Innate Antifungal Immune Receptor, Dectin-1, Undergoes Ligand-Induced Oligomerization with Highly Structured β-glucans and at Fungal Cell Contact Sites

Abstract

Dectin-1A is a C-type Lectin innate immunoreceptor that recognizes β-(1,3)-glucan, a structural component of Candida species cell walls. The higher order structure of β-glucans ranges from random coil to insoluble fiber due to varying degrees of tertiary (helical) and quaternary structure. Model Saccharomyces cerevisiae β-glucans of medium and high molecular weight (MMW and HMW, respectively) are highly structured. In contrast, low MW glucan (LMW) are much less structured. Despite similar affinity for Dectin-1, the ability of glucans to induce Dectin-1A mediated calcium influx and Syk phosphorylation positively correlates with their degree of higher order structure. Chemical denaturation and renaturation of MMW glucan showed that glucan structure determines agonistic potential, but not binding affinity, for Dectin-1A. We explored the role of glucan structure on Dectin-1A oligomerization, which is thought to be required for Dectin-1 signaling. Decreased Dectin-1A diffusion coefficient inversely proportional to glucan structural content was consistent with Dectin-1A aggregation. Förster Resonance Energy Transfer (FRET) measurements revealed that molecular aggregation of Dectin-1 occurs in a manner dependent upon glucan higher order structure. Numbers and Brightness analysis specifically confirmed an increase in the Dectin-1A dimer and oligomer populations that is correlated with glucan structure content. Receptor modeling data confirms that in resting cells, Dectin-1A is predominantly in a monomeric state. Super Resolution Microscopy revealed that glucan-stimulated Dectin-1 aggregates are small (<15 nm) collections of engaged receptor. Finally, FRET measurements confirmed increased molecular aggregation of Dectin-1A at fungal particle contact sites in a manner that positively correlated with the degree of exposed glucan on the particle surface. These results indicate that Dectin-1A senses the solution conformation of β-glucans through their varying ability to drive receptor dimer/oligomer formation and activation of membrane proximal signaling events.