Abstract
Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. The connections between Siglecs and human disease motivate improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively. Using these Siglec-Fc proteins, Siglec ligands are systematically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. Additional features enable the production of small, homogenous Siglec fragments and development of a quantitative ligand-binding mass spectrometry assay. Using this assay, the ligand specificities of several Siglecs are clarified. For CD33 (Siglec-3), we demonstrate that it recognizes both α2-3 and α2-6 sialosides in solution and on cells, which has implications for its link to Alzheimer’s disease susceptibility. These soluble Siglecs reveal the abundance of their glycan ligands on host cells as self-associated molecular patterns.
Highlights
Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands
Aiming to generate soluble Siglecs that are compatible with a variety of assays, four features were incorporated: (1) a His6-tag for purification that is directly C-terminal to the Fc domain; (2) a Streptag II for purification and detection; (3) mutations in the Fc domain (L234A, L235A, G237A, H268A, P238S, A330S, P331S)
Interactions between Siglecs and their sialic acid-containing glycan ligands are proposed to be a form of ‘self’ recognition used by immune cells
Summary
Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively Using these Siglec-Fc proteins, Siglec ligands are systematically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. For CD33 (Siglec-3), we demonstrate that it recognizes both α2-3 and α2-6 sialosides in solution and on cells, which has implications for its link to Alzheimer’s disease susceptibility These soluble Siglecs reveal the abundance of their glycan ligands on host cells as selfassociated molecular patterns. To better understand Siglec-mediated regulation of immune cell function and develop more targeted approaches to block Siglec–ligand interactions, better methods are needed to probe the glycan ligands of Siglecs. Avoiding a secondary antibody would be desirable for detection of cellular Siglec ligands
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