Molecular and immunochemical characterization of profilin as major allergen from Platanus acerifolia pollen.

BACKGROUND: The Platanus acerifolia (P. acerifolia) pollen is one of the most common causes of allergic respiratory symptoms in China. However, the allergenic components in P. acerifolia are not fully studied yet. The study aimed to determine the molecular and immunochemical characterization of the profilin from P. acerifolia pollen. METHODS: The coding sequence of profilin was amplified, cloned, and then expressed in Escherichia coli BL21 cells and purified by nickel affinity chromatography. Protein refolding was followed by structural characterization and homology 3D model building. The allergenicity and cross-reactivity were assessed by ELISA, immunoblotting, or basophil activation test (BAT) using the sera of P. acerifolia allergic patients. RESULTS: The cDNA sequence of profilin was cloned with a 396 bp open reading frame coding for 131 amino acids. The molecular weight of the profilin was approximately 14 kDa, and the predicted structure consisted of 3 α-helixes and 7 ß-sheets. Physicochemical analysis indicated the profilin was a stable, relatively thermostable, and relatively conserved protein. The allergenicity determined by ELISA, western blot, and BAT suggested 76.9% (30/39) of the P. acerifolia pollen allergic patients displayed specific IgE recognition of the profilin. The profilin shared > 80% sequence identity with Pop n 2, the profilin from Populus nigra, and observed a significant cross-reactivity with Pop n 2 in IgE-inhibition assay. CONCLUSION: Profilin, as one of the major component allergens in P. acerifolia pollen, was identified and characterized at molecular and immunochemical levels in this study. These findings would contribute to developing diagnostic and therapeutic strategies for P. acerifolia pollen allergic patients.

Profilin promotes formin-mediated actin filament assembly and vesicle transport during polarity formation in pollen.

Pollen germination is critical for the reproduction of flowering plants. Formin-dependent actin polymerization plays vital roles in vesicle trafficking and polarity establishment during this process. However, how formin-mediated actin assembly is regulated in vivo remains poorly understood. Here, we investigated the function of reproductive profilin 4 and 5 (PRF4 and PRF5) in polarity establishment during pollen germination in Arabidopsis thaliana. Our data showed that the actin filament content was reduced in the prf4 prf5 double mutant and substantially increased in both PRF4- and PRF5-overexpressing pollen grains. By contrast, the positive effect of profilin in promoting actin polymerization was abolished in a formin mutant, atfh5. In addition, the interaction between Arabidopsis formin homology 5 (AtFH5) and actin filaments was attenuated and the trafficking of AtFH5-labeled vesicles was slowed in prf4 prf5 pollen grains. Formation of the collar-like structure at the germination pore was also defective in prf4 prf5 pollen grains as the fast assembly of actin filaments was impaired. Together, our results suggest that PRF4 and PRF5 regulate vesicle trafficking and polarity establishment during pollen germination by promoting AtFH5-mediated actin polymerization and enhancing the interaction between AtFH5 and actin filaments.

Identification of profilin 1 as the primary target for the anti-cancer activities of Furowanin A in colorectal cancer.

BACKGROUND: Furowanin A (Fur A) is a flavonoid compound isolated from medicinal plant Millettia pachycarpa Benth. This study aims to explore the effect of Fur A on Colorectal cancer (CRC) and its molecular mechanisms. METHODS: Cell proliferative capacity of CRC cells was assessed by CCK-8 assay. Cell apoptosis and cell cycle distribution were detected by flow cytometry. Cell migration and invasion were detected by wound healing and Transwell assay, respectively. EMT markers, apoptosis and profilin 1(Pfn1) expression were detected by immunohistochemistry (IHC). The protein expression levels were examined by western blotting. i-TRAQ analyses were conducted to identify the differentially expressed genes in CRC cells. CRC xenograft model was also used to validate the in vivo anti-cancer activity of Fur A. RESULTS: Fur A exhibited anti-prolifertive, blocked cell cycle progression and promoted apoptotic cell death in CRC cells. Fur A suppressed the migration, invasion and epithelial-to-mesenchymal transition (EMT) in vitro, and tumor growth and pulmonary metastasis in vivo, without causing obvious toxicity. iTRAQ analysis identified Pfn1 as a gene up-regulated by Fur A. In xenograft tumor tissue, the expression of Pfn1 was also elevated by Fur A treatment. In clinical CRC samples, high expression of Pfn1 was correlated with lower stage and longer survival. Knockdown of Pfn1 significantly dampened the pro-apoptotic and anti-metastatic activities of Fur A in CRC cells. Ectopic Pfn1 expression augmented the anti-neoplastic activities of Fur A. CONCLUSION: Fur A exhibited anti-cancer activities in vitro and in vivo in CRC by up-regulating Pfn1.

Role of GSPE in improving early cerebral vascular damage by inhibition of Profilin-1 expression in a ouabain-induced hypertension model.

OBJECTIVE: Grape seed proanthocyanidin extract (GSPE), as one of the most popular natural drug extracted from the grape, has been reported to improve endothelial function and arteriosclerosis. However, little is known about the influence of GSPE on hypertension and vascular remodeling. Profilin-1, an Actin-binding protein, is closely involved in the remodeling of large vessels in ouabain-induced hypertension. To date, there is no effective prevention or treatment in place for the high incidence of ischemic stroke associated with hypertension. In this study, we aimed to determine the role of GSPE via inhibition Profilin-1 in ischemic cerebral cortices of ouabain-hypertension rats and potentially provide a new target to prevent stroke associated with hypertension. MATERIALS AND METHODS: The blood pressure of male Sprague-Dawley (SD) rats was measured during a period of ouabain-induced hypertension. The expression of Profilin-1, vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the cerebral cortex were determined by quantitative Real Time-PCR (qRT-PCR) and Western blot. Histopathological and behavioral tests were also conducted. RESULTS: Blood pressure elevation started at week 5 and reached clinical standards for hypertension at week 8. GSPE was proved to suppress Profilin-1 and VEGF levels through inhibition of Profilin-1-protein kinase B (AKT)-hypoxia inducible factor-1α (HIF-1α) signal pathway and promote eNOS expression. Moreover, the histopathological and ethiological improvement was observed in GSPE over-expression and Profilin-1 inhibition groups. CONCLUSIONS: We detected that GSPE could improve cerebral vascular damage through inhibiting Profilin-1 in an ouabain-induced hypertension model.

Structure-based virtual screening identifies a small-molecule inhibitor of the profilin 1-actin interaction.

Profilin 1 (Pfn1) is an important regulator of the actin cytoskeleton and plays a vital role in many actin-based cellular processes. Therefore, identification of a small-molecule intervention strategy targeted against the Pfn1-actin interaction could have broad utility in cytoskeletal research and further our understanding of the role of Pfn1 in actin-mediated biological processes. Based on an already resolved Pfn1-actin complex crystal structure, we performed structure-based virtual screening of small-molecule libraries to seek inhibitors of the Pfn1-actin interaction. We identified compounds that match the pharmacophore of the key actin residues of Pfn1-actin interaction and therefore have the potential to act as competitive inhibitors of this interaction. Subsequent biochemical assays identified two candidate compounds with nearly identical structures that can mitigate the effect of Pfn1 on actin polymerization in vitro As a further proof-of-concept test for cellular effects of these compounds, we performed proximity ligation assays in endothelial cells (ECs) to demonstrate compound-induced inhibition of Pfn1-actin interaction. Consistent with the important role of Pfn1 in regulating actin polymerization and various fundamental actin-based cellular activities (migration and proliferation), treatment of these compounds reduced the overall level of cellular filamentous (F) actin, slowed EC migration and proliferation, and inhibited the angiogenic ability of ECs both in vitro and ex vivo In summary, this study provides the first proof of principle of small-molecule-mediated interference with the Pfn1-actin interaction. Our findings may have potential general utility for perturbing actin-mediated cellular activities and biological processes.

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation.

Profilin 1, cofilin 1, and vasodialator-stimulated phosphoprotein (VASP) are actin-binding proteins (ABP) that regulate actin remodeling and facilitate cancer cell metastases. miR-17-92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. In addition, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1, and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by Western blot and confocal analysis. miR-17-92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASP(pS157), but no changes in cofilin1/VASP(pS239) in the human malignant tissues compared with normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASP(pS157) or cofilin1/VASP(pS239) suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR-17-92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR-17-92, and DHA as a novel therapeutic. Mol Cancer Ther; 15(9); 2220-31. ©2016 AACR.

Is the ISAC 112 Microarray Useful in the Diagnosis of Pollinosis in Spain?

BACKGROUND: Multiple sensitization is frequent among pollen-allergic patients. The goal of this study was to determine the diagnostic accuracy of the ImmunoCAP ISAC 112 (ISAC112) microarray in allergy to pollen from several taxa and its clinical utility in a Spanish population. METHODS: Specific IgE was determined in 390 pollen-allergic patients using the ISAC112 microarray. Diagnostic accuracy (sensitivity, specificity, predictive values, and area under the ROC curve) was calculated for the diagnosis of allergy to pollen from grass (n=49), cypress (n=75), olive tree (n=33), plane tree (n=63), and pellitory of the wall (n=17) and compared with that of the singleplex ImmunoCAP immunoassay. RESULTS: The sensitivity of the ISAC112 microarray ranged from 68.2% for allergy to plane tree pollen to 93.9% for allergy to grass pollen. The specificity was >90%. The AUC for the diagnosis of allergy to plane tree pollen was 0.798, whereas the AUC for the remaining cases was ≥0.876. The accuracy of ISAC112 was higher than that of ImmunoCAP for plane tree pollen and similar for the remaining pollens. The frequency of sensitization to most species-specific allergenic components and profilins varied between the different geographical regions studied. A total of 73% of pollen-allergic patients were sensitized to species-specific components of more than 1 pollen type. CONCLUSIONS: The ISAC112 microarray is an accurate tool for the diagnosis of allergy to pollen from grass, cypress, olive tree, plane tree, and pellitory of the wall. The features of the ISAC112 microarray are similar or superior (in the case of plane tree pollen) to those of ImmunoCAP. This microarray is particularly useful for the etiologic diagnosis of pollinosis in patients sensitized to multiple pollen species whose pollination periods overlap.

Pro j 2 is mesquite profilin: molecular characteristics and specific IgE binding activity.

BACKGROUND: Pollens from mesquite (Prosopis juliflora) are potent allergen responsible in causing immediate hypersensitivity reactions in susceptible people in tropical countries. OBJECTIVE: This study aimed to clone, express and purify the mesquite pollen profilin (Pro j 2) as well as evaluating its nucleotide sequence homology in order to predict allergenic cross-reactivity with profilins of common allergenic plants. METHODS: Immunoblotting assay and specific ELISA were applied to determine the immunoreactivity of sera from 35 patients who were allergic to mesquite pollen. The mesquite profilin-coding sequence was cloned into PTZ57R/T vector and amplified. The cDNA of mesquite pollen profilin was then expressed in Escherichia coli using pET-21b (+) vector and puri?ed by one-step Ni2+ a?nity chromatography. IgE binding capacity of the recombinant mesquite profiling (rPro j 2) was analyzed by specific ELISA, immunoblotting, and inhibition assays. RESULTS: cDNA nucleotide sequencing revealed an open reading frame of 399bp encoding for 133 amino acids which belongs to the profilin family. Seventeen patients (17/35, 48.57%) had significant specific IgE level for rPro j 2. Immunodetection and inhibition assays indicated that puri?ed rPro j 2 might be similar as that in the crude extract. CONCLUSION: Pro j 2, as a new allergen from mesquite pollen, was produced in E. coli with an IgE-reactivity similar to that of its natural counterpart. The amino acid sequences homology analysis of mesquite profilin and several profilin molecules from other plants showed high degree of cross-reactivity among plant-derived profilins from unrelated families.

Variability of Corylus avellana, L. CorA and profilin pollen allergens expression.

Corylus avellana is the source of inhalant allergies induced by hazel pollen as well as food allergies induced after ingestion of hazelnuts. In this study, real-time PCR approach was used to analyse expression of hazel pollen allergens on the molecular level. Relative quantity of hazelnut allergens Corylus avellana, L. CorA and Corylus avellana, L. pollen profiling in samples from different Ukraine areas were determining and comparing. Differences among the levels of both analysed allergen transcripts were found for hazel CorA and profillin. In both cases, the expression within the urbanized growth conditions was higher when compared to the sample from village area. The average expression for CorA was 0.84 times higher than for profilin and the results are very variable depending on the place of growth. Expression levels here were within the range of 2.957 up to the 52.936. Profilin expression was the highest in the sample from the polluted place of growth-cement plant area with the value of 52 times higher when compared to the sample from the village area. In this study, comparison of expression levels of hazel CorA and profiling pollen allergens was performed for the first time. Real-time PCR assay developed in this study proved the sensitivity for detection of the changes of the hazel pollen allergens expression levels and could benefit labs by fast and reproducible detection method of these allergens.

The effect of component-resolved diagnosis on specific immunotherapy prescription in children with hay fever.

BACKGROUND: Sensitization to profilins and other cross-reacting molecules might hinder proper specific immunotherapy (SIT) prescription in polysensitized patients with pollen-related allergic rhinitis (AR). In these patients, component-resolved diagnosis (CRD) might modify SIT prescription by improving the identification of the disease-eliciting pollen sources. OBJECTIVES: We sought to measure the effect of CRD on SIT prescription in children with pollen-related AR. METHODS: Children (n = 651) with moderate-to-severe pollen-related AR were recruited between May 2009 and June 2011 in 16 Italian outpatient clinics. Skin prick test (SPT) reactivity to grass, cypress, olive, mugwort, pellitory, and/or Betulaceae pollen was considered clinically relevant if symptoms occurred during the corresponding peak pollen season. IgE sensitization to Phl p 1, Phl p 5, Bet v 1, Cup a 1, Art v 1, Ole e 1, Par j 2, and Phl p 12 (profilin) was measured by using ImmunoCAP. SIT prescription was modeled on SPT responses first and then remodeled considering also CRD according to GA(2)LEN-European Academy of Allergology and Clinical Immunology guidelines and the opinions of 14 pediatric allergists. RESULTS: No IgE to the respective major allergens was detected in significant proportions of patients with supposed clinically relevant sensitization to mugwort (45/65 [69%]), Betulaceae (146/252 [60%]), pellitory (78/257 [30%]), olive (111/390 [28%]), cypress (28/184 [15%]), and grass (56/568 [10%]). IgE to profilins, polcalcins, or both could justify 173 (37%) of 464 of these SPT reactions. After CRD, the SPT-based decision on SIT prescription or composition was changed in 277 (42%) of 651 or 315 (48%) of 651 children according to the European or American approach, respectively, and in 305 (47%) of 651 children according to the opinion of the 14 local pediatric allergists. CONCLUSIONS: In children with pollen-related AR, applying CRD leads to changes in a large proportion of SIT prescriptions as opposed to relying on clinical history and SPT alone. The hypothesis that CRD-guided prescription improves SIT efficacy deserves to be tested.

Cloning, expression in E. coli and immunological characterization of Par j 3.0201, a Parietaria pollen profilin variant.

Parietaria judaica pollen is one of the main sources of allergens in the Mediterranean area. Its allergenic composition has been studied in detail showing the presence of two major allergens (Par j 1 and Par j 2) and two minor allergens belonging to the profilin and calcium binding protein families of allergens (Par j 3 and Par j 4, respectively). Clinical reports support the hypothesis of a limited cross-reactivity between profilin from Parietaria and unrelated sources. We screened a P. judaica cDNA library to identify novel forms of profilins with allergenic activity. This strategy allowed us to isolate a 767 bp cDNA containing the information for a 131 amino acids protein with homology to profilins from unrelated sources greater than that observed with the already published Parietaria profilins. This profilin was expressed in Escherichia coli as a recombinant protein and its immunological prevalence was studied in a population of Parietaria allergic patients from Southern Europe. Immunoblotting analysis showed that the Parietaria profilin was recognized by IgE from 6.5% of the allergic population. Finally, a selected population of profilin allergic patients was enrolled to demonstrate the cross-reactivity of this novel variant with other profilins from grass and date palm. In conclusion, molecular cloning and immunological studies have allowed the isolation, expression and immunological characterization of a novel cross-reactive profilin allergen from P. judaica pollen named Par j 3.0201.

Analysis of the effects of polymorphism on pollen profilin structural functionality and the generation of conformational, T- and B-cell epitopes.

An extensive polymorphism analysis of pollen profilin, a fundamental regulator of the actin cytoskeleton dynamics, has been performed with a major focus in 3D-folding maintenance, changes in the 2-D structural elements, surface residues involved in ligands-profilin interactions and functionality, and the generation of conformational and lineal B- and T-cell epitopes variability. Our results revealed that while the general fold is conserved among profilins, substantial structural differences were found, particularly affecting the special distribution and length of different 2-D structural elements (i.e. cysteine residues), characteristic loops and coils, and numerous micro-heterogeneities present in fundamental residues directly involved in the interacting motifs, and to some extension these residues nearby to the ligand-interacting areas. Differential changes as result of polymorphism might contribute to generate functional variability among the plethora of profilin isoforms present in the olive pollen from different genetic background (olive cultivars), and between plant species, since biochemical interacting properties and binding affinities to natural ligands may be affected, particularly the interactions with different actin isoforms and phosphoinositides lipids species. Furthermore, conspicuous variability in lineal and conformational epitopes was found between profilins belonging to the same olive cultivar, and among different cultivars as direct implication of sequences polymorphism. The variability of the residues taking part of IgE-binding epitopes might be the final responsible of the differences in cross-reactivity among olive pollen cultivars, among pollen and plant-derived food allergens, as well as between distantly related pollen species, leading to a variable range of allergy reactions among atopic patients. Identification and analysis of commonly shared and specific epitopes in profilin isoforms is essential to gain knowledge about the interacting surface of these epitopes, and for a better understanding of immune responses, helping design and development of rational and effective immunotherapy strategies for the treatment of allergy diseases.

Characterization of profilin polymorphism in pollen with a focus on multifunctionality.

Profilin, a multigene family involved in actin dynamics, is a multiple partners-interacting protein, as regard of the presence of at least of three binding domains encompassing actin, phosphoinositide lipids, and poly-L-proline interacting patches. In addition, pollen profilins are important allergens in several species like Olea europaea L. (Ole e 2), Betula pendula (Bet v 2), Phleum pratense (Phl p 12), Zea mays (Zea m 12) and Corylus avellana (Cor a 2). In spite of the biological and clinical importance of these molecules, variability in pollen profilin sequences has been poorly pointed out up until now. In this work, a relatively high number of pollen profilin sequences have been cloned, with the aim of carrying out an extensive characterization of their polymorphism among 24 olive cultivars and the above mentioned plant species. Our results indicate a high level of variability in the sequences analyzed. Quantitative intra-specific/varietal polymorphism was higher in comparison to inter-specific/cultivars comparisons. Multi-optional posttranslational modifications, e.g. phosphorylation sites, physicochemical properties, and partners-interacting functional residues have been shown to be affected by profilin polymorphism. As a result of this variability, profilins yielded a clear taxonomic separation between the five plant species. Profilin family multifunctionality might be inferred by natural variation through profilin isovariants generated among olive germplasm, as a result of polymorphism. The high variability might result in both differential profilin properties and differences in the regulation of the interaction with natural partners, affecting the mechanisms underlying the transmission of signals throughout signaling pathways in response to different stress environments. Moreover, elucidating the effect of profilin polymorphism in adaptive responses like actin dynamics, and cellular behavior, represents an exciting research goal for the future.

Pollen and plant food profilin allergens show equivalent IgE reactivity.

BACKGROUND: Profilins are commonly involved in polysensitization of allergic patients; therefore, appropriate markers should be used in component-resolved diagnosis. OBJECTIVE: To evaluate the immunological equivalence between profilins from pollens and plant-derived foods, to be used in component-resolved diagnosis. METHODS: Specific immunoglobulin (Ig) G antibodies against pollen and fruit profilins, as well as sera from patients allergic to mustard, melon, or olive pollen, were used. Purified profilins from mustard seeds, fruit melon, and chenopod and birch pollen were assayed in immunoblotting, enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition assays. RESULTS: Significant correlation was found in the response of purified profilins by ELISA and immunoblotting for both specific IgG and IgE. The highest levels of IgE binding were obtained for olive pollen-allergic patients, which could be related to the route of sensitization. The responses of individual patients to profilins were also similar and independent of the sensitizing source. The inhibition between pairs of allergens was generally higher than 70%, indicating that profilins share most of the IgE epitopes. Modeling of mimotopes in the conformational structure of the implicated profilins supports their strong cross-reactivity obtained experimentally. CONCLUSIONS: No correlation exists between the level of IgE response of individual patients to specific profilins and the corresponding theoretical sensitizing source, suggesting that the sensitization could be attributable to any profilin present in the environment of the patients. This would bear out the use of most profilins as a common marker for polysensitization in component-resolved diagnosis and for therapeutic approaches.

Identification of a new allergen from Amaranthus retroflexus pollen, Ama r 2.

BACKGROUND: Pollinosis from Amaranthus retroflexus pollen is a common cause of respiratory allergy in Iran with a high positive rate (68.8%) among Iranian allergic patients. The aim of the present study was to evaluate the allergenicity of the A. retroflexus pollen profilin. METHODS: Using sera from twelve patients allergic to A. retroflexus pollen, IgE-binding proteins from the A. retroflexus pollen extract was identified by immunoblotting. The cDNA of A. retroflexus pollen profilin was amplified, then cloned into the pET-21b (+) vector, expressed in Escherichia coli, and finally purified by metal affinity chromatography. The IgE-binding capacity of the recombinant protein was then analyzed by the ELISA, immunoblotting, and inhibition assays, as well as by the skin prick test (SPT). RESULTS: Immunoblotting results indicated a 14.6kDa protein with IgE-reactivity to 33% (4/12) among A. retroflexus pollen-allergic patients. Nucleotide sequencing of the cDNA revealed an open reading frame of 399 bp encoding for 133 amino acid residues which was belonged to the profilin family and designated as Ama r 2. A recombinant Ama r 2 (rAma r 2) was then produced in E. coli as a soluble protein which showed a strong IgE-reactivity via ELISA confirmed by the SPT. Inhibition experiments revealed high IgE cross-reactivities with the profilins from other plants. CONCLUSIONS: The profilin from the A. retroflexus pollen, Ama r 2, was firstly identified as an allergen. Moreover, rAma r 2 was produced in E. coli as a soluble immunoreactive protein with an IgE-reactivity similar to that of its natural counterpart.

Chenopodium album pollen profilin (Che a 2): homology modeling and evaluation of cross-reactivity with allergenic profilins based on predicted potential IgE epitopes and IgE reactivity analysis.

The inhalation of Chenopodium album (C. album) pollen has been reported as an important cause of allergic respiratory symptoms. The aim of this study was to produce the recombinant profilin of C. album (rChe a 2) pollen and to investigate its cross-reactivity with other plant-derived profilins based on potential conformational epitopes and IgE reactivity analysis. Che a 2-coding sequence was cloned, expressed, and purified using one step metal affinity chromatography to recover high-purity target protein. We assessed cross-reactivity and predicted IgE potential epitopes among rChe a 2 and other plant-derived profilins. Immunodetection and inhibition assays using sixteen individual sera from C. album allergic patients demonstrated that purified rChe a 2 could be the same as that in the crude extract. The results of inhibition assays among rChe a 2 and other plant-derived profilins were in accordance with those of the homology of predicted conserved conformational regions. In this study, amino acid sequence homology analysis showed that a high degree of IgE cross-reactivity among plant-derived profilins may depend on predicted potential IgE epitopes.

Cloning and expression of the allergen Cro s 2 profilin from saffron (Crocus sativus).

BACKGROUND: Profilin is a panallergen that is recognized by IgE in allergic patients. Allergy to saffron (Crocus sativus) pollen has been described in people exposed to its pollen. Saffron contains a profilin that may cause allergic reactions in atopic subjects. The aim of this study was to describe the cloning, expression and purification of saffron profilin from pollen. METHODS: Cloning of saffron profilin was performed by polymerase chain reaction using specific primers from saffron pollen RNA. Expression was carried out in Escherichia coli BL21 (DE3) using a vector pET-102- TOPO. A recombinant fusion protein was expressed and the recombinant profilin was purified by metal precipitation. Immunological characterization was performed by immunoblotting experiments. RESULTS: The 34kDa- recombinant saffron profilin, Cro s 2, as a fusion protein was purified. Immunoblotting tested with the sera of allergic patients showed a specific reaction with the recombinant Cro s 2 band. CONCLUSIONS: The sequence of Cro s 2 showed a high degree of identity and similarity to other plant profilins and the recombinant saffron profilin, Cro s 2, may be used for target-specific diagnosis and structural analyses and investigation of cross reactivity of Cro s 2 with other plant profilins.

Molecular cloning and characterization of a profilin gene BnPFN from Brassica nigra that expressing in a pollen-specific manner.

Brassica nigra is a newly found invasive species in Zhejiang Province, China. It distributes alongside the roads, in vegetable fields and on riversides. When it blooms, some natives there will suffer from allergic rhinitis. We designed gene-specific primer pairs according to reported profilin genes and successfully isolated their homolog from flower bud cDNA of B. nigra. The gene, designated BnPFN, was submitted to GenBank under accession number EU004073. BnPFN was 405 bp in length encoding 134 amino acids. Expression analysis of BnPFN gene was carried out by means of RT-PCR. The results showed that BnPFN express only in anthers and pollens, and there was no detection in roots, leaves, stems, sepals, petals and pistils. We suggest that BnPFN is a pollen-specific gene and may be responsible for pollen anaphylactic reactions in those invading areas when B. nigra blooms.

Molecular and immunological characterization of novel weed pollen pan-allergens.

BACKGROUND: Pan-allergens like profilins, calcium-binding proteins (CBPs), and nonspecific lipid transfer proteins have been suggested as possible specific markers for multiple pollen sensitizations, and could be used to predict cross-sensitization/poly-sensitization to several pollen allergens. Therefore, the purification and characterization of cross-reacting allergens in pollen is an extremely important task towards correct allergy diagnosis. METHODS: New pan-allergens were identified by screening a ragweed pollen cDNA library with sera of patients allergic to mugwort pollen. Resulting proteins were cloned, expressed, purified and characterized. RESULTS: We report complete cDNA sequences of two profilin isoforms (Amb a 8.01 and Amb a 8.02), two isoforms of a 2EF-hand CBP (Amb a 9.01 and Amb a 9.02), a new 3EF-hand CBP (Amb a 10) from ragweed pollen and a 2EF-hand CBP from mugwort (Art v 5). All these proteins were expressed in Escherichia coli, purified to homogeneity and characterized by biochemical and immunological means. CONCLUSIONS: The identified proteins are novel pan-allergens and can be used as diagnostic markers for polysensitization and used in component-resolved diagnosis.

Mango profilin: cloning, expression and cross-reactivity with birch pollen profilin Bet v 2.

Mango can cause severe anaphylactic reactions. Profilin has been assumed partly responsible for the cross-reactivity between mango fruit and other allergens but has not been finally clarified. In this study, two isoforms of mango fruits profilin were amplified by RT-PCR and 3'RACE from total RNA. Each mango profilin cDNA includes an open reading frame coding for 131 amino acids. The deduced amino acid sequence of the corresponding protein show high identity with other allergenic profilins. Expression of the recombinant mango profilin was carried out in Escherichia coli BL21(DE3) using vector PET28a and the purification of the recombinant protein was performed via affinity chromatography with Ni+ coupled to sepharose. IgE reactivity of recombinant mango profilin was investigated by immunoblot and 8 of 18 mango-allergic patients tested presented specific IgE-antibodies to recombinant mango profilin. IgE-inhibition and ELISA inhibition experiments were performed to analyze mango profilin cross-reactivity with profilins from birch pollen and high cross-reactivities have been found.