Effects of Ser47-Point Mutation on Conformation Structure and Allergenicity of the Allergen of Der p 2, a Major House Dust Mite Allergen.
10.4168/aair.2019.11.1.129
- Author:
Bhakkawarat KULWANICH
1
;
Sasipa THANYARATSRISAKUL
;
Orathai JIRAPONGSANANURUK
;
Belinda J. HALES
;
Wayne R. THOMAS
;
Surapon PIBOONPOCANUN
Author Information
1. Institute of Molecular Biosciences, Mahidol University, Salaya, NakornPathom, Thailand. piboons@gmail.com
- Publication Type:Original Article
- Keywords:
Airway allergic reaction;
point mutated Der p 2;
hypoallergenic allergen
- MeSH:
Animals;
Antibodies, Blocking;
Asian Continental Ancestry Group;
Basophils;
Dust*;
Enzyme-Linked Immunosorbent Assay;
Humans;
Immunoglobulin E;
Immunoglobulin G;
Immunoglobulins;
Interleukin-8;
Interleukins;
Mice;
Pichia;
Point Mutation;
Pyroglyphidae*
- From:Allergy, Asthma & Immunology Research
2019;11(1):129-142
- CountryRepublic of Korea
- Language:English
-
Abstract:
PURPOSE: Hypoallergenic recombinant Der p 2 has been produced by various genetic manipulations, but mutation of a naturally polymorphic amino acid residue known to affect IgE binding has not been studied. This study aimed to determine the effect of a point mutation (S47W) of residue 47 of Der p 2 on its structure and immunoglobulin (Ig) E binding. Its ability to induce pro-inflammatory responses and to induce blocking IgG antibody was also determined. METHODS: S47 of recombinant Der p 2.0110, one of the predominant variants in Bangkok, was mutated to W (S47W). S47W secreted from Pichia pastoris was examined for secondary structure and for the formation of a hydrophobic cavity by 8-Anilino-1-naphthalenesulfonic acid (ANS) staining. Monoclonal and human IgE-antibody binding was determined by enzyme-linked immunosorbent assay. Allergen-induced degranulation by human epsilon receptor expressed-rat basophil was determined. Stimulation of the pro-inflammatory cytokine interleukin (IL)-8 release from human bronchial epithelial (BEAS2B) cells and inhibition of IgE binding to the wild type allergen by S47W-induced IgG were determined. RESULTS: S47W reduced secondary structure and failed to bind the hydrophobic ANS ligand as well as a monoclonal antibody known to be dependent on the nature of the side chain of residue 114 in an adjacent loop. It could also not stimulate IL-8 release from BEAS2B cells. IgE from house dust mite (HDM)-allergic Thais bound S47W with 100-fold weaker avidity, whereas IgE of HDM-allergic Australians did not. S47W still induced basophil degranulation, although requiring higher concentrations for some subjects. Anti-S47W antiserum-immunized mice blocked the binding of human IgE to wild type Der p 2. CONCLUSIONS: The mutant S47W had altered structure and reduced ability to stimulate pro-inflammatory responses and to bind IgE, but retained its ability to induce blocking antibodies. It thus represents a hypoallergen produced by a single mutation of a non-solvent-accessible amino acid.
