Co-introduction of an antisense gene for an endogenous seed storage protein can increase expression of a transgene in Arabidopsis thaliana seeds.
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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. (+info)
Direct kinetic evidence for folding via a highly compact, misfolded state.
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The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.7 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m(M) = -0.95 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein. (+info)
GerN, an antiporter homologue important in germination of Bacillus cereus endospores.
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A homologue of the grmA spore germination gene of Bacillus megaterium and of a NaH-antiporter gene (napA) of Enterococcus hirae has been identified in Bacillus cereus 569 (ATCC 10876). The putative protein product has 58 and 43% amino acid identity with GrmA and NapA, respectively. Insertional inactivation of this B. cereus gene, named gerN, did not affect vegetative growth or sporulation. The null mutant spores were 30-fold slower to germinate in inosine (5 mM) but germinated almost normally in response to L-alanine (10 mM). The null mutant spores germinated after several hours with inosine as the sole germinant, but germination was asynchronous and the normal order of germination events was perturbed. At a suboptimal germinant concentration (50 microM), inosine germination was completely blocked in the mutant, while the rate of germination in 50 microM L-alanine was reduced to one-third of that of the wild type. The requirement for GerN function in the response to a particular germinant suggests that a germination receptor may have a specifically associated antiporter, which is required at the initiation of germination and which, in the case of the inosine receptor, is GerN. Since germination in suboptimal concentrations of L-alanine shows a delay, additional germination transporters may be required for optimal response at low germinant concentrations. (+info)
Calcium-mediated association of a putative vacuolar sorting receptor PV72 with a propeptide of 2S albumin.
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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. (+info)
Recombinant pronapin precursor produced in Pichia pastoris displays structural and immunologic equivalent properties to its mature product isolated from rapeseed.
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2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor. The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced. The amino-acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser-Glu-Asn. Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris. The induced protein was secreted to the extracellular medium at a yield of 80 mg.L(-1) of culture and was purified by means of size-exclusion chromatography and reverse phase-HPLC. Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein. As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity. The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed. In conclusion, the recombinant expression of napin precursors in P. pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability. (+info)
Protein structure plays a critical role in peanut allergen stability and may determine immunodominant IgE-binding epitopes.
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Hypersensitivity to peanuts is a reaction mediated by IgE Abs in response to several peanut protein allergens. Among these allergenic proteins, Ara h 2 is one of the most commonly recognized allergens. Ara h 2 is a 17-kDa protein that has eight cysteine residues that could form up to four disulfide bonds. Circular dichroism studies showed substantial changes in the secondary and tertiary structures of the reduced Ara h 2 as compared with the native protein. Upon treatment with trypsin, chymotrypsin, or pepsin, a number of relatively large fragments are produced that are resistant to further enzymatic digestion. These resistant Ara h 2 peptide fragments contain intact IgE-binding epitopes and several potential enzyme cut sites that are protected from the enzymes by the compact structure of the protein. The enzyme-treated allergen remains essentially intact despite the action of proteases until the fragments are dissociated when the disulfide linkages are reduced. Amino acid sequence analysis of the resistant protein fragments indicates that they contain most of the immunodominant IgE-binding epitopes. These results provide a link between allergen structure and the immunodominant IgE-binding epitopes within a population of food-allergic individuals. (+info)
Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model.
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Peanut allergy (PNA) is the major cause of fatal and near-fatal anaphylactic reactions to foods. Traditional immunotherapy using peanut (PN) protein is not an option for PNA therapy because of the high incidence of adverse reactions. We investigated the effects of s.c. injections of engineered (modified) recombinant PN proteins and heat-killed Listeria monocytogenes (HKLM) as an adjuvant on anaphylactic reactions in a mouse model of PN allergy. PN-allergic C3H/HeJ mice were treated s.c. with a mixture of the three major PN allergens and HKLM (modified (m)Ara h 1-3 plus HKLM). The effects on anaphylactic reactions following PN challenge and the association with Ab levels and cytokine profiles were determined. Although all mice in the sham-treated groups exhibited anaphylactic symptoms with a median symptom score of 3, only 31% of mice in the mAra h 1-3 plus HKLM group developed mild anaphylaxis, with a low median symptom score of 0.5. Alterations in core body temperature, bronchial constriction, plasma histamine, and PN-specific IgE levels were all significantly reduced. This protective effect was markedly more potent than in the mAra h 1-3 protein alone-treated group. HKLM alone did not have any protective effect. Reduced IL-5 and IL-13, and increased IFN-gamma levels were observed only in splenocytes cultures from mAra h 1-3 plus HKLM-treated mice. These results show that immunotherapy with modified PN proteins and HKLM is effective for treating PN allergy in this model, and may be a potential approach for treating PNA. (+info)
A plant-based allergy vaccine suppresses experimental asthma via an IFN-gamma and CD4+CD45RBlow T cell-dependent mechanism.
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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. (+info)