Determination of the primary structure of two lipid transfer proteins from apricot (Prunus armeniaca).

It has been recently demonstrated that the major allergen of apricot is a protein of molecular mass (Mr) 9000 belonging to the family of Lipid Transfer Protein. The aim of this study was the determination of the primary structure of apricot LTP by micro-sequencing and mass spectrometric analyses. Apricot LTP is a 91 amino acids protein like peach and almond LTPs with a sequence identity of 91% and 94%, respectively. Like for the peach LTP, out of the 25 amino acids forming the inner surface of the tunnel-like hydrophobic cavity in maize ns-LTP, 16 are identical and 7 similar in the apricot LTP, supporting the hypothesis of a similar function.

Isolation of food allergens.

The identification of food allergens is a priority in the management of food allergy, because of the need to obtain standardized extracts and pure allergens for diagnosis and therapy. It is thus important to develop methods for purification of allergenic molecules in order to study their biological and immunological characteristics. Protocols for protein extraction from foods and for allergen purification are reviewed in this paper. We report published methods for extraction of allergens from either animal and vegetable foods and detailed purification methodologies including ion-exchange, gel filtration and reversed-phase chromatography of well known allergens.

The major allergen of sesame seeds (Sesamum indicum) is a 2S albumin.

BACKGROUND: Allergic reactions induced by ingestion of foods containing sesame seeds are a well recognized cause of severe food-induced anaphylaxis. OBJECTIVE: This study aimed to identify and characterize the clinically most important major allergen of sesame seeds. METHODS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and IgE immunoblotting were performed on sera of 10 patients selected for severe and documented allergic reaction after eating food containing sesame. The major allergen was purified by gel filtration and characterized by isoelectric point (pI), glycosylation and amino acid sequencing. RESULTS: All the patients had positive IgE antibodies and skin prick tests (SPTs) to sesame. The major, clinically most important allergen was a protein with molecular mass of about 9000. It was not glycosylated, the amino acid sequence showed it was a 2S albumin with a pI of 7.3; the small and the large subunits, forming the whole protein, showed pI values of 6.5 and 6.0.

Characterization of the major allergen of plum as a lipid transfer protein.

BACKGROUND: Allergy to Prunoideae fruit (plum, peach, cherry and apricot) is one of the most frequent food allergies in southern Europe. All these fruits cross-react in vivo and in vitro, as they share their major allergen, a 9 kD lipid transfer protein (LTP). OBJECTIVE: The aim of the study was the identification and molecular characterization of the major allergen of plum. METHODS: The IgE pattern of reactivity to plums was investigated by SDS-PAGE and immunoblotting with the sera of 23 patients. The identified major allergen was purified by HPLC, using a cationic-exchange column followed by gel-filtration. Further characterization was achieved by periodic-Schiff stain, isoelectrofocusing and N-terminal amino acid sequencing. RESULTS AND CONCLUSIONS: The major allergen of plum is a 9 kD lipid transfer protein, not glycosylated and with a basic character (pI>9), highly homologous to the major allergen of peach.

Lipid transfer proteins and 2S albumins as allergens.

Plant lipid transfer proteins, a widespread family of proteins, have been recently identified as important food allergens. Their common structural features, such as eight conserved cysteines forming disulfide bridges, basic isoelectric point and high similarity in amino acid sequence, are the basis of allergic clinical cross-reactivity. This has been demonstrated for the LTP allergens of the Prunoideae subfamily, whose similarity is about 95% as demonstrated for the purified allergens of peach, apricot, plum and apple. A relevant aspect is the existence of sequence homology of LTPs of botanically unrelated foods, as demonstrated for LTPs of maize and peach. A class of food allergens of well recognized clinical importance is that of seed storage 2S albumins. They have been identified in the most diffused edible seeds and nuts, such as mustard, sesame, Brazil nut, walnut and peanut. In particular, a strong correlation between IgE-binding to these proteins and food-induced anaphylaxis has been demonstrated for Brazil nut and sesame seeds.

The maize major allergen, which is responsible for food-induced allergic reactions, is a lipid transfer protein.

BACKGROUND: Cereals are the most important nutritional component in the human diet. Food-induced allergic reactions to these substances therefore have serious implications, and exhaustive diagnosis is required. Such diagnosis is still difficult because of the incomplete knowledge about major cereal allergens. In particular, few food-induced allergic reactions to maize have been reported, and no information on the allergenic proteins is available. OBJECTIVE: Having observed several anaphylactic reactions to maize, we planned a study to identify maize major allergens and cross-reactivity with other cereals, as well as to peach because the majority of patients also reacted to Prunoideae fruits. METHODS: Twenty-two patients with systemic symptoms after maize ingestion and positive skin prick test responses and serum-specific IgE antibodies to maize were selected. The IgE-reactivity pattern was identified by SDS-PAGE and immunoblotting. The major allergen identified was then purified by HPLC and characterized by mass spectrometry, determination of the isoelectric point value, and N-terminal amino acid sequencing. RESULTS: Sera from 19 (86%) of the 22 patients recognized a 9-kd protein, thus confirming this as the maize major allergen. This protein had an isoelectric point of greater than 9, a molecular mass of 9047.0 d, and no glycosylation. Determination of its N-terminal sequence showed that it was a lipid transfer protein (LTP). By using immunoblotting-inhibition experiments, we demonstrated that the LTP cross-reacts completely with rice and peach LTPs but not with wheat or barley LTPs. N-terminal sequence of the 16-kd allergen (recognized by 36% of patients) showed it to be the maize inhibitor of trypsin. This protein cross-reacts completely with grass, wheat, barley, and rice trypsin inhibitors. CONCLUSION: The major allergen of maize is an LTP with a molecular weight of 9 kd that is highly homologous with the peach LTP, the major allergen of the Prunoideae subfamily.

Sensitization to the major allergen of Brazil nut is correlated with the clinical expression of allergy.

BACKGROUND: Only a few studies have investigated the clinical role of food allergens, especially the relationship between sensitization to a given allergen and occurrence of adverse reactions when eating the relevant food item. OBJECTIVE: This study evaluated the clinical role of the allergens of Brazil nut by comparing the patterns of IgE binding in sera from 11 patients with anaphylaxis after eating Brazil nuts with those from 10 subjects with no symptoms to this food item. Both groups had specific IgE to Brazil nut. METHODS: Allergens in the in-house extract of Brazil nut were identified by SDS-PAGE/immunoblotting, the major allergen was purified by HPLC, and its N-terminal sequence was determined by a protein sequencer. RESULTS: SDS-PAGE/immunoblotting detected a number of allergenic components with molecular weights ranging from 4 to 58 kd. All sera from symptomatic patients recognized a 9-kd allergen corresponding (as established by amino acid sequencing) to a 2S albumin already described as a major allergen of Brazil nut, whereas the other allergens each bound IgE from less than 50% of sera. No sera from asymptomatic subjects showed IgE binding to the 9-kd allergen, but they did recognize components from 25 to 58 kd, which are minor allergens. CONCLUSIONS: These findings indicate that the allergen underlying clinical reactions to Brazil nut is a 2S albumin of 9 kd and that in vitro reactivity to this allergen identifies subjects who react in vivo to ingestion of this food.