Reversible Modulation of Glycan Recognition in C-Type Lectins by Calcium/Lanthanide Exchange.

C-type lectins are Ca2+-dependent glycan-binding proteins involved in immune recognition and host-pathogen interactions. Previous structural studies suggested that lanthanide ions can replace Ca2+ while preserving protein structure, raising the possibility of exploiting paramagnetic lanthanides as NMR probes for studying glycan recognition. Here, we investigated Ca2+/Ln3+ exchange in the immune lectins DC-SIGN and MGL using T2-filtered 19F-NMR binding assays with monofluorinated monosaccharides, together with heteronuclear NMR analysis of 15N-labeled proteins. Chemical shift perturbations, pseudocontact shifts, and paramagnetic relaxation enhancements showed preferential replacement of the Ca2+ ion at the glycan-binding site, while the remaining Ca2+-binding sites were less susceptible to exchange. Unexpectedly, substitution with La3+ or Yb3+ suppresses carbohydrate binding in both lectins, despite preserving the folded structure of the carbohydrate-recognition domains. Glycan recognition was restored upon EDTA-mediated removal of lanthanides in excess Ca2+, demonstrating the reversibility of the process. These findings show that lanthanides do not necessarily behave as functional Ca2+ mimetics in C-type lectins but instead act as reversible disruptors of glycan recognition. Beyond highlighting the need to assess the functional consequences of Ca2+ substitution, this work establishes Ca2+/Ln3+ exchange as a strategy for controlled modulation of lectin activity and for developing new tools to investigate C-type lectin-mediated biological processes.