Langerin-heparin interaction: two binding sites for small and large ligands as revealed by a combination of NMR spectroscopy and cross-linking mapping experiments.
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.
†Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.
Juan C
JC
Muñoz-García
‡Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France.|§CNRS, IBS, F-38044, Grenoble, France.|∥CEA, IBS, F-38044 Grenoble, France.
Eric
E
Chabrol
‡Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France.|§CNRS, IBS, F-38044, Grenoble, France.|∥CEA, IBS, F-38044 Grenoble, France.
Romain R
RR
Vivès
⊥CERMAV UPR5301, CNRS and Université Grenoble Alpes, BP 53, 38041 Grenoble cedex 9, France.
Aline
A
Thomas
†Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.
José L
JL
de Paz
†Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.
Javier
J
Rojo
⊥CERMAV UPR5301, CNRS and Université Grenoble Alpes, BP 53, 38041 Grenoble cedex 9, France.
Anne
A
Imberty
‡Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France.|§CNRS, IBS, F-38044, Grenoble, France.|∥CEA, IBS, F-38044 Grenoble, France.
Franck
F
Fieschi
†Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.
Pedro M
PM
Nieto
†Glycosystems Laboratory, Instituto de Investigaciones Químicas (IIQ), Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.|#School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.
Jesús
J
Angulo
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D006493:Heparin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D037181:Lectins, C-Type / Q000737:chemistry / Q000378:metabolism*; D037241:Mannose-Binding Lectins / Q000737:chemistry / Q000378:metabolism*; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D014312:Trisaccharides / Q000737:chemistry / Q000378:metabolism
D015703:Antigens, CD; C121916:CD207 protein, human; D002413:Cations, Divalent; D037181:Lectins, C-Type; D037241:Mannose-Binding Lectins; D009844:Oligosaccharides; D014312:Trisaccharides; D006493:Heparin; D002118:Calcium