Unraveling Molecular Recognition of Glycan Ligands by Siglec-9 via NMR Spectroscopy and Molecular Dynamics Modeling.

Human sialic-acid-binding immunoglobulin-like lectin-9 (Siglec-9) is a glycoimmune checkpoint receptor expressed on several immune cells. Binding of Siglec-9 to sialic acid containing glycans (sialoglycans) is well documented to modulate its functions as an inhibitory receptor. Here, we first assigned the amino acid backbone of the Siglec-9 V-set domain (Siglec-9d1), using well-established triple resonance three-dimensional nuclear magnetic resonance (NMR) methods. Then, we combined solution NMR and molecular dynamic simulation methods to decipher the molecular details of the interaction of Siglec-9 with the natural ligands α2,3 and α2,6 sialyl lactosamines (SLN), sialyl Lewis X (sLeX), and 6-O sulfated sLeX and with two synthetically modified sialoglycans that bind with high affinity. As expected, Neu5Ac is accommodated between the F and G ß-strands at the canonical sialic acid binding site. Addition of a heteroaromatic scaffold 9N-5-(2-methylthiazol-4-yl)thiophene sulfonamide (MTTS) at the C9 position of Neu5Ac generates new interactions with the hydrophobic residues located at the G-G' loop and the N-terminal region of Siglec-9. Similarly, the addition of the aromatic substituent (5-N-(1-benzhydryl-1H-1,2,3-triazol-4-yl)methyl (BTC)) at the C5 position of Neu5Ac stabilizes the conformation of the long and flexible B'-C loop present in Siglec-9. These results expose the underlying mechanism responsible for the enhanced affinity and specificity for Siglec-9 for these two modified sialoglycans and sheds light on the rational design of the next generation of modified sialoglycans targeting Siglec-9.

Structural basis for sulfation-dependent self-glycan recognition by the human immune-inhibitory receptor Siglec-8.

Siglec-8 is a human immune-inhibitory receptor that, when engaged by specific self-glycans, triggers eosinophil apoptosis and inhibits mast cell degranulation, providing an endogenous mechanism to down-regulate immune responses of these central inflammatory effector cells. Here we used solution NMR spectroscopy to dissect the fine specificity of Siglec-8 toward different sialylated and sulfated carbohydrate ligands and determined the structure of the Siglec-8 lectin domain in complex with its prime glycan target 6'-sulfo sialyl Lewis(x) A canonical motif for sialic acid recognition, extended by a secondary motif formed by unique loop regions, recognizing 6-O-sulfated galactose dictates tight specificity distinct from other Siglec family members and any other endogenous glycan recognition receptors. Structure-guided mutagenesis revealed key contacts of both interfaces to be equally essential for 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.

Functional Siglec lectin domains from soluble expression in the cytoplasm of Escherichia coli.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are a family of mammalian cell-surface receptors that are involved in cell-cell interactions and signaling functions, primarily expressed on cells of the immune system. Key to their function is their specific binding of distinct sialylated glycan ligands mediated via an N-terminal carbohydrate recognition (lectin) domain. Studies concerning the molecular basis of their individual carbohydrate specificities are rare due to the absence of suitable recombinant expression methods for producing these disulfide-containing proteins in sufficient quantities required for their in-depth in vitro characterization. We established an efficient E. coli-based expression and purification method for Siglec lectin domains, utilizing the trxB gor suppressor strain Rosetta-gami B (DE3) in which proper folding with intact disulfide bonds was achieved in the cytoplasm. The approach is demonstrated for human Siglec-7, -8 and -9 lectin domains and works equally well for expression in nutrient-rich (LB) or minimal growth medium, allowing stable-isotope labeling for NMR studies. The recombinant proteins were properly folded as proven by 2D (1)H-(15)N HSQC NMR spectroscopy and by thermal unfolding followed by CD spectroscopy, and functionally active as confirmed by monitoring ligand binding using NMR titration experiments. Our method enables efficient production of homogeneous and active protein samples in milligram quantities. Its implementation will significantly enhance future structure-function studies of this important class of immune-modulating receptors and will support a variety of applications including screening for natural and synthetic ligands or the development of fluorescently-labeled molecular tools for glycan ligand detection or flow-cytometric cell sorting.