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

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.

Allergens and their associated small molecule ligands—their dual role in sensitization

“…It is possible that Bla g 1 ligands could also have immunomodulatory properties which have not been fully explored. Lastly, Bla g 1 has a very high affinity for phosphatidylcholine (PC) that exceeds the affinity for the natural fatty acids 15 . PC is a major component of lung surfactant so it is possible that Bla g 1 would exchange fatty acids for PC disrupting the natural balance of surfactant in the lung, leading to barrier defects.…”

“…The protein structure of Bla g 1 corresponding to a tandem repeat is spherical with twelve α‐helices that enclose a large cavity capable of enclosing up to four diacyl‐phospholipids. 15 , 28 Recombinant Bla g 1 contained a variety of phospholipids whose nature depended on the expression system (Figure 5D ), whereas natural Bla g 1 bound a mixture of the fatty acids palmitate, oleate, and stearate. When loaded with these natural fatty acids, Bla g 1 was both more proteolytically resistant and thermally stable than when loaded with lipids with shorter or longer fatty acid chains.…”

“… 150 The stabilizing effect of the ligands anticorrelated with the generation of known T‐cell epitopes using an in vitro assay. 15 The implication is that the stabilizing property of the lipids could modulate the generation of T‐cell epitopes and subsequent allergenicity. As precedent, isoforms of Bet v 1 that display enhanced stability are able to avoid premature processing in the endosome, yielding a stronger Th2 response than less allergenic variants.…”

“…14 Likewise, the binding of fatty acids by the cockroach allergen Bla g 1 was found to significantly enhance the allergen's thermostability while inhibiting cleavage by cathepsin S, an endosomal protease essential for antigen processing and presentation. 15 These topics are discussed in more details below. This review summarizes our current knowledge on allergenic proteins that bind small molecule ligands.…”

“… Availability of experimental molecular structures for a range of allergens with hydrophobic cavities 16 , 17 Establishment of biophysical and biochemical methods to study ligand‐protein interaction and its impact on local structural changes 18 , 19 , 20 , 21 , 22 , 23 Identification of group 2 house dust mite (HDM) allergens as ligand‐binding proteins 16 , 17 Discovery of the natural ligand of Bet v 1 24 , 25 , 26 Discovery of the natural ligand of Pru p 3 12 Elucidation of the role of the phytosphingosin part of the Pru p 3 ligand in the sensitization process 27 Discovery of Bla g 1 ligands 15 , 28 Discovery of the natural ligand of Cor a 1 29 Allergens from the PR‐10 family bind a broader spectrum of ligands in vitro than in vivo 25 , 26 , 30 Ligand enhances proteolytic resistance of Bet v 1 and inhibits endolysosomal cathepsin S protease activity 14 …”