Langerin–Heparin Interaction: Two Binding Sites for Small and Large Ligands As Revealed by a Combination of NMR Spectroscopy and Cross-Linking Mapping Experiments

Abstract

Langerin is a C-type lectin present on Langerhans cells that mediates capture of pathogens in a carbohydrate-dependent manner, leading to subsequent internalization and elimination in the cellular organelles called Birbeck granules. This mechanism mediated by langerin was shown to constitute a natural barrier for HIV-1 particle transmission. Besides interacting specifically with high mannose and fucosylated neutral carbohydrate structures, langerin has the ability to bind sulfated carbohydrate ligands as 6-sulfated galactosides in the Ca(2+)-dependent binding site. Very recently langerin was demonstrated to interact with sulfated glycosaminoglycans (GAGs), in a Ca(2+)-independent way, resulting in the proposal of a new binding site for GAGs. On the basis of those results, we have conducted a structural study of the interactions of small heparin (HEP)-like oligosaccharides with langerin in solution. Heparin bead cross-linking experiments, an approach specifically designed to identify HEP/heparan sulfate binding sites in proteins were first carried out and experimentally validated the previously proposed model for the interaction of langerin extracellular domain with 6 kDa HEP. High-resolution NMR studies of a set of eight synthetic HEP-like trisaccharides harboring different sulfation patterns demonstrated that all of them bound to langerin in a Ca(2+)-dependent way. The binding epitopes were determined by saturation transfer difference NMR and the bound conformations by transferred NOESY experiments. These experimental data were combined with docking and molecular dynamics and resulted in the proposal of a binding mode characterized by the coordination of calcium by the two equatorial hydroxyl groups, OH3 and OH4, at the non-reducing end. The binding also includes the carboxylate group at the adjacent iduronate residue. This epitope is shared by all eight ligands, explaining the absence of any impact on binding from differences in their substitution patterns. Finally, in contrast to the small trisaccharides, we demonstrated that a longer HEP-like hexasaccharide, bearing an additional O-sulfate group at the non-reducing end, which precludes binding to the Ca(2+) site, interacts with langerin in the previously identified Ca(2+)-independent binding site.

Current structural biology of the heparin interactome

“…A recent NMR study has shown that while heparin trisaccharides bind langerin at the calcium-dependent site, a hexasaccharide binds to the GAG-binding site in the same mode as fulllength heparin [32].…”

CarbBuilder: Software for building molecular models of complex oligo‐ and polysaccharide structures

“…Knowledge of the three‐dimensional (3D) structures of carbohydrates can greatly assist in a mechanistic understanding of these processes. As the characterization of carbohydrate molecular conformation remains a central problem in glycobiology, 3D theoretical models are often useful for interpreting experimental results . However, there remain few software tools for building models of complex, possibly branched, polysaccharides from the specification of the primary sequence.…”

Recent Advances in NMR Studies of Carbohydrates

“…These basic components are further varied by epimerization, sulfation, and deacetylation. The study of heparin-protein interaction has been focused mainly on heparin-antithrombin interactions [81][82][83], but there are also several other example such as fibroblast growth factor (FGF) [84] or recently langerin [85]. Sapay and coworkers [86] prepared a 13 C-labeled HS-like oligosaccharide with a defined oligosaccharide sequence.…”