TETRALEC, Artificial Tetrameric Lectins: A Tool to Screen Ligand and Pathogen Interactions

Abstract

C-type lectin receptor (CLR)/carbohydrate recognition occurs through low affinity interactions. Nature compensates that weakness by multivalent display of the lectin carbohydrate recognition domain (CRD) at the cell surface. Mimicking these low affinity interactions in vitro is essential to better understand CLR/glycan interactions. Here, we present a strategy to create a generic construct with a tetrameric presentation of the CRD for any CLR, termed TETRALEC. We applied our strategy to a naturally occurring tetrameric CRD, DC-SIGNR, and compared the TETRALEC ligand binding capacity by synthetic N- and O-glycans microarray using three different DC-SIGNR constructs i) its natural tetrameric counterpart, ii) the monomeric CRD and iii) a dimeric Fc-CRD fusion. DC-SIGNR TETRALEC construct showed a similar binding profile to that of its natural tetrameric counterpart. However, differences observed in recognition of low affinity ligands underlined the importance of the CRD spatial arrangement. Moreover, we further extended the applications of DC-SIGNR TETRALEC to evaluate CLR/pathogens interactions. This construct was able to recognize heat-killed Candida albicans by flow cytometry and confocal microscopy, a so far unreported specificity of DC-SIGNR. In summary, the newly developed DC-SIGNR TETRALEC tool proved to be useful to unravel novel CLR/glycan interactions, an approach which could be applied to other CLRs.

DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist

“…The extracellular domains (ECD) of DC-SIGN (residues: 66-404), L-SIGN (residues: 78-399), Langerin (residues: 68-328) and MGL (residues: 61-292) were produced and purified as already described [88][89][90][91] while SARS-CoV-2 Spike protein was expressed and purified as follows. The mammalian expression vectors used for the S ectodomain, derived from a pαH vector, was a kind gift from J. McLellan [30].…”

Fc‐conjugated C‐type lectin receptors: Tools for understanding host–pathogen interactions

“…Many of these binding sites would not be exposed when the CRDs are clustered into HMW complexes. In general, binding profiles of either DC-SIGN or DC-SIGNR when used as pre-complexed Fc proteins, dimeric Fc protein or bacterial expressed proteins are comparable, with some differences in binding to low-affinity ligands observed (Achilli et al, 2020;Geissner et al, 2019;Tateno et al, 2008).…”

“…Alternative approaches such as the use of the fusion proteins precomplexed to fluorescent beads or antibodies, or harnessing systems using variations on fluorescently labeled streptavidin-binding biotin tagging have been successfully used (Achilli et al, 2020) (See Figure 1). These examples highlight some of the challenges using Fc CLRs, instead of cell membrane expressed receptors, as tools for probing ligand content on pathogens.…”

Complex Carbohydrates and Glycoconjugates: Structure, Functions and Applications

“…Artificial tetrameric lectins were constructed by Achilli et al [9]. C-type lectin receptor (CLR)/carbohydrate recognition occurs through low affinity interactions.…”

“…DC-SIGNR TETRALEC was applied to evaluate CLR/pathogens interactions. This construct was able to recognize heat-killed Candida albicans by flow cytometry and confocal microscopy, a so far unreported specificity of DC-SIGNR [9].…”