Discovery of a new sialic acid binding region that regulates Siglec-7

Abstract

Siglec-7 is a human CD33-like siglec, and is localised predominantly on human natural killer (NK) cells and monocytes. Siglec-7 is considered to function as an immunoreceptor in a sialic acid-dependent manner. However, the underlying mechanisms linking sialic acid-binding and function remain unknown. Here, to gain new insights into the ligand-binding properties of Siglec-7, we carried out in silico analysis and site-directed mutagenesis, and found a new sialic acid-binding region (site 2 containing R67) in addition to the well-known primary ligand-binding region (site 1 containing R124). This was supported by equilibrium dialysis, STD-NMR experiments, and inhibition analysis of GD3-binding toward Siglec-7 using synthetic sialoglycoconjugates and a comprehensive set of ganglioside-based glycoconjugates. Our results suggest that the two ligand-binding sites are potentially controlled by each other due to the flexible conformation of the C-C′ loop of Siglec-7. Siglecs are sialic acid (Sia)-binding immunoglobulin-like lectins and the majority of them function as transmembrane receptors in the immune system via recognition of Sia residues 1. The expression pattern of siglecs is often cell type-specific. Through cytosolic regions or adaptor proteins, they are able to regulate inhibitory or activating signals. Siglecs contain a Sia-binding N-terminal V-set domain followed by 1 to 16 C2-set Ig-domains that are considered to act as spacers or regulators of oligomerisation. Siglecs differ from most other mammalian Sia-binding lectins, such as selectins, with respect to the absolute requirement for Sia, but the details of their molecular interactions, including specific natural ligands and ligand-binding mechanisms, remain largely unclear. These are thought to be important for immune cell functions via Sia-binding because disrupting the interactions between Sias and siglecs can alter responses to infection, inflammation, autoimmunity, and cancer 1. Therefore, understanding the role of ligand-binding in biological functions of siglecs is important. The binding affinity between siglecs and naturally occurring sialylated glycoconjugates is known to be relatively weak (0.1-3 mM) 2 , and therefore biological interactions between siglecs and their ligands depend on clustering and multivalent binding. For example, mucins can display multiple ligands, and gangliosides can be clustered within lipid microdomains. To conquer the low affinity of Sia ligands for siglecs, recent research in Sia chemistry has focussed on the development of biochemical probes toward siglecs with high affinity and specificity 2-6. Attempts to increase the affinity were successful, and in some cases materials were used for clinical therapy 7,8. However, the precise details of how siglecs engage their ligands remain unclear. Siglec-7 (CDw328) is the major siglec expressed by NK cells where it can function as an inhibitory receptor 4,9,10. Siglec-7 consists of an extracellular N-terminal V-set Ig domain that is responsible for a Sia-binding, two

Identification and functional characterization of a Siglec-7 counter-receptor on K562 cells

“…Recently, we demonstrated the presence of a secondary Siglec-7 binding site (site 2) in addition to the previously known binding site (site 1) ( 19 ). It has been suggested that Siglec-7 has several Sia-binding sites and that its counter-receptor for Siglec-7 has polyvalent glycans ( e.g.…”

“…1 A ). In addition, the Siglec-7 R124A mutant, in which the Arg residue essential for Sia binding is replaced by Ala ( 18 , 19 , 20 ), showed no binding, indicating that binding occurred with the V-set domain of Siglec-7. The binding signal of Siglec-7 to K562 was stronger than that of HeLa cells, indicating that K562 expressed the ligands at a higher level than HeLa cells.…”

“…Complex ligand specificity has also been shown by several sets of glycan array experiments [ http://www.functionalglycomics.org/glycomics/publicdata/primaryscreen.jsp ]. Although it is still unknown why such complex specificity occurs in the region of the Siglec-7 V-set domain containing residue R124, which was proven by X-ray crystallography as necessary for ligand binding ( 17 , 18 ), the existence of a new Sia-binding region (site 2 containing R67) was recently demonstrated based on in silico docking simulation and subsequent mutation experiments ( 19 ).…”

Siglecs Modulate Activities of Immune Cells Through Positive and Negative Regulation of ROS Generation

“…Siglec-sialoglycan binding is weak and transient (24). Interaction between Siglecs with multivalent ligands leads to Siglec clustering, which increases the strength of Siglec-ligand binding and initiates cellular signaling (22,(25)(26)(27).…”

Sialic Acids and Their Influence on Human NK Cell Function

“…The binding domain of Siglec-7 is well characterized, and different crystal structures are available that can help to understand the binding specificity and to identify possible substrates and inhibitors [ 72 , 73 ]. In addition to the sialic acid-binding site around the essential arginine residue (R124), Siglec-7 has another binding site in the V-set domain, and both binding sites contribute to glycan recognition [ 74 ]. The signaling of Siglec-7 is similar to other inhibitory Siglecs, and is mediated through the membrane-proximal ITIM.…”