Mannose glycoconjugates functionalized at positions 1 and 6. Binding analysis to DC-SIGN using biosensors

The design of glycoconjugates to allow the generation of multivalent ligands capable of interacting with the receptor DC-SIGN is a topic of high interest due to the role played by this lectin in pathogen infections. Mannose, a ligand of this lectin, could be conjugated at two different positions, 1 and 6, not implicated in the binding process. We have prepared mannose conjugates at these two positions with a long spacer to allow their attachment to a biosensor chip surface. Analysis of the interaction between these surfaces and the tetravalent extracellular domain (ECD) of DC-SIGN by SPR biosensor has demonstrated that both positions are available for this conjugation without affecting the protein binding process. These results emphasize the possibility to conjugate mannose at position 6, allowing the incorporation of hydrophobic groups at the anomeric position to interact with hydrophobic residues in the carbohydrate recognition domain of DC-SIGN, increasing binding affinities. This fact is relevant for the future design of new ligands and the corresponding multivalent systems for DC-SIGN.     

Genetically-encoded fragment-based discovery of glycopeptide ligands for DC-SIGN

We have employed genetically-encoded fragment-based discovery to identify novel glycopeptides with affinity for the dendritic cell receptor DC-SIGN. Starting from libraries of 10⁸ mannose-conjugated peptides, we identified glycopeptides that exhibited up to a 650-fold increase in multivalent binding affinity for DC-SIGN, which is also preserved in cells. Monovalently, our most potent glycopeptides have a similar potency to a Man₃ oligosaccharide, representing a 15-fold increase in activity compared to mannose. These compounds represent the first examples of glycopeptide ligands that target the CRD of DC-SIGN. The natural framework of glycopeptide conjugates and the simplicity of orthogonal conjugation to make these glycopeptides anticipates a promising future for development of DC-SIGN-targeting moieties.     

Measles virus targets DC-SIGN to enhance dendritic cell infection

Dendritic cells (DCs) are involved in the pathogenesis of measles virus (MV) infection by inducing immune suppression and possibly spreading the virus from the respiratory tract to lymphatic tissues. It is becoming evident that DC function can be modulated by the involvement of different receptors in pathogen interaction. Therefore, we have investigated the relative contributions of different MV-specific receptors on DCs to MV uptake into and infection of these cells. DCs express the MV receptors CD46 and CD150, and we demonstrate that the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is a novel receptor for laboratory-adapted and wild-type MV strains. The ligands for DC-SIGN are both MV glycoproteins F and H. In contrast to CD46 and CD150, DC-SIGN does not support MV entry, since DC-SIGN does not confer susceptibility when stably expressed in CHO cells. However, DC-SIGN is important for the infection of immature DCs with MV, since both attachment and infection of immature DCs with MV are blocked in the presence of DC-SIGN inhibitors. Our data demonstrate that DC-SIGN is crucial as an attachment receptor to enhance CD46/CD150-mediated infection of DCs in cis. Moreover, MV might not only target DC-SIGN to infect DCs but may also use DC-SIGN for viral transmission and immune suppression.     

cis Expression of DC-SIGN Allows for More Efficient Entry of Human and Simian Immunodeficiency Viruses via CD4 and a Coreceptor

DC-SIGN is a C-type lectin expressed on dendritic cells and restricted macrophage populations in vivo that binds gp120 and acts in trans to enable efficient infection of T cells by human immunodeficiency virus type 1 (HIV-1). We report here that DC-SIGN, when expressed in cis with CD4 and coreceptors, allowed more efficient infection by both HIV and simian immunodeficiency virus (SIV) strains, although the extent varied from 2- to 40-fold, depending on the virus strain. Expression of DC-SIGN on target cells did not alleviate the requirement for CD4 or coreceptor for viral entry. Stable expression of DC-SIGN on multiple lymphoid lines enabled more efficient entry and replication of R5X4 and X4 viruses. Thus, 10- and 100-fold less 89.6 (R5/X4) and NL4-3 (X4), respectively, were required to achieve productive replication in DC-SIGN-transduced Jurkat cells when compared to the parental cell line. In addition, DC-SIGN expression on T-cell lines that express very low levels of CCR5 enabled entry and replication of R5 viruses in a CCR5-dependent manner, a property not exhibited by the parental cell lines. Therefore, DC-SIGN expression can boost virus infection in cis and can expand viral tropism without affecting coreceptor preference. In addition, coexpression of DC-SIGN enabled some viruses to use alternate coreceptors like STRL33 to infect cells, whereas in its absence, infection was not observed. Immunohistochemical and confocal microscopy data indicated that DC-SIGN was coexpressed and colocalized with CD4 and CCR5 on alveolar macrophages, underscoring the physiological significance of these cis enhancement effects.     

Oligolysine-based oligosaccharide clusters: selective recognition and endocytosis by the mannose receptor and dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin

Dendritic cells are potent antigen-presenting cells that express several membrane lectins, including the mannose receptor and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin). To identify highly specific ligands for these dendritic cell receptors, oligosaccharides were converted into glycosynthons (Os1) and were used to prepare oligolysine-based glycoclusters, Os-[Lys(Os)]n-Ala-Cys-NH2. Clusters containing two to six dimannosides as well as clusters containing four or five pentasaccharides (Lewisa or Lewisx) or hexasaccharides (Lewisb) were synthesized. The thiol group of the appended cysteine residue allows easy tagging by a fluorescent probe or convenient substitution with an antigen. Surface plasmon resonance was used to determine the affinity of the different glycoclusters for purified mannose receptor and DC-SIGN, whereas flow cytometry and confocal microscopy analysis allowed assessment of cell uptake of fluoresceinyl-labeled glycoclusters. Dimannoside clusters are recognized by the mannose receptor with an affinity constant close to 106 liter.mol-1 but have a very low affinity for DC-SIGN (less than 104 liter x mol-1). Conversely, Lewis clusters have a higher affinity toward DC-SIGN than toward the mannose receptor. Dimannoside clusters are efficiently taken up by human dendritic cells as well as by rat fibroblasts expressing the mannose receptor but not by HeLa cells or rat fibroblasts expressing DC-SIGN; DC-SIGN-expressing cells take up Lewis clusters. The results suggest that ligands containing dimannoside clusters can be used specifically to target the mannose receptor, whereas ligands containing Lewis clusters will be targeted to DC-SIGN.     

Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR

Both the dendritic cell receptor DC-SIGN and the closely related endothelial cell receptor DC-SIGNR bind human immunodeficiency virus and enhance infection. However, biochemical and structural comparison of these receptors now reveals that they have very different physiological functions. By screening an extensive glycan array, we demonstrated that DC-SIGN and DC-SIGNR have distinct ligand-binding properties. Our structural and mutagenesis data explain how both receptors bind high-mannose oligosaccharides on enveloped viruses and why only DC-SIGN binds blood group antigens, including those present on microorganisms. DC-SIGN mediates endocytosis, trafficking as a recycling receptor and releasing ligand at endosomal pH, whereas DC-SIGNR does not release ligand at low pH or mediate endocytosis. Thus, whereas DC-SIGN has dual ligand-binding properties and functions both in adhesion and in endocytosis of pathogens, DC-SIGNR binds a restricted set of ligands and has only the properties of an adhesion receptor.     

Hepatitis C virus targets DC-SIGN and L-SIGN to escape lysosomal degradation

Hepatitis C virus (HCV) is a major health problem. However, the mechanism of hepatocyte infection is largely unknown. We demonstrate that the dendritic cell (DC)-specific C-type lectin DC-SIGN and its liver-expressed homologue L-SIGN/DC-SIGNR are important receptors for HCV envelope glycoproteins E1 and E2. Mutagenesis analyses demonstrated that both HCV E1 and E2 bind the same binding site on DC-SIGN as the pathogens human immunodeficiency virus type 1 (HIV-1) and mycobacteria, which is distinct from the cellular ligand ICAM-3. HCV virus-like particles are efficiently captured and internalized by DCs through binding of DC-SIGN. Antibodies against DC-SIGN specifically block HCV capture by both immature and mature DCs, demonstrating that DC-SIGN is the major receptor on DCs. Interestingly, internalized HCV virus-like particles were targeted to nonlysosomal compartments within immature DCs, where they are protected from lysosomal degradation in a manner similar to that demonstrated for HIV-1. Lewis X antigen, another ligand of DC-SIGN, was internalized to lysosomes, demonstrating that the internalization pathway of DC-SIGN-captured ligands may depend on the structure of the ligand. Our results suggest that HCV may target DC-SIGN to "hide" within DCs and facilitate viral dissemination. L-SIGN, expressed by THP-1 cells, internalized HCV particles into similar nonlysosomal compartments, suggesting that L-SIGN on liver sinusoidal endothelial cells may capture HCV from blood and transmit it to hepatocytes, the primary target for HCV. We therefore conclude that both DCs and liver sinusoidal endothelial cells may act as reservoirs for HCV and that the C-type lectins DC-SIGN and L-SIGN, as important HCV receptors, may represent a molecular target for clinical intervention in HCV infection.     

Identification of Novel Contributions to High-affinity Glycoprotein-Receptor Interactions using Engineered Ligands

Engineered receptor fragments and glycoprotein ligands employed in different assay formats have been used to dissect the basis for the dramatic enhancement of binding of two model membrane receptors, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the macrophage galactose lectin, to glycoprotein ligands compared to simple sugars. These approaches make it possible to quantify the importance of two major factors that combine to enhance the affinity of single carbohydrate-recognition domains (CRDs) for glycoprotein ligands by 100-to 300-fold. First, the presence of extended binding sites within a single CRD can enhance interaction with branched glycans, resulting in increases of fivefold to 20-fold in affinity. Second, presentation of glycans on a glycoprotein surface increases affinity by 15-to 20-fold, possibly due to low-specificity interactions with the surface of the protein or restriction in the conformation of the glycans. In contrast, when solution-phase networking is avoided, enhancement due to binding of multiple branches of a glycan to multiple CRDs in the oligomeric forms of these receptors is minimal and binding of a receptor oligomer to multiple glycans on a single glycoprotein makes only a twofold contribution to overall affinity. Thus, in these cases, multivalent interactions of individual glycoproteins with individual receptor oligomers have a limited role in achieving high affinity. These findings, combined with considerations of membrane receptor geometry, are consistent with the idea that further enhancement of the binding to multivalent glycoprotein ligands requires interaction of multiple receptor oligomers with the ligands.     

DC-SIGN mediated sphingomyelinase-activation and ceramide generation is essential for enhancement of viral uptake in dendritic cells

As pattern recognition receptor on dendritic cells (DCs), DC-SIGN binds carbohydrate structures on its pathogen ligands and essentially determines host pathogen interactions because it both skews T cell responses and enhances pathogen uptake for cis infection and/or T cell trans-infection. How these processes are initiated at the plasma membrane level is poorly understood. We now show that DC-SIGN ligation on DCs by antibodies, mannan or measles virus (MV) causes rapid activation of neutral and acid sphingomyelinases followed by accumulation of ceramides in the outer membrane leaflet. SMase activation is important in promoting DC-SIGN signaling, but also for enhancement of MV uptake into DCs. DC-SIGN-dependent SMase activation induces efficient, transient recruitment of CD150, which functions both as MV uptake receptor and microbial sensor, from an intracellular Lamp-1+ storage compartment shared with acid sphingomyelinase (ASM) within a few minutes. Subsequently, CD150 is displayed at the cell surface and co-clusters with DC-SIGN. Thus, DC-SIGN ligation initiates SMase-dependent formation of ceramide-enriched membrane microdomains which promote vertical segregation of CD150 from intracellular storage compartments along with ASM. Given the ability to promote receptor and signalosome co-segration into (or exclusion from) ceramide enriched microdomains which provide a favorable environment for membrane fusion, DC-SIGN-dependent SMase activation may be of general importance for modes and efficiency of pathogen uptake into DCs, and their routing to specific compartments, but also for modulating T cell responses.     

Semen clusterin is a novel DC-SIGN ligand

The C-type lectin receptor dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is an important player in the recognition of pathogens by dendritic cells. A plethora of pathogens including viruses, bacteria, parasites, and fungi are recognized by DC-SIGN through both mannose and fucose-containing glycans expressed on the pathogen surface. In this study, we identified semen clusterin as a novel DC-SIGN ligand. Semen clusterin, but not serum clusterin, expresses an extreme abundance of fucose-containing blood-type Ags such as Le(x) and Le(y), which are both excellent DC-SIGN ligands. These motifs enable semen clusterin to bind DC-SIGN with very high affinity (K(d) 76 nM) and abrogate the binding of HIV-1 to DC-SIGN. Depletion of clusterin from semen samples, however, did not completely prevent the ability of semen to inhibit the capture of HIV-1 by DC-SIGN, supporting that besides clusterin, semen contains other DC-SIGN ligands. Further studies are needed to characterize these ligands and define their contribution to the DC-SIGN-blocking activity mediated by semen. Clusterin is an enigmatic protein involved in a variety of physiologic and pathologic processes including inflammation, atherosclerosis, and cancer. Our results uncover an unexpected heterogeneity in the glycosylation pattern of clusterin and suggest that the expression of high concentrations of fucose-containing glycans enables semen clusterin to display a unique set of biological functions that might affect the early course of sexually transmitted infectious diseases.     

The dendritic cell-specific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells

Dendritic cells (DCs) capture Ags or viruses in peripheral tissue to transport them to lymphoid organs to induce cellular T cell responses. Recently, a DC-specific C-type lectin was identified, DC-specific ICAM-grabbing non-integrin (DC-SIGN), that functions as cell adhesion receptor mediating both DC migration and T cell activation. DC-SIGN also functions as an HIV-1R that captures HIVgp120 and facilitates DC-induced HIV transmission of T cells. Internalization motifs in the cytoplasmic tail of DC-SIGN hint to a function of DC-SIGN as endocytic receptor. In this study we demonstrate that on DCs DC-SIGN is rapidly internalized upon binding of soluble ligand. Mutating a putative internalization motif in the cytoplasmic tail reduces ligand-induced internalization. Detailed analysis using ratio fluorescence imaging and electron microscopy showed that DC-SIGN-ligand complexes are targeted to late endosomes/lysosomes. Moreover, ligands internalized by DC-SIGN are efficiently processed and presented to CD4+ T cells. The distinct pattern of expression of C-type lectins on DCs in situ and their nonoverlapping Ag recognition profile hint to selective functions of these receptors to allow a DC to recognize a wide variety of Ags and to process these to induce T cell activation. These data point to a novel function of the adhesion receptor DC-SIGN as an efficient DC-specific Ag receptor that can be used as a target to induce viral and antitumor immunity.     

Identification of four novel DC-SIGN ligands on Mycobacterium bovis BCG

Dendritic-cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN; CD209) has an important role in mediating adherence of Mycobacteria species, including M. tuberculosis and M. bovis BCG to human dendritic cells and macrophages, in which these bacteria can survive intracellularly. DC-SIGN is a C-type lectin, and interactions with mycobacterial cells are believed to occur via mannosylated structures on the mycobacterial surface. Recent studies suggest more varied modes of binding to multiple mycobacterial ligands. Here we identify, by affinity chromatography and mass-spectrometry, four novel ligands of M. bovis BCG that bind to DC-SIGN. The novel ligands are chaperone protein DnaK, 60 kDa chaperonin-1 (Cpn60.1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and lipoprotein lprG. Other published work strongly suggests that these are on the cell surface. Of these ligands, lprG appears to bind DC-SIGN via typical protein-glycan interactions, but DnaK and Cpn60.1 binding do not show evidence of carbohydrate-dependent interactions. LprG was also identified as a ligand for DC-SIGNR (L-SIGN; CD299) and the M. tuberculosis orthologue of lprG has been found previously to interact with human toll-like receptor 2. Collectively, these findings offer new targets for combating mycobacterial adhesion and within-host survival, and reinforce the role of DC-SIGN as an important host ligand in mycobacterial infection.     

Cellular uptake of gold nanoparticles bearing HIV gp120 oligomannosides

Dendritic cells are the most potent of the professional antigen-presenting cells which display a pivotal role in the generation and regulation of adaptive immune responses against HIV-1. The migratory nature of dendritic cells is subverted by HIV-1 to gain access to lymph nodes where viral replication occurs. Dendritic cells express several calcium-dependent C-type lectin receptors including dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN), which constitute a major receptor for HIV-1. DC-SIGN recognizes N-linked high-mannose glycan clusters on HIV gp120 through multivalent and Ca(2+)-dependent protein-carbohydrate interactions. Therefore, mimicking the cluster presentation of oligomannosides from the virus surface is a strategic approach for carbohydrate-based microbicides. We have shown that gold nanoparticles (mannoGNPs) displaying multiple copies of structural motifs (di-, tri-, tetra-, penta-, or heptaoligomanosides) of the N-linked high-mannose glycan of viral gp120 are efficient inhibitors of DC-SIGN-mediated trans-infection of human T cells. We have now prepared the corresponding fluorescent-labeled glyconanoparticles (FITC-mannoGNPs) and studied their uptake by DC-SIGN expressing Burkitt lymphoma cells (Raji DC-SIGN cell line) and monocyte-derived immature dendritic cells (iDCs) by flow cytometry and confocal laser scanning microscopy. We demonstrate that the 1.8 nm oligomannoside coated nanoparticles are endocytosed following both DC-SIGN-dependent and -independent pathways and part of them colocalize with DC-SIGN in early endosomes. The blocking and sequestration of DC-SIGN receptors by mannoGNPs could explain their ability to inhibit HIV-1 trans-infection of human T cells in vitro.     

A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands

DC-SIGN and DC-SIGNR are cell-surface receptors that mediate cell-cell interactions within the immune system by binding to intercellular adhesion molecule-3. The receptor polypeptides share 77% amino acid sequence identity and are type II transmembrane proteins. The extracellular domain of each comprises seven 23-residue tandem repeats and a C-terminal C-type carbohydrate-recognition domain (CRD). Cross-linking, equilibrium ultracentrifugation, and circular dichroism studies of soluble recombinant fragments of DC-SIGN and DC-SIGNR have been used to show that the extracellular domain of each receptor is a tetramer stabilized by an alpha-helical stalk. Both DC-SIGN and DC-SIGNR bind ligands bearing mannose and related sugars through the CRDs. The CRDs of DC-SIGN and DC-SIGNR bind Man(9)GlcNAc(2) oligosaccharide 130- and 17-fold more tightly than mannose, and affinity for a glycopeptide bearing two such oligosaccharides is increased by a further factor of 5- to 25-fold. These results indicate that the CRDs contain extended or secondary oligosaccharide binding sites that accommodate mammalian-type glycan structures. When the CRDs are clustered in the tetrameric extracellular domain, their arrangement provides a means of amplifying specificity for multiple glycans on host molecules targeted by DC-SIGN and DC-SIGNR. Binding to clustered oligosaccharides may also explain the interaction of these receptors with the gp120 envelope protein of human immunodeficiency virus-1, which contributes to virus infection.     

Tumor biomarker glycoproteins in the seminal plasma of healthy human males are endogenous ligands for DC-SIGN

DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewis(x) and Lewis(y) carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewis(x) and Lewis(y) sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.     

N-linked glycosylation facilitates sialic acid-independent attachment and entry of influenza A viruses into cells expressing DC-SIGN or L-SIGN

It is widely recognized that sialic acid (SA) can mediate attachment of influenza virus to the cell surface, and yet the specific receptors that mediate virus entry are not known. For many viruses, a definitive demonstration of receptor function has been achieved when nonpermissive cells are rendered susceptible to infection following transfection of the gene encoding a putative receptor. For influenza virus, such approaches have been confounded by the abundance of SA on mammalian cells so that it has been difficult to identify cell lines that are not susceptible to infection. We examined influenza virus infection of Lec2 Chinese hamster ovary (CHO) cells, a mutant cell line deficient in SA. Lec2 CHO cells were resistant to influenza virus infection, and stable cell lines expressing either DC-SIGN or L-SIGN were generated to assess the potential of each molecule to function as SA-independent receptors for influenza A viruses. Virus strain BJx109 (H3N2) bound to Lec2 CHO cells expressing DC-SIGN or L-SIGN in a Ca(2+)-dependent manner, and transfected cells were susceptible to virus infection. Treatment of Lec2-DC-SIGN and Lec2-L-SIGN cells with mannan, but not bacterial neuraminidase, blocked infection, a finding consistent with SA-independent virus attachment and entry. Moreover, virus strain PR8 (H1N1) bears low levels of mannose-rich glycans and was inefficient at infecting Lec2 CHO cells expressing either DC-SIGN or L-SIGN, whereas other glycosylated H1N1 subtype viruses could infect cells efficiently. Together, these data indicate that human C-type lectins (DC-SIGN and L-SIGN) can mediate attachment and entry of influenza viruses independently of cell surface SA.     

Design,functional evaluation and biomedical applications of carbohydrate dendrimers (glycodendrimers)

Receptors for carbohydrates of the lectin type are multisubunit and multivalent proteins with many important biological functions. In order to put their unique biological activities into use in biotechnology and biomedicine, efficient carbohydrate ligands of the glycodendrimer type have been constructed. Although these compounds may be branched into the multiple generations, structures bearing four to 16 terminal carbohydrate substituents have proved to be efficient ligands in most lectin systems. These compounds are rapidly finding important practical applications as antitumor and antiinfective compounds.     

DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages

Human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma and pleural effusion lymphoma. In this study, we show that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) is a receptor for HHV-8 infection of myeloid DCs and macrophages. DC-SIGN was required for virus attachment to these cells and DC-SIGN-expressing cell lines. HHV-8 binding and infection were blocked by anti-DC-SIGN mAb and soluble DC-SIGN, and mannan, a natural ligand for DC-SIGN. Infection of DCs and macrophages with HHV-8 led to production of viral proteins, with little production of viral DNA, similar to HHV-8 infection of vascular endothelial cells. Infection of DCs resulted in down-regulation of DC-SIGN, a decrease in endocytic activity, and an inhibition of Ag stimulation of CD8+ T cells. We propose that DC-SIGN serves as a portal for immune dysfunction and oncogenesis caused by HHV-8 infection.     

Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN)-mediated enhancement of dengue virus infection is independent of DC-SIGN internalization signals

Dengue virus (DV) is a mosquito-borne flavivirus that causes hemorrhagic fever in humans. In the natural infection, DV is introduced into human skin by an infected mosquito vector where it is believed to target immature dendritic cells (DCs) and Langerhans cells (LCs). We found that DV productively infects DCs but not LCs. We show here that the interactions between DV E protein, the sole mannosylated glycoprotein present on DV particles, and the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) are essential for DV infection of DCs. Binding of mannosylated N-glycans on DV E protein to DC-SIGN triggers a rapid and efficient internalization of the viral glycoprotein. However, we observed that endocytosis-defective DC-SIGN molecules allow efficient DV replication, indicating that DC-SIGN endocytosis is dispensable for the internalization step in DV entry. Together, these results argue in favor of a mechanism by which DC-SIGN enhances DV entry and infection in cis. We propose that DC-SIGN concentrates mosquito-derived DV particles at the cell surface to allow efficient interaction with an as yet unidentified entry factor that is ultimately responsible for DV internalization and pH-dependent fusion into DCs.     

Noncarbohydrate Glycomimetics and Glycoprotein Surrogates as DC-SIGN Antagonists and Agonists

An understanding of the biological roles of lectins will be advanced by ligands that can inhibit or even recruit lectin function. To this end, glycomimetics, noncarbohydrate ligands that function analogously to endogenous carbohydrates, are being sought. The advantage of having such ligands is illustrated by the many roles of the protein DC-SIGN. DC-SIGN is a C-type lectin displayed on dendritic cells, where it binds to mannosides and fucosides to mediate interactions with other host cells or bacterial or viral pathogens. DC-SIGN engagement can modulate host immune responses (e.g., suppress autoimmunity) or benefit pathogens (e.g., promote HIV dissemination). DC-SIGN can bind to glycoconjugates, internalize glycosylated cargo for antigen processing, and transduce signals. DC-SIGN ligands can serve as inhibitors as well as probes of the lectin's function, so they are especially valuable for elucidating and controlling DC-SIGN's roles in immunity. We previously reported a small molecule that embodies key features of the carbohydrates that bind DC-SIGN. Here, we demonstrate that this noncarbohydrate ligand acts as a true glycomimetic. Using NMR HSQC experiments, we found that the compound mimics saccharide ligands: It occupies the same carbohydrate-binding site and interacts with the same amino acid residues on DC-SIGN. The glycomimetic also is functional. It had been shown previously to antagonize DC-SIGN function, but here we use it to generate DC-SIGN agonists. Specifically, appending this glycomimetic to a protein scaffold affords a conjugate that elicits key cellular signaling responses. Thus, the glycomimetic can give rise to functional glycoprotein surrogates that elicit lectin-mediated signaling.