Identification of the Immunoglobulin E Epitope of Arginine Kinase, an Important Allergen from Crassostrea angulata

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Sarcoplasmic calcium-binding protein (Cra a 4) from Crassostrea angulata belongs to the EF-hand superfamily, and understanding of its structure−allergenicity relationship is still insufficient. In this study, chemical denaturants were used to destroy the structure of Cra a 4, showing that disruption of the structure reduced its IgG-/IgE-binding activity. To explore which critical amino acid site affects the allergenicity of Cra a 4, the mutants were obtained by site-directed mutations in the disulfide bonds site (C 97 ), conformational epitopes (I 105 , D 114 ), or Ca 2+ -binding region (D 106 , D 110 ) and their IgG-/IgE-binding activity was reduced significantly using serological tests. Notably, C 97 A had the lowest immunoreactivity. In addition, two conformational epitopes of Cra 4 were verified. Meanwhile, the increase of the α-helical content, surface hydrophobicity, and surface electrostatic potential of C 97 A affected its allergenicity. Overall, the understanding of the structure−allergenicity relationship of Cra a 4 allowed the development of a hypoallergenic mutant.

Section: Discussionmentioning

confidence: 99%

Mutation of Disulfide Bond Sites Reduces the Immunoreactivity of Cra a 4 by Changing the Structural Characteristics

Gao,

Huan,

et al. 2024

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“…15−17 Sodium dodecyl sulfate (SDS) changed the tertiary structures of protein molecules by destroying the hydrogen bonds and hydrophobicity of proteins. 18 Dithiothreitol (DTT) and βmercaptoethanol (β-Me) are typical disulfide bond reducing agents that can destroy the disulfide bond in protein. Guanidinium chloride (GdnHCl) is a kind of effective denaturant that can destroy the hydrogen bond.…”

Section: ■ Introductionmentioning

confidence: 99%

“…22,23 Because of the rapid development of computer informatics and molecular biology, the combination of the two research methods had gradually become the mainstream. 9, 24 Huan et al 18 utilized bioinformatics combined with peptide synthesis technology in oysters to validate the nine IgE linear epitopes of AK through serological experiments. Chen et al 25 tional epitope by mutating the critical amino acid in the conformational epitope predicted by SCP into alanine and observing the changes in the IgG-/IgE-binding capacity of the mutant.…”

Section: ■ Introductionmentioning

confidence: 99%

“…At present, chemical denaturants are used to study the relationship between the destruction of allergen structure and the change of IgG-/IgE-binding capacity. Meanwhile, hypoallergenic derivatives treated with denaturants could be used for allergen specific immunotherapy. − Sodium dodecyl sulfate (SDS) changed the tertiary structures of protein molecules by destroying the hydrogen bonds and hydrophobicity of proteins . Dithiothreitol (DTT) and β-mercaptoethanol (β-Me) are typical disulfide bond reducing agents that can destroy the disulfide bond in protein.…”

Section: Introductionmentioning

confidence: 99%

“…They are generally located on the surface of antigen molecules to facilitate direct contact with antibodies or cell surface receptors . The study of allergen IgE epitopes would not only help to enrich the comprehensive understanding of allergens but also help to diagnose and treat food allergy in the clinical environment. , Because of the rapid development of computer informatics and molecular biology, the combination of the two research methods had gradually become the mainstream. , Huan et al utilized bioinformatics combined with peptide synthesis technology in oysters to validate the nine IgE linear epitopes of AK through serological experiments. Chen et al verified the conformational epitope by mutating the critical amino acid in the conformational epitope predicted by SCP into alanine and observing the changes in the IgG-/IgE-binding capacity of the mutant.…”

Section: Introductionmentioning

confidence: 99%

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Immunoglobulin E Epitope Mapping and Structure–Allergenicity Relationship Analysis of Crab Allergen Scy p 9

He,

Yang,

Chen

et al. 2023

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Filamin C is an allergen of Scylla paramamosain (Scy p 9), and six IgE linear epitopes of the allergenic predominant region had previously been validated. However, the IgE epitope and structure–allergenicity relationship of Scy p 9 are unclear. In this study, a hydrophobic bond was found to be an important factor of conformation maintaining. The critical amino acids in the six predicted conformational epitopes were mutated, and the IgE-binding capacity and surface hydrophobicity of four mutants (E216A, T270A, Y699A, and V704A) were reduced compared to Scy p 9. Ten linear epitopes were verified with synthetic peptides, among which L-AA187–205 had the strongest IgE-binding capacity. In addition, IgE epitopes were mapped in the protruding surface of the tertiary structure, which were conducive to binding with IgE and exhibited high conservation among filamin genes. Overall, these data provided a basis for IgE epitope mapping and structure–allergenicity relationship of Scy p 9.

Development of Hypoallergenic Derivatives of Cra a 1 with B Cell Epitope Deletion and T Cell Epitope Retention

Huan,

Gao,

et al. 2024

Tropomyosin was reported as an important allergen in Crassostrea angulata and designated as Cra a 1. The localization of the T cell epitopes and the reduction of the immunoreactivity of Cra a 1 are still lacking. In this study, four T cell epitopes were identified by using wild-type Cra a 1 (wtCra a 1)-immunized mouse splenocytes cultured with synthetic peptides. The immunoreactivity was maintained after chemical denaturation treatment, indicating that the linear epitope is an immunodominant epitope of wtCra a 1. Furthermore, the hypoallergenic derivative (mCra a 1) was developed by the deletion of linear B cell epitopes and retention of T cell epitopes. mCra a 1 could stimulate CD4 + T cell proliferation and upregulate interleukin-10 secretion. Overall, basophil activation by mCra a 1 was low, but its ability to induce T cell proliferation was retained, suggesting that mCra a 1 may serve as a viable candidate for treating oyster allergy.