Dataset Information

Crosslinked Recombinant-Ara h 1 Catalyzed by Microbial Transglutaminase: Preparation, Structural Characterization and Allergic Assessment.

ABSTRACT: As the one of the major allergens in peanut, the allergenicity of Ara h 1 is influenced by its intrinsic structure, which can be modified by different processing. However, molecular information in this modification has not been clarified to date. Here, we detected the influence of microbial transglutaminase (MTG) catalyzed cross-linking on the recombinant peanut protein Ara h 1 (rAra h 1). Electrophoresis and spectroscopic methods were used to analysis the structural changes. The immunoreactivity alterations were characterized by enzyme linked immunosorbent assay (ELISA), immunoblotting and degranulation test. Structural features of cross-linked rAra h 1 varied at different reaction stages. Hydrogen bonds and disulfide bonds were the main molecular forces in polymers induced by heating and reducing. In MTG-catalyzed cross-linking, ?-(?-glutamyl) lysine isopeptide bonds were formed, thus inducing a relatively stable structure in polymers. MTG catalyzed cross-linking could modestly but significantly reduce the immunoreactivity of rAra h 1. Decreased content of conserved secondary structures led to a loss of protection of linear epitopes. Besides, the reduced surface hydrophobic index and increased steric hindrance of rAra h 1 made it more difficult to bind with antibodies, thus hindering the subsequent allergic reaction.

SUBMITTER: Tian Y

PROVIDER: S-EPMC7590132 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Crosslinked Recombinant-Ara h 1 Catalyzed by Microbial Transglutaminase: Preparation, Structural Characterization and Allergic Assessment.

Tian Yang Y Liu Chenglong C Xue Wentong W Wang Zhongfu Z

Foods (Basel, Switzerland) 20201021 10

As the one of the major allergens in peanut, the allergenicity of Ara h 1 is influenced by its intrinsic structure, which can be modified by different processing. However, molecular information in this modification has not been clarified to date. Here, we detected the influence of microbial transglutaminase (MTG) catalyzed cross-linking on the recombinant peanut protein Ara h 1 (rAra h 1). Electrophoresis and spectroscopic methods were used to analysis the structural changes. The immunoreactivit ...[more]

PMID: 33096617

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Project description:Background Peanut-allergic patients from the Mediterranean region are predominantly sensitized to the lipid transfer protein (LTP) Ara h 9, and the peach LTP Pru p 3 seems to be the primary sensitizer. However, LTP sensitization in peanut allergy is not a predictive marker for clinically relevant symptoms. Objective We aimed to identify sequential epitopes of IgE and IgG4 from Pru p 3 and Ara h 9 in peach-allergic patients sensitized to peanuts. We also sought to determine the differences in IgE and IgG4 binding between patients who had developed peanut allergy and those tolerating peanuts. Methods A total of 46 peach-allergic patients sensitized to peanuts were selected. A total of 35 patients were allergic to peanuts (peanut-allergic group) and 11 were tolerant to peanuts (peanut-tolerant group). We measured sIgE and sIgG4 in peanut, peach, and their recombinant allergen (Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9) with fluorescence enzyme immunoassay. We examined the IgE and IgG4 binding to sequential epitopes using a peptide microarray corresponding to linear sequences of the LTPs Ara h 9 and Pru p 3 with a library of overlapping peptides with a length of 20 amino acids (aa) and an offset of 3 aa. Results The frequency and the intensity of IgE recognition of Ara h 9 and Pru p 3 peptides were higher in the peanut-tolerant group than in the peanut-allergic group. We found four Ara h 9 peptides (p4, p14, p21, and p25) and four Pru p 3 peptides (p1, p3, p21, and p24) with a significantly elevated IgE recognition in peanut-tolerant patients. Only one peptide of Ara h 9 (p4) recognized by IgG4 was significantly elevated in the peanut-tolerant group. The IgG4/IgE ratio of Ara h 9 peptide 4 was significantly higher in peanut-tolerant patients than in peanut-allergic patients, while no significant differences were observed in the IgG4/IgE ratio of this peptide in Pru p 3. Conclusion Although we found significant differences in IgE and IgG4 recognition of Ara h 9 and Pru p 3 between peanut-tolerant and peanut-allergic patients (all of whom were allergic to peach), polyclonal IgE peptide recognition of both LTPs was observed in peach-allergic patients tolerating peanuts. However, the IgG4 blocking antibodies against Ara h 9 peptide 4 could provide an explanation for the absence of clinical reactivity in peanut-tolerant peach-allergic patients. Further studies are needed to validate the usefulness of IgG4 antibodies against Ara h 9 peptide 4 for peanut allergy diagnosis.

Project description:BackgroundNatural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis.ResultsA synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N-terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2.ConclusionRecombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics.

Dataset Information

Crosslinked Recombinant-Ara h 1 Catalyzed by Microbial Transglutaminase: Preparation, Structural Characterization and Allergic Assessment.

Publications

Crosslinked Recombinant-Ara h 1 Catalyzed by Microbial Transglutaminase: Preparation, Structural Characterization and Allergic Assessment.

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