Defining Glycan Recognition by DC-SIGN Using Native Mass Spectrometry

Abstract

C-type lectins (CTLs), many of which bind carbohydrates (glycans) in a Ca²⁺-dependent manner, are an important family of glycan-binding proteins that play essential roles in immune recognition and regulation. Among them, dendritic cell–specific ICAM-3-grabbing nonintegrin (DC-SIGN) is notable for its ability to recognize a broad range of self- and pathogen-derived glycans. Despite extensive study, the glycan binding specificity of DC-SIGN remains incompletely understood, in part because accurately quantifying intrinsic lectin–glycan binding affinities across diverse glycan repertoires remains challenging. Here, we employ native mass spectrometry (nMS), a label- and immobilization-free approach well suited for probing weak lectin–glycan interactions, to characterize the glycan-binding preferences of the carbohydrate recognition domain (CRD) of DC-SIGN. Native MS, applied directly or via a catch-and-release (CaR) strategy, enabled quantitative measurements and high-throughput screening under physiologically relevant, nonvolatile salt conditions. We further examined the binding of several divalent metal ions to the DC-SIGN CRD and their influence on glycan recognition. Although divalent cations other than Ca²⁺ bind the CRD with comparable affinity, they exert only modest effects on ligand binding. Screening a defined glycan library, including oligomannoses, human milk oligosaccharides, Lewis and blood group antigens, and ganglioside oligosaccharides, revealed a preference for oligomannoses and Lewis antigens, with affinity positively correlated with the number of mannose or fucose residues. Finally, application of CaR-nMS to a natural N-glycan library identified fucosylated N-glycans as DC-SIGN ligands. Together, these results define the glycan specificity of DC-SIGN and establish nMS as a platform for comprehensive interactome profiling of CTLs.