Langerin Carbohydrate Recognition Domain Research Articles

A Common Genetic Variation in Langerin (CD207) Compromises Cellular Uptake of Staphylococcus aureus

Langerhans cells are key sentinel cells of the skin and mucosal lining. They sense microorganisms through their repertoire of pattern-recognition receptors to mount and direct appropriate immune responses. We recently demonstrated that human Langerhans cells interact with the Gram-positive pathogen Staphylococcus aureus through the Langerhans cell-specific receptor langerin (CD207). It was previously hypothesized that two linked single nucleotide polymorphisms (SNPs; N288D and K313I) in the carbohydrate recognition domain of langerin would affect interaction with microorganisms. We show that recognition of S. aureus by recombinant langerin molecules is abrogated in the co-inheriting SNP variant, which is mainly explained by the N288D SNP and further enhanced by K313I. Moreover, introduction of SNP N288D in ectopically-expressed langerin affected cellular distribution of the receptor such that langerin displayed enhanced plasma membraneexpression. Despite this increased binding of S. aureus by the langerin double SNP variant, uptake of bacteria by this langerin variant was compromised. Our findings indicate that in a proportion of the human population, the recognition and uptake of S. aureus by Langerhans cells may be affected, which could have important consequences for proper immune activation and S. aureus-associated disease.

Intradomain Allosteric Network Modulates Calcium Affinity of the C-Type Lectin Receptor Langerin.

Antigen uptake and processing by innate immune cells is crucial to initiate the immune response. Therein, the endocytic C-type lectin receptors serve as pattern recognition receptors, detecting pathogens by their glycan structures. Herein, we studied the carbohydrate recognition domain of Langerin, a C-type lectin receptor involved in the host defense against viruses such as HIV and influenza as well as bacteria and fungi. Using a combination of nuclear magnetic resonance and molecular dynamics simulations, we unraveled the molecular determinants underlying cargo capture and release encoded in the receptor architecture. Our findings revealed receptor dynamics over several time scales associated with binding and release of the essential cofactor Ca(2+) controlled by the coupled motions of two loops. Applying mutual information theory and site-directed mutagenesis, we identified an allosteric intradomain network that modulates the Ca(2+) affinity depending on the pH, thereby promoting fast ligand release.

Structural Studies of Langerin and Birbeck Granule: A Macromolecular Organization Model

Dendritic cells, a sentinel immunity cell lineage, include different cell subsets that express various C-type lectins. For example, epidermal Langerhans cells express langerin, and some dermal dendritic cells express DC-SIGN. Langerin is a crucial component of Birbeck granules, the Langerhans cell hallmark organelle, and may have a preventive role toward HIV, by its internalization into Birbeck granules. Since langerin carbohydrate recognition domain (CRD) is crucial for HIV interaction and Birbeck granule formation, we produced the CRD of human langerin and solved its structure at 1.5 A resolution. On this basis gp120 high-mannose oligosaccharide binding has been evaluated by molecular modeling. Hydrodynamic studies reveal a very elongated shape of recombinant langerin extracellular domain (ECD). A molecular model of the langerin ECD, integrating the CRD structure, has been generated and validated by comparison with hydrodynamic parameters. In parallel, Langerhans cells were isolated from human skin. From their analysis by electron microscopy and the langerin ECD model, an ultrastructural organization is proposed for Birbeck granules. To delineate the role of the different langerin domains in Birbeck granule formation, we generated truncated and mutated langerin constructs. After transfection into a fibroblastic cell line, we highlighted, in accordance with our model, the role of the CRD in the membrane zipping occurring in BG formation as well as some contribution of the cytoplasmic domain. Finally, we have shown that langerin ECD triggering with a specific mAb promotes global rearrangements of LC morphology. Our results open the way to the definition of a new membrane deformation mechanism.

Overproduction, purification and preliminary crystallographic analysis of the carbohydrate-recognition domain of human langerin.

Langerin, a lectin that is specific to Langerhans cells, interacts with glycoconjugates through its carbohydrate-recognition domain (CRD). This carbohydrate binding occurs by an avidity-based mechanism that is enabled by the neck domain responsible for trimerization. Langerin binds HIV through its CRD and thus plays a protective role against its propagation by the internalization of virions in Birbeck granules. Here, the overproduction, purification and crystallization of the langerin CRD is reported. Crystals obtained by the hanging-drop vapour-diffusion method allowed the collection of a complete data set to 1.5 A resolution and belonged to the tetragonal space group P4(2), with unit-cell parameters a = b = 79.55, c = 90.14 A.

The carbohydrate recognition domain of Langerin reveals high structural similarity with the one of DC-SIGN but an additional, calcium-independent sugar-binding site

Langerin is a type II transmembrane oligosaccharide receptor on Langerhans cells (LCs), a prominent subclass of dendritic cells (DCs) that mediate immune responses in epithelia and play a role in HIV degradation. Its extracellular moiety comprises a neck region with several heptad repeats and an exposed carboxy-terminal calcium-type carbohydrate-recognition domain (CRD). The CRD of human Langerin, which was expressed as a soluble protein in the periplasm of E. coli, was crystallized both alone and in the presence of two sugars, followed by X-ray analyses to resolutions of 2.5 Å for apo-Langerin and to 1.6 Å and 2.1 Å for the complexes with mannose and maltose, respectively. The fold of the Langerin CRD (dubbed LangA) resembles that of other typical C-type lectins such as DC-SIGN. However, especially in the long loop region (LLR), which is responsible for carbohydrate-binding, two additional secondary structure elements are present: a 3 10 helix and a small β-sheet arising from the extended β-strand 2, which enters into a hairpin and a new strand β2′. Unexpectedly, the crystal structures in the presence of maltose and mannose reveal two sugar-binding sites. One is calcium-dependent and structurally conserved in the C-type lectin family whereas the second one represents a novel, calcium-independent type. Based on these data, a model for the binding of mannan, a component of many endogenous as well as viral glycoproteins, is proposed and the differences in binding behavior between Langerin and DC-SIGN with respect to the Lewis X carbohydrate antigen and its derivatives can be explained. Therefore, the crystal structure of LangA should be helpful for the development of new marker reagents selective for LCs and also of therapeutic compounds that may enhance the inhibitory role of Langerin towards HIV infection.

Polymorphisms in Human Langerin Affect Stability and Sugar Binding Activity

Langerhans cells are specialized skin dendritic cells that take up and degrade antigens for presentation to the immune system. Langerin, a cell surface C-type lectin of Langerhans cells, can be internalized and accumulates in Birbeck granules, subdomains of the endosomal recycling compartment that are specific to Langerhans cells. Langerin binds and mediates uptake and degradation of glycoconjugates containing mannose and related sugars. Analysis of the human genome has identified three single nucleotide polymorphisms that result in amino acid changes in the carbohydrate-recognition domain of langerin. The effects of the amino acid changes on the activity of langerin were examined by expressing each of the polymorphic forms. Expression of full-length versions of the four common langerin haplotypes in fibroblasts revealed that all of these forms can mediate endocytosis of neoglycoprotein ligands. However, sugar binding assays and differential scanning calorimetry performed on fragments from the extracellular domain showed that two of the amino acid changes reduce the affinity of the carbohydrate-recognition domain for mannose and decrease the stability of the extracellular domain. In addition, analysis of sugar binding by langerin containing the rare W264R mutation, previously identified in an individual lacking Birbeck granules, shows that this mutation abolishes sugar binding activity. These findings suggest that certain langerin haplotypes may differ in their binding to pathogens and thus might be associated with susceptibility to infection.

Analysis of binding of mannosides in relation to Langerin (CD207) in Langerhans cells of normal and transformed epithelia.

Tandem-repeat C-type lectins (pattern-recognition receptors) with specificity for mannosides are intimately involved in antigen recognition, uptake, routing and presentation in macrophages and dendritic cells. In Langerhans cells, Langerin (CD207), a type-II transmembrane protein with a single C-type carbohydrate recognition domain attached to a heptad repeat in the neck region, which is likely to establish oligomers with an alpha-coiled-coil stalk, has been implicated in endocytosis and the formation of Birbeck granules. The structure of Langerin harbours essential motifs for Ca2+-binding and sugar accommodation. Lectin activity has previously been inferred by diminished antibody binding to cells in the presence of the glycan ligand mannan. In view of the complexity of the C-type lectin/lectin-like network, it is unclear what role Langerin plays for Langerhans cells in binding mannosides. In order to reveal in frozen tissue sections to what extent mannose-binding activity co-localizes with Langerin, we have used a synthetic marker, i.e. a neoglycoprotein carrying mannose maxiclusters, as a histochemical ligand, and computer-assisted fluorescence monitoring in a double-labelling procedure. Mannoside-binding capacity was detected in normal epithelial cells. Double labelling ensured the unambiguous assessment of the binding of the neoglycoprotein in Langerhans cells. Light-microscopically, its localization profile resembled the pattern of immunohistochemical detection of Langerin. This result has implications for suggesting rigorous controls in histochemical analysis of this cell type, because binding of kit reagents, i.e. mannose-rich glycoproteins horseradish peroxidase or avidin, to Langerin (or a spatially closely associated lectin) could yield false-positive signals. To show that recognition of carbohydrate ligands in dendritic cells is not restricted to mannose clusters, we have also documented binding of carrier-immobilized histo-blood group A trisaccharide, a ligand of galectin-3, which was not affected by the presence of a blocking antibody to Langerin. Remarkably, access to the carbohydrate recognition domain of Langerin appeared to be impaired in proliferatively active environments (malignancies, hair follicles), indicating presence of an endogenous ligand with high affinity to saturate the C-type lectin under these conditions.