific IgE concentrations were measured in fluorescence enzyme immunoassays (CAP, Pharmacia, Uppsala, Sweden). Further, potato patatin was chromatographically isolated to perform auto-inhibition tests. Stability of patatin to degradation was determined by digestion in vitro. RESULTS: Patatin was identified as major cross-reactive potato allergen by N-terminal sequencing. Seventy-five percent of the potato-sensitized people reacted with patatin in 1-D immunoblots, and 25% of the positive reactions to Hev b 7 could be blocked by preincubation of the patients' sera with purified potato patatin. Examination of children with atopic dermatitis showed that most sera contained patatin-specific IgE, whereas no Hev b 7-specific IgE was detected. Finally, patatin has been found partially stable to digestion in vitro. CONCLUSIONS: Patatin was identified as a major cross-reactive protein in latex-associated potato allergy and appears to be relevant for atopic dermatitis. Therefore, patatin could be a suitable marker for the determination of potato sensitization, and it may also constitute an important food allergen. Cross-reactivity between Hev b 7 and patatin was restricted to primarily latex-sensitized adults, suggesting a different mechanism of sensitization in children with atopic dermatitis.

Effect of heat-induced aggregation on the IgE binding of patatin (Sol t 1) is dominated by other potato proteins.

The interaction of the major potato allergen patatin, Sol t 1, with IgE was investigated on a quantitative level as a function of heat treatment at different temperatures. On the basis of a number of publications, potato is considered to be a heat-labile allergen, but the molecular explanation for this behavior was not given. In this work, heat treatment of patatin in the absence and presence of other potato proteins mimicking the proteinaceous environment of the potato was studied. Using far-UV circular dichrosim spectroscopy, tryptophan fluorescence spectroscopy, and differential scanning calorimetry, the molecular transitions during heating of patatin were investigated. It was found that as long as patatin is not aggregated, denaturation of patatin on a secondary or tertiairy folding level is reversible with only a minor effect on the IgE affinity. Aggregation of patatin results in a nonreversible unfolding and a concomitant important decrease in affinity for IgE (25-fold). Aggregation of patatin in the presence of other potato proteins results in a less condensed aggregate compared to the situation of isolated patatin, resulting in a more pronounced decrease of affinity for IgE (110-fold). It is concluded that the heat lability of patatin-IgE interaction is explained by aggregation of patatin with other potato proteins rather than by denaturation of patatin itself.

Tomato allergy in children and young adults: cross-reactivity with latex and potato.

BACKGROUND: Several studies have shown that allergy to natural rubber latex is associated with cross-reactivity to certain foods such as tomato and potato. The objective was to investigate the clinical and immunologic differences between a group of patients with clinical allergy to tomato and latex and another which had only clinical allergy to tomato. We also aimed to assess, in vitro, the relationship of tomato and latex allergens, which could explain the cross-reactivity. METHODS: Forty patients with histories of adverse reactions to tomato and IgE-mediated hypersensitivity were enrolled in the study. Tomato, latex, and potato components were analyzed by SDS-PAGE immunoblotting. CAP and immunoblot inhibition were used to study allergen cross-reactivity. RESULTS: Patients from group A had a mean age of 13.2 years, and in group B the mean age was 21.7 years. In group B, 9/10 patients belonged to the latex-fruits syndrome. All patients of both groups tolerated potato. Immunoblotting patterns obtained with patients' sera from pool A showed IgE-binding bands to tomato ranging from 44 to 46 kDa and a triple band at 67 kDa. For latex, there was a strong binding at 44 kDa, and potato showed a strong band of 44 kDa and a 67-kDa triple band. In pool B, the binding to the band of 44 kDa in latex and tomato was more intense than in pool A. In pool A, immunoblot inhibition with potato allergen showed an intense inhibition of the three allergens (potato, latex, and tomato); with latex, inhibition was partial and with tomato, a complete inhibition of tomato and latex was observed, and a partial inhibition of potato. In pool B, the inhibition pattern followed a similar tendency to pool A. The CAP inhibition confirmed the high rate of cross-reactivity between tomato, potato, and latex. CONCLUSIONS: In our study, tomato, potato, and latex showed a common band of 44-46 kDa probably corresponding to patatin. This protein could be implicated in the high cross-reactivity between tomato, latex, and potato observed in the immunoblot and CAP inhibition.

Positive skin and oral challenge responses to potato and occurrence of immunoglobulin E antibodies to patatin (Sol t 1) in infants with atopic dermatitis.

The clinical significance and molecular specificity of hypersensitivity reactions to raw and cooked potatoes remain ambiguous. We therefore investigated the clinical hypersensitivity to raw and cooked potato in infants suspected to have potato allergy and compared the findings with the occurrence of immunoglobulin E (IgE) antibodies to patatin (Sol t 1), characterized as the primary allergen of potato. Twelve infants (10 to 24 months of age) suffering from atopic dermatitis (AD) and suspected to have adverse reactions to potato, were examined. As a skin exposure test we used rubbing with both raw and cooked potato, and used open oral challenge with cooked potato for 7 days. A special eczema scoring system (SCORAD) was used to assess the severity of symptoms and signs of AD. Skin-prick tests (SPTs) were performed with raw potato and natural Sol t 1, and serological studies included measurement of total serum IgE and IgE antibodies to Sol t 1, and potato radioallergosorbent testing (RAST). The skin-rubbing test with raw potato was positive in seven (58%) and the oral challenge positive in eight (67%) infants. One infant presented with an immediate reaction and seven with a delayed reaction, i.e. exacerbation of AD, after oral challenge responses to cooked potato. Nine (75%) infants had IgE antibodies to Sol t 1 in enzyme-linked immunosorbent assay (ELISA), and SPT to natural Sol t 1 was positive in six (50%) potato-allergic infants. In conclusion, we observed positive challenge responses to both raw and cooked potato in food-allergic atopic infants. The presence of IgE antibodies and concomitant positive SPTs to the heat-stable potato allergen, Sol t 1, suggest that cooked potato can be an allergenic food for infants suffering from AD.

Overproduction in Escherichia coli of the pectin methylesterase A from Erwinia chrysanthemi 3937: one-step purification, biochemical characterization, and production of polyclonal antibodies.

Pectin methylesterase A (EC 3.1.1.11), one of the pathogenicity factors of Erwinia chrysanthemi strain 3937, was purified to homogeneity using one-step chromatography on cross-linked pectate. The purified protein showed maximum activity at pH 8-9, 50 degrees C, 50-100 mM monovalent cations or 5-10 mM divalent cations, and on a 50% esterified pectin. A particular effect of Ca2+ and Zn2+ on PMEA activity, due to the formation of a pectin gel, was observed. A Km value of 0.03% and 0.051% was determined at pH 6 and 7.6, respectively, using the same substrate. Polyclonal antibodies raised against the PMEA from E. chrysanthemi strain 3937 were produced. It recognized PMEs from Erwinia species, but did not cross-react with PME of fungal or plant origin, and will therefore be a useful tool to immunolocalize the protein during plant-pathogen interactions.

Improving scFv antibody expression levels in the plant cytosol.

Expression of single-chain antibody fragments (scFvs) in the plant cytosol is often cumbersome. It was unexpectedly shown that addition at the C-terminus of the ER retention signal KDEL resulted in significantly improved expression levels. In this report the cytosolic location of the scFv-CK was confirmed, excluding possible mistranslocation to other subcellular compartments. It was shown that expression of several other scFvs was also improved in tobacco protoplasts. In addition expression was improved in transgenic potato. Changing from KDEL to KDEI did not affect the enhanced protein expression level. Addition of the KDEL motif is a simple and straightforward tool to stabilize in planta cytosolic expression of many scFvs.