Hydrophobic ligands influence the structure, stability, and processing of the major cockroach allergen Bla g 1

Alexander C Y Foo,Lalith Perera,Simrat Arora,Jason Williams,Geoffrey A Mueller,Peter M Thompson,Eugene F Derose

doi:10.1038/s41598-019-54689-8

Alexander C Y Foo, Lalith Perera + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-54689-8

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Abstract

The cockroach allergen Bla g 1 forms a novel fold consisting of 12 amphipathic alpha-helices enclosing an exceptionally large hydrophobic cavity which was previously demonstrated to bind a variety of lipids. Since lipid-dependent immunoactivity is observed in numerous allergens, understanding the structural basis of this interaction could yield insights into the molecular determinants of allergenicity. Here, we report atomic modelling of Bla g 1 bound to both fatty-acid and phospholipids ligands, with 8 acyl chains suggested to represent full stoichiometric binding. This unusually high occupancy was verified experimentally, though both modelling and circular dichroism indicate that the general alpha-helical structure is maintained regardless of cargo loading. Fatty-acid cargoes significantly enhanced thermostability while inhibiting cleavage by cathepsin S, an endosomal protease essential for antigen processing and presentation; the latter of which was found to correlate to a decreased production of known T-cell epitopes. Both effects were strongly dependent on acyl chain length, with 18–20 carbons providing the maximal increase in melting temperature (~20 °C) while completely abolishing proteolysis. Diacyl chain cargoes provided similar enhancements to thermostability, but yielded reduced levels of proteolytic resistance. This study describes how the biophysical properties of Bla g 1 ligand binding and digestion may relate to antigen processing, with potential downstream implications for immunogenicity.

Highlights

The cockroach allergen Bla g 1 forms a novel fold consisting of 12 amphipathic alpha-helices enclosing an exceptionally large hydrophobic cavity which was previously demonstrated to bind a variety of lipids
To generate samples of Bla g 1 that were uniformly loaded with a defined cargo the annealing process was carried out in the presence of >16:1 molar excess of the desired molecules. 31P NMR spectra obtained for Bla g 1 loaded with distearoyl phosphatidylcholine (DSPC) lipid in this manner show the presence of the desired phosphatidylcholine (PC) cargo while no peaks from the recombinant expression system are detected, suggesting that the cleaning and loading protocol was successful (Fig. 1a); a conclusion supported by thin layer chromatography studies coupled with iodine staining, or CuSO4-phosphoric acid charring (Supplementary Fig. S1)
Circular dichroism (CD) spectra obtained for Bla g 1 prepared using this method displays the characteristic minima at ~208 and 222 nm indicative of an alpha-helical protein consistent with the available crystal structure[6], suggesting that the structure of Bla g 1 is recovered after the purification and annealing process (Fig. 1c)

Summary

Introduction

The cockroach allergen Bla g 1 forms a novel fold consisting of 12 amphipathic alpha-helices enclosing an exceptionally large hydrophobic cavity which was previously demonstrated to bind a variety of lipids. Previous studies show that Bla g 1 is able to bind a range of phospholipids and fatty acids, and the available X-ray structure of Bla g 1 reports electron density within the central cavity suggesting it may act as a lipid -binding site.

Results

Conclusion