Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of mammalian cell-surface receptors, primarily expressed on immune cells and involved in modulating immune functions. Key to their function is an N-terminal lectin domain that mediates the specific binding of distinct sialylated glycan ligands. The elucidation of the molecular basis of their individual carbohydrate specificities has been hampered by the lack of suitable recombinant expression methods for producing these disulfide-containing proteins in sufficient quantities for their in-depth in vitro characterization. To overcome this hurdle, we have established an E. coli-based cytoplasmatic expression protocol for Siglec lectin domains, yielding properly folded protein with intact disulfide bonds in good quantities. Using this expression system, we produced isotopically enriched Siglec-8 and determined its three-dimensional structure in complex with its preferred natural ligand 6′-sulfo sialyl Lewisx by NMR spectroscopy. Siglec-8 contains a canonical recognition motif for sialic acid, extended by a secondary motif formed by unique loop regions, that enables binding of 6-O-sulfated galactose. These structural features, which dictate tight specificity, are distinct from those exhibited by other Siglec family members and any other endogenous glycan-recognition receptors. Structure-guided mutagenesis revealed key interactions essential for ligand binding. Our work provides critical structural and mechanistic insights into how Siglec-8 selectively recognizes its glycan target, rationalizes the functional impact of site-specific glycan-sulfation in modulating this lectin-glycan interaction, and will enable the rational design of Siglec-8-targeted agonists to treat eosinophil- and mast cell-related allergic and inflammatory diseases, such as asthma.

Molecular basis of Siglec-carbohydrate interaction

254th National Meeting (2017)

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