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At least 19 recordsLinked to original sources

PubMed2002

The disulphide mapping, folding and characterisation of recombinant Ber e 1, an allergenic protein, and SFA8, two sulphur-rich 2S plant albumins.

We have cloned and expressed genes encoding the allergenic brazil nut 2S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.

2S Albumins, Plant↗

PubMed2006

Differential polarization of immune responses by plant 2S seed albumins, Ber e 1, and SFA8.

The plant 2S seed albumins Ber e 1 and SFA8, although structurally very similar, vary with respect to their allergenic properties. Whereas the former represents a major allergen, the latter appears to promote only weak allergenic responses. The aim of this investigation was to determine whether the allergenic properties of Ber e 1 and SFA8 reflected differential polarization of dendritic cell (DC) and Th cell responses. We thus investigated the effect of recombinant forms of both allergens on DC and Th cell responses as indicated by cell surface phenotype and cytokine production. Exposure of murine DCs to SFA8, but not Ber e 1, resulted in production of the cytokines IL-12 p40 and TNF-alpha by a mechanism independent of recognition by TLRs. Furthermore, depending on the mouse strain used, increased expression of MHC class II and costimulatory molecules such as CD40, CD80, and CD86 was associated with exposure to SFA8, but not Ber e 1. In coculture experiments using the DO11.10 transgenic T cell that recognizes OVA peptide, DCs exposed to both allergens induced T cells to produce IFN-gamma, but only Ber e 1 could induce significant production of IL-4 and IL-5. Likewise, analysis of transcription factors shows increased T-bet with respect to both allergens, but also GATA-3 with respect to Ber e 1. Overall, our data are consistent with the idea that the ability of Ber e 1, but not SFA8, to act as a potent allergen may reflect differences in their ability to induce IL-12 production.

2S Albumins, Plant↗

PubMed2003

In vitro stability and immunoreactivity of the native and recombinant plant food 2S albumins Ber e 1 and SFA-8.

BACKGROUND: The ability of an intact protein to reach the circulatory system may be a prerequisite to allergenicity and many allergens, particularly those from plant foods, have been found to be consistently more resistant to digestion by pepsin than other proteins. OBJECTIVE: This study assessed the pepsinolytic stability of native 2S albumins from Brazil nut and sunflower seed and their recombinant versions produced in Pichia pastoris. The physicochemical stability of native and recombinant Brazil nut 2S albumins and recombinant sunflower seed 2S albumin was also assessed. The immunoreactivity of native Brazil nut 2S albumin and recombinant 2S albumins was compared using serum from patients allergic to Brazil nuts and animals immunized with native 2S albumins. METHODS: Digestibility was measured in simulated gastric fluid followed by SDS-PAGE. Circular dichroism spectra were used to analyse unfolding, as proteins were denatured by temperature, pH and guanidinium chloride. Immunoreactivity was assessed by immunoblot, RAST and ELISA. RESULTS: Brazil nut 2S albumin was significantly more resistant to proteolytic digestion than other Brazil nut proteins. It was also resistant to thermally and chemically induced denaturation. Equally high resistance to proteolytic digestion was observed with sunflower seed 2S albumin. The recombinant albumins mirrored their native counterparts in stability and immunoreactivity. CONCLUSION: The important food allergen Brazil nut 2S albumin is as stable to digestion as is sunflower seed 2S albumin, whose allergenicity has yet to be determined. The 2S albumins and their recombinant counterparts could not be easily denatured by physicochemical treatments. The results suggest that 2S albumin is the only Brazil nut protein to reach the gut immune system intact. The production of properly folded recombinant proteins will facilitate mechanistic studies as well as diagnostic testing and antigen-based therapies.

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PubMed2001

Isolation and characterization of 2S cocoa seed albumin storage polypeptide and the corresponding cDNA.

The amine pool of cocoa is known to be an essential component for the development of the typical cocoa flavor. To better understand and to produce an intense in vitro cocoa flavor, identification of the polypeptides that are the source of the amine flavor precursor pool is essential. Chromatographic analysis of the polypeptide profile of unfermented cocoa resulted in identification of a novel storage polypeptide of M(r) 8515. The N-terminal sequence of the first 34 residues of the purified polypeptide shows similarity to 2S storage albumins of cotton and Brazil nut and sweet protein, Mabinlin. To identify the corresponding cDNA of the putative cocoa 2S albumin, 18 randomly chosen clones from the cDNA library of immature Theobroma cacao seed mRNA were sequenced, and a full-length cDNA clone encoding a protein harboring the N-terminal sequence of the novel polypeptide was selected. The open reading frame of the clone encodes a polypeptide of M(r) 17125. Comparison of the translated amino acid sequence of the precursor protein or the mature polypeptide against the Swiss-Prot and TrEMBL databases shows high sequence similarity (>52%) and identity (>38%) to many plant 2S albumins. Tryptic peptide mass fingerprinting of the purified polypeptide by high-performance liquid chromatography-electrospray ionization mass spectrometry shows 10 masses that match the expected tryptic peptides of the deduced sequence. Together with the published work on plant 2S albumin processing, the results presented here suggest that post-translational processing yields a 73-residue polypeptide (residue positions 78-150) corresponding to the 9 kDa subunit of the mature cocoa 2S albumin protein.

Amino Acids↗

PubMed1996

Modified 2S albumins with improved tryptophan content are correctly expressed in transgenic tobacco plants.

Brazil nut 2S albumins lack the essential amino acid tryptophan. In order to improve the protein's nutritional value and create a basis for structural investigations, three separate modified Brazil nut 2S albumin genes were constructed. The first mutant contains five consecutive tryptophan codons, while the other two modified genes encode proteins carrying single tryptophan residues at sites that will allow confirmation of the predicted protein structure through fluorescence quenching techniques. The modified genes, under the regulation of the CaMV 35S promoter, were introduced into Nicotiana tabacum. All three modified genes were correctly transcribed and the 2S albumin accumulated in the seeds of transgenic plants.

Agrobacterium tumefaciens↗

PubMed1991

Homology of placental protein 11 and pea seed albumin 2 with vitronectin.

Vitronectin (complement S-protein), a plasma and tissue glycoprotein of 75 kDa, shares the amino-terminal somatomedin B domain with the membrane glycoprotein PC1 of plasma cells and several hemopexin-type repeats with hemopexin and the non-catalytic carboxy-terminal domain of collagenases. It serves as a ligand for certain integrin receptors, binds to distinct members of the serpin family and inhibits the pore-forming cytolytic reaction of the terminal complement pathway. Computer-assisted data base searches revealed the presence of a single somatomedin B domain in the recently cloned placental protein 11, and four hemopexin-type repeats in the cytosolic plant protein PA2, the major albumin of pea seeds, whose function is unknown. Our finding shows that hemopexin-type repeats are present in extracellular as well as in cytosolic proteins and most likely originated before the divergence of the animal and plant kindoms.

2S Albumins, Plant↗

PubMed1999

Co-introduction of an antisense gene for an endogenous seed storage protein can increase expression of a transgene in Arabidopsis thaliana seeds.

We have investigated whether the expression in Arabidopsis thaliana seeds of a transgene (the Phaseolus vulgaris arcelin (arc)5-I gene) could be enhanced by the simultaneous introduction of an antisense gene for an endogenous seed storage protein (2S albumin). Seeds of plants transformed with both the arc5-I gene and a 2S albumin antisense gene contained reduced amounts of 2S albumins and increased arcelin-5 (Arc5) accumulation levels compared to lines harboring the arc5-I gene only. Arc5 production could be enhanced to more than 24% of the total seed protein content, suggesting that antisense technology could be of great utility to favor high expression of transgenes.

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PubMed2001

Lipid transfer proteins and 2S albumins as allergens.

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

Albumins↗

PubMed2001

Calcium-mediated association of a putative vacuolar sorting receptor PV72 with a propeptide of 2S albumin.

PV72, a type I membrane protein with three epidermal-growth factor (EGF)-like motifs, was found to be localized on the membranes of the precursor-accumulating (PAC) vesicles that accumulated precursors of various seed storage proteins. To clarify the function of PV72 as a sorting receptor, we expressed four modified PV72s and analyzed their ability to bind the internal propeptide (the 2S-I peptide) of pro2S albumin by affinity chromatography and surface plasmon resonance. The recombinant PV72 specifically bound to the 2S-I peptide with a K(D) value of 0.2 microm, which was low enough for it to function as a receptor. The EGF-like motifs modulated the Ca(2+)-dependent conformational change of PV72 to form a functional pocket for the ligand binding. The binding of Ca(2+) stabilizes the receptor-ligand complex even at pH 4.0. The association and dissociation of PV72 with the ligand is modulated by the Ca(2+) concentration (EC(50) value = 40 microm) rather than the environmental pH. Overall results suggest that Ca(2+) regulates the vacuolar sorting mechanism in higher plants.

2S Albumins, Plant↗

PubMed1996

Purification and sequencing of napin-like protein small and large chains from Momordica charantia and Ricinus communis seeds and determination of sites phosphorylated by plant Ca(2+)-dependent protein kinase.

The basic protein fraction from seeds of castor bean (Ricinus communis L.) contains 4732 Da and 4603 Da proteins phosphorylated in vitro by plant Ca(2+)-dependent protein kinase (CDPK). These proteins, RS1A and RS1B respectively, were purified by cation-exchange HPLC (SP5PW column) and reverse-phase HPLC (C18 column) and identified as napin-like protein small chains by Edman sequencing and electrospray ionization mass spectrometry (ESMS). The other R. communis 4 kDa small chains (RS2A, RS2B, RS2C and RS2D) are not phosphorylated by CDPK and neither is the corresponding 7332 Da large chain (RL) that forms 1:1 disulfide-linked complexes with RS2(A-D). RS1A/B is one of the best substrates found for plant CDPK (K(m) = 1.8 +/- 0.8 microM). RS2(A-D) (but not RL or RS1A/B) strongly inhibit calmodulin (CaM)-dependent myosin light chain protein kinase (MLCK) (IC50 = 0.25 microM) and inhibit the Ca(2+)-dependent enhancement of dansyl-CaM fluorescence. The basic protein fraction from seeds of bitter melon (Momordica charantia) also contains napin-like proteins that are 1:1 disulfide-linked complexes of a small chain (MS1, MS2, MS3 or MS4) and a large chain (ML). The M. charantia small chains were purified and completely sequenced by Edman degradation and ESMS. M. charantia small chains MS1, MS2, and MS4 (but not MS3) are phosphorylated by CDPK to unit stoichiometry on S21 within the sequence R17SCES21FLR. The R. communis small chain RS1A is phosphorylated on S34 within the sequence R31QSS34SRR. Both of these phosphorylation site motifs are consistent with those found for other plant CDPK substrates.

2S Albumins, Plant↗

PubMedDate not supplied

[Plant proteinase inhibitors as polyfunctional proteins (a review)].

Literature data on plant proteinase inhibitors as multifunctional proteins are reviewed. In addition to the direct inhibitory effect on enzymes, these proteins may function in other processes, particularly under biotic and environmental stressful conditions. A special section discusses the relationships of plant proteinase inhibitors and storage proteins.

2S Albumins, Plant↗

PubMed1992

Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A.

Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated beta-glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting beta-glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.

2S Albumins, Plant↗

PubMed2003

A plant-based allergy vaccine suppresses experimental asthma via an IFN-gamma and CD4+CD45RBlow T cell-dependent mechanism.

Allergic asthma is currently considered a chronic airway inflammatory disorder associated with the presence of activated CD4(+) Th2-type lymphocytes, eosinophils, and mast cells. Interestingly, therapeutic strategies based on immune deviation and suppression have been shown to successfully attenuate the development of the asthma phenotype. In this investigation, we have for the first time used a genetically modified (GM) plant, narrow leaf lupin (Lupinus angustifolius L.), expressing a gene for a potential allergen (sunflower seed albumin) (SSA-lupin) to examine whether a GM plant/food-based vaccine strategy can be used to suppress the development of experimental asthma. We show that oral consumption of SSA-lupin promoted the induction of an Ag-specific IgG2a Ab response. Furthermore, we demonstrate that the plant-based vaccine attenuated the induction of delayed-type hypersensitivity responses and pathological features of experimental asthma (mucus hypersecretion, eosinophilic inflammation, and enhanced bronchial reactivity (airways hyperreactivity). The suppression of experimental asthma by SSA-lupin was associated with the production of CD4(+) T cell-derived IFN-gamma and IL-10. Furthermore, we show that the specific inhibition of experimental asthma was mediated via CD4(+)CD45RB(low) regulatory T cells and IFN-gamma. Thus, our data demonstrate that a GM plant-based vaccine can promote a protective immune response and attenuate experimental asthma, suggesting that plant-based vaccines may be potentially therapeutic for the protection against allergic diseases.

2S Albumins, Plant↗

PubMed2000

Transgenic Trifolium repens with foliage accumulating the high sulphur protein, sunflower seed albumin.

With the aim of increasing the rumen-protected level of the sulphur amino acids cysteine and methionine in Trifolium repens, we introduced the coding sequence of the sunflower seed albumin (SSA) into T. repens by Agrobacterium tumefaciens-mediated transformation. The SSA gene was modified such that the protein would be localised to the endoplasmic reticulum (ER). Four different T-DNA constructions all containing the SSA gene driven by either the promoter of a gene encoding the small subunit of ribulose bisphosphate carboxylase (Rubisco) from Arabidopsis thaliana (Assu), the promoter of the gene encoding the small subunit of Rubisco of Medicago sativa (Lssu), or the Cauliflower Mosaic Virus 35S promoter (CaMV35S), were transferred to T. repens cv. Haifa. Transgenic T0-plants and inter-transgenic hybrids were analysed for the level of SSA accumulation in the leaves by western blotting. The highest observed level of SSA accumulation was 0.1% of total extractable leaf protein. We observed that the promoter had a substantive effect on the level of SSA accumulation with Assu > CaMV35S > Lssu. Results from the inter-transgenic hybrids showed that the capacity to synthesise SSA was inherited. However the level of SSA accumulation in the leaves generally appears not to be additive with extra transgenic loci. During this work, we attempted to improve the efficiency of A. tumefaciens-mediated transformation of T. repens using the SAAT-method (Sonication Assisted Agrobacterium-mediated Transformation) on cotyledons of T. repens. T-DNA transfer was in general not enhanced by sonication compared to traditional A. tumefaciens-mediated transformation. Furthermore, Southern blot analyses of plants regenerated from the same cotyledon after A. tumefaciens treatment and under selection, indicated that multiple shoots were usually derived from the same transformation event. We concluded from these results that only one plant from each A. tumefaciens-treated cotyledon should be taken to avoid transgenic clones.

2S Albumins, Plant↗

PubMed2002

Solution structure of allergenic 2 S albumins.

The NMR solution structures at different levels of refinement of three different 2 S albumin seed proteins, the recombinant pronapin precursor from Brassica napus, the recombinant RicC3 from Ricinus communis and the methionine-rich protein from sunflower ( Helianthus annuus ), are described. The resulting common structure consists of a bundle of five alpha-helices, folded in a right-handed superhelix. The structure is very similar to that of other plant proteins: the hydrophobic protein from soybean, non-specific lipid transfer proteins and amylase/trypsin inhibitors. Analogies and differences in the structures of these families, as well as their possible relationship to allergenicity, are discussed.

2S Albumins, Plant↗

PubMed1987

Sequence and expression of a gene encoding an albumin storage protein in sunflower.

The complete sequence of a sunflower (Helianthus annuus) gene, HaG5, encoding a 2 S albumin storage protein was determined. The predicted unprocessed precursor has 295 amino acids, is rich in glutamine residues (24%) and contains a hydrophobic amino-terminus that is similar to the consensus signal peptide. Amino acid sequencing of the mature protein revealed extensive post-translational processing. Nuclease protection and primer extension analysis indicated a major transcriptional start 30 nucleotides 5' of the predicted ATG start codon. Additional sequence data, determined from a nearly full length cDNA recombinant, indicate that HaG5 is a member of a small gene family comprised of at least two divergent genes. Comparison of the predicted HaG5 gene product with sequences of other known plant proteins revealed distant but significant homology with the napins of Brassica and other heterogeneous seed proteins in the albumin superfamily.

2S Albumins, Plant↗

PubMed2004

Protein patterns of the Brassica rapa ovules and seeds under altered gravity.

Electrophoretic investigation of protein patterns of Brassica rapa L. ovules and seeds from plants grown under clinorotation and in the laboratory control was carried out. Ovules at different stages (7 and 18 days after pollination) and mature seeds were analyzed. Polymorphism of seed storage proteins of B. rapa was taken into consideration in analysis of changes in ovule protein patterns under clinorotation. The appearance of a protein component in the region of about 43 kDa was detected in protein patterns of 7-day-old and 18-day-old ovules in the clinostat variants. Under altered gravity, in 18-day-old ovules, the appearance of a protein in the region of about 70 kDa was also revealed. The appearance of the protein component with the similar mobility (about 43 kDa) in ovules of different age from plants grown at clinorotation suggests that synthesis of this protein may be associated with the plant response to altered gravity. However, the investigation of the nature of this protein and its role requires further research to rule out its appearance because of genotypic differences between ovules of the control and experimental variants.

2S Albumins, Plant↗

PubMed1994

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis.

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-beta-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.

2S Albumins, Plant↗