Par j 1 and Par j 2, the two major allergens in Parietaria judaica, bind preferentially to monoacylated negative lipids.

Par j 1 and Par j 2 proteins are the two major allergens in Parietaria judaica pollen, one of the main causes of allergic diseases in the Mediterranean area. Each of them contains eight cysteine residues organized in a pattern identical to that found in plant nonspecific lipid transfer proteins. The 139- and 102-residue recombinant allergens, corresponding respectively to Par j 1 and Par j 2, refold properly to fully functional forms, whose immunological properties resemble those of the molecules purified from the natural source. Molecular modeling shows that, despite the lack of extensive primary structure homology with nonspecific lipid transfer proteins, both allergens contain a hydrophobic cavity suited to accommodate a lipid ligand. In the present study, we present novel evidence for the formation of complexes of these natural and recombinant proteins from Parietaria pollen with lipidic molecules. The dissociation constant of oleyl-lyso-phosphatidylcholine is 9.1 +/- 1.2 microm for recombinant Par j 1, whereas pyrenedodecanoic acid shows a much higher affinity, with a dissociation constant of approximately 1 microm for both recombinant proteins, as well as for the natural mixture. Lipid binding does not alter the secondary structure content of the protein but is very efficient in protecting disulfide bonds from reduction by dithiothreitol. We show that Par j 1 and Par j 2 not only bind lipids from micellar dispersions, but also are able to extract and transfer negative phospholipids from bilayers.

Genetically engineered hybrid proteins from Parietaria judaica pollen for allergen-specific immunotherapy.

BACKGROUND: Despite the use of conventional allergen-specific immunotherapy in clinical practice, more defined, efficient, and safer allergy vaccines are required. OBJECTIVE: The aim of the study was to obtain hypoallergenic molecules by deleting B-cell epitopes, which could potentially be applied to Parietaria judaica pollen allergy treatment. METHODS: Three hybrid molecules (Q1, Q2, and Q3) derived from fragments of the 2 major P judaica pollen allergens, Par j 1 and Par j 2, were engineered by means of PCR. Hybrid structures were compared with their natural components by means of circular dichroism, and their biologic activities were compared by using T-cell proliferation assays. Their IgE-binding activity was determined with Western blotting, skin prick tests, and enzyme allergosorbent and ELISA inhibition tests. RESULTS: The hybrid proteins, especially Q2 and Q3, revealed significantly reduced IgE reactivity compared with the natural allergens, as well as with the whole P judaica extract. Furthermore, in vivo skin prick tests showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the whole P judaica extract. Two (Q1 and Q2) of the 3 hybrid proteins induced a comparable T-cell proliferation response as that produced by the whole extract and natural allergens. CONCLUSION: Considering its reduced anaphylactogenic potential, together with its conserved T-cell reactivity, the engineered Q2 protein could be used in safe and shortened schedules of allergen-specific immunotherapy against P judaica pollen allergy. CLINICAL IMPLICATIONS: Recombinant hybrid Q2 is able to induce T-cell proliferation, thus evidencing a potential therapeutic effect. Its reduced IgE-binding capacity envisages an excellent safety profile.

A sensitive two-site enzyme-linked immunosorbent assay for measurement of the major Alternaria alternata allergen Alt a 1.

BACKGROUND: Alt a 1 is the major allergen in Alternaria alternata, one of the most important fungi associated with allergic diseases. Mold allergenic extracts show considerable heterogeneity, and thus accurate standardization of these extracts is essential to guarantee their quality. OBJECTIVE: To develop an Alt a 1-specific assay and to evaluate the correlation of Alt a 1 content with the IgE-binding activity of A. alternata extracts. METHODS: Recombinant Alt a 1 was produced as nonfusion protein from a polymerase chain reaction-cloned complementary DNA Alt a 1 sequence. Natural Alt a 1 was purified from spent culture medium. Monoclonal and polyclonal antibodies directed to Alt a 1 were produced and used to construct a specific Alt a 1 enzyme-linked immunosorbent assay (ELISA). RESULTS: The ELISA developed was highly reproducible and sensitive, with a detection limit lower than 0.5 ng/mL and a practical working range of 0.5 to 50 ng/mL. The assay was able to detect an Alt a 1-like protein in Stemphylium extracts. Identical parallel dose-response curves were observed when natural Alt a 1 and recombinant Alt a 1 were used as standard. A good correlation was obtained between Alt a 1 content of 13 A. alternata extracts and their IgE-binding activity. Alt a 1 was responsible for 70% of the IgE-binding activity of the whole extract. CONCLUSIONS: This sensitive and specific Alt a 1 assay allows the quantification of this major mold allergen and represents a useful tool for the standardization of A. alternata extracts in mass units. It also provides a reliable indication of the allergenic activity of the whole extract.