Regulation of mast cells by overlapping but distinct protein interactions of Siglec‐6 and Siglec‐8

Abstract Background Sialic acid‐binding immunoglobulin‐like lectin (Siglec)‐6 and Siglec‐8 are closely related mast cell (MC) receptors with broad inhibitory activity, but whose functional differences are incompletely understood. Methods Proteomic profiling using quantitative mass spectrometry was performed on primary mouse MCs to identify proteins associated with Siglec‐6 and Siglec‐8. For functional characterization, each receptor was evaluated biochemically and in ex vivo and in vivo inhibition models of IgE and non‐IgE‐mediated MC activation in Siglec‐6‐ or Siglec‐8‐expressing transgenic mice. Results Siglec‐6 and Siglec‐8 were found in MCs within large complexes, interacting with 66 and 86 proteins, respectively. Strikingly, Siglec‐6 and Siglec‐8 interacted with a large cluster of proteins involved in IgE and non‐IgE‐mediated MC activation, including the high affinity IgE receptor, stem cell factor (SCF) receptor KIT/CD117, IL‐4 and IL‐33 receptors, and intracellular kinases LYN and JAK1. Protein interaction networks revealed Siglec‐6 and Siglec‐8 had overlapping yet distinct MC functions, with a potentially broader regulatory role for Siglec‐6. Indeed, Siglec‐6 preferentially interacted with the mature form of KIT at the cell surface, and treatment with an anti‐Siglec‐6 antibody significantly inhibited SCF‐mediated MC activation more in comparison to targeting Siglec‐8. Conclusion These data demonstrate a central role for Siglec‐6 and Siglec‐8 in controlling MC activation through interactions with multiple activating receptors and key signaling molecules. Our findings suggest that Siglec‐6 has a role distinct from that of Siglec‐8 in regulating MC function and represents a distinct potential therapeutic target in mast cell‐driven diseases.