Protein quaternary structure and aggregation in relation to allergenicity

In order to induce systemic food allergic reactions in humans, proteins after digestion in the human gastro-intestinal tract should still be able to bind IgE. The aim of the work presented in this thesis was to determine the effects of heating on the structure and digestibility of cupin and prolamin food allergens from peanuts, Brazil nuts and soybeans and to determine the effects of digestion on the IgE binding capacity of these allergens. Representative allergen preparations were purified prior to the investigations. To this end, a large-scale purification method was developed for the purification of Brazil nut allergen Ber e 1, comprising its complete isoform pool. The denaturation temperature of the latter allergen at the common pH values of foods (5-7) appeared to be very high. As a result, this protein is assumed not to be denatured during common food processing and to be digested (mainly) in its native form. The native form of Ber e 1 is known to have a high stability towards peptic digestion, which likely explains the allergenicity of this protein. The representative quaternary structure of vicilin allergen Ara h 1 from peanuts appeared to be an oligomeric structure, in which the protein is assumed to interact with proanthocyanidins via a specific distribution of proline residues on its surface. Heat-induced aggregation prior to peptic hydrolysis under in vitro conditions did not affect the digestibility of Ara h 1, whereas heat-induced aggregation of legumin allergens Ara h 3 from peanuts and glycinin from soybeans (slightly) decreased the digestion rates. Regardless of pre-heating, the IgE binding capacity of the latter allergens diminished fast during pepsin digestion. As a result, in terms of systemic food allergic reactions, legumin allergens from peanuts and soybeans might not be very important allergens. Vicilin allergen Ara h 1 could still bind IgE after prolonged peptic digestion. Two pepsin-resistant epitopes were deduced and indications for four additional pepsin-resistant epitopes are presented. These epitopes are all situated on the unique N-terminal part of the allergen, which might explain why allergic reactions to peanuts are often severe, compared to other legume allergies

Legumin allergens from peanuts and soybeans : Effects of denaturation and aggregation on allergenicity

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°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

Legumin allergens from peanuts and soybeans : Effects of denaturation and aggregation on allergenicity

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°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

Heat denaturation of Brazil nut allergen Ber e 1 in relation to food processing

Ber e 1, a major allergen from Brazil nuts, is very stable to in vitro peptic digestion. As heat-induced denaturation may affect protein digestibility, the denaturation behaviour of Ber e 1 was investigated. The denaturation temperature of Ber e 1 varies from approximately 80-110 °C, depending on the pH. Upon heating above its denaturation temperature at pH 7.0, the protein partly forms insoluble aggregates and partly dissociates into its polypeptides, whereas heating at pH 5.0 does neither induce aggregation, nor dissociation of the protein. The denaturation temperature of approximately 110 °C at pH values corresponding to the general pH values of foods (pH 5-7) is very high and is expected to be even higher in Brazil nuts themselves. As a result, it is unlikely that heat processing causes the denaturation of all Ber e 1 present in food products. Consequently, the allergen is assumed to be consumed (mainly) in its native form, having a high stability towards pepsin digestion. © 2008 Elsevier Ltd. All rights reserved

Peanut Allergen Ara h 1 Interacts with Proanthocyanidins into Higher Molecular Weight Complexes

Mildly extracted peanut allergen Ara h 1 was previously reported to occur as an oligomeric complex. In this paper we describe how the protein in this oligomeric complex interacts noncovalently with phenolic compounds of the proanthocyanidin type. These interactions are being disrupted during anion exchange chromatography, resulting in the dissociation of the oligomeric Ara h 1 complex into protein trimers. By use of the known three-dimensional structure of ß-conglycinin, a soy protein homologous to Ara h 1, proline-rich regions were observed in silico on both faces of its trimeric structure, which are conserved in Ara h 1. These proline-rich regions could explain the binding of proanthocyanidins to Ara h 1 and the formation of multiple Ara h 1 trimer complexes. This was supported by the observation that the addition of peanut proanthocyanidins to trimeric Ara h 1 and to ß-conglycinin resulted in the formation of soluble oligomeric protein complexes. The structurally related legumin proteins do not contain such proline-rich regions on both sides of the protein, and proanthocyanidins were shown to have a lower affinity for legumin proteins from peanuts and soybeans (peanut allergen Ara h 3 and soy glycinin, respectively). Ara h 1 present as the oligomeric complex is assumed to be the representative form of the allergen in which it is consumed by humans. Keywords: Peanut allergy; Ara h 1; protein-polyphenol interaction; proanthocyanidins

Allergen Ara h 1 Occurs in Peanuts as a Large Oligomer Rather Than as a Trimer

Ara h 1, a major peanut allergen, is known as a stable trimeric protein. Nevertheless, upon purification of native Ara h 1 from peanuts using only size exclusion chromatography, the allergen appeared to exist in an oligomeric structure, rather than as a trimeric structure. The oligomeric structure was independent of the salt concentration applied. Subjecting the allergen to anion exchange chromatography induced the allergen to dissociate into trimers. Ammonium sulfate precipitation did not bring about any structural changes, whereas exposing the allergen to hydrophobic interaction chromatography caused it to partly dissociate into trimers, with increasing amounts of trimers at higher ionic strengths. The (partial) dissociation into trimers led to a change in the tertiary structure of the monomeric subunits of the allergen, with the monomers in Ara h 1 oligomers having a more compact tertiary structure compared with the monomers in Ara h 1 trimers. As structural characteristics are important for a protein's allergenicity, this finding may imply a different allergenicity for Ara h 1 than previously described

Allergen Ara h 1 occurs in peanuts as a large oligomer rather than as a trimer

Ara h 1, a major peanut allergen, is known as a stable trimeric protein. Nevertheless, upon purification of native Ara h 1 from peanuts using only size exclusion chromatography, the allergen appeared to exist in an oligomeric structure, rather than as a trimeric structure. The oligomeric structure was independent of the salt concentration applied. Subjecting the allergen to anion exchange chromatography induced the allergen to dissociate into trimers. Ammonium sulfate precipitation did not bring about any structural changes, whereas exposing the allergen to hydrophobic interaction chromatography caused it to partly dissociate into trimers, with increasing amounts of trimers at higher ionic strengths. The (partial) dissociation into trimers led to a change in the tertiary structure of the monomeric subunits of the allergen, with the monomers in Ara h 1 oligomers having a more compact tertiary structure compared with the monomers in Ara h 1 trimers. As structural characteristics are important for a protein's allergenicity, this finding may imply a different allergenicity for Ara h 1 than previously described. © 2006 American Chemical Society

Peanut Allergen Ara h 1 Interacts with Proanthocyanidins into Higher Molecular Weight Complexes

Mildly extracted peanut allergen Ara h 1 was previously reported to occur as an oligomeric complex. In this paper we describe how the protein in this oligomeric complex interacts noncovalently with phenolic compounds of the proanthocyanidin type. These interactions are being disrupted during anion exchange chromatography, resulting in the dissociation of the oligomeric Ara h 1 complex into protein trimers. By use of the known three-dimensional structure of ß-conglycinin, a soy protein homologous to Ara h 1, proline-rich regions were observed in silico on both faces of its trimeric structure, which are conserved in Ara h 1. These proline-rich regions could explain the binding of proanthocyanidins to Ara h 1 and the formation of multiple Ara h 1 trimer complexes. This was supported by the observation that the addition of peanut proanthocyanidins to trimeric Ara h 1 and to ß-conglycinin resulted in the formation of soluble oligomeric protein complexes. The structurally related legumin proteins do not contain such proline-rich regions on both sides of the protein, and proanthocyanidins were shown to have a lower affinity for legumin proteins from peanuts and soybeans (peanut allergen Ara h 3 and soy glycinin, respectively). Ara h 1 present as the oligomeric complex is assumed to be the representative form of the allergen in which it is consumed by humans. Keywords: Peanut allergy; Ara h 1; protein-polyphenol interaction; proanthocyanidins

Expanded bed adsorption as a fast technique for the large-scale purification of the complete isoform pool of Ber e 1, the major allergen from Brazil nuts

A new, fast, large-scale purification method for Ber e 1, the major allergen from Brazil nuts, using expanded bed adsorption (EBA) chromatography, is presented. Using EBA, crude extracts can be applied to a fluidized column, which allows the unhindered passage of particulate impurities, thereby avoiding time-consuming centrifugation or filtration steps. With this new purification method, 2.8 g of Ber e 1 was obtained from 85 g defatted Brazil nut meal, essentially within 1 day. Various structural as well as immunochemical characteristics of the purified protein were determined, and compared to those of Ber e 1 purified using conventional chromatographic techniques. The complete pool of Ber e 1 isoforms was collected using EBA. The most abundant isoforms were observed to have pI around 8 and heterogeneity was observed in both the large and the small subunit of the heterodimeric protein. Ber e 1 has a highly ordered secondary structure. No apparent differences in immune reactivity were observed between EBA purified Ber e 1 and conventionally purified Ber e 1, using IgE-binding experiments. Thus, using EBA, Bere 1 can be purified fast and on gram-scale, while having purity equal to that of conventionally purified Ber e 1. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA

Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083.

Clones of a genomic library of Bifidobacterium adolescentis were grown in minimal medium with sucrose as sole carbon source. An enzymatic fructose dehydrogenase assay was used to identify sucrose-degrading enzymes. Plasmids were isolated from the positive colonies and sequence analysis revealed that two types of insert were present, which only differed with respect to their orientation in the plasmid. An open reading frame of 1,515 nucleotides with high homology for sucrose phosphorylases was detected on these inserts. The gene was designated SucP and encoded a protein of 56,189 Da. SucP was heterologously expressed in Escherichia coli, purified, and characterized. The molecular mass of SucP was 58 kDa, as estimated by SDS-PAGE, while 129 kDa was found with gel permeation, suggesting that the native enzyme was a dimer. The enzyme showed high activity towards sucrose and a lower extent towards -glucose-1-phosphate. The transglucosylation properties were investigated using a broad range of monomeric sugars as acceptor substrate for the recombinant enzyme, while -glucose-1-phosphate served as donor. d- and l-arabinose, d- and l-arabitol, and xylitol showed the highest production of transglucosylation products. The investigated disaccharides and trisaccharides were not suitable as acceptors. The structure of the transglucosylation product obtained with d-arabinose as acceptor was elucidated by NMR. The structure of the synthesized non-reducing dimer was -Glcp(11)-Araf