Structures of the Inhibitory Receptor Siglec‑8 in Complex with a High-Affinity Sialoside Analogue and a Therapeutic Antibody

Human sialic acid binding immunoglobulin-like
lectin-8 (Siglec-8) is an inhibitory receptor that triggers eosinophil
apoptosis and can inhibit mast cell degranulation when engaged by
specific monoclonal antibodies (mAbs) or sialylated ligands. Thus,
Siglec-8 has emerged as a critical negative regulator of
inflammatory responses in diverse diseases, such as allergic airway
inflammation. Herein, we have deciphered the molecular
recognition features of the interaction of Siglec-8 with the mAb
lirentelimab (2C4, under clinical development) and with a sialoside
mimetic with the potential to suppress mast cell degranulation. The
three-dimensional structure of Siglec-8 and the fragment antigen
binding (Fab) portion of the anti-Siglec-8 mAb 2C4, solved by Xray
crystallography, reveal that 2C4 binds close to the carbohydrate
recognition domain (V-type Ig domain) on Siglec-8. We have also deduced the binding mode of a high-affinity analogue of its sialic
acid ligand (9-N-napthylsufonimide-Neu5Ac, NSANeuAc) using a combination of NMR spectroscopy and X-ray crystallography.
Our results show that the sialoside ring of NSANeuAc binds to the canonical sialyl binding pocket of the Siglec receptor family and
that the high affinity arises from the accommodation of the NSA aromatic group in a nearby hydrophobic patch formed by the Nterminal
tail and the unique G−G′ loop. The results reveal the basis for the observed high affinity of this ligand and provide clues for
the rational design of the next generation of Siglec-8 inhibitors. Additionally, the specific interactions between Siglec-8 and the Nlinked
glycans present on the high-affinity receptor FcεRIα have also been explored by NMR.