Vicilin

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Yuzhu Zhang - One of the best experts on this subject based on the ideXlab platform.

  • almond prunus dulcis allergen pru du 8 the first member of a new family of food allergens
    Journal of Agricultural and Food Chemistry, 2019
    Co-Authors: Huilian Che, Yuzhu Zhang, Shu-chen Lyu, Kari C. Nadeau, Songsong Jiang, Tengchuan Jin, Tara H. Mchugh
    Abstract:

    An almond allergen with two known short peptide sequences was reported as the almond 2S albumin but was later suspected to be almond Vicilin. However, this allergen was not designated by the World Health Organization/International Union of Immunological Societies. This study aimed to determine the true identity of this elusive almond allergen. cDNAs were synthesized from total RNA of the Nonpareil almond. The complete sequence of the previously reported almond allergen was determined from its coding sequence. The deduced protein was produced recombinantly and was confirmed to be a food allergen by testing with 18 almond-allergic sera. The allergen is a potential cysteine-rich antimicrobial protein with characteristic C[X]3C-[X]10-12-C[X]3C motifs of the hairpinin antimicrobial protein. This first member of a novel family of food allergens was named Pru du 8. The signature motif of the hairpinin antimicrobial protein can be found in the N-terminal region of some Vicilin allergens (e.g., Ara h 1). It can also be found in the signal peptide of other Vicilin allergens (e.g., Car i 2). In many species, however, Vicilins do not contain such a motif, indicating that the presence of the signature motifs of the hairpinin antimicrobial protein in Vicilins might be a result of translocation during evolution.

  • Identification of Almond (Prunus dulcis) Vicilin As a Food Allergen
    Journal of agricultural and food chemistry, 2018
    Co-Authors: Huilian Che, Yuzhu Zhang, Shu-chen Lyu, Kari C. Nadeau, Tara H. Mchugh
    Abstract:

    Almond is one of the tree nuts listed by U.S. FDA as a food allergen source. A food allergen identified with patient sera has been debated to be the 2S albumin or the 7S Vicilin. However, neither of these proteins has been defined as a food allergen. The purpose of this study was to clone, express, and purify almond Vicilin and test whether it is a food allergen. Western blot experiment was performed with 18 individual sera from patients with double-blind, placebo-controlled clinical almond allergy. The results showed that 44% of the sera contained IgE antibodies that recognized the recombinant almond Vicilin, indicating that it is an almond allergen. Identifying this and additional almond allergens will facilitate the understanding of the allergenicity of seed proteins in tree nuts and their cross-reactivity.

  • Identification of Almond (Prunus dulcis) Vicilin As a Food Allergen
    2018
    Co-Authors: Huilian Che, Yuzhu Zhang, Shu-chen Lyu, Kari C. Nadeau, Tara Mchugh
    Abstract:

    Almond is one of the tree nuts listed by U.S. FDA as a food allergen source. A food allergen identified with patient sera has been debated to be the 2S albumin or the 7S Vicilin. However, neither of these proteins has been defined as a food allergen. The purpose of this study was to clone, express, and purify almond Vicilin and test whether it is a food allergen. Western blot experiment was performed with 18 individual sera from patients with double-blind, placebo-controlled clinical almond allergy. The results showed that 44% of the sera contained IgE antibodies that recognized the recombinant almond Vicilin, indicating that it is an almond allergen. Identifying this and additional almond allergens will facilitate the understanding of the allergenicity of seed proteins in tree nuts and their cross-reactivity

  • identification characterization and initial epitope mapping of pine nut allergen pin k 2
    Food Research International, 2016
    Co-Authors: Yuzhu Zhang, Kari C. Nadeau, Wenxian Du, Jiang Yi, Tara H. Mchugh
    Abstract:

    Abstract The aims of this study were to predict, identify and characterize pine nut allergens. Korean pine ( Pinus koraiensis ) Vicilin was predicted to be a pine nut allergen. Recombinant Korean pine Vicilin was expressed in E. coli and purified. Natural Korean pine Vicilin isolated from pine nuts (which displayed multiple bands in SDS-gels due to posttranslational digestion) and its full length recombinant counterpart were used to test whether it is a food allergen. The recognition of the protein (and its fragments) by patient serum IgE was analyzed by Western blot. The study included fourteen patients diagnosed with clinical pine nut allergy. Twenty nine percent of the patient sera recognized both the natural and recombinant pine nut Vicilin, indicating that Korean pine Vicilin is a bona fide food allergen. The serum recognition patterns of the naturally occurring protein fragments suggested that some of linear IgE epitopes may be mapped to the fragment boundaries. The chemical and thermal stability of the recombinant protein was investigated. It underwent a thermal transition with a Tm = 76.6 °C. The transition was accompanied by an increase in the amplitude of the circular dichroism signal at 220 nm. Urea induced unfolding of the recombinant protein had a Cm of 4.6 M.

  • identification and characterization of a new pecan carya illinoinensis wangenh k koch allergen car i 2
    Journal of Agricultural and Food Chemistry, 2016
    Co-Authors: Yuzhu Zhang, Kari C. Nadeau, L.j. Grauke, Wenxian Du, Yan Zhang, Shuo Wang, Jiang Yi, Tara H. Mchugh
    Abstract:

    The 7S Vicilin and 11S legumin seed storage globulins belong to the cupin protein superfamily and are major food allergens in many foods from the “big eight” food allergen groups. Here, for the first time, pecan Vicilin was found to be a food allergen. Western blot experiments revealed that 30% of 27 sera used in this study and 24% of the sera from 25 patients with double-blind, placebo controlled clinical pecan allergy contained IgE antibodies specific to pecan Vicilin. This allergen consists of a low-complexity region at its N-terminal and a structured domain at the C-terminal that contains two cupin motifs and forms homotrimers. The crystal structure of recombinant pecan Vicilin was determined. The refined structure gave R/Rfree values of 0.218/0.262 for all data to 2.65 A. There were two trimeric biological units in the crystallographic asymmetric unit. Pecan Vicilin is also a copper protein. These data may facilitate the understanding of the nutritional value and the allergenicity relevance of the co...

Tara H. Mchugh - One of the best experts on this subject based on the ideXlab platform.

  • almond prunus dulcis allergen pru du 8 the first member of a new family of food allergens
    Journal of Agricultural and Food Chemistry, 2019
    Co-Authors: Huilian Che, Yuzhu Zhang, Shu-chen Lyu, Kari C. Nadeau, Songsong Jiang, Tengchuan Jin, Tara H. Mchugh
    Abstract:

    An almond allergen with two known short peptide sequences was reported as the almond 2S albumin but was later suspected to be almond Vicilin. However, this allergen was not designated by the World Health Organization/International Union of Immunological Societies. This study aimed to determine the true identity of this elusive almond allergen. cDNAs were synthesized from total RNA of the Nonpareil almond. The complete sequence of the previously reported almond allergen was determined from its coding sequence. The deduced protein was produced recombinantly and was confirmed to be a food allergen by testing with 18 almond-allergic sera. The allergen is a potential cysteine-rich antimicrobial protein with characteristic C[X]3C-[X]10-12-C[X]3C motifs of the hairpinin antimicrobial protein. This first member of a novel family of food allergens was named Pru du 8. The signature motif of the hairpinin antimicrobial protein can be found in the N-terminal region of some Vicilin allergens (e.g., Ara h 1). It can also be found in the signal peptide of other Vicilin allergens (e.g., Car i 2). In many species, however, Vicilins do not contain such a motif, indicating that the presence of the signature motifs of the hairpinin antimicrobial protein in Vicilins might be a result of translocation during evolution.

  • Identification of Almond (Prunus dulcis) Vicilin As a Food Allergen
    Journal of agricultural and food chemistry, 2018
    Co-Authors: Huilian Che, Yuzhu Zhang, Shu-chen Lyu, Kari C. Nadeau, Tara H. Mchugh
    Abstract:

    Almond is one of the tree nuts listed by U.S. FDA as a food allergen source. A food allergen identified with patient sera has been debated to be the 2S albumin or the 7S Vicilin. However, neither of these proteins has been defined as a food allergen. The purpose of this study was to clone, express, and purify almond Vicilin and test whether it is a food allergen. Western blot experiment was performed with 18 individual sera from patients with double-blind, placebo-controlled clinical almond allergy. The results showed that 44% of the sera contained IgE antibodies that recognized the recombinant almond Vicilin, indicating that it is an almond allergen. Identifying this and additional almond allergens will facilitate the understanding of the allergenicity of seed proteins in tree nuts and their cross-reactivity.

  • identification characterization and initial epitope mapping of pine nut allergen pin k 2
    Food Research International, 2016
    Co-Authors: Yuzhu Zhang, Kari C. Nadeau, Wenxian Du, Jiang Yi, Tara H. Mchugh
    Abstract:

    Abstract The aims of this study were to predict, identify and characterize pine nut allergens. Korean pine ( Pinus koraiensis ) Vicilin was predicted to be a pine nut allergen. Recombinant Korean pine Vicilin was expressed in E. coli and purified. Natural Korean pine Vicilin isolated from pine nuts (which displayed multiple bands in SDS-gels due to posttranslational digestion) and its full length recombinant counterpart were used to test whether it is a food allergen. The recognition of the protein (and its fragments) by patient serum IgE was analyzed by Western blot. The study included fourteen patients diagnosed with clinical pine nut allergy. Twenty nine percent of the patient sera recognized both the natural and recombinant pine nut Vicilin, indicating that Korean pine Vicilin is a bona fide food allergen. The serum recognition patterns of the naturally occurring protein fragments suggested that some of linear IgE epitopes may be mapped to the fragment boundaries. The chemical and thermal stability of the recombinant protein was investigated. It underwent a thermal transition with a Tm = 76.6 °C. The transition was accompanied by an increase in the amplitude of the circular dichroism signal at 220 nm. Urea induced unfolding of the recombinant protein had a Cm of 4.6 M.

  • identification and characterization of a new pecan carya illinoinensis wangenh k koch allergen car i 2
    Journal of Agricultural and Food Chemistry, 2016
    Co-Authors: Yuzhu Zhang, Kari C. Nadeau, L.j. Grauke, Wenxian Du, Yan Zhang, Shuo Wang, Jiang Yi, Tara H. Mchugh
    Abstract:

    The 7S Vicilin and 11S legumin seed storage globulins belong to the cupin protein superfamily and are major food allergens in many foods from the “big eight” food allergen groups. Here, for the first time, pecan Vicilin was found to be a food allergen. Western blot experiments revealed that 30% of 27 sera used in this study and 24% of the sera from 25 patients with double-blind, placebo controlled clinical pecan allergy contained IgE antibodies specific to pecan Vicilin. This allergen consists of a low-complexity region at its N-terminal and a structured domain at the C-terminal that contains two cupin motifs and forms homotrimers. The crystal structure of recombinant pecan Vicilin was determined. The refined structure gave R/Rfree values of 0.218/0.262 for all data to 2.65 A. There were two trimeric biological units in the crystallographic asymmetric unit. Pecan Vicilin is also a copper protein. These data may facilitate the understanding of the nutritional value and the allergenicity relevance of the co...

  • expression purification and crystallization of pecan carya illinoinensis Vicilin
    Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2014
    Co-Authors: Boram Lee, L.j. Grauke, Tara H. Mchugh, Renhao Zhang, Yuzhu Zhang
    Abstract:

    Tree nuts are responsible for many cases of severe food allergies. The 7S seed storage protein Vicilin has been identified as a food allergen in many kinds of tree nuts. The Vicilin protein consists of an N-terminal low-complexity region with antimicrobial activity and a C-terminal domain that forms a trimeric structure that belongs to the cupin superfamily. In this study, Vicilin from pecan (Carya illinoinensis) was isolated and was expressed in bacteria for the first time. The cupin structural core of the protein, residues 369–792, was purified by metal-affinity and gel-filtration chromatography to high purity. Vicilin crystals were obtained and the best crystal diffracted to 2.65 A resolution in space group P212121.

Jose Xavierfilho - One of the best experts on this subject based on the ideXlab platform.

  • binding of vigna unguiculata Vicilins to the peritrophic membrane of tenebrio molitor affects larval development
    Entomologia Experimentalis Et Applicata, 2008
    Co-Authors: E V Paes, Adriana F Uchoa, Carlos P Silva, Marcio Dos Santos Teixeira Pinto, Katia Valevski Sales Fernandes, Antonia Elenir A Oliveira, Jose Xavierfilho
    Abstract:

    We investigated the effects of Vicilins (7S storage proteins) from Vigna unguiculata (L.) Walp. (Fabaceae), cultivars EPACE-10 [genotype susceptible to the cowpea weevil, Callosobruchus maculatus (Fabricius)] and IT81D-1045 [cowpea weevil-resistant genotype], seeds on Tenebrio molitor L. (Coleoptera: Tenebrionidae) larval development. Toxicity of Vicilins was investigated through the incorporation of these proteins in artificial diet offered to the larvae. Binding tests of Vicilins to the peritrophic membranes (PM) were carried out by in vitro incubation of PM with solutions of Vicilins. Bound proteins were desorbed from PM with 100 m m HCl. Desorbed Vicilins were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by immunoprobing on Western blotting using an anti-Vicilin cv. EPACE-10 antibody. The chitin content of the T. molitor PM was evaluated by the Von Wisselingh color test and presence of chitin in the larval PM was confirmed. Bioassays showed that both Vicilins from EPACE-10 and IT81D-1045 genotypes were toxic to T. molitor larvae, and in vitro binding assays showed that these seed-storage proteins were capable of binding to the larval PM.

  • performance of bean bruchids callosobruchus maculatus and zabrotes subfasciatus coleoptera bruchidae reared on resistant it81d 1045 and susceptible epace 10 vigna unguiculata seeds relationship with trypsin inhibitor and Vicilin excretion
    Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2005
    Co-Authors: Mauricio P Sales, Katia Valevski Sales Fernandes, Adeliana S Oliveira, L B S Andrade, M B Ary, Maria Raquel Alcântara De Miranda, Fabiano M Teixeira, Jose Xavierfilho
    Abstract:

    Abstract Callosobruchus maculatus ( Cm ) and Zabrotes subfasciatus ( Zs ) were reared on resistant (IT81D-1045) and on susceptible (Epace 10) cowpea seeds. The emergence of adult insects, total developmental period (TDP) and excretion of trypsin inhibitor and Vicilin were determined for both bruchid populations. Parameter evaluation showed that the Zs populations emerged from both seeds had no significant differences in emergence and TDP. The Cm population raised from resistant seeds had lower emergence (5.6 ± 1.3%) and delayed TDP (46 ± 1.25 days) than those emerged from susceptible seeds. The excretion of defense proteins showed that Zs reared in resistant seeds excreted 1.7 times more trypsin inhibitor, but this did not affect emergence or TDP. Furthermore, Cm population emerged from resistant seeds excreted 7 times higher Vicilin and 0.4 times less trypsin inhibitor than that emerged from susceptible seeds. These results indicate that Vicilins from resistant seeds are involved to significantly longer TDP (46 days) and also drastic reduction of insect emergence (∼5%) of C. maculatus .

  • cowpea vigna unguiculata Vicilins bind to the peritrophic membrane of larval sugarcane stalk borer diatraea saccharalis
    Journal of Insect Physiology, 2003
    Co-Authors: A C Mota, Carlos P Silva, Renato A Damatta, Lima M Filho, Jose Xavierfilho
    Abstract:

    Abstract In this work, we show that Vicilins from two Vigna unguiculata (cowpea) genotypes, Epace-10 and IT 81D-1045, which are susceptible and resistant to attack by the cowpea weevil Callosobruchus maculatus , respectively, associate with the peritrophic membrane (PM) from larvae of Diatraea saccharalis. Solutions with increasing concentrations of Vicilins were incubated with PM of the larvae and subsequently analysed by electrophoresis with SDS. It was observed that the majority of the bands of approximately 50,000 Da (characteristic of Vicilins) did not appear in the separating gel and only lower molecular weight polypeptides were seen. When Vicilins were incubated with PM, and the solution was then heated after the incubation, the band pattern in the gel appeared completely different. It was observed that the Vicilins were being hydrolysed by proteinases associated with the PM. When the incubated samples were heated after the reaction, the major bands reappeared, demonstrating that most of the Vicilin molecules had bound to the PM of D. saccharalis . These results suggest that when the Vicilins are in contact with the PM they are bound and also digested by the PM of this insect. The major and several minor proteinases from the PM were extracted with Triton X-100 and their activity and the inhibition of this activity were analysed by in gel assays. Based on the effects of proteinase inhibitors, the PM-associated activity is due to serine class proteinases. Larvae of D. saccharalis were fed on artificial diets containing purified Vicilins from Epace-10 or IT 81D-1045 seeds. Vicilins from Epace-10 did not affect the larval development, while IT 81D-1045 Vicilins reduced significantly the survival rate of the sugar cane borer.

  • effect of sugars on the association between cowpea Vicilin 7s storage proteins and fungal cells
    Biocell, 2003
    Co-Authors: Tatiana L Rose, Katia Valevski Sales Fernandes, V M Gomes, M Da Cunha, Jose Xavierfilho
    Abstract:

    Vicilins (7S storage proteins) found in various legume seeds have been previously shown to interfere with the germination of spores or conidia of phytopathogenic fungi and inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. In the present work Vicilins from cowpea (Vigna unguiculata) seeds were added to the growth medium of Saccharomyces cerevisiae cells and Fusarium oxysporum conidia. Helix pomatia lectin, wheat germ agglutinin and Ulex europaeus lectin were used to identify differences in the binding of the Vicilins to the surface of cells of S. cerevisiae and F. oxysporum treated with this protein. After the growth period, the material in suspension (yeast cells) was centrifuged and the final pellet was also treated with different sugar (glucose, sucrose, glucosamine, N-acetyl-glucosamine) concentrations and 0.1 M HCl for extraction of Vicilins associated to chitinous structures present in yeast cells. Our results showed that Vicilin sub-units were present in the different sugar extracts of yeast cells pretreated with the Vicilins and these proteins were eluted by 0.5 M solutions of sugars in the following order of efficiency of elution: N-acetyl-glucosamine, sucrose/glucose and glucosamine.

  • Vicilin storage proteins from vigna unguiculata legume seeds inhibit fungal growth
    Journal of Agricultural and Food Chemistry, 1997
    Co-Authors: Valdirene Moreira Gomes, Katia Valevski Sales Fernandes, Mariaisabel Mosqueda, Alejandro Blancolabra, Mauricio P Sales, Rossana De Aguiar Cordeiro, Jose Xavierfilho
    Abstract:

    Vicilin storage proteins (7S globulins) isolated from Vigna unguiculata (cowpea) seeds were shown to interfere with the germination of spores or conidia of the fungi Fusarium solani, Fusarium oxysporum, Colletotrichum musae, Phytophtora capsici, Neurospora crassa, and Ustilago maydis sporidia. Cowpea Vicilins have been shown to bind to fungal structures, possibly chitin-containing structures of the cell wall, and can be desorbed by strong acid. The results presented in this paper are in agreement with data previously obtained on the chitin-binding properties of cowpea Vicilins and the effect they exert on the development and survival of the storage pest insect Callosobruchus maculatus (Coleoptera: Bruchidae).

Carlos P Silva - One of the best experts on this subject based on the ideXlab platform.

  • phaseolin ingestion affects vesicular traffic causing oxidative stress in the midgut of callosobruchus maculatus larvae
    Comparative Biochemistry and Physiology B, 2019
    Co-Authors: Daniele Kunz, Richard Ian Samuels, Maria Ligia Rodrigues Macedo, Gabriel B Oliveira, Theo C Brascher, Luiz Felipe De Souza, Alcir Luiz Dafre, Carlos P Silva
    Abstract:

    Abstract It has been reported that phaseolin, the major storage globulin of the common bean (Phaseolus vulgaris), is toxic to Callosobruchus maculatus larvae, an Old World bruchid beetle that is not capable of infesting this New World edible bean. It has also been demonstrated that Vicilin, the major storage globulin found in cowpea (Vigna unguiculata) seeds, is absorbed through receptor-mediated endocytosis in the insect midgut. A putative Vicilin receptor has been purified and showed high homology to α-tocopherol transfer protein. However, the ingestion of a variant Vicilin purified from C. maculatus resistant seeds inhibits transcytosis, resulting in the accumulation of Vicilins in the midgut cells and ultimately antibiosis. In the present work, we studied the cellular up-take of phaseolin in C. maculatus larvae with the aim of discovering if this protein is also capable of inhibiting endocytic traffic in the enterocytes. FITC-labelled Vicilin and FITC-labelled phaseolin were incorporated into the diet of the larvae at a physiological concentration of 0.5% w/w. The fate of labelled and non-labelled globulins was monitored by confocal microscopy. Here we demonstrated that phaseolin is also endocytosed by enterocytes causing an accumulation of endocytic vesicles in the midgut when compared to the ingestion of Vicilin obtained from a susceptible V. unguiculata cultivar. From the results obtained for HNE, MDA and TBARS, a pro-oxidative scenario was established in the intestinal epithelial cells of the larvae, which may explain the deleterious effect observed in larvae developing inside P. vulgaris seeds.

  • receptor mediated endocytosis of Vicilin in callosobruchus maculatus coleoptera chrysomelidae larval midgut epithelial cells
    Comparative Biochemistry and Physiology B, 2017
    Co-Authors: Daniele Kunz, Adriana F Uchoa, Maria Ligia Rodrigues Macedo, Gabriel B Oliveira, Richard Samuels, Carlos P Silva
    Abstract:

    The transport of proteins across the intestinal epithelium of insects is still not well understood. There is evidence that Vicilin, a major storage protein of cowpea seeds (Vigna unguiculata), is internalized in larvae of the seed-beetle Callosobruchus maculatus. It has been reported that this Vicilin interacts with proteins present in the microvillar membranes of columnar cells along the digestive tract of the larvae. In the present work, we studied the cellular pathway involved in endocytosis of Vicilin in larval C. maculatus by employing ex vivo experiments. In the ex vivo approach, we incubated FITC-labelled Vicilin with isolated midgut wholemounts in the absence or in the presence of endocytosis inhibitors. The fate of labelled or non-labelled globulins was monitored by confocal microscopy and fluorescence measurement. Our results suggest that the internalization of Vicilins is due to receptor-mediated endocytosis. Here we report the identity of a microvillar Vicilin-binding protein that was purified using affinity chromatography on a Vicilin-sepharose column. The putative Vicilin receptor showed high homology to proteins with the CRAL-TRIO domain, specifically the Sec14 superfamily member α-tocopherol transfer protein. The precise mechanism involved in Vicilin internalization was defined through the use of specific inhibitors of the endocytosis pathway. The inhibitors filipin III and nystatin significantly inhibited the endocytosis of Vicilin, while chlorpromazine and phenylarsine oxide had a much lower effect on endocytosis, suggesting that the endocytic pathway is predominantly mediated by caveolin.

  • the fate of Vicilins 7s storage globulins in larvae and adult callosobruchus maculatus coleoptera chrysomelidae bruchinae
    Journal of Insect Physiology, 2010
    Co-Authors: Sheila M Souza, Adriana F Uchoa, Jose R Silva, Richard Ian Samuels, Antonia E A Oliveira, Eliana De Medeiros Oliveira, Ricardo T Linhares, Daniel Alexandre, Carlos P Silva
    Abstract:

    Abstract The fate of Vicilins ingested by Callosobruchus maculatus and the physiological importance of these proteins in larvae and adults were investigated. Vicilins were quantified by ELISA in the haemolymph and fat body during larval development (2nd to 4th instars), in pupae and adults, as well as in ovaries and eggs. Western blot analysis demonstrated that the majority of absorbed Vicilins were degraded in the fat body. Tracing the fate of Vicilins using FITC revealed that the FITC–Vicilin complex was present inside cells of the fat body of the larvae and in the fat bodies of both male and female adult C. maculatus. Labelled Vicilin was also detected in ovocytes and eggs. Based on the results presented here, we propose that following absorption, Vicilins accumulate in the fat body, where they are partially degraded. These peptides are retained throughout the development of the insects and eventually are sequestered by the eggs. It is possible that accumulation in the eggs is a defensive strategy against pathogen attack as these peptides are known to have antimicrobial activity. Quantifications performed on internal organs from larvae of C. maculatus exposed to extremely dry seeds demonstrated that the Vicilin concentration in the haemolymph and fat body was significantly higher when compared to larvae fed on control seeds. These results suggest that absorbed Vicilins may also be involved in the survival of larvae in dry environments.

  • binding of vigna unguiculata Vicilins to the peritrophic membrane of tenebrio molitor affects larval development
    Entomologia Experimentalis Et Applicata, 2008
    Co-Authors: E V Paes, Adriana F Uchoa, Carlos P Silva, Marcio Dos Santos Teixeira Pinto, Katia Valevski Sales Fernandes, Antonia Elenir A Oliveira, Jose Xavierfilho
    Abstract:

    We investigated the effects of Vicilins (7S storage proteins) from Vigna unguiculata (L.) Walp. (Fabaceae), cultivars EPACE-10 [genotype susceptible to the cowpea weevil, Callosobruchus maculatus (Fabricius)] and IT81D-1045 [cowpea weevil-resistant genotype], seeds on Tenebrio molitor L. (Coleoptera: Tenebrionidae) larval development. Toxicity of Vicilins was investigated through the incorporation of these proteins in artificial diet offered to the larvae. Binding tests of Vicilins to the peritrophic membranes (PM) were carried out by in vitro incubation of PM with solutions of Vicilins. Bound proteins were desorbed from PM with 100 m m HCl. Desorbed Vicilins were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by immunoprobing on Western blotting using an anti-Vicilin cv. EPACE-10 antibody. The chitin content of the T. molitor PM was evaluated by the Von Wisselingh color test and presence of chitin in the larval PM was confirmed. Bioassays showed that both Vicilins from EPACE-10 and IT81D-1045 genotypes were toxic to T. molitor larvae, and in vitro binding assays showed that these seed-storage proteins were capable of binding to the larval PM.

  • cowpea vigna unguiculata Vicilins bind to the peritrophic membrane of larval sugarcane stalk borer diatraea saccharalis
    Journal of Insect Physiology, 2003
    Co-Authors: A C Mota, Carlos P Silva, Renato A Damatta, Lima M Filho, Jose Xavierfilho
    Abstract:

    Abstract In this work, we show that Vicilins from two Vigna unguiculata (cowpea) genotypes, Epace-10 and IT 81D-1045, which are susceptible and resistant to attack by the cowpea weevil Callosobruchus maculatus , respectively, associate with the peritrophic membrane (PM) from larvae of Diatraea saccharalis. Solutions with increasing concentrations of Vicilins were incubated with PM of the larvae and subsequently analysed by electrophoresis with SDS. It was observed that the majority of the bands of approximately 50,000 Da (characteristic of Vicilins) did not appear in the separating gel and only lower molecular weight polypeptides were seen. When Vicilins were incubated with PM, and the solution was then heated after the incubation, the band pattern in the gel appeared completely different. It was observed that the Vicilins were being hydrolysed by proteinases associated with the PM. When the incubated samples were heated after the reaction, the major bands reappeared, demonstrating that most of the Vicilin molecules had bound to the PM of D. saccharalis . These results suggest that when the Vicilins are in contact with the PM they are bound and also digested by the PM of this insect. The major and several minor proteinases from the PM were extracted with Triton X-100 and their activity and the inhibition of this activity were analysed by in gel assays. Based on the effects of proteinase inhibitors, the PM-associated activity is due to serine class proteinases. Larvae of D. saccharalis were fed on artificial diets containing purified Vicilins from Epace-10 or IT 81D-1045 seeds. Vicilins from Epace-10 did not affect the larval development, while IT 81D-1045 Vicilins reduced significantly the survival rate of the sugar cane borer.

Sergio T Ferreira - One of the best experts on this subject based on the ideXlab platform.

  • functional properties of purified Vicilins from cowpea vigna unguiculata and pea pisum sativum and cowpea protein isolate
    Journal of Agricultural and Food Chemistry, 2003
    Co-Authors: Alessandra Rangel, Gilberto B Domont, Cristiana Pedrosa, Sergio T Ferreira
    Abstract:

    The major storage globulins (Vicilins) of cowpea (Vigna unguiculata) and pea (Pisum sativum) seeds were purified by ammonium sulfate precipitation, and a semipurified cowpea protein isolate (CPI) was prepared by isoelectric precipitation. Some of the functional properties of these proteins, including solubility, foaming, and emulsifying capacities, were investigated and compared. The solubility of purified cowpea Vicilin was reduced at pH 5.0, increasing markedly below and above this value. Pea Vicilin exhibited poor solubility between pH 5.0 and pH 6.0, and CPI was little soluble in the pH range from 4.0 to 6.0. At neutral pH, the emulsifying activity indexes (EAI) of purified pea Vicilin and CPI were 194 and 291 m2/g, respectively, which compare quite favorably to EAIs of 110 and 133 m2/g for casein and albumin, respectively. Remarkably, purified cowpea Vicilin exhibited an EAI of 490 m2/g, indicating a very high emulsifying activity. Purified cowpea and pea Vicilins exhibited lower foaming capacities a...

  • selective neoglycosylation increases the structural stability of Vicilin the 7s storage globulin from pea seeds
    Archives of Biochemistry and Biophysics, 2000
    Co-Authors: Cristiana Pedrosa, Fernanda G De Felice, Cristina Trisciuzzi, Sergio T Ferreira
    Abstract:

    Abstract The effects of glycosylation on the stability and subunit interactions of Vicilin, the major storage protein in pea seeds, were investigated. Glycosylated Vicilin derivatives were prepared by alkylation of lysine ϵ-amino groups with various carbohydrates. Average modification levels of 13.4 ± 3.0, 11.1 ± 3.6, 7.5 ± 4.2, and 4.7 ± 0.3 moles of carbohydrate/mol of Vicilin were obtained with glucose, galactose, galacturonic acid, and lactose, respectively. Nondenaturing polyacrylamide gel electrophoresis and size-exclusion chromatography indicated that the quaternary structure and hydrodynamic radius of Vicilin were not affected by glycosylation at the levels used. We have previously shown that application of hydrostatic pressure causes dissociation of Vicilin subunits [C. Pedrosa and S. T. Ferreira (1994) Biochemistry 33, 4046–4055]. Analysis of pressure dissociation data allowed determination of the Gibbs free energy change (Δ G diss ) and molar volume change (Δ V diss ) of dissociation of Vicilin subunits. For unmodified Vicilin, Δ G diss = 18.2 kcal/mol and Δ V diss = −102 ml/mol. Glycosylated Vicilin derivatives were significantly stabilized against subunit dissociation, with Δ G diss of 19.4, 19.2, 20.6, and 22.1 kcal/mol for glucose, galactose, lactose, and galacturonic acid derivatives, respectively. No changes in Δ V diss were found for the glucose and galactose derivatives, whereas Δ V diss of −128 and −135 ml/mol, respectively, were found for the lactose and galacturonic acid derivatives. The glycosylated derivatives also appeared more resistant to unfolding by guanidine hydrochloride than unmodified Vicilin. Intrinsic fluorescence lifetime measurements showed that glycosylation caused a significant increase in heterogeneity of the fluorescence decay, possibly reflecting increased conformational heterogeneity of glycosylated derivatives relative to unmodified Vicilin. These results indicate that the stability and subunit interactions of Vicilin may be modulated by mild, selective glycosylation at low modification levels, an effect that may be of interest in the study of other oligomeric proteins.

  • effects of glycosylation on functional properties of Vicilin the 7s storage globulin from pea pisum sativum
    Journal of Agricultural and Food Chemistry, 1997
    Co-Authors: Cristiana Pedrosa, Cristina Trisciuzzi, Sergio T Ferreira
    Abstract:

    Functional properties (solubility, foaming, and emulsifying capacities) of neoglycosylated derivatives of pea Vicilin were investigated and compared with unmodified Vicilin. Glysosylation was achieved by reductive alkylation of e-amino groups of lysine residues using different carbohydrates (lactose, galactose, glucose, or galacturonic acid). Levels of carbohydrate incorporation ranged from 5 to 13 mol of carbohydrate/mol of Vicilin. Solubilities of glycosylated derivatives (notably derivatives modified with glucose or galacturonic acid) were significantly improved in the pH range 5−6 compared to unmodified Vicilin. Emulsifying capacity and emulsion stability were also significantly increased in glycosylated Vicilin derivatives. Modification with galacturonic acid was the most effective in increasing both emulsifying capacity and stability. Derivatives containing lactose or galactose displayed enhanced foaming capacity relative to unmodified Vicilin. However, foam stability was found to decrease by glycos...

  • deterministic pressure induced dissociation of Vicilin the 7s storage globulin from pea seeds effects of ph and cosolvents on oligomer stability
    Biochemistry, 1994
    Co-Authors: Cristiana Pedrosa, Sergio T Ferreira
    Abstract:

    A thermodynamic characterization of subunit association of Vicilin, a storage protein from pea seeds, was performed using a combination of hydrostatic pressure and fluorescence spectroscopy. Application of pressure up to 2.4 kbar caused dissociation of Vicilin subunits, as revealed by (1) size-exclusion PPLC of pressurized samples, (2) fluorescence anisotropy measurements of a dansyl-Vicilin conjugate under pressure, and (3) quenching of the intrinsic fluorescence of Vicilin. Pressure dissociation data were well described by a model for dissociation of a trimer. This enabled calculation of the standard molar volume change of association (delta Vo) and the equilibrium dissociation constant at atmospheric pressure (Ko); at pH 10 these were found to be delta Vo = 146 mL/mol and Ko = 2.2 x 10(-15) M2, respectively, corresponding to C1/2 (the concentration of protein at 50% dissociation at atmospheric pressure) = 18 nM and a stabilization free energy of -19.6 kcal/mol for the oligomer. Vicilin exhibited an anomalously low dependence on protein concentration for pressure dissociation. This appeared related to conformational changes in the dissociated subunits, which caused a loss of ca. 5 kcal/mol in the free energy of association and to structural/energetic heterogeneity in the population of oligomers. Pressure dissociation was markedly pH-dependent, with a stabilization free energy loss of 3.4 kcal/mol upon raising pH from 9 to 10. Circular dichroism and intrinsic fluorescence lifetime measurements at atmospheric pressure showed that the structure of Vicilin was largely unaffected by pH in the range investigated. These results suggest that the effect of pH may involve deprotonation of lysine residues participating in salt bridges between Vicilin subunits. Pressure dissociation of Vicilin was significantly inhibited by addition of salts (NaCl, KCl, LiCl) or glycerol. Dissociation curves obtained in the presence of salts enabled calculation of the free energies of stabilization (ranging from ca. -1.2 to -2.4 kcal/mol) of the Vicilin oligomers by these cosolvents. The similar effects of salts or glycerol suggest a common mechanism of stabilization of the oligomer involving exclusion of the cosolvents from the protein interface and preferential hydration of the protein.