Characterization of a Phl p 6 mutant with increased structural stability

Wai Tuck Soh, Stefan Stubenvoll, Andreas Brückl, Sandra Scheiblhofer, Josef Laimer, P Lackner, Josef Thalhamer, Hans Brandstetter, Richard Weiss

Publikation: KonferenzbeitragPoster

Abstract

Introduction: Protein fold stability has been proposed to represent an intrinsic feature contributing to allergenicity. It was recently demonstrated that increased structural stability of an allergen can lead to prolonged survival of endolysosomal degradation, resulting in altered presentation of immunodominant T cell epitopes, thereby promoting T helper 2 type immune responses 1. In ongoing work, we investigate fold stability mutants of the Timothy grass pollen allergen Phl p 6 with respect to changes in their antigen processing and presentation.
Methods: By employing MAESTRO algorithm, Phl p 6 point mutant S46Y was predicted to have a G of -1,27 kcal/mol, indicating an increase in structural stability. The mutant was expressed in E. coli, purified by chromatography, and analyzed for thermal stability by circular dichroism (CD). To evaluate its capacity to stimulate T cell proliferation, co-cultures of bone-marrow derived dendritic cells (BMDCs) pulsed with various concentrations of the wild-type and the mutated Phl p 6 together with a T cell hybridoma specific for the immunodominant epitope of Phl p 6 were performed. Finally, the structure of the mutant protein was obtained by X-ray crystallography.
Results: Circular dichroism analysis confirmed the MAESTRO prediction as Phl p 6 S46Y mutant displayed a higher melting temperature of 72.5 °C compared to the wild-type allergen (58.5 °C). The high resolution crystal structure revealed an overall high structural similarity of the mutant and the wild-type protein (PDB: 1nlx). The Tyr46 was found to complement the aromatic stacking and thereby to stabilize the hydrophobic core interaction of the four-helix bundle architecture of Phl p 6, explaining the increased thermal stability found by CD. IL-2 production of Phl p 6-specific T cells was weaker following culture with BMDCs pulsed with the mutant, pointing to reduced presentation of the immunodominant epitope.
Conclusion: The replacement of serine 46 to tyrosine maintains the overall structure of Phl p 6 while stabilizing its fold as compared to the natural allergen. Rigidification of the molecule led to reduced presentation of the immunodominant T cell epitope in vitro, suggesting reduced or delayed processing. Whether this also translates to decreased in vivo immunogenicity and/or allergenicity remains to be investigated.
OriginalspracheEnglisch
PublikationsstatusVeröffentlicht - 17 Jun 2017
VeranstaltungEAACI Congress 2017 - Helsinki, Finnland
Dauer: 17 Jun 201721 Jun 2017

Konferenz

KonferenzEAACI Congress 2017
LandFinnland
OrtHelsinki
Zeitraum17/06/1721/06/17

Systematik der Wissenschaftszweige 2012

  • 106 Biologie
  • 301 Medizinisch-theoretische Wissenschaften, Pharmazie

Zitieren

Soh, W. T., Stubenvoll, S., Brückl, A., Scheiblhofer, S., Laimer, J., Lackner, P., Thalhamer, J., Brandstetter, H., & Weiss, R. (2017). Characterization of a Phl p 6 mutant with increased structural stability. Postersitzung präsentiert bei EAACI Congress 2017, Helsinki, Finnland.