Legumin allergens from peanuts and soybeans: Effects of denaturation and aggregation on allergenicity
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Legumin proteins Ara h 3 from peanuts and glycinin from soybeans are increasingly described as important allergens. The stability of an allergen's IgE binding capacity towards heating and digestion is considered an important characteristic for food allergens. We investigated the effects of heating and digestion on the IgE binding of Ara h 3 and glycinin. Both proteins are relatively stable to denaturation, having denaturation temperatures ranging from 70 to 92 degrees C, depending on their quaternary structure and the ionic strength. Aggregates were formed upon heating, which were partly soluble for glycinin. Heating slightly decreased the pepsin digestion rate of both allergens. However, heating did not affect the IgE binding capacity of the hydrolyzates, as after only 10 min of hydrolysis no IgE binding could be detected any more in all samples. Peanut allergen Ara h 1, when digested under equal conditions, still showed IgE binding after 2 h of hydrolysis. Our results indicate that the IgE binding capacity of legumin allergens from peanuts and soybeans does not withstand peptic digestion. Consequently, these allergens are likely unable to sensitize via the gastro-intestinal tract and cause systemic food allergy symptoms. These proteins might thus be less important allergens than was previously assumed.Keywords:
Legumin
Denaturation (fissile materials)
Digestion
Food allergens
Pepsin
Legumin
Vicilin
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The major storage protein of buckwheat seed is the 13S globulin. Separation of buckwheat seed proteins by sucrose density gradient revealed the existence of an additional new minor storage protein. Analysis of 13S and the new minor class storage proteins by two systems of two-dimensional gel electrophoresis showed that the 13S globulin resembles a structure of legumin-like seed storage proteins, but the new protein appears to be a vicilin-like storage protein. The 57−58 kDa polypeptides, previously described as the unusual subunits of the 13S storage protein, in fact are the subunits of the minor class of buckwheat seed storage proteins. The major and minor classes of storage protein represent about 33 and 6.5% of total seed proteins, respectively. Keywords: Fagopyrum esculentum Moench; buckwheat; seed storage protein
Legumin
Vicilin
Fagopyrum
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Legumin
Vicilin
Immunogold labelling
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Legumin
Vicilin
Cotyledon
Transcription
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Suspensor
Vicilin
Legumin
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Vicilin
Legumin
Epicotyl
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The tissue-specific syntheses of seed storage proteins in the cotyledons of developing pea (Pisum sativum L.) seeds have been demonstrated by estimates of their qualitative and quantitative accumulation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and rocket immunoelectrophoresis respectively. Vicilin-fraction proteins initially accumulated faster than legumin, but whereas legumin was accumulated throughout development, different components of the vicilin fraction had their predominant periods of synthesis at different stages of development. The translation products in vitro of polysomes isolated from cotyledons at different stages of development reflected the synthesis in vivo of storage-protein polypeptides at corresponding times. The levels of storage-protein mRNA species during development were estimated by ‘Northern’ hybridization using cloned complementary-DNA probes. This technique showed that the levels of legumin and vicilin (47000-Mr precursors) mRNA species increased and decreased in agreement with estimated rates of synthesis of the respective polypeptides. The relative amounts of these messages, estimated by kinetic hybridization were also consistent. Legumin mRNA was present in leaf poly(A)+ RNA at less than one-thousandth of the level in cotyledon poly(A)+ (polyadenylated) RNA, demonstrating tissue-specific expression. Evidence is presented that storage-protein mRNA species are relatively long-lived, and it is suggested that storage-protein synthesis is regulated primarily at the transcriptional level.
Legumin
Vicilin
Polysome
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An 8S storage globulin from buckwheat seed, which resembles the structure and features common to the vicilin-like family of seed storage proteins, was analyzed for this paper. It was found that expression of the 8S globulin gene precedes that of the 13S globulin (the main buckwheat storage protein) and starts from an early stage of buckwheat seed development (9−11 days after flowering), continuing to accumulate throughout seed development to contribute ∼7% of total seed proteins. This protein fraction might be more interesting for biotechnological application than the 13S buckwheat legumin consisting of 23−25 kDa subunits reported to be the major buckwheat allergen. A partial cDNA was also isolated, showing high homology with cDNAs coding for vicilin-like storage proteins from various plant species, and its expression profile throughout seed development as well as in different buckwheat tissues was analyzed. Keywords: Buckwheat; Fagopyrum esculentum; storage proteins; vicilin-like; biosynthesis; expression
Vicilin
Legumin
Fagopyrum
Glutelin
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The seeds of lugumes are eaten throughout the world, constituting a major input of dietary protein. There can be problems of malnutrition in the more deprived areas where they provide the main source of protein, for though legume seeds can be very rich in protein, they are deficient in some amino acids, mainly methionine, that are essential for a healthy diet. Recombinant DMA technology may offer a solution to this problem by introducing DNA encoding these deficient amino acids into seed storage protein genes, which on reintroduction into the host plant- could be grown as a more nutritional crop. The 2s storage proteins of the Brazil nut contain a very high proportion of methionine, therefore DNA encoding 2s protein could be introduced into the sequence of legume storage protein genes. It was proposed that this be attempted, and any constructions that should be produced could be cloned into yeast to detect expression of the mutated genes. Attempts were made to construct and isolate pUCl8 vector clones of Brazil nut DNA, to determine and attempt a rationale for the insertion of DNA from these clones into sequences encoding legumin A by site directed mutagenesis, and to detect the formation of Brazil nut - legumin DNA constructs with radiolabelled probes DNA probes and agarose gel electrophoresis. Initial steps were taken to perform a similar mutation of a vicilin cDNA. Two pUCl8 clones of Brazil nut DNA; - pBnA and pBnB were created and isolated. Clones of legumin - Brazil nut DNA;- pGPBl were constructed based on an insertional mutation of the legumin cDNA construct pJYB with Brazil nut DNA from the clone pBnA. They were isolated from the in – situ hybridisation of transformed cells with radiolabelled DNA. Clones of pGPBl were found by gel electrophoresis and Southern hybridisation to contain the BnA insertion in the correct orientation. Time constraints prevented the cloning of pGPBl into yeast. The choice of legumin mutation sites was discussed and the rationale adopted justified on the grounds of restriction site analysis and the restraints imposed by legumin solubility and protein structure. The Results and problems encountered were discussed in some detail. It was suggested that further work should attempt the expression of pGPBl in yeast.
Legumin
Vicilin
Brazil nut
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