Maize food allergy: lipid-transfer proteins, endochitinases, and alpha-zein precursor are relevant maize allergens in double-blind placebo-controlled maize-challenge-positive patients.

Italian patients with maize anaphylaxis have been shown to have IgE toward two major maize allergens: an alpha-amylase inhibitor and a 9-kDa LTP. A complete study on maize food allergens in patients with positive maize double-blind, placebo-controlled food challenge (DBPCFC) is lacking. The objective was to utilize the three maize protein fractions to identify and characterize the most relevant IgE-binding proteins recognized by the sera of Italian and Swiss patients with either a positive maize-DBPCFC or a history of maize-induced anaphylaxis. Osborne's protein fractions of maize were extracted to obtain water-soluble, total zein, and total protein fractions. Protein IgE-binding capacity was investigated by SDS-PAGE immunoblotting using the sera from DBPCFC-positive patients and from patients with maize-induced anaphylaxis. Purified maize LTP was used to inhibit the IgE immunoblotting of the three protein fractions. IgE immunoblotting demonstrated that the 9-kDa LTP was recognized by all the Italian patients and by none of the Swiss patients. Other allergens were: 14-kDa alpha-amylase inhibitor, 30-kDa endochitinases A and -B, 19 kDa zein-beta precursor, and 26 kDa zein-alpha precursor; a newly described allergen, the globulin-2 precursor, identified in the total protein fraction. It is noteworthy that maize LTP and endochitinase were cross-reactive with grape LTP and one grape endochitinase. LTP was found to be the only major allergen in Italian patients with either positive maize challenge or a history of maize-induced anaphylaxis. We have identified other maize allergens in subjects with maize food allergy, as grape cross-reactive endochitinase, however, the clinical significance of these proteins needs to be investigated in larger groups of patients with allergy to these food items.

Molecular characterisation of Lac s 1, the major allergen from lettuce (Lactuca sativa).

BACKGROUND: IgE sensitisation to non-specific lipid transfer proteins (nsLTP), e.g., Pru p 3 the major allergen from peach and most important allergenic LTP, is strongly associated with severe symptoms in food allergic patients. Lac s 1, a member of the nsLTP protein family, was recently identified as major allergen in lettuce (Lactuca sativa), but has not yet been investigated on the molecular basis. OBJECTIVE: Molecular characterisation and immunological comparison of Lac s 1 to peach allergen Pru p 3. METHODS: Lac s 1 cDNA was cloned by RT-PCR and natural (n) Lac s 1 was purified by a two-step chromatography. Protein structure was verified by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy. Immunoblotting, ImmunoCAP, and competitive IgE binding experiments were performed to study the IgE sensitisation pattern and cross-reactivity with Pru p 3. Allergenic potency was analysed by histamine release assay. RESULTS: Twenty-nine lettuce allergic patients, with or without concomitant peach allergy, and 19 peach allergic patients without lettuce allergy were included in this study. IgE reactivity to lettuce was due to mono-sensitisation to Lac s 1 or cross-reactive glycan structures. Two Lac s 1 isoforms were identified which showed amino acid identity (aa-id) of 62% to each other, up to 66% to Pru p 3, and 72% to the N-terminal peptide of plane pollen LTP Pla a 3. The prevalence of IgE binding to nLac s 1 was 90% using lettuce extract in immunoblotting experiments. Enhanced sensitivity was observed in ImmunoCAP using purified nLac s 1 in comparison to extracts (93% versus 76%). Although IgE sensitisation to Lac s 1 and Pru p 3 was strongly associated, the two LTPs showed different IgE binding properties. Sensitisation to LTPs does not necessarily reflect the clinical disease, but Lac s 1 was capable of triggering histamine release as shown by positive skin test results in Lac s 1 mono-sensitised patients and by in vitro mediator release assays. CONCLUSION: Purified nLac s 1 will enhance the sensitivity in component resolved diagnosis of lettuce allergy. Similar to other cross-reactive food allergies, exclusive testing of IgE reactivities to LTP cannot be used as biomarker for clinical relevance. Our data provide indirect evidence that Pru p 3 might act as the primary sensitising agent in patients allergic to both lettuce and peach.

Wheat IgE-mediated food allergy in European patients: alpha-amylase inhibitors, lipid transfer proteins and low-molecular-weight glutenins. Allergenic molecules recognized by double-blind, placebo-controlled food challenge.

BACKGROUND: Three main problems hamper the identification of wheat food allergens: (1) lack of a standardized procedure for extracting all of the wheat protein fractions; (2) absence of double-blind, placebo-controlled food challenge studies that compare the allergenic profile of Osborne's three protein fractions in subjects with real wheat allergy, and (3) lack of data on the differences in IgE-binding capacity between raw and cooked wheat. METHODS: Sera of 16 wheat-challenge-positive patients and 6 patients with wheat anaphylaxis, recruited from Italy, Denmark and Switzerland, were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblotting of the three Osborne's protein fractions (albumin/globulin, gliadins and glutenins) of raw and cooked wheat. Thermal sensitivity of wheat lipid transfer protein (LTP) was investigated by spectroscopic approaches. IgE cross-reactivity between wheat and grass pollen was studied by blot inhibition. RESULTS: The most important wheat allergens were the alpha-amylase/trypsin inhibitor subunits, which were present in all three protein fractions of raw and cooked wheat. Other important allergens were a 9-kDa LTP in the albumin/globulin fraction and several low-molecular-weight (LMW) glutenin subunits in the gluten fraction. All these allergens showed heat resistance and lack of cross-reactivity to grass pollen allergens. LTP was a major allergen only in Italian patients. CONCLUSIONS: The alpha-amylase inhibitor was confirmed to be the most important wheat allergen in food allergy and to play a role in wheat-dependent exercise-induced anaphylaxis, too. Other important allergens were LTP and the LMW glutenin subunits.

Detailed proteomic analysis on DM: insight into its hypoallergenicity.

Successful therapy in cow milk (CM) protein allergy rests upon completely eliminating CM proteins from the child's diet: it is thus necessary to provide a replacement food. Donkey milk (DM) has recently aroused scientific and clinical interest, above all among paediatric allergologists. A deeper knowledge of proteins in DM is necessary to evaluate the immunological and physiological properties of this natural substitute for cow's milk. The paper offers a detailed comparative analysis among the protein fractions of DM, CM and human milk, following an extensive proteomic study of the casein and whey proteins of DM performed by narrow pH range 2-DE. The detailed protein composition and structural features reported in this study provide insight into the molecular reasons for the hypoallergenicity of DM. Whole DM might constitute a valid substitute of CM in feeding children with CM protein allergy and it might also constitute the basis for formulas suitable for allergic subjects in the first year of life.

Efficacy of donkey's milk in treating highly problematic cow's milk allergic children: an in vivo and in vitro study.

Successful therapy in cow's milk protein allergy rests on completely eliminating cow's milk proteins from the child's diet: it is thus necessary to provide a replacement food. This prospective study investigated tolerance of donkey's milk in a population of 46 selected children with cow's milk protein allergy, for whom it was not possible to use any cow's milk substitute. Thirty-eight children (82.6%) liked and tolerated donkey's milk at the challenge and for the entire duration of follow-up. Catch-up growth was observed in all subjects with growth deficit during cow's milk proteins challenge. The degree of cross-reactivity of immunoglobulin E (IgE) with donkey's milk proteins was very weak and aspecific. Donkey's milk was found to be a valid alternative to both IgE-mediated and non-IgE-mediated cow's milk proteins allergy, including in terms of palatability and weight-height gain.

Analysis of the composition of an immunoglobulin E reactive high molecular weight protein complex of peanut extract containing Ara h 1 and Ara h 3/4.

Peanuts (Arachis hypogaea) contain some of the most potent food allergens. In recent years an increasing prevalence of peanut allergies both in children and adults has been observed in the USA and in Europe. In vitro identification and characterization of allergens including those from peanut have been frequently performed by Western blotting. However this method may alter the immunoglobulin E (IgE) antibody reactivity since the proteins are denatured by detergent treatment and/or reduction of disulfide bonds by reducing reagents and does not answer the question how peanut allergens interact with the human digestive apparatus and immune system. Size exclusion chromatography of peanut extract shows that approximately 90% of the total protein content is eluted as one peak in the exclusion volume with a molecular mass of over 200 kDa. The proteins of this fraction were analyzed by blue-native polyacrylamide gel electrophoresis (PAGE), immunoblotting, two-dimensional PAGE and Western blotting. A complex of Ara h 1 (Acc. no. P43237), Ara h 3/4 (AAM46958), Ara h 3 (AAC63045), Ara h 4 (AF086821), Gly 1 (AAG01363) and iso-Ara h 3 (AAT39430) was identified using patients' IgE and allergen-specific monoclonal antibodies; N-terminal sequencing and matrix-assisted laser desorption/ionisation-time of flight analysis verified these findings. A comparison of the peanut allergen sequences of Ara h 3/4, Ara h 3, Ara h 4 and peanut trypsin inhibitor (AF487543) and the proteins Gly 1 and iso-Ara h 3, not yet described as allergens, leads to the conclusion that these proteins are isoallergens of each other. It was shown that these isoallergens are post-translationally cleaved and held together by disulfide bonds in accordance to the 11S plant seed storage proteins signature.

Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics.

Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.

Towards royal jelly proteome.

The recent availability of the honey-bee Apis mellifera genome and trascriptome of both the female castes, has stimulated new efforts in investigating the protein composition of royal jelly (RJ), its role in caste differentiation and its quality and typicality by a proteomic approach. This study is aimed both to separate and identify proteins of royal jelly and to detect some of them in honey-bee pollen-bread by using two-dimensional gel electrophoresis, mass spectrometry and by de novo sequencing. All the identified proteins belonged to the Apis mellifera genome. Apalbumin 1 was also confirmed to be present in honey-bee pollen-bread where the presence of apalbumin 2 was also found. In addition several fragments of apalbumin 1 and apalbumin 3 were also found in RJ. These could be the result of protease activity other than that of serine-protease. This study is a contribution to the description of royal jelly proteome.

Membrane proteome of Acinetobacter radioresistens S13 during aromatic exposure.

Study of the bacterial membrane proteome, though in its early stages, is a field of growing interest in the search for information about nutrient transport and processing. We tested different strategies and chemical compounds to extract proteins from the membranes (inner and outer) of Acinetobacter radioresistens S13, a Gram-negative bacterium selected for its ability to degrade aromatics. A. radioresistens S13 was monitored under different growth substrate conditions, using acetate, benzoate or phenol as sole carbon source. Two-dimensional gel electrophoresis map analysis of membrane extracts from benzoate- and phenol-grown cells reveals differences versus controls (acetate-grown cultures). Primarily, a different pattern of spots was observed and, in particular, some proteins were only expressed in the presence of aromatic substrate. Among these, we detected a Na(+)/H(+) antiporter, whose function is likely to be regulation of intracellular pH, and an ABC type sugar transport system, probably involved in capsular polysaccharide translocation. We also identified other proteins, detectable in acetate-grown but over-expressed in aromatic-grown cells. These include: (1) an outer membrane protein ascribable to an OmpA-like protein, recently described in the literature as "alasan", a bioemulsifying agent involved in solubilizing and enhancing bioavailability of hydrocarbons; (2) a trimeric porin of the PhoE family also belonging to the outer membrane and involved in facilitating the transport of anions (especially phosphate); and (3) two glycosyl transferases probably involved in capsules and/or lipopolysaccharide biosynthesis. Study of the bacterial membrane proteome helps to elucidate the role of the membrane as modulable site enabling communication between internal and external environments.

Lipid transfer protein and vicilin are important walnut allergens in patients not allergic to pollen.

BACKGROUND: Walnut is the most common cause of allergic reactions to tree nuts, as reported by large population studies. Two major allergens of walnut have been identified up until now: a 2S albumin and a vicilin-like protein. OBJECTIVE: This study was designed to identify the walnut major allergens in the Italian population and to compare the walnut IgE-binding profile in patients with or without pollen allergy. METHODS: We selected 46 patients either with oral allergy syndrome confirmed by open oral challenge or with systemic symptoms after ingestion of walnut. These patients' sera were used for the immunoblotting of walnut extract; the identified allergens were purified by HPLC and sequenced. A peach-walnut cross-inhibition study was then performed. RESULTS: The only major allergen recognized by our study population was a 9-kd lipid transfer protein (LTP), recognized by 37 patients. Two other minor allergens of approximately 9-kd molecular weight, both belonging to the vicilin family, were recognized by 10 patients. IgE binding to walnut LTP was completely inhibited by peach LTP. CONCLUSION: In Italian patients with walnut allergy confirmed by documented history of severe systemic reactions or by open oral food challenge, the major allergen is an LTP. The sensitization to this protein seems to be secondary to the sensitization to peach LTP, which acts as the primary sensitizer. LTP and vicilins were able to sensitize patients not allergic to pollen.

Structural proteome of human colostral fat globule membrane proteins.

Milk fat globule membrane (MFGM) contains proteins derived from the apical membrane of secreting epithelial cells of the mammary gland. Between 2-4% of total human milk protein content is associated with the fat globule fraction, as MFGM proteins. While MFGM proteins have very low classical nutritional value, they play important roles in various cell processes and defence mechanisms for the newborn. To date, fewer than 30 human MFGM proteins have been identified and characterized, either by immunological methods or by Edman sequencing and mass spectrometry. This study aimed to update the structural proteome of human colostral MFGM proteins and to create an annotated two-dimensional electrophoresis (2-DE) MFGM protein database available on-line. More than one hundred 2-DE spots derived from human colostral MFGM proteins were investigated by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and proteins were identified by three different software packages available on the web (PeptIdent, MS-Fit and ProFound); uncertain identifications were solved by nanoelectrospray ionization-ion trap mass spectrometry using SEQUEST software.

Identification of grape and wine allergens as an endochitinase 4, a lipid-transfer protein, and a thaumatin.

BACKGROUND: Few allergic reactions to grape are reported in the literature. In some cases an association with peach and cherry allergy was observed. No IgE-mediated reactions to wine have been described, and no grape major allergens have yet been identified. OBJECTIVE: We describe several severe reactions to grape or wine. We characterized the grape major allergens and tried to identify the allergen in wine. METHODS: We collected documented histories of allergic reactions to grape and wine. Grape allergens were identified by means of SDS-PAGE and immunoblotting and purified by means of HPLC. Using amino acid sequencing and mass spectrometry, we identified the family of proteins to which the allergens belong. Cross-reactivity with peach and cherry was evaluated by means of cross-wise inhibition experiments. RESULTS: Eleven patients with reactions to grape and 3 with anaphylactic reactions to wine were recruited. The major allergens were an endochitinase 4A and a lipid-transfer protein (LTP) that was homologous to and cross-reactive with peach LTP. A 24-kd protein homologous to the cherry thaumatin-like allergen was a minor allergen. Endochitinase 4A is very likely the allergen in vino novello and in vino Fragolino. CONCLUSIONS: Grape and wine might cause severe allergic reactions in sensitive patients. The major allergens of grape are endochitinase 4A, which is also the allergen of wine, and an LTP cross-reacting with the peach major allergen.

Hypersensitivity to mugwort (Artemisia vulgaris) in patients with peach allergy is due to a common lipid transfer protein allergen and is often without clinical expression.

BACKGROUND: The observation of mugwort-specific IgE antibodies in patients with peach allergy suggests that mugwort sensitization might play a role in sensitization to peach. OBJECTIVE: We sought to study the clinical manifestations of mugwort hypersensitivity in patients with peach allergy, identify the common allergens, and evaluate their IgE crossreactivity. METHODS: Patients with oral allergy syndrome for peach and specific IgE antibodies to mugwort were investigated for respiratory symptoms during the mugwort season. Peach and mugwort allergens were identified by means of SDS-PAGE and IgE immunoblotting. Immunoblotting inhibition experiments were done to study cross-reactivity between peach and mugwort and other pollens. RESULTS: Seventeen patients were studied, 10 with no seasonal respiratory symptoms and 7 with clear late summer respiratory symptoms. In IgE immunoblotting the 10 asymptomatic patients reacted only to a 9-kd allergen of both mugwort and peach, whereas the 7 patients with pollinosis reacted to other allergens. Ten patients with mugwort allergy, no history of allergy to peach, and negative results for peach-specific IgE antibodies were also studied. The mugwort 9-kd protein was identified as a lipid transfer protein (LTP) homologous to peach LTP. Immunoblotting inhibition showed that IgE binding to the peach 9-kd band was totally inhibited by 4 microg of peach LTP but only by 400 microg of mugwort LTP, whereas 4 microg of both mugwort and peach LTP totally inhibited the mugwort immunoblotting. The results were similar with other pollens. CONCLUSIONS: Patients sensitized only to the 9-kd LTP of mugwort do not present hay fever symptoms, and this sensitization is a consequence of the peach sensitization.

A proteomic approach to evaluate the butyrophilin gene family expression in human milk fat globule membrane.

Human butyrophilin (BTN) expression in milk fat globule (MFGM) was evaluated using two dimensional electrophoresis (2-DE) as the separation technique, and peptide mass mapping by matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) as the identification tool. Since milk composition changes throughout lactation time, 2-DE maps in the pH range 4-7 of colostral MFGM and mature MFGM were compared, showing only slight differences in BTN spot distribution. The BTN gene family codes for seven proteins (BTN, BTN2A1, BTN2A2, BTN2A3, BTN3A1, BTN3A2, BTN3A3), their presence in human tissues has to date been evaluated only at a transcriptional level. Among 70 spots, analyzed and identified by MALDI-MS, 13 spots were identified as BTN spots and only one as a fragment of BTN2A1. BTN was present in multiple glycoforms, and two smaller BTN forms of about 45 kDa were also identified. We propose an array of BTNs on human MFGM, which could provide breast-fed infants with immune molecules during the neonatal period.

Lipid-transfer protein is the major maize allergen maintaining IgE-binding activity after cooking at 100 degrees C, as demonstrated in anaphylactic patients and patients with positive double-blind, placebo-controlled food challenge results.

BACKGROUND: In a previous study a 9-kd lipid-transfer protein (LTP) was identified as the major allergen of raw maize in a population of 22 anaphylactic patients. However, the stability of this protein in cooked maize is unknown. OBJECTIVE: We investigated the allergenicity of 5 maize hybrids and its modification after different thermal treatments by using sera from anaphylactic patients and patients with positive double-blind, placebo-controlled food challenges. METHODS: Five maize hybrids were extracted by using different methods, obtaining the water-soluble, zein, total zein, glutelin, and total protein fractions. The IgE-binding capacity of the different extracts, both raw and after thermal treatment, was investigated by means of SDS-PAGE immunoblotting. A 9-kd heat-stable allergen was purified by means of HPLC and sequenced. Changes in its secondary structure during and after heating from 25 degrees C to 100 degrees C were monitored by means of circular dichroism. RESULTS: All raw maize hybrids showed similar protein and IgE-binding profiles. The SDS-PAGE of all the heat-treated hybrids demonstrated a decreased number of stained bands in respect to the raw samples. The IgE immunoblotting demonstrated that the major allergen of the water-soluble, total zein, total protein, and glutelin fractions was a 9-kd protein identified by means of amino acid sequence as an LTP and a sub-tilisin-chymotrypsin inhibitor (in total zein fraction). The IgE-binding capacity of this 9-kd protein remained unchanged after thermal treatments, even though circular dichroism demonstrated an altered secondary structure. CONCLUSIONS: Maize LTP maintains its IgE-binding capacity after heat treatment, thus being the most eligible candidate for a causative role in severe anaphylactic reactions to both raw and cooked maize.

Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results.

BACKGROUND: The hazelnut major allergens identified to date are an 18-kd protein homologous to Bet v 1 and a 14-kd allergen homologous to Bet v 2. No studies have reported hazelnut allergens recognized in patients with positive double-blind, placebo-controlled food challenge (DBPCFC) results or in patients allergic to hazelnut but not to birch. OBJECTIVE: We characterized the hazelnut allergens by studying the IgE reactivity of 65 patients with positive DBPCFC results and 7 patients with severe anaphylaxis to hazelnut. METHODS: Hazelnut allergens were identified by means of SDS-PAGE and IgE immunoblotting. Further characterization was done with amino acid sequencing, evaluation of the IgE-binding properties of raw and roasted hazelnut with enzyme allergosorbent test inhibition, assessment of cross-reactivity with different allergens by means of immunoblotting inhibition, and purification by means of HPLC. RESULTS: All the sera from the patients with positive DBPCFC results recognized an 18- and a 47-kd allergen; other major allergens were at molecular weights of 32 and 35 kd. Binding to the 18-kd band was inhibited by birch extract, indicating its homology with the birch major allergen, and abolished in roasted hazelnut. The 47-kd allergen is a sucrose-binding protein, the 35-kd allergen is a legumin, and the 32-kd allergen is a 2S albumin. Patients with severe anaphylactic reactions to hazelnut showed specific IgE reactivity to a 9-kd allergen, totally inhibited by purified peach lipid-transfer protein (LTP), which was heat stable and, when purified, corresponded to an LTP. CONCLUSIONS: The major allergen of hazelnut is an 18-kd protein homologous to Bet v 1, and the 9-kd allergen is presumably an LTP. Other major allergens have molecular weights of 47, 32, and 35 kd.