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Jeetain Mittal - One of the best experts on this subject based on the ideXlab platform.

  • computational modeling highlights the role of the disordered formin homology 1 domain in profilin actin transfer
    FEBS Letters, 2018
    Co-Authors: Brandon Gregory Horan, Gül H Zerze, Dimitrios Vavylonis, Youngchan Kim, Jeetain Mittal
    Abstract:

    Formins accelerate actin polymerization, assumed to occur through flexible Formin Homology 1 (FH1) domain-mediated transfer of profilin-actin to the barbed end. To study FH1 properties and address sequence effects, including varying length/distribution of profilin-binding proline-rich motifs, we performed all-atom simulations of a set of representative FH1 domains of formins: mouse mDia1 and mDia2, budding yeast Bni1 and Bnr1, and fission yeast Cdc12, For3, and Fus1. We find FH1 has flexible regions between high-propensity polyproline helix regions. A coarse-grained model retaining sequence specificity, assuming rigid polyproline segments, describes their size. Multiple bound profilins or profilin-actin complexes expand mDia1-FH1, which may be important in cells. Simulations of the barbed end bound to Bni1-FH1-FH2 dimer show that the leading FH1 can better transfer profilin or profilin-actin, with decreasing probability as the distance from FH2 increases.

  • computational modeling highlights disordered formin homology 1 domain s role in profilin actin transfer
    bioRxiv, 2018
    Co-Authors: Brandon Gregory Horan, Gül H Zerze, Dimitrios Vavylonis, Youngchan Kim, Jeetain Mittal
    Abstract:

    Formins accelerate actin polymerization, assumed to occur through flexible FH1 domain mediated transfer of profilin-actin to the barbed end. To study FH1 properties and address sequence effects including varying length/distribution of profilin-binding proline-rich motifs, we performed all-atom simulations of mouse mDia1, mDia2; budding yeast Bni1, Bnr1; fission yeast Cdc12, For3, and Fus1 FH1s. We find FH1 has flexible regions between high propensity polyproline helix regions. A coarse-grained model retaining sequence-specificity, assuming rigid polyproline segments, describes their size. Multiple profilins and profilin-actin complexes can simultaneously bind, expanding mDia1-FH1, which may be important in cells. Simulations of the barbed end bound to Bni1-FH1-FH2 dimer show the leading FH1 can better transfer profilin or profilin-actin, having decreasing probability with increasing distance from FH2.

  • Dataset for: Computational modeling highlights disordered Formin Homology 1 domain's role in profilin-actin transfer
    2018
    Co-Authors: Brandon Gregory Horan, Gül H Zerze, Young C. Kim, Dimitrios Vavylonis, Jeetain Mittal
    Abstract:

    Formins accelerate actin polymerization, assumed to occur through flexible FH1 domain mediated transfer of profilin-actin to the barbed end. To study FH1 properties and address sequence effects including varying length/distribution of profilin-binding proline-rich motifs, we performed all-atom simulations of mouse mDia1, mDia2; budding yeast Bni1, Bnr1; fission yeast Cdc12, For3, and Fus1 FH1s. We find FH1 has flexible regions between high propensity polyproline helix regions. A coarse-grained model retaining sequence-specificity, assuming rigid polyproline segments, describes their size. Multiple bound profilins or profilin-actin complexes expand mDia1-FH1, which may be important in cells. Simulations of the barbed end bound to Bni1-FH1-FH2 dimer show the leading FH1 can better transfer profilin or profilin-actin, having decreasing probability with increasing distance from FH2

Stefan Vieths - One of the best experts on this subject based on the ideXlab platform.

  • germin like protein cit s 1 and profilin cit s 2 are major allergens in orange citrus sinensis fruits
    Molecular Nutrition & Food Research, 2006
    Co-Authors: Jesus F Crespo, Kay Foetisch, Mechthild Retzek, Elena Sierramaestro, Ana B Cidsanchez, C Pascual, Amedeo Conti, Angelica Feliu, Julia Rodriguez, Stefan Vieths
    Abstract:

    Oranges are clinically relevant allergenic foods. To date, orange allergens have not been characterized in detail. The study is aimed at analyzing the sensitization profile in orange-sensitized subjects with and without clinical allergy, and to identify orange allergens. Fifty-six sensitized subjects with self-reported reactions to orange were grouped into reactors (anaphylaxis or multiple episodes of immediate reactions and/or positive challenge tests) and non-reactors (negative open food challenge tests). Allergens were characterized by IgE immunoblotting, N-terminal sequencing, IgE-inhibition assays, and mediator release assays were performed to determine the allergenic potency of orange profilin. Of 56 subjects, 23 were classified as orange allergic showing mainly an oral allergy syndrome. Of 23 subjects classified as orange allergic, 22 were sensitized to profilin, Cit s 2. In patients with mono-sensitization to profilin in vitro histamine releases up to 75% from basophils were induced using orange extract and purified plant profilins. Of the allergic patients 78% were sensitized to germin-like protein, Cit s 1. Both allergens showed retained IgE reactivity in heat-processed orange juice. Interestingly, subjects with and without clinical allergy showed a comparable sensitization profile. Profilin and germin-like proteins are major orange allergens. The potential clinical relevance of orange profilin was indicated by its strong capacity to release histamine from basophils. However, a predominant sensitization to both allergens in subjects without symptoms also indicates a high frequency of clinically insignificant sensitization.

  • tomato profilin lyc e 1 ige cross reactivity and allergenic potency
    Allergy, 2004
    Co-Authors: Sandra Westphal, Stefan Vieths, W Kempf, Kay Foetisch, Mechthild Retzek, Stephan Scheurer
    Abstract:

    Background:  To date, very little data are available about the nature of tomato allergens. Immunoglobulin E (IgE) cross-reactive profilins have been suggested to account for allergic symptoms in patients suffering from tomato allergy. Methods:  The cDNA of tomato profilin was amplified by reversely transcribed polymerase chain reaction (RT-PCR) from total RNA extracted from ripe tomato fruit. The gene was cloned into the pET101D expression plasmid and the protein was produced in Escherichia coli BL21. Purification was performed via poly-l-proline (PLP) affinity chromatography. IgE reactivity of recombinant tomato profilin was investigated by immunoblot and enzyme-linked immunosorbent assay. IgE-inhibition studies were performed to analyse cross-reactivity with other profilins. To determine the allergenic activity of the recombinant protein, basophil histamine release assays using sera of patients with adverse reactions to tomato were performed. Results:  Profilin was identified as a new minor allergen in tomato fruits. The recombinant tomato profilin comprises 131 amino acids and high sequence identity to other allergenic food and pollen profilins. It was shown to be IgE-reactive with a prevalence of 22% (11/50) in tomato-allergic patients. In patients with tomato allergy and multiple sensitization to other foods and birch pollen, IgE directed against tomato profilin showed a strong cross-reactivity with profilins from plant food sources and birch pollen. The tomato profilin was able to induce mediator release from human basophils. Conclusion:  The tomato profilin is a minor allergen in tomato fruit. Thus, it shows biological activity, as confirmed by in vitro histamine release assays with human basophils and thereby has the potential to account for clinical symptoms in tomato-allergic patients.

  • cross reactivity within the profilin panallergen family investigated by comparision of recombinant profilins from pear pyr c 4 cherry pru av 4 and celery api g 4 with birch pollen profilin bet v 2
    Journal of Chromatography B: Biomedical Sciences and Applications, 2001
    Co-Authors: Stephan Scheurer, Andrea Wangorsch, Joerg Nerkamp, Per Stahl Skov, Barbara Ballmerweber, B Wuthrich, D Haustein, Stefan Vieths
    Abstract:

    Profilin is a panallergen which is recognised by IgE from about 20% of birch pollen- and plant food-allergic patients. Little is known about epitope diversity among these homologous proteins, and about the correlation between IgE-cross-reactivity and allergenic reactivity. Plant food profilins from pear (Pyr c 4) and cherry (Pru av 4) were cloned by polymerase chain reaction and produced in Escherichia coli BL21. The profilins were purified as non-fusion proteins by affinity chromatography on poly-(L-proline)-Sepharose and characterized by immunoblotting, IgE-inhibition experiments and histamine release assays. The coding regions of the cDNA of pear and cherry profilin were identified as a 393 bp open reading frame. The deduced amino acid sequences showed high identities with birch pollen profilin Bet v 2 (76-83%) and other allergenic plant profilins. Pyr c 4 and Pru av 4 were investigated for their immunological properties in comparison with profilins from celery (Api g 4) and birch pollen (Bet v 2). Fourty-three of 49 patients (88%), preselected for an IgE-reactivity with Bet v 2 showed specific IgE-antibodies to the recombinant pear protein, 92% of the sera were positive with the recombinant cherry allergen and 80% of the sera were reactive with the celery protein. Inhibition experiments showed a strong cross-reactivity of IgE with profilins from plant food and birch pollen. However, IgE binding profiles also indicated the presence of epitope differences among related profilins. All investigated profilins, Pyr c 4, Pru av 4, Api g 4 and Bet v 2, presented almost identical allergenic properties in cellular mediator release tests. Therefore, cross-reactivities between related profilins may explain pollen-related allergy to food in a minority of patients. The nucleotide sequences reported have been submitted to the Genbank database under accession numbers AF129424 (Pyr c 4) and AF129425 (Pru av 4).

  • patient tailored cloning of allergens by phage display peanut arachis hypogaea profilin a food allergen derived from a rare mrna
    Journal of Chromatography B: Biomedical Sciences and Applications, 2001
    Co-Authors: Tamara Kleberjanke, Stefan Vieths, Reto Crameri, Stephan Scheurer, Wolfmeinhard Becker
    Abstract:

    Abstract A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10 11 cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-( l -proline)-Sepharose and yielded 30 mg l −1 culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.

  • cloning of the minor allergen api g 4 profilin from celery apium graveolens and its cross reactivity with birch pollen profilin bet v 2
    Clinical & Experimental Allergy, 2000
    Co-Authors: Stephan Scheurer, Andrea Wangorsch, D Haustein, Stefan Vieths
    Abstract:

    Background Profilin is a panallergen that is recognized by IgE from about 20% of birch pollen- and plant food-allergic patients. A subgroup of celery-allergic patients shows IgE-reactivity with this minor allergen. To investigate the IgE-binding potential and cross-reactivity of celery profilin at the molecular level, this study was aimed at the cloning and immunological characterization of this allergen. Objectives Cloning, expression and purification of profilin from celery tuber to characterize its immunological properties and its cross-reactivity with birch pollen profilin. Methods Cloning of celery profilin was performed by polymerase chain reaction using degenerated primers and a 5′RACE method for the identification of the unknown 5′-end of the cDNA. Expression was carried out in Escherichia coli BL21 (DE3) using a modified vector pET-30a. The recombinant profilin was purified by affinity chromatography on poly l-proline coupled to sepharose. Immunological characterization was performed by immunoblotting, EAST and IgE-inhibition experiments. Results The coding region of the cDNA of celery profilin was identified as a 399-bp open reading frame, coding for a protein of 133 amino acids with a calculated molecular weight of 14.3 kDa. The deduced amino acid sequence of the corresponding protein showed high identity with other plant profilins (71–82%) recently described as allergens. Celery profilin was isolated as highly pure nonfusion protein. The IgE-reactivity of celery profilin was similar to that of natural protein. Seven of 17 celery-allergic patients tested presented specific IgE-antibodies to the recombinant protein tested by immunoblotting. Inhibition experiments showed high cross-reactivity of IgE with both profilins from celery and birch pollen. Moreover, the biological activity of recombinant celery profilin was demonstrated by a histamine release assay. Conclusions Celery profilin is an important allergenic compound in celery and shows high homology to birch pollen profilin, Bet v 2. According to the revised IUIS allergen nomenclature, we suggest naming the celery profilin Api g 4. In addition to the cross-reacting major allergens Api g 1 and Bet v 1, birch pollinosis and associated allergies to celery can therefore additionally be explained by the cross-reactivity between homologous profilins. Moreover, recombinant Api g 4 may be used for target-specific diagnosis and structural analyses.

Robert D Burke - One of the best experts on this subject based on the ideXlab platform.

  • an ectromelia virus profilin homolog interacts with cellular tropomyosin and viral a type inclusion protein
    Virology Journal, 2007
    Co-Authors: Christine Butlercole, Mary J Wagner, Melissa Da Silva, Gordon D Brown, Robert D Burke
    Abstract:

    Background Profilins are critical to cytoskeletal dynamics in eukaryotes; however, little is known about their viral counterparts. In this study, a poxviral profilin homolog, ectromelia virus strain Moscow gene 141 (ECTV-PH), was investigated by a variety of experimental and bioinformatics techniques to characterize its interactions with cellular and viral proteins.

  • an ectromelia virus profilin homolog interacts with cellular tropomyosin and viral a type inclusion protein
    Virology Journal, 2007
    Co-Authors: Christine Butlercole, Mary J Wagner, Melissa Da Silva, Gordon D Brown, Robert D Burke
    Abstract:

    Profilins are critical to cytoskeletal dynamics in eukaryotes; however, little is known about their viral counterparts. In this study, a poxviral profilin homolog, ectromelia virus strain Moscow gene 141 (ECTV-PH), was investigated by a variety of experimental and bioinformatics techniques to characterize its interactions with cellular and viral proteins. Profilin-like proteins are encoded by all orthopoxviruses sequenced to date, and share over 90% amino acid (aa) identity. Sequence comparisons show highest similarity to mammalian type 1 profilins; however, a conserved 3 aa deletion in mammalian type 3 and poxviral profilins suggests that these homologs may be more closely related. Structural analysis shows that ECTV-PH can be successfully modelled onto both the profilin 1 crystal structure and profilin 3 homology model, though few of the surface residues thought to be required for binding actin, poly(L-proline), and PIP2 are conserved. Immunoprecipitation and mass spectrometry identified two proteins that interact with ECTV-PH within infected cells: alpha-tropomyosin, a 38 kDa cellular actin-binding protein, and the 84 kDa product of vaccinia virus strain Western Reserve (VACV-WR) 148, which is the truncated VACV counterpart of the orthopoxvirus A-type inclusion (ATI) protein. Western and far-western blots demonstrated that the interaction with alpha-tropomyosin is direct, and immunofluorescence experiments suggest that ECTV-PH and alpha-tropomyosin may colocalize to structures that resemble actin tails and cellular protrusions. Sequence comparisons of the poxviral ATI proteins show that although full-length orthologs are only present in cowpox and ectromelia viruses, an ~ 700 aa truncated ATI protein is conserved in over 90% of sequenced orthopoxviruses. Immunofluorescence studies indicate that ECTV-PH localizes to cytoplasmic inclusion bodies formed by both truncated and full-length versions of the viral ATI protein. Furthermore, colocalization of ECTV-PH and truncated ATI protein to protrusions from the cell surface was observed. These results suggest a role for ECTV-PH in intracellular transport of viral proteins or intercellular spread of the virus. Broader implications include better understanding of the virus-host relationship and mechanisms by which cells organize and control the actin cytoskeleton.

Abdolreza Varasteh - One of the best experts on this subject based on the ideXlab platform.

  • 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
    Molecular Biology Reports, 2011
    Co-Authors: Akram Amini, Mojtaba Sankian, Mohammadali Assarehzadegan, Fatemeh Vahedi, Abdolreza Varasteh
    Abstract:

    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.

  • identification of a new allergen from amaranthus retroflexus pollen ama r 2
    Allergology International, 2011
    Co-Authors: Mohsen Tehrani, Mojtaba Sankian, Mohammadali Assarehzadegan, Reza Falak, Reihaneh Noorbakhsh, Maliheh Dadgar Moghadam, Farahzad Jabbari, Abdolreza Varasteh
    Abstract:

    ABSTRACT 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 14.6 kDa 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 IgEreactivity 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.

  • Sequence homology: A poor predictive value for profilins cross-reactivity
    Clinical and Molecular Allergy, 2005
    Co-Authors: Mojtaba Sankian, Nazanin Pazouki, Abdolreza Varasteh, Mahmoud Mahmoudi
    Abstract:

    Background Profilins are highly cross-reactive allergens which bind IgE antibodies of almost 20% of plant-allergic patients. This study is aimed at investigating cross-reactivity of melon profilin with other plant profilins and the role of the linear and conformational epitopes in human IgE cross-reactivity.

Stephan Scheurer - One of the best experts on this subject based on the ideXlab platform.

  • tomato profilin lyc e 1 ige cross reactivity and allergenic potency
    Allergy, 2004
    Co-Authors: Sandra Westphal, Stefan Vieths, W Kempf, Kay Foetisch, Mechthild Retzek, Stephan Scheurer
    Abstract:

    Background:  To date, very little data are available about the nature of tomato allergens. Immunoglobulin E (IgE) cross-reactive profilins have been suggested to account for allergic symptoms in patients suffering from tomato allergy. Methods:  The cDNA of tomato profilin was amplified by reversely transcribed polymerase chain reaction (RT-PCR) from total RNA extracted from ripe tomato fruit. The gene was cloned into the pET101D expression plasmid and the protein was produced in Escherichia coli BL21. Purification was performed via poly-l-proline (PLP) affinity chromatography. IgE reactivity of recombinant tomato profilin was investigated by immunoblot and enzyme-linked immunosorbent assay. IgE-inhibition studies were performed to analyse cross-reactivity with other profilins. To determine the allergenic activity of the recombinant protein, basophil histamine release assays using sera of patients with adverse reactions to tomato were performed. Results:  Profilin was identified as a new minor allergen in tomato fruits. The recombinant tomato profilin comprises 131 amino acids and high sequence identity to other allergenic food and pollen profilins. It was shown to be IgE-reactive with a prevalence of 22% (11/50) in tomato-allergic patients. In patients with tomato allergy and multiple sensitization to other foods and birch pollen, IgE directed against tomato profilin showed a strong cross-reactivity with profilins from plant food sources and birch pollen. The tomato profilin was able to induce mediator release from human basophils. Conclusion:  The tomato profilin is a minor allergen in tomato fruit. Thus, it shows biological activity, as confirmed by in vitro histamine release assays with human basophils and thereby has the potential to account for clinical symptoms in tomato-allergic patients.

  • cross reactivity within the profilin panallergen family investigated by comparision of recombinant profilins from pear pyr c 4 cherry pru av 4 and celery api g 4 with birch pollen profilin bet v 2
    Journal of Chromatography B: Biomedical Sciences and Applications, 2001
    Co-Authors: Stephan Scheurer, Andrea Wangorsch, Joerg Nerkamp, Per Stahl Skov, Barbara Ballmerweber, B Wuthrich, D Haustein, Stefan Vieths
    Abstract:

    Profilin is a panallergen which is recognised by IgE from about 20% of birch pollen- and plant food-allergic patients. Little is known about epitope diversity among these homologous proteins, and about the correlation between IgE-cross-reactivity and allergenic reactivity. Plant food profilins from pear (Pyr c 4) and cherry (Pru av 4) were cloned by polymerase chain reaction and produced in Escherichia coli BL21. The profilins were purified as non-fusion proteins by affinity chromatography on poly-(L-proline)-Sepharose and characterized by immunoblotting, IgE-inhibition experiments and histamine release assays. The coding regions of the cDNA of pear and cherry profilin were identified as a 393 bp open reading frame. The deduced amino acid sequences showed high identities with birch pollen profilin Bet v 2 (76-83%) and other allergenic plant profilins. Pyr c 4 and Pru av 4 were investigated for their immunological properties in comparison with profilins from celery (Api g 4) and birch pollen (Bet v 2). Fourty-three of 49 patients (88%), preselected for an IgE-reactivity with Bet v 2 showed specific IgE-antibodies to the recombinant pear protein, 92% of the sera were positive with the recombinant cherry allergen and 80% of the sera were reactive with the celery protein. Inhibition experiments showed a strong cross-reactivity of IgE with profilins from plant food and birch pollen. However, IgE binding profiles also indicated the presence of epitope differences among related profilins. All investigated profilins, Pyr c 4, Pru av 4, Api g 4 and Bet v 2, presented almost identical allergenic properties in cellular mediator release tests. Therefore, cross-reactivities between related profilins may explain pollen-related allergy to food in a minority of patients. The nucleotide sequences reported have been submitted to the Genbank database under accession numbers AF129424 (Pyr c 4) and AF129425 (Pru av 4).

  • patient tailored cloning of allergens by phage display peanut arachis hypogaea profilin a food allergen derived from a rare mrna
    Journal of Chromatography B: Biomedical Sciences and Applications, 2001
    Co-Authors: Tamara Kleberjanke, Stefan Vieths, Reto Crameri, Stephan Scheurer, Wolfmeinhard Becker
    Abstract:

    Abstract A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10 11 cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-( l -proline)-Sepharose and yielded 30 mg l −1 culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.

  • cloning of the minor allergen api g 4 profilin from celery apium graveolens and its cross reactivity with birch pollen profilin bet v 2
    Clinical & Experimental Allergy, 2000
    Co-Authors: Stephan Scheurer, Andrea Wangorsch, D Haustein, Stefan Vieths
    Abstract:

    Background Profilin is a panallergen that is recognized by IgE from about 20% of birch pollen- and plant food-allergic patients. A subgroup of celery-allergic patients shows IgE-reactivity with this minor allergen. To investigate the IgE-binding potential and cross-reactivity of celery profilin at the molecular level, this study was aimed at the cloning and immunological characterization of this allergen. Objectives Cloning, expression and purification of profilin from celery tuber to characterize its immunological properties and its cross-reactivity with birch pollen profilin. Methods Cloning of celery profilin was performed by polymerase chain reaction using degenerated primers and a 5′RACE method for the identification of the unknown 5′-end of the cDNA. Expression was carried out in Escherichia coli BL21 (DE3) using a modified vector pET-30a. The recombinant profilin was purified by affinity chromatography on poly l-proline coupled to sepharose. Immunological characterization was performed by immunoblotting, EAST and IgE-inhibition experiments. Results The coding region of the cDNA of celery profilin was identified as a 399-bp open reading frame, coding for a protein of 133 amino acids with a calculated molecular weight of 14.3 kDa. The deduced amino acid sequence of the corresponding protein showed high identity with other plant profilins (71–82%) recently described as allergens. Celery profilin was isolated as highly pure nonfusion protein. The IgE-reactivity of celery profilin was similar to that of natural protein. Seven of 17 celery-allergic patients tested presented specific IgE-antibodies to the recombinant protein tested by immunoblotting. Inhibition experiments showed high cross-reactivity of IgE with both profilins from celery and birch pollen. Moreover, the biological activity of recombinant celery profilin was demonstrated by a histamine release assay. Conclusions Celery profilin is an important allergenic compound in celery and shows high homology to birch pollen profilin, Bet v 2. According to the revised IUIS allergen nomenclature, we suggest naming the celery profilin Api g 4. In addition to the cross-reacting major allergens Api g 1 and Bet v 1, birch pollinosis and associated allergies to celery can therefore additionally be explained by the cross-reactivity between homologous profilins. Moreover, recombinant Api g 4 may be used for target-specific diagnosis and structural analyses.