Allergy Asthma Immunol Res.  2019 Jan;11(1):129-142. 10.4168/aair.2019.11.1.129.

Effects of Ser47-Point Mutation on Conformation Structure and Allergenicity of the Allergen of Der p 2, a Major House Dust Mite Allergen

• Kulwanich
• Thanyaratsrisakul S
• Jirapongsananuruk O
• Hales BJ
• Thomas WR
• Piboonpocanun S

• KMID: 2426791
• DOI: http://doi.org/10.4168/aair.2019.11.1.129

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.

Keyword

Airway allergic reaction; point mutated Der p 2; hypoallergenic allergen

MeSH Terms

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*
Antibodies, Blocking
Dust
Immunoglobulin E
Immunoglobulin G
Immunoglobulins
Interleukin-8
Interleukins

Figure

• 

  Fig. 1 (A) CD spectra of nDer p 2, D20110 and S47W. (B) Fluorescent spectra of ANS stained with (a) nDer p 2, (b) D20110, and (c) S47W. The fluorescent spectra were recorded from 400–550 nm, with excitation at 390 nm. The λmax of each protein and the respective intensity are showed in Tables 1 and 2. (C) Indirect binding of 1D8 mAb to Der p 2 isoforms: D20101, D20104, D20110 and S47W. Results represent mean ± SE of 5–6 experiments. CD, circular dichroism; ANS, 8-Anilino-1-naphthalenesulfonic acid; SE, standard error. *P < 0.0001.

• 

  Fig. 2 Binding of specific IgE to D20110 and S47W. (A) Serum IgE of 8 Thai donors binding to D20110 and S47W. (B) Serum IgE of 6 Perth donors binding to D20110 and S47W. Results represent the mean ± SE. Dashed line indicates background. Inhibition of specific IgE binding using D20110 and S47W. (C) Serum IgE of 8 HDM-allergic Thai donors. (D) Serum IgE of 6 HDM-allergic Perth donors. Results represent the mean ± SE. The IC50 was showed in the inlet. Dotted line indicates 50% inhibition. Ig, immunoglobulin; SE, standard error; HDM, house dust mite.

• 

  Fig. 3 Degranulation of rat RBL-SX38 basophils stimulated with D20110 and S47W. The percentage of degranulation was calculated from the released β-hexosaminidase and the total amount of β-hexosaminidase in the cells. (A) Degranulation experiments with 1 µg/mL allergen showing controls for the presence or absence of atopic serum and allergen and anti-IgE-mediated degranulation. Results show mean±SE using sera from 4 donors. (B) Degranulation of RBL-SX38 basophils primed with 1:20 dilutions of sera from 4 allergic Thai donors. Closed squares for D20110, open squares for S47W; dashed line background degranulation of cells incubated with sera, but not allergens. Ig, immunoglobulin; SE, standard error.

• 

  Fig. 4 IL-8 was secreted from BEAS-2B cells after stimulation with D20110 and S47W. Data show mean ± SE of the 3–6 experiments. IL, interleukin; SE, standard error. *P < 0.05, †P < 0.005, ‡P < 0.001, §P < 0.0001.

• 

  Fig. 5 (A) Direct binding of mouse anti-D20110 antiserum and anti-S47W antiserum. Results represent mean ± SE of the 4–6 experiments. (B) Inhibition of serum IgE binding to Der p 2 by mouse antiserum against D20110 or -S47W. Results represent mean ± SE of D20110 antiserum blocking IgE from 8 individual sera and of -S47W antiserum blocking IgE from 8 individual sera. SE, standard error; Ig, immunoglobulin.

• 

  Fig. 6 Structural modelling of D20110 using the Der p 2 pdb file accession code 1KTJ and program PyMOL (Molecular Graphics System, DeLano Scientific, San Carlos,CA, USA). The ANS-accessible area of the cavity is shown as mesh; C71–C74 represents disulfide bond; L40, L110 and N114 are dominant polymorphic residues found in variants: D20103, D20104, D20109 and D20110. W92 is located at the entrance of the hydrophobic cavity. ANS, 8-Anilino-1-naphthalenesulfonic acid.

Reference

1. Vrtala S, Huber H, Thomas WR. Recombinant house dust mite allergens. Methods. 2014; 66:67–74.

2. Marth K, Focke-Tejkl M, Lupinek C, Valenta R, Niederberger V. Allergen peptides, recombinant allergens and hypoallergens for allergen-specific immunotherapy. Curr Treat Options Allergy. 2014; 1:91–106.

3. Derewenda U, Li J, Derewenda Z, Dauter Z, Mueller GA, Rule GS, et al. The crystal structure of a major dust mite allergen Der p 2, and its biological implications. J Mol Biol. 2002; 318:189–197.

4. Chen KW, Blatt K, Thomas WR, Swoboda I, Valent P, Valenta R, et al. Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy. J Allergy Clin Immunol. 2012; 130:435–443.e4.

5. Asturias JA, Ibarrola I, Arilla MC, Vidal C, Ferrer A, Gamboa PM, et al. Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen-specific immunotherapy. Clin Exp Allergy. 2009; 39:1088–1098.

6. Smith AM, Chapman MD. Reduction in IgE binding to allergen variants generated by site-directed mutagenesis: contribution of disulfide bonds to the antigenic structure of the major house dust mite allergen Der p 2. Mol Immunol. 1996; 33:399–405.

7. Hakkaart GA, Aalberse RC, van Ree R. Epitope mapping of the house-dust-mite allergen Der p 2 by means of site-directed mutagenesis. Allergy. 1998; 53:165–172.

8. Smith AM, Chapman MD. Localization of antigenic sites on Der p 2 using oligonucleotide-directed mutagenesis targeted to predicted surface residues. Clin Exp Allergy. 1997; 27:593–599.

9. Reginald K, Chew FT. Conformational IgE epitope mapping of Der p 2 and the evaluations of two candidate hypoallergens for immunotherapy. Sci Rep. 2018; 8:3391.

10. Smith WA, Hales BJ, Jarnicki AG, Thomas WR. Allergens of wild house dust mites: environmental Der p 1 and Der p 2 sequence polymorphisms. J Allergy Clin Immunol. 2001; 107:985–992.

11. Piboonpocanun S, Malainual N, Jirapongsananuruk O, Vichyanond P, Thomas WR. Genetic polymorphisms of major house dust mite allergens. Clin Exp Allergy. 2006; 36:510–516.

12. Smith AM, Benjamin DC, Hozic N, Derewenda U, Smith WA, Thomas WR, et al. The molecular basis of antigenic cross-reactivity between the group 2 mite allergens. J Allergy Clin Immunol. 2001; 107:977–984.

13. Hales BJ, Hazell LA, Smith W, Thomas WR. Genetic variation of Der p 2 allergens: effects on T cell responses and immunoglobulin E binding. Clin Exp Allergy. 2002; 32:1461–1467.

14. Park JW, Kim KS, Jin HS, Kim CW, Kang DB, Choi SY, et al. Der p 2 isoallergens have different allergenicity, and quantification with 2-site ELISA using monoclonal antibodies is influenced by the isoallergens. Clin Exp Allergy. 2002; 32:1042–1047.

15. Jeong KY, Lee IY, Yong TS, Lee JH, Kim EJ, Lee JS, et al. Sequence polymorphisms of Der f 1, Der p 1, Der f 2 and Der p 2 from Korean house dust mite isolates. Exp Appl Acarol. 2012; 58:35–42.

16. Tanyaratsrisakul S, Malainual N, Jirapongsananuruk O, Smith WA, Thomas WR, Piboonpocanun S. Structural and IgE binding analyses of recombinant Der p 2 expressed from the hosts Escherichia coli and Pichia pastoris . Int Arch Allergy Immunol. 2010; 151:190–198.

17. Gray VE, Hause RJ, Fowler DM. Analysis of large-scale mutagenesis data yo assess the impact of single amino acid substitutions. Genetics. 2017; 207:53–61.

18. Manček-Keber M, Jerala R. Structural similarity between the hydrophobic fluorescent probe and lipid A as a ligand of MD-2. FASEB J. 2006; 20:1836–1842.

19. Österlund C, Grönlund H, Polovic N, Sundström S, Gafvelin G, Bucht A. The non-proteolytic house dust mite allergen Der p 2 induce NF-κB and MAPK dependent activation of bronchial epithelial cells. Clin Exp Allergy. 2009; 39:1199–1208.

20. Chiou YL, Lin CY. Der p2 activates airway smooth muscle cells in a TLR2/MyD88-dependent manner to induce an inflammatory response. J Cell Physiol. 2009; 220:311–318.

21. Mueller GA, Benjamin DC, Rule GS. Tertiary structure of the major house dust mite allergen Der p 2: sequential and structural homologies. Biochemistry. 1998; 37:12707–12714.

22. Mueller GA, Smith AM, Chapman MD, Rule GS, Benjamin DC. Hydrogen exchange nuclear magnetic resonance spectroscopy mapping of antibody epitopes on the house dust mite allergen Der p 2. J Biol Chem. 2001; 276:9359–9365.

23. Tanyaratsrisakul S, Jirapongsananuruk O, Kulwanich B, Hales BJ, Thomas WR, Piboonpocanun S. Effect of amino acid polymorphisms of house dust mite Der p 2 variants on allergic sensitization. Allergy Asthma Immunol Res. 2016; 8:55–62.

24. Christensen LH, Holm J, Lund G, Riise E, Lund K. Several distinct properties of the IgE repertoire determine effector cell degranulation in response to allergen challenge. J Allergy Clin Immunol. 2008; 122:298–304.

25. Trompette A, Divanovic S, Visintin A, Blanchard C, Hegde RS, Madan R, et al. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. Nature. 2009; 457:585–588.