Dendritic Cell-specific Intracellular Adhesion Molecule-3-grabbing Nonintegrin Research Articles

Immunohistochemical Study of SARS-CoV-2 Viral Entry Factors in the Cornea and Ocular Surface.

To confirm the ocular tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by evaluating the expression of viral entry factors in human ocular tissues using immunohistochemistry. Fresh donor corneas and primary explant cultures of corneal, limbal, and conjunctival epithelial cells were evaluated for the expression of viral entry factors. Using immunohistochemistry, the samples were tested for the expression of angiotension-converting enzyme 2 (ACE2), dendritic cell-specific intracellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), DC-SIGN-related protein (DC-SIGNR), and transmembrane serine protease 2 (TMPRSS2). In total, 5 donor corneas were evaluated for the expression of viral entry factors. In all specimens, both ACE2 and TMPRSS2 were expressed throughout the surface epithelium (corneal, limbal, and conjunctival) and corneal endothelium. In corneal stromal cells, ACE2 was sporadically expressed, whereas TMPRSS2 was absent. DC-SIGN/DC-SIGNR expression varied between donor specimens. Four specimens expressed DC-SIGN/DC-SIGNR in a similar distribution to ACE2, but 1 specimen from a young donor showed no expression of DC-SIGN/DC-SIGNR. ACE2, TMPRSS2, and DC-SIGN/DC-SIGNR were all expressed in the cultured corneal, limbal, and conjunctival epithelial cells. Both corneal and conjunctival epithelia express ACE2, DC-SIGN/DC-SIGNR, and TMPRSS2, suggesting that the ocular surface is a potential route for the transmission of SARS-CoV-2. The risk of viral transmission with corneal transplantation cannot be ruled out, given the presence of ACE2 in corneal epithelium and endothelium. Cultured corneal, limbal, and conjunctival epithelial cells mimic the expression of viral entry factors in fresh donor tissue and may be useful for future in vitro SARS-CoV-2 infection studies.

The mTOR-RUNX1 pathway regulates DC-SIGN expression in renal tubular epithelial cells

Renal tubular epithelial cells (RTECs) play pivotal roles in the innate immune response in kidneys. Dendritic cell specific intracellular adhesion molecule–3 grabbing nonintegrin (DC-SIGN) functions as both the innate immune recognition receptor and the adhesion molecule. In our previous study, we found that DC-SIGN expression was induced in RTECs during renal inflammation. However, the underlying mechanism remains unclear. Here, we used the human renal proximal tubular epithelial cell lines (HK-2) to investigate the mechanism of TNF-α-induced expression of DC-SIGN. Our results showed that TNF-α up-regulated the expressions of DC-SIGN and Runt-related transcription factor 1 (RUNX1) in a time-dependent manner and that it up-regulated DC-SIGN promoter-driven luciferase activity in a dose-dependent manner. The mTOR inhibitor rapamycin and mTOR siRNA blocked the TNF-α-induced up-regulation of DC-SIGN expression. Meanwhile, DC-SIGN expression was also inhibited by RUNX1 siRNA and its inhibitor Ro5-3335. In addition, both mTOR and RUNX1 inhibitors attenuated TNF-α-induced the increase in DC-SIGN promoter-driven luciferase activity. Finally, we found that HK-2 cells exposed to rapamycin or mTOR siRNA reduced the TNF-α-induced up-regulation of RUNX1. In conclusion, these results indicated that the mTOR-RUNX1 pathway participates in the regulation of TNF-α-induced DC-SIGN expression in RTECs.

Molecular Insights into DC-SIGN Binding to Self-Antigens: The Interaction with the Blood Group A/B Antigens.

The dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is an important receptor of the immune system. Besides its role as pathogen recognition receptor (PRR), it also interacts with endogenous glycoproteins through the specific recognition of self-glycan epitopes, like LeX. However, this lectin represents a paradigmatic case of glycan binding promiscuity, and it also has been shown to recognize antigens with α1−α2 linked fucose, such as the histo blood group antigens, with similar affinities to LeX. Herein, we have studied the interaction in solution between DC-SIGN and the blood group A and B antigens, to get insights into the atomic details of such interaction. With a combination of different NMR experiments, we demonstrate that the Fuc coordinates the primary Ca2+ ion with a single binding mode through 3-OH and 4-OH. The terminal αGal/αGalNAc affords marginal direct polar contacts with the protein, but provides a hydrophobic hook in which V351 of the lectin perfectly fits. Moreover, we have found that αGal, but not αGalNAc, is a weak binder itself for DC-SIGN, which could endow an additional binding mode for the blood group B antigen, but not for blood group A.

Hydroponic Treatment of Nicotiana benthamiana with Kifunensine Modifies the N-glycans of Recombinant Glycoprotein Antigens to Predominantly Man9 High-Mannose Type upon Transient Overexpression.

Nicotiana benthamiana transient overexpression systems offer unique advantages for rapid and scalable biopharmaceuticals production, including high scalability and eukaryotic post-translational modifications such as N-glycosylation. High-mannose-type glycans (HMGs) of glycoprotein antigens have been implicated in the effectiveness of some subunit vaccines. In particular, Man9GlcNAc2 (Man9) has high binding affinity to mannose-specific C-type lectin receptors such as the mannose receptor and dendritic cell-specific intracellular adhesion molecule 3-grabbing non-integrin (DC-SIGN). Here, we investigated the effect of kifunensine, an α-mannosidase I inhibitor, supplemented in a hydroponic culture of N. benthamiana for the production of Man9-rich HMG glycoproteins, using N-glycosylated cholera toxin B subunit (gCTB) and human immunodeficiency virus gp120 that are tagged with a H/KDEL endoplasmic reticulum retention signal as model vaccine antigens. Biochemical analysis using anti-fucose and anti-xylose antibodies as well as Endo H and PNGase F digestion showed that kifunensine treatment effectively reduced plant-specific glycoforms while increasing HMGs in the N-glycan compositions of gCTB. Detailed glycan profiling revealed that plant-produced gp120 had a glycan profile bearing mostly HMGs regardless of kifunensine treatment. However, the gp120 produced under kifunensine-treatment conditions showed Man9 being the most prominent glycoform (64.5%), while the protein produced without kifunensine had a substantially lower Man9 composition (20.3%). Our results open up possibilities for efficient production of highly mannosylated recombinant vaccine antigens in plants.

Structural characterization of the DC-SIGN-Lewis(X) complex.

Dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a C-type lectin highly expressed on the surface of antigen-presenting dendritic cells. DC-SIGN mediates interactions among dendritic cells, pathogens, and a variety of epithelia, myeloid cells, and endothelia by binding to high mannose residues on pathogenic invaders or fucosylated residues on the membranes of other immune cells. Although these interactions are normally beneficial, they can also contribute to disease. The structural characterization of binding geometries is therefore of interest as a basis for the construction of mimetics that can mediate the effects of abnormal immune response. Here, we report the structural characteristics of the interaction of the DC-SIGN carbohydrate recognition domain (CRD) with a common fucosylated entity, the LewisX trisaccharide (LeX), using NMR methods. Titration of the monomeric DC-SIGN CRD with LeX monitored by 2D NMR revealed significant perturbations of DC-SIGN cross-peak positions in 1H–15N heteronuclear single quantum coherence (HSQC) spectra and identified residues near the binding site. Additionally, saturation transfer difference (STD) and transferred nuclear Overhauser effect (trNOE) NMR experiments, using a tetrameric form of DC-SIGN, identified binding epitopes and bound conformations of the LeX ligand. The restraints derived from these multiple experiments were used to generate models for the binding of LeX to the DC-SIGN CRD. Ranking of the models based on the fit of model-based simulations of the trNOE data and STD buildup curves suggested conformations distinct from those seen in previous crystal structures. The new conformations offer insight into how differences between binding of LewisX and mannose-terminated saccharides may be propagated.

Detection and isolation of dendritic cells using Lewis X-functionalized magnetic nanoparticles.

dendritic cell (DC)-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) is a receptor found on DCs that recognizes antigens bearing mannose-rich or fucosylated glycans, including Lewis X (Le(X)). Here, we report the fabrication of magnetic nanoparticles coated with multivalent Le(X) glycans using Cu (I)-catalyzed azide-alkyne cycloaddition. The resulting nanoparticles are selective and biocompatible, serving as a highly efficient tool for DC detection and enrichment.

Association between the CD209 promoter −336A/G polymorphism and susceptibility to tuberculosis: A meta‐analysis

Dendritic cell-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN), encoded by the CD209 gene, is a major Mycobacterium tuberculosis receptor on human dendritic cells. The potentially functional -336A/G polymorphism in the CD209 promoter region has been associated with susceptibility to tuberculosis (TB), but the results have been inconclusive. We performed a meta-analysis to clarify the relationship between the CD209-336A/G variant and the risk of TB. Ten studies involving a total of 2598 TB patients and 2614 control subjects were systematically reviewed, and the data were quantitatively synthesized by meta-analysis. The Q-test was applied to assess the heterogeneity of associations among the studies, and Egger's regression test was used to assess potential publication bias. No significant association was identified between the CD209-336A/G polymorphism and risk of TB (G allele vs A allele: odds ratio (OR) 1.02, 95% confidence interval (CI) 0.90-1.15). Moreover, no significant association was observed in populations of African ethnicity (OR 1.01, 95% CI 0.87-1.17) or among individuals who were negative for the human immunodeficiency virus (OR 0.98, 95% CI 0.84-1.15). This meta-analysis has indicated that the CD209-336A/G polymorphism may not contribute to susceptibility to TB.

Relationship between polymorphism of DC-SIGN (CD209) gene and the susceptibility to pulmonary tuberculosis in an eastern Chinese population

Dendritic cell–specific intracellular adhesion molecule–3–grabbing nonintegrin (DC-SIGN) is an important receptor for Mycobacterium tuberculosis on human dendritic cells. Previous studies have shown that the variation, especially the −871A/G and −336A/G in DC-SIGN promoter influenced the susceptibility to tuberculosis. We therefore investigated whether polymorphisms in the DC-SIGN gene were associated with susceptibility to tuberculosis in an eastern Chinese population. A total of 237 culture-positive pulmonary tuberculosis case patients and 244 controls were genotyped for −871A/G and −336A/G by pyrosequencing. Our results suggested that the 2 promoter variants of DC-SIGN gene were not associated with susceptibility to tuberculosis in Chinese. Further analysis showed that the allele -336G was associated with a protective effect against fever in pulmonary tuberculosis patients, but not against cavity formation. In addition, we compared the allelic frequencies of −871A/G and −336A/G in African, Caucasian, and Asian groups. The results showed that the tw forms of allelic frequencies detected Chinese individuals in our study were similar to the reported frequencies in other Asian populations but differed significantly from those in the African and Caucasian groups studied.

Frontal affinity chromatography analysis of constructs of DC‐SIGN, DC‐SIGNR and LSECtin extend evidence for affinity to agalactosylated N‐glycans

Dendritic cell‐specific intracellular adhesion molecule‐3‐grabbing nonintegrin (DC‐SIGN) is a member of the C‐type lectin family selectively expressed on immune‐related cells. In the present study, we performed a systematic interaction analysis of DC‐SIGN and its related receptors, DC‐SIGN‐related protein (DC‐SIGNR) and liver and lymph node sinusoidal endothelial cell C‐type lectin (LSECtin) using frontal affinity chromatography (FAC). Carbohydrate‐recognition domains of the lectins, expressed as Fc–fusion chimeras, were immobilized to Protein A–Sepharose and subjected to quantitative FAC analysis using 157 pyridylaminated glycans. Both DC‐SIGN–Fc and DC‐SIGNR–Fc showed similar specificities for glycans containing terminal mannose and fucose, but great difference in affinity under the given experimental conditions. By contrast, LSECtin–Fc showed no affinity to these glycans. As a common feature, the DC‐SIGN‐related lectin–Fc chimeras, including LSECtin, exhibited binding affinity to mono‐ and/or bi‐antennary agalactosylated N‐glycans. The detailed FAC analysis further implied that the presence of terminal GlcNAc at the N‐acetylglucosaminyltransferase I position is a key determinant for the binding of these lectins to agalactosylated N‐glycans. By contrast, none of the lectins showed significant affinity to highly branched agalactosylated N‐glycans. All of the lectins expressed on the cells were able to mediate cellular adhesion to agalactosylated cells and endocytosis of a model glycoprotein, agalactosylated α1‐acid glycoprotein. In this context, we also identified three agalactosylated serum glycoproteins recognized by DC‐SIGN‐Fc (i.e. α‐2‐macroglobulin, serotransferrin and IgG heavy chain), by lectin blotting and MS analysis. Hence, we propose that ‘agalactosylated N‐glycans’ are candidate ligands common to these lectins.

Tumor necrosis factor–alpha, transforming growth factor–β1, and interleukin-10 gene polymorphisms: implication in protection or susceptibility to dengue hemorrhagic fever

Dengue virus infection has emerged as one of the most important arthropod-borne viral diseases. Some dengue infected individuals develop the severe, life-threatening form of the disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Host genetic factors may be relevant and may predispose some individuals to the severe illness. Human leukocyte antigen (HLA), FcγR, tumor necrosis factor (TNF)–α, and dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), among others genes have been associated with the pathogenesis of dengue. Little is known, however, about the predictive value of cytokine genotypes for the clinical outcome of dengue infection. In this study, the TNF-α, interleukin (IL)–6, interferon (IFN)–γ, IL-10 and transforming growth factor (TGF)–β1 gene single nucleotide polymorphisms (SNP) were studied by polymerase chain reaction–sequence-specific primer in a group of individuals with the antecedent of DHF during a secondary infection in the sequence dengue 1/dengue 2. A control group was also included. TNF-α (−308) A allele and IL-10 (−1082/−819/−592) ACC/ATA haplotype were significantly associated with DHF. TNF-α (−308) GG and TGF-β1 (c25) GG genotypes were associated with protection. Our results suggest that genetic predisposition to a high TNF-α production and a low IL-10 production seems to increase the susceptibility to DHF during a secondary dengue 2 infection, whereas TGF-β1 high producers might be protected for developing DHF.

N-Glycan Targeted Gene Delivery to the Dendritic Cell SIGN Receptor

A novel nonviral gene delivery vector composed of a high-mannose N-glycan conjugated to a polyacridine peptide was prepared. The glycopeptide was designed to bind to plasmid DNA by a combination of polyintercalation and ionic binding, and to the DC-SIGN (dendritic cell-specific intracellular adhesion molecule-3 grabbing nonintegrin) receptor expressed on CHO cells by recognition of the high-mannose N-glycan. The glycopeptide conjugate was prepared by purification of a high-mannose N-glycan from affinity fractionated soybean agglutinin (SBA). The SBA was proteolyzed to release the N-glycan which was then modified on its N-terminus with Tyr and a propionate maleimide. A DNA binding polyacridine peptide, Cys-(Acr-Lys)(4), was prepared by solid-phase peptide synthesis using Fmoc-Lys(Acr), then conjugated to the maleimide on the N-glycan to produce a glycopeptide. The glycopeptide bound to DNA with high affinity as determined by fluorophore displacement assay and DNA band shift on agarose gel. When bound to Cy5 labeled DNA, the glycopeptide mediated specific uptake in DC-SIGN CHO (+) cells as determined by FACS analysis. In vitro gene transfer studies established that the glycopeptide increased the specificity of gene transfer in DC-SIGN CHO (+) cells 100-fold relative to CHO (-) cells. These studies suggest that a high-mannose N-glycan conjugated to a polyacridine peptide may also facilitate receptor mediated gene delivery in dendritic cells and thereby find utility in the delivery of DNA vaccines.

Gold Manno‐Glyconanoparticles: Multivalent Systems to Block HIV‐1 gp120 Binding to the Lectin DC‐SIGN

The HIV envelope glycoprotein gp120 takes advantage of the high-mannose clusters on its surface to target the C-type lectin dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) on dendritic cells. Mimicking the cluster presentation of oligomannosides on the virus surface is a strategy for designing carbohydrate-based antiviral agents. Bio-inspired by the cluster presentation of gp120, we have designed and prepared a small library of multivalent water-soluble gold glyconanoparticles (manno-GNPs) presenting truncated (oligo)mannosides of the high-mannose undecasaccharide Man(9)GlcNAc(2) and have tested them as inhibitors of DC-SIGN binding to gp120. These glyconanoparticles are ligands for DC-SIGN, which also interacts in the early steps of infection with a large number of pathogens through specific recognition of associated glycans. (Oligo)mannosides endowed with different spacers ending in thiol groups, which enable attachment of the glycoconjugates to the gold surface, have been prepared. manno-GNPs with different spacers and variable density of mannose (oligo)saccharides have been obtained and characterized. Surface plasmon resonance (SPR) experiments with selected manno-GNPs have been performed to study their inhibition potency towards DC-SIGN binding to gp120. The tested manno-GNPs completely inhibit the binding from the micro- to the nanomolar range, while the corresponding monovalent mannosides require millimolar concentrations. manno-GNPs containing the disaccharide Manalpha1-2Manalpha are the best inhibitors, showing more than 20 000-fold increased activity (100 % inhibition at 115 nM) compared to the corresponding monomeric disaccharide (100 % inhibition at 2.2 mM). Furthermore, increasing the density of dimannoside on the gold platform from 50 to 100 % does not improve the level of inhibition.

Galectins in innate immunity: dual functions of host soluble β‐galactoside‐binding lectins as damage‐associated molecular patterns (DAMPs) and as receptors for pathogen‐associated molecular patterns (PAMPs)

The glycocalyx is a glycan layer found on the surfaces of host cells as well as microorganisms and enveloped virus. Its thickness may easily exceed 50 nm. The glycocalyx does not only serve as a physical protective barrier but also contains various structurally different glycans, which provide cell- or microorganism-specific 'glycoinformation'. This information is decoded by host glycan-binding proteins, lectins. The roles of lectins in innate immunity are well established, as exemplified by collectins, dectin-1, and dendritic cell (DC)-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). These mammalian lectins are synthesized in the secretory pathway and presented on the cell surface to bind to specific glycan 'epitopes'. As they recognize non-self glycans presented by microorganisms, they can be considered as receptors for pathogen-associated molecular patterns (PAMPs), i.e. pattern recognition receptors (PRRs). One notable exception is the galectin family. Galectins are synthesized and stored in the cytoplasm, but upon infection-initiated tissue damage and/or following prolonged infection, cytosolic galectins are either passively released by dying cells or actively secreted by inflammatory activated cells through a non-classical pathway, the 'leaderless' secretory pathway. Once exported, galectins act as PRR, as well as immunomodulators (or cytokine-like modulators) in the innate response to some infectious diseases. As galectins are dominantly found in the lesions where pathogen-initiated tissue damage signals appear, this lectin family is also considered as potential damage-associated molecular pattern (DAMP) candidates that orchestrate innate immune responses alongside the PAMP system.

DC-SIGN and DC-SIGNR genetic diversity among different ethnic populations: Potential implications for pathogen recognition and disease susceptibility

SummaryDendritic cell–specific intracellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN) and DC-SIGNR are C-type lectins that serve both as cell adhesion and pathogen recognition receptors. Because of the essential role of the these molecules in the immune response, the implication of their alleles in human disease states, and the possible genetic variation at these loci among ethnically diverse populations, we undertook a study to analyze the full extent of DC-SIGN and DC-SIGNR polymorphisms in Caucasian Canadian and indigenous African populations. We report several novel nucleotide variants within regulatory 5′- and 3′-untranslated regions of the genes that could affect their transcription and translation. There were significant differences in the distribution of DC-SIGN and DC-SIGNR alleles among African and non-African populations. Finally, our study clearly demonstrates that Africans show greater genetic diversity at these two closely-related immune loci than observed in other major population groups. The differences may reflect evolutionary pressures generated by environmental factors, such as prevalent pathogens in these geographically distinct regions. Further studies will be needed to determine the net impact of DC-SIGN and DC-SIGNR genetic variants on the expression, translation, and function of the proteins and to understand how these functional polymorphisms may affect immune responses or immune escape.

Proteomic Analysis of DC-SIGN on Dendritic Cells Detects Tetramers Required for Ligand Binding but No Association with CD4

DC-SIGN (dendritic cell specific intracellular adhesion molecule 3 grabbing non-integrin) or CD209 is a type II transmembrane protein and one of several C-type lectin receptors expressed by dendritic cell subsets, which bind to high mannose glycoproteins promoting their endocytosis and potential degradation. DC-SIGN also mediates attachment of HIV to dendritic cells and binding to this receptor can subsequently lead to endocytosis or enhancement of CD4/CCR5-dependent infection. The latter was proposed to be facilitated by an interaction between DC-SIGN and CD4. Endocytosis of HIV virions does not necessarily lead to their complete degradation. A proportion of the virions remain infective and can be later presented to T cells mediating their infection in trans. Previously, the extracellular domain of recombinant DC-SIGN has been shown to assemble as tetramers and in the current study we use a short range covalent cross-linker and show that DC-SIGN exists as tetramers on the surface of immature monocyte-derived dendritic cells. There was no evidence of direct binding between DC-SIGN and CD4 either by cross-linking or by fluorescence resonance energy transfer measurements suggesting that there is no constitutive association of the majority of these proteins in the membrane. Importantly we also show that the tetrameric complexes, in contrast to DC-SIGN monomers, bind with high affinity to high mannose glycoproteins such as mannan or HIV gp120 suggesting that such an assembly is required for high affinity binding of glycoproteins to DC-SIGN, providing the first direct evidence that DC-SIGN tetramers are essential for high affinity interactions with pathogens like HIV.