Abstract
Multivalent interactions between complex carbohydrates and oligomeric C-type lectins govern a wide range of immune responses. Up to date, standard SPR (surface plasmon resonance) competitive assays have largely been to evaluate binding properties from monosaccharide units (low affinity, mM) to multivalent elemental antagonists (moderate affinity, μM). Herein, we report typical case-studies of SPR competitive assays showing that they underestimate the potency of glycoclusters to inhibit the interaction between DC-SIGN and immobilized glycoconjugates. This paper describes the design and implementation of a SPR direct interaction over DC-SIGN oriented surfaces, extendable to other C-type lectin surfaces as such Langerin. This setup provides an overview of intrinsic avidity generation emanating simultaneously from multivalent glycoclusters and from DC-SIGN tetramers organized in nanoclusters at the cell membrane. To do so, covalent biospecific capture of DC-SIGN via StreptagII/StrepTactin interaction preserves tetrameric DC-SIGN, accessibility and topology of its active sites, that would have been dissociated using standard EDC-NHS procedure under acidic conditions. From the tested glycoclusters libraries, we demonstrated that the scaffold architecture, the valency and the glycomimetic-based ligand are crucial to reach nanomolar affinities for DC-SIGN. The glycocluster 3·D illustrates the tightest binding partner in this set for a DC-SIGN surface (KD = 18 nM). Moreover, the selectivity at monovalent scale of glycomimetic D can be easily analyzed at multivalent scale comparing its binding over different C-type lectin immobilized surfaces. This approach may give rise to novel insights into the multivalent binding mechanisms responsible for avidity and make a major contribution to the full characterization of the binding potency of promising specific and multivalent immodulators.
Highlights
DC-SIGN extracellular domain (ECD) at pH 7.4 is largely negatively charged and is repulsed from the negatively charge surface of the sensor preventing its functionalization. It confirms that it is the biospecific interaction between the StreptagII, at the N-ter of the S-ECD construct, and StrepTactin that preconcentrate the protein at the surface allowing its reaction with activated groups
The multivalent interaction in glycosciences is considered as one of the more complex interaction due to the unpredictable avidity generated by carbohydrate multi presentation
DCSIGN S-ECD oriented surface, that can be extended to other Ctype lectins, as shown with Langerin, were designed, characterized and validated as method to evaluate reliable affinities for promising multivalent immunomodulators
Summary
(8) To overcome these limits of the competition assay, monitoring the direct interaction between multivalent compounds and a DC-SIGN surface would be the best approach to evaluate surface-generated avidity (Figure 1C). The bio-specific capture of DC-SIGN, in a buffer at physiological pH yields undisturbed tetrameric DC-SIGN ECD surfaces with well-oriented CRDs. In that setting, we are able to accurately estimate the affinity and the avidity of multivalent optimized compounds.
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