Abstract
A dense and diverse array of glycans on glycoproteins and glycolipids decorate all cell surfaces. In vertebrates, many of these carry sialic acid, in a variety of linkages and glycan contexts, as their outermost sugar moiety. Among their functions, glycans engage complementary glycan binding proteins (lectins) to regulate cell physiology. Among the glycan binding proteins are the Siglecs, sialic acid binding immunoglobulin-like lectins. In humans, there are 14 Siglecs, most of which are expressed on overlapping subsets of immune system cells. Each Siglec engages distinct, endogenous sialylated glycans that initiate signaling programs and regulate cellular responses. Here, we explore the emerging science of Siglec ligands, including endogenous sialoglycoproteins and glycolipids and synthetic sialomimetics. Knowledge in this field promises to reveal new molecular pathways controlling cell physiology and new opportunities for therapeutic intervention.
Highlights
Siglecs, sialic acid binding immunoglobulin-like lectins, are a family of structurally related animal cell surface glycan binding proteins [1,2,3]
Enhanced binding was found when the terminal ganglioside trisaccharide was further substituted with an α2-6-linked sialic acid on the GalNAc, Neu5Acα2-3Galβ13(Neu5Acα2-6)GalNAc, the terminus of so-called “α-series” gangliosides such as GQ1bα that are selectively expressed on cholinergic neurons [58]
This same terminus is attached to proteins as the disialyl T antigen, suggesting that in some circumstances O-linked glycoproteins may engage myelin-associated glycoprotein (MAG) [59]
Summary
Sialic acid binding immunoglobulin-like lectins, are a family of structurally related animal cell surface glycan binding proteins [1,2,3]. We first describe some of the experimental approaches used to identify Siglec sialoglycan binding, including glycan arrays, structural elucidation, design and screening of synthetic glycomimetics, cell-based expression systems and extraction, and purification and characterization of endogenous Siglec ligands. Fc chimeras have the advantage that they spontaneously form dimers and can be further complexed with commercial fluorescent or enzyme-tagged anti-human-Fc antibodies to increase their multivalency This is technically important, since the site affinities of Siglecs for monovalent glycans are typically modest (KD > 100 μM), with rapid off rates that preclude many types of binding experiments. By engineering in a Strep-tag sequence, the Fc chimeras were efficiently tetramerized using Strep-Tactin to create octameric multimers that increased binding to cellular targets by nearly 1000-fold compared to the dimeric Fc. In an alternate approach, the extracellular domains of Siglecs were expressed with a C-terminal pentamerizing domain from cartilage oligomeric matrix protein to create high-avidity multivalent probes [19]. The utility of soluble tagged versions of many human and mouse Siglecs is well established
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