Dendritic Cell-specific Intercellular Adhesion Molecule-3-grabbing Non-integrin Research Articles

Thermodynamics-Guided Design Reveals a Cooperative Hydrogen Bond in DC-SIGN-targeted Glycomimetics.

Due to the shallow and hydrophilic binding sites of carbohydrate-binding proteins, the design of glycomimetics is often complicated by high desolvation costs as well as competition with solvent. Therefore, a careful optimization of interaction vectors and ligand properties is required in the design and optimization of glycomimetics. Here, we employ thermodynamics-guided design to optimize mannose-based glycomimetics targeting the human C-type lectin receptor dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN), a pathogenic host factor in viral infections. By exploring ligand rigidification and hydrogen bond engineering, a monovalent glycomimetic with an unprecedented affinity for DC-SIGN in the low μM range was discovered. A matched molecular pair analysis based on microcalorimetric data revealed a stereospecific hydrogen bond interaction with Glu358/Ser360 as the origin of this cooperative and enthalpically dominated interaction. This detailed insight into the binding mechanism paves the way for an improvement of monovalent glycomimetics targeting DC-SIGN.

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is acellularreceptor for delta inulin adjuvant.

Delta inulin, or Advax, is a polysaccharide vaccine adjuvant that significantly enhances vaccine-mediated immune responses against multiple pathogens and was recently licensed for use in the coronavirus disease 2019 (COVID-19) vaccine SpikoGen. Although Advax has proven effective as an immune adjuvant, its specific binding targets have not been characterized. In this report, we identify a cellular receptor for Advax recognition. In vitro uptake of Advax particles by macrophage cell lines was substantially greater than that of latex beads of comparable size, suggesting an active uptake mechanism by phagocytic cells. Using a lectin array, Advax particles were recognized by lectins specific for various carbohydrate structures including mannosyl, N-acetylgalactosamine and galactose moieties. Expression in nonphagocytic cells of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a C-type lectin receptor, resulted in enhanced uptake of fluorescent Advax particles compared with mock-transfected cells. Advax uptake was reduced with the addition of ethylenediaminetetraacetic acid and mannan to cells, which are known inhibitors of DC-SIGN function. Finally, a specific blockade of DC-SIGN using a neutralizing antibody abrogated Advax uptake in DC-SIGN-expressing cells. Together, these results identify DC-SIGN as a putative receptor for Advax. Given the known immunomodulatory role of DC-SIGN, the findings described here have implications for the use of Advax adjuvants in humans and inform future mechanistic studies.

A cleaved adhesin DNA vaccine targeting dendritic cell against Porphyromonas gingivalis-induced periodontal disease.

Arg-gingipain A (RgpA) is the primary virulence factor of Porphyromonas gingivalis and contains hemagglutinin adhesin (HA), which helps bacteria adhere to cells and proteins. Hemagglutinin's functional domains include cleaved adhesin (CA), which acts as a hemagglutination and hemoglobin-binding actor. Here, we confirmed that the HA and CA genes are immunogenic, and using adjuvant chemokine to target dendritic cells (DCs) enhanced protective autoimmunity against P. gingivalis-induced periodontal disease. C57 mice were immunized prophylactically with pVAX1-CA, pVAX1-HA, pVAX1, and phosphate-buffered saline (PBS) through intramuscular injection every 2 weeks for a total of three administrations before P. gingivalis-induced periodontitis. The DCs were analyzed using flow cytometry and ribonucleic acid sequencing (RNA-seq) transcriptomic assays following transfection with CA lentivirus. The efficacy of the co-delivered molecular adjuvant CA DNA vaccine was evaluated in vivo using flow cytometry, immunofluorescence techniques, and micro-computed tomography. After the immunization, both the pVAX1-CA and pVAX1-HA groups exhibited significantly elevated P. gingivalis-specific IgG and IgG1, as well as a reduction in bone loss around periodontitis-affected teeth, compared to the pVAX1 and PBS groups (p<0.05). The expression of CA promoted the secretion of HLA, CD86, CD83, and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) in DCs. Furthermore, the RNA-seq analysis revealed a significant increase in the chemokine (C-C motif) ligand 19 (p<0.05). A notable elevation in the quantities of DCs co-labeled with CD11c and major histocompatibility complex class II, along with an increase in interferon-gamma (IFN-γ) cells, was observed in the inguinal lymph nodes of mice subjected to CCL19-CA immunization. This outcome effectively illustrated the preservation of peri-implant bone mass in rats afflicted with P. gingivalis-induced peri-implantitis (p<0.05). The co-administration of a CCL19-conjugated CA DNA vaccine holds promise as an innovative and targeted immunization strategy against P. gingivalis-induced periodontitis and peri-implantitis.

The S1 spike protein of SARS-CoV-2 upregulates the ERK/MAPK signaling pathway in DC-SIGN-expressing THP-1 cells.

Dendritic cells, macrophages, neutrophils, and other antigen-presenting cells express various C-type lectin receptors that function to recognize the glycans associated with pathogens. The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds various pathogens such as HIV glycoprotein 120, the Ebola glycoprotein, hemagglutinin, and the dengue virus glycoprotein in addition to the SARS-CoV-2 spike protein, and also triggers antigen-presenting cell endocytosis and immune escape from systemic infections. Many studies on the binding of SARS-CoV-2 spike protein with glycans have been published, but the underlying mechanism by which intracellular signaling occurs remains unclear. In this study, we report that the S1 spike protein of SARS-CoV-2 induces the phosphorylation of extracellular signal-regulated kinases (ERKs) in THP-1 cells, a DC-SIGN-expressing human monocytic leukemic cell line. On the other hand, the phosphorylation level of NF-κB remained unchanged under the same conditions. These data suggest that the major cell signaling pathway regulated by the S1 spike protein is the ERK pathway, which is superior to the NF-κB pathway in these DC-SIGN-expressing THP-1 cells and may contribute to immune hyperactivation in SARS-CoV-2 infections. Additionally, several glycans such as mannans, mannosylatedbovine serum albumin, the serum amyloid beta protein, andintracellular adhesion molecule 3 suppressed ERKphosphorylation,suggesting that these molecules are target molecules for SARS-CoV-2 infection by suppressing immune hyperactivation that occurs in the ERK signaling pathway.

Aberrant Mannosylated and Highly Fucosylated Glycoepitopes of Prostatic Acid Phosphatase as Potential Ligands for Dendritic-Cell Specific ICAM-Grabbing Nonintegrin (DC-SIGN) in Human Seminal Plasma-A Step towards Explaining Idiopathic Infertility.

Semen prostatic acid phosphatase (PAP) has been proposed as an endogenous ligand for dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), which plays a critical immuno-modulating role in maintaining homeostasis in the female reproductive tracts. In the current study, we assumed that semen PAP bears a set of fucosylated and mannosylated glycans, which may mediate the efficient binding of PAP to DC-SIGN. To investigate this hypothesis, we developed ELISA assays using Galanthus nivalis and Lotus tetragonolobus lectins capable of binding mannose-containing glycans or LewisX and LewisY motifs, respectively. In our assay with Galanthus nivalis, we detected that the relative reactivity of PAP mannose-presenting glycans in the normozoospermic idiopathic group was significantly higher than in the asthenozoospermic, oligozoospermic and oligoasthenozoospermic groups. Simultaneously, we observed slight differences in the relative reactivities of PAP glycans with Lotus tetragonolobus lectin among groups of patients with abnormal semen parameters. Subsequently, we examined whether DC-SIGN interacts with seminal plasma PAP glycans, and we detected a significantly higher relative reactivity in the normozoospermic group compared to the oligozoospermic group. Finally, we concluded that the significantly aberrant abundance of mannosylated functional groups of PAP among patients with semen disorders can suggest that PAP may thereby be engaged in modulating the immune response and promoting a tolerogenic response to male antigens in the female reproductive system.

Combating DC-SIGN-mediated SARS-CoV-2 dissemination by glycan-mimicking polymers.

Many viruses exploit the human C-type lectin receptor dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) for cell entry and virus dissemination. An inhibition of DC-SIGN-mediated virus attachment by glycan-derived ligands has, thus, emerged as a promising strategy towardbroad-spectrum antiviral therapeutics. In this contribution, several cognate fragments of oligomannose- and complex-type glycans grafted onto a poly-l-lysine scaffold are evaluated as polyvalent DC-SIGN ligands. The range of selected carbohydrate epitopes encompasses linear (α- d-Man-(1→2)-α- d-Man, α- d-Man-(1→2)-α- d-Man-(1→2)-α- d-Man-(1→3)-α- d-Man) and branched (α- d-Man-(1→6)-[α- d-Man-(1→3)]-α- d-Man) oligomannosides, as well as α- l-Fuc. The thermodynamics of binding are investigated on a mono- and multivalent level to shed light on the molecular details of the interactions with the tetravalent receptor. Cellular models of virus attachment and DC-SIGN-mediated virus dissemination reveal a high potency of the presented glycopolymers in the low pico- and nanomolar ranges, respectively. The high activity of oligomannose epitopes in combination with the biocompatible properties of the poly- l-lysine scaffold highlights the potential for further preclinical development of polyvalent DC-SIGN ligands.

Localization of DC-SIGN Expressing Cells in Follicular Lymphoma

The high incidence of unusual mannose-rich glycans on the expressed surface immunoglobulins of follicular lymphoma (FL) suggests a pathogenic role for this modification. While lectin-mediated signaling has been proposed to provide trophic support to FL cells, the localization of the lectins has not been extensively characterized. Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) is a mannose-selective lectin named for its expression on classic marrow-derived dendritic cells. In vitro, recombinant DC-SIGN induces signaling in primary FL cells. The nodules of Follicular lymphoma (nFL) are the neoplastic counterparts of normal germinal centers (GC). Both GC and nFL contain variable numbers of centrocytic and/or centroblastic B cells, T-cells, and follicular dendritic cells (FDCs), but macrophages are typically found only in GC. Although termed “follicular dendritic cells”, FDCs are thought to be derived from non-blood stem-cells, potentially pericytes. Therefore, nFL lack classic dendritic cells and macrophages so it is unclear what cells in FL could express DC-SIGN. Therefore, we systematically characterized FDC and DC- SIGN localization in sixteen FL and nine Follicular hyperplasia (FH) lymph nodes. Using flow cytometry of disaggregated FL nodes, we were not able to identify a specific cell population expressing DC-SIGN. However, using immunohistochemistry and Immunofluorescence methods, we identified nine cases of FH and five cases of FL (n=5/16) with DC-SIGN and FDC co-localization. Immunohistochemical stains of the FDCs using CD23 and CD21 showed that the follicular dendritic structures were disrupted in almost all the FL cases assessed showing either a peripheral crescentic staining or broken FDC meshwork within the follicle. We identified five cases of FL with an average of more than four well visualized and complete FDC structures staining per low power field (4X) while the remaining cases had an average of less than two complete FDC network staining. There was near complete absence of CD21 positive FDCs meshwork identified in 50% of the FL cases compared to only one case lacking CD23 positive FDCs structure staining. In contrast, FH cases (n=9) showed complete staining of the FDC meshwork within the follicles with both CD21/CD23. For FH, the average number of complete FDC staining with CD23 was 18 while an average of 10 complete FDC meshworks stained with CD21. Furthermore, immunofluorescence imaging analysis showed different pattern of localization of CD23 positive FDC with DC-SIGN with between the two conditions. In FH, DC-SIGN and CD23 colocalization was identified mainly in the light zone. For FL, we observed that DC-SIGN overlap with CD23 localized in the intra-follicular areas and co-localized with CD163 (M2 macrophages) in the inter-follicular zones. Confocal imaging confirmed colocalization of CD23 and DC-SIGN on FDCs with close association to MUM1-positive B cells in both GC and nFL. Additional analysis also showed that 80% (n=5) of the cases which had more than four complete FDC structures staining with CD23 co-localized with DC-SIGN in the intra-follicular areas and the cases with complete FDC network disruption did not. There was no significant relationship identified between the extent of FDC networks and the fraction of non-neoplastic B cells in the FL cases. In summary, our data show that DC-SIGN is expressed on FDCs in FL and FH even though FDCs are not the marrow-derived DCs for which DC-SIGN is named. Furthermore, localization of DC-SIGN to the follicle suggests a potential role for the microenvironment in the pathogenesis of FL and possible therapeutic target.

Oligomannose-Type Glycans in the Antigen Binding Site Identify Origin and Prognosis of Diffuse Large B Cell Lymphoma

The acquisition of N-glycosylation sites (AGS) in the complementarity-determining region (CDR) of the tumor immunoglobulin (Ig) is an early clonal requirement of classic follicular lymphoma (FL). In FL, the AGS are occupied by oligomannose-type glycans, which mediate lymphoma Ig interaction with the microenvironmental lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). This interaction induces a persistent low-level signal, favouring adhesion and ultimately survival and growth of the lymphoma cells in their protected environment . Oligomannose-type glycans can also occupy the CDR-located AGS of a subset of germinal-centre B cell-like (GCB) diffuse large B-cell lymphomas (DLBCL) but the consequences of AGS in DLBCL are less known. We determined the frequency, distribution, and composition of the glycan structures occupying the AGS in DLBCL using a discovery-validation approach with two large independent international public cohorts (BCC, dataset ID: EGAD00001003783; NCI accession phs001444.v1.p1), following our RNAseq Ig assembly pipeline (Ig-Seq-R) and high-resolution mass-spectrometry. Glycan types were compared to our FL cohort (12 samples) and correlated with DLBCL cell of origin (COO), genetic, clinical characteristics and outcome. The full IGHV-IGHD-IGHJ-IGHC transcript sequences were obtained from 251 DLBCL of the BCC, and 339 of the NCI cohort. Sample distribution by COO, clinical or molecular features, LymphGen subtype, progression-free survival (PFS) and overall survival (OS) were not different from the extended public cohorts. AGS were observed in 48-55% GCB-DLBCL, with 84-85% of them located in the CDR. Overall, frequency of CDR-located AGS contrasted dramatically between GCB-DLBCL (41-46%) and activated B-cell like (ABC)-DLBCL (7-10%). Also, within GCB-DLBCL, CDR-located AGS associated mostly with the EZB subtype (68-73%). However, only 66-61% EZB were AGS+ve, all of which were CDR+ve, while the remaining 34-39% had no AGS (AGS-ve EZB). The glycan structures of the synthetic F(ab)s from 35 AGS+ve DLBCL and 12 FL were analyzed by site-specific mass spectrometry. The DLBCL comprised 12 AGS+ve EZB and 23 AGS+ve non-EZB (14 CDR+ve, 9 located in the framework region). Glycan analysis revealed that all AGS+ve EZB and FL were invariably occupied by oligomannose-type glycans (Ig-Mann+ve DLBCL). In contrast, the other AGS+ve DLBCL were not occupied by oligomannose type glycans. They were either occupied by complex glycans (the remaining non-EZB GCB-DLBCL) or not occupied (preferentially the ABC-DLBCL), irrespective of AGS location. These data indicated that CDR+ve EZB Ig were universally and exclusively Ig-Mann+ve DLBCL, share the COO of FL, and have likely been influenced by an environmental driver distinct from the other GCB-DLBCL and ABC-DLBCL. We compared the clinical behavior of Ig-Mann+ve DLBCL versus other DLBCL subtypes including AGS-ve EZB and the other non-EZB GCB-DLBCL and ABC-DLBCL. In both the BCC and NCI cohorts, Ig-Mann+ve DLBCL status identified the subset with the worst PFS and OS of all GCB-DLBCL, not different from ABC-DLBCL (Figure 1A). This contrasted with the other GCB-DLBCL (AGS-ve/EZB and AGS+ve/non-EZB), which all shared an extremely good PFS and OS (Figure 1A). Univariate and multivariate analyses revealed that Ig-Mann+ve status was an independent prognostic factor for PFS and OS (Figure 1B). Gene expression profile analysis revealed that Ig-Mann+ve status associated with an enrichment of MYC, PI3K-Akt-mTORC1, pro-survival, and cell cycle pathways, while proinflammatory and apoptotic pathways were decreased in the Ig-Mann+ve DLBCL compared to AGS-ve/EZB and AGS+ve/non-EZB, independently of MYC translocations and double-hit signature. These data indicate identical COO of a EZB subset with FL cells, and point to a highly selective chronic environmental pressure on Ig-Mann+ve DLBCL negatively affecting patients survival. The combination of Ig gene analysis with the lymphGen classification can predict Ig glycan structure and provide a new fundamental approach to identifying the most aggressive GCB-DLBCL subtype, and the precise environmental tumour interaction (DC-SIGN:Ig-Mann) to intercept therapeutically early in the natural history of Ig-Mann+ve DLBCL.

Identification of a new DC-SIGN binding pentamannoside epitope within the complex structure of Candida albicans mannan

The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is an innate immune C-type lectin receptor that recognizes carbohydrate-based pathogen associated with molecular patterns of various bacteria, fungi, viruses and protozoa. Although a range of highly mannosylated glycoproteins have been shown to induce signaling via DC-SIGN, precise structure of the recognized oligosaccharide epitope is still unclear. Using the array of oligosaccharides related to selected fragments of main fungal antigenic polysaccharides we revealed a highly specific pentamannoside ligand of DC-SIGN, consisting of α-(1 → 2)-linked mannose chains with one inner α-(1 → 3)-linked unit. This structural motif is present in Candida albicans cell wall mannan and corresponds to its antigenic factors 4 and 13b. This epitope is not ubiquitous in other yeast species and may account for the species-specific nature of fungal recognition via DC-SIGN. The discovered highly specific oligosaccharide ligands of DC-SIGN are tractable tools for interdisciplinary investigations of mechanisms of fungal innate immunity and anti-Candida defense. Ligand- and receptor-based NMR data demonstrated the pentasaccharide-to-DC-SIGN interaction in solution and enabled the deciphering of the interaction topology.

Nanoscale clustering of mycobacterial ligands and DC-SIGN host receptors are key determinants for pathogen recognition.

The bacterial pathogen Mycobacterium tuberculosis binds to the C-type lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) on dendritic cells to evade the immune system. While DC-SIGN glycoconjugate ligands are ubiquitous among mycobacterial species, the receptor selectively binds pathogenic species from the M. tuberculosis complex (MTBC). Here, we unravel the molecular mechanism behind this intriguing selective recognition by means of a multidisciplinary approach combining single-molecule atomic force microscopy with Förster resonance energy transfer and bioassays. Molecular recognition imaging of mycobacteria demonstrates that the distribution of DC-SIGN ligands markedly differs between Mycobacterium bovis Bacille Calmette-Guérin (BCG) (model MTBC species) and Mycobacterium smegmatis (non-MTBC species), the ligands being concentrated into dense nanodomains on M. bovis BCG. Upon bacteria-host cell adhesion, ligand nanodomains induce the recruitment and clustering of DC-SIGN. Our study highlights the key role of clustering of both ligands on MTBC species and DC-SIGN host receptors in pathogen recognition, a mechanism that might be widespread in host-pathogen interactions.

Investigation of TNF-&amp;alpha; and DC-SIGN promoter polymorphisms in patients with dengue fever in Lahore city of Pakistan

&lt;p&gt;&lt;strong&gt;Background and Objective:&lt;/strong&gt; Dengue fever (DF) has been a major health concern globally. Pakistan is also combating this infection for the last decade. Cytokine genes play an important role in DF pathogenesis. This study aimed to analyze dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and tumor necrosis factor &amp;alpha; (TNF-&amp;alpha;) genes promoter polymorphisms in DF patients.&lt;/p&gt;&#x0D; &lt;p&gt;&lt;strong&gt;Methods:&lt;/strong&gt; A total of 140 (n = 140) dDF patients were recruited for study at the Department of Human Genetics and Molecular Biology of University of Health Sciences, Lahore, Pakistan over a period of 3 years. Simple DF was noted in 105 patients (75%) while 35 (25%) showed bleeding complications. All patients were found positive for dengue non-structural protein or dengue IgM. All patients were tested for two polymorphisms in TNF-&amp;alpha; (-238G/A, and -308G/A) and one polymorphism in DC-SIGN (-336G/A) using restriction fragment length polymorphism technique. A single nucleotide polymorphism stats program was used for statistical analysis.&lt;/p&gt;&#x0D; &lt;p&gt;&lt;strong&gt;Results:&lt;/strong&gt; Susceptibility to develop dengue infection in the presence of -336G allele odds ratio (OR = 27.95, p = &amp;lt;0.0001) and GG genotype (OR = 183.77, p = &amp;lt;0.0001) was found to be significantly associated in this study. Presence of a combination of alleles -336G/-238A/-308A was noted in 59.4% of DF cases and 7.6% healthy controls, a difference with statistical significance (OR = 31.46, p = &amp;lt;0.0001). Moreover, prevalence of DF symptoms showed a trend higher in G-carriers versus non-G-carriers of DC-SIGN -336 polymorphism.&lt;/p&gt;&#x0D; &lt;p&gt;&lt;strong&gt;Conclusion:&lt;/strong&gt; This work suggests a potential association of DC-SIGN -336 polymorphism with susceptibility to develop symptomatic dengue illness. However, no potential association was found between TNF-&amp;alpha; promoter polymorphisms and dengue infection in this study.&lt;/p&gt;

Information transfer in mammalian glycan-based communication.

Glycan-binding proteins, so-called lectins, are exposed on mammalian cell surfaces and decipher the information encoded within glycans translating it into biochemical signal transduction pathways in the cell. These glycan-lectin communication pathways are complex and difficult to analyze. However, quantitative data with single-cell resolution provide means to disentangle the associated signaling cascades. We chose C-type lectin receptors (CTLs) expressed on immune cells as a model system to study their capacity to transmit information encoded in glycans of incoming particles. In particular, we used nuclear factor kappa-B-reporter cell lines expressing DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), macrophage C-type lectin (MCL), dectin-1, dectin-2, and macrophage-inducible C-type lectin (MINCLE), as well as TNFαR and TLR-1&2 in monocytic cell lines and compared their transmission of glycan-encoded information. All receptors transmit information with similar signaling capacity, except dectin-2. This lectin was identified to be less efficient in information transmission compared to the other CTLs, and even when the sensitivity of the dectin-2 pathway was enhanced by overexpression of its co-receptor FcRγ, its transmitted information was not. Next, we expanded our investigation toward the integration of multiple signal transduction pathways including synergistic lectins, which is crucial during pathogen recognition. We show how the signaling capacity of lectin receptors using a similar signal transduction pathway (dectin-1 and dectin-2) is being integrated by compromising between the lectins. In contrast, co-expression of MCL synergistically enhanced the dectin-2 signaling capacity, particularly at low-glycan stimulant concentration. By using dectin-2 and other lectins as examples, we demonstrate how signaling capacity of dectin-2 is modulated in the presence of other lectins, and therefore, the findings provide insight into how immune cells translate glycan information using multivalent interactions.

Single-Molecule Analysis of SARS-CoV-2 Binding to C-Type Lectin Receptors.

Despite intense scrutiny throughout the pandemic, development of efficacious drugs against SARS-CoV-2 spread remains hindered. Understanding the underlying mechanisms of viral infection is fundamental for developing novel treatments. While angiotensin converting enzyme 2 (ACE2) is accepted as the key entry receptor of the virus, other infection mechanisms exist. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and its counterpart DC-SIGN-related (DC-SIGNR, also known as L-SIGN) have been recognized as possessing functional roles in COVID-19 disease and binding to SARS-CoV-2 has been demonstrated previously with ensemble and qualitative techniques. Here we examine the thermodynamic and kinetic parameters of the ligand-receptor interaction between these C-type lectins and the SARS-CoV-2 S1 protein using force-distance curve-based AFM and biolayer interferometry. We evidence that the S1 receptor binding domain is likely involved in this bond formation. Further, we employed deglycosidases and examined a nonglycosylated S1 variant to confirm the significance of glycosylation in this interaction. We demonstrate that the high affinity interactions observed occur through a mechanism distinct from that of ACE2.

Sialylated IVIg binding to DC-SIGN+ Hofbauer cells induces immune tolerance through the caveolin-1/NF-kB pathway and IL-10 secretion

Although the use of IVIg has increased in various immune-driven diseases and even in pregnancy, the exact action mechanisms of IVIg are not fully understood. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) is a known receptor for α-2,6-sialylated IgG (sIVIg), which is responsible for the anti-inflammatory effect of IVIg. DC-SIGN is expressed on Hofbauer cells (HBCs) of the fetal villi of the placenta which act as an innate immune modulator at the maternal-fetal interface. Preeclampsia is a major complication in pregnancy and is related to IL-10, a cytokine with an important role in immune tolerance. DC-SIGN interaction with sIVIg in HBCs promoted IL-10 secretion through the activation of the caveolin-1/NF-κB pathway, especially in plasma lipid rafts. Consistent results were obtained for HBCs from patients with preeclampsia. Collectively, the stimulation of DC-SIGN+ HBCs with sIVIg enhanced immune tolerance in the feto-maternal environment, suggesting the therapeutic application of sIVIg to prevent preeclampsia.

DC-SIGN Binding to the Oligomannose-Type Glycans on the Surface Immunoglobulin Variable Region of Follicular Lymphoma Promotes Selective Adhesion to Microenvironmental VCAM-1

The selective acquisition of oligomannose-type glycans at the antigen-binding site of the surface immunoglobulin (sIg-Mann) is a tumor-specific post-translational modification of all follicular lymphomas (FL). Our immunogenetic and structural analyses have revealed that these oligomannoses at N-glycosylation sites are universally acquired and persist during the entire natural history of FL from the early stages throughout transformation into EZB lymphoma (Chiodin G, Blood 2021). If a FL cell loses these sites, which is rare, the progeny disappears unless a new glycosylation site is acquired (Odabashian M, Blood 2020). These data indicate that sIg-Mann is a fundamental functional requirement for the entire clonal population of all FLs. The low-affinity interaction of sIg-Mann with its specific ligand dendritic cell-specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), expressed on interfollicular M2-macrophages and follicular dendritic cells (FDC) of FL (RadtkeA, BioRxiv, 2022), distinguishes from conventional high-affinity antigen:Ig protein interactions for promoting prolonged low-level growth and prosurvival signals via AKT and MYC and not endocytosis (Linley A, Blood 2015). However, the function of DC-SIGN:Ig-Mann remains to be further elucidated. In this study, we investigated the hypothesis that the specific engagement of DC-SIGN:Ig-Mann interaction functions to promote homing, while maintaining selective growth and survival signals. We found that DC-SIGN induced adhesion of sIg-Mann+ve lymphoma cells to VCAM-1 and inhibited migration towards SDF-1 in vitro. Blocking of DC-SIGN with an anti-DC-SIGN antibody specific to the carbohydrate-recognition domain completely abrogated DC-SIGN-induced adhesion, confirming that adhesion was mediated by the selective interaction with the oligomannose of sIg-Mann. Although intracellular signals were significantly lower than those by soluble anti-IgM F(ab)2 (here used to mimic the binding of cross-linking polyvalent antigen to Ig protein), the level of adhesion induced by DC-SIGN was similar to that induced by anti-IgM F(ab)2. Adhesion to VCAM-1 was observed at concentrations from 20 µg/ml down to 20 ng/ml, even when AKT and ERK phosphorylation was not detectable by immunoblotting. However, either proximal inhibition of the PI3K/AKT pathway or distal inhibition of ARP2/3, formin, Rac and Cdc42 for lamellipodium formation at the surface membrane, suppressed adhesion. This suggested that Ig-Mann-mediated adhesion required both low-level intracellular signals and membrane adaptation (Valle-Argos, Sci Rep 2021). Remarkably, unlike anti-IgM F(ab)2, adhesion was specific to VCAM-1 but not to other stromal elements including fibronectin (FN). Instead, using the sIg-Mann+ve (IGHV4-34+ve) WSU-FSCCL cells, DC-SIGN appeared to mimic the interaction of monovalent ligands, including Fab fragments to either the Ig constant (anti-IgM Fab) or Ig variable region (9G4 Fab, which recognizes an epitope in the framework region 1 of IGHV4-34). Like DC-SIGN, Fab fragments induced selective adhesion to VCAM-1 and low-level signals via AKT, but neither endocytosis nor adhesion to FN. These results identify an important functional role of the DC-SIGN:sIg-Mann in mediating a signal that is tuned at the right threshold to promote selective and, in the absence of Ig endocytosis, persistent adhesion to environmental VCAM-1, determining homing of the FL tumor cells in the lymph node. This tumor-specific antigen-independent low-affinity DC:SIGN-Ig-Mann interaction contrasts to conventional high-affinity antigen:Ig protein interaction, which is prevented in FL (Schneider D, Blood 2015) and would instead lead to endocytosis and death. Therapeutic interruption of the interaction of DC-SIGN+ve cells with sIg-Mann+ve FL cells would provide a new way to displace the tumor cells from their prosurvival microenvironment.

In-silico Characterization of Target Protein in Dengue Infection

Infection of humans with the virus is primarily mediated by the Aedes mosquito. The virus grows in the mosquito gut and migrates to aq 1 the salivary glands.. Dengue virus (DENV), which includes four serotypes (DENV1–4), is transmitted to humans by Aedes mosquitos and is the etiological agent of dengue fever and dengue hemorrhagic fever. DENV causes an estimated 50–100 million cases of dengue fever, 500,000 cases of severe dengue (dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS)), and more than 20,000 deaths each year in tropical and subtropical regions, representing a considerable public health threat in over 100 countries worldwide. However, there are still no specific antiviral drugs or licensed vaccines against DENV infection. Dengue virus primarily propagates in skin dendritic cells, and subsequently virus proliferation is thought to occur in target cells such as those of the monocyte/macrophage lineage. Second, carbohydrate binding proteins, termed lectins, expressed on dendritic cells (DCs) and macrophages under the human skin are involved in initial contact of DENV introduced by mosquito bite. Among these lectins, dendritic cell-specific intercellular adhesion molecule-3-grabbing non integrin (DCSIGN) has been best characterized in virus-DC interaction.

Danshensu prevents thrombosis by inhibiting platelet activation via SIRT1/ROS/mtDNA pathways without increasing bleeding risk

BackgroundCoronary thrombosis and its correlated disorders are main healthcare problems globally. The therapeutic effects of current treatments involving antiplatelet drugs are not fully satisfactory. Danshensu (DSS) is an important monomer obtained from Salvia miltiorrhiza roots that have been widely employed for vascular diseases in medicinal practices. Nonetheless, the underlying mechanisms of DSS are not fully unraveled. PurposeThe objective of this study was to penetrate the antithrombotic and antiplatelet mechanisms of DSS. MethodsNetwork pharmacology assay was used to forecast the cellular mechanisms of DSS for treating thrombosis. The work focused the impacts of DSS on platelet activation by analyzing aggregation and adhesion in vitro. Flow cytometry, western blotting, CM-H2DCFDA staining and mitochondrial function assays were performed to reveal the molecular mechanisms. The model of common carotid artery thrombus induced by ferric chloride was established. The wet weight of thrombus was measured, and the thrombosis was observed by hematoxylin and eosin (H&E) staining, in order to support the inhibitory effect of DSS on thrombosis. ResultsData mining found the antithrombotic effect of DSS is related to platelet activation and the core target is silent information regulator 1 (SIRT1). We confirmed that DSS dose-dependently inhibited platelet activation in vitro. DSS was further demonstrated to induce the expression of SIRT1 and decreased reactive oxygen species (ROS) burden and thereby prevented mitochondrial dysfunction. Mitochondrial function tests further indicated that DSS prevented mitochondrial DNA (mtDNA) release, which induced activation of platelet in a dendritic cell specific intercellular-adhesion-molecule-3 grabbing non-integrin (DC-SIGN)-dependent manner. In carotid artery injury model induced by ferric chloride, DSS inhibited the development of carotid arterial thrombosis. More encouragingly, in tail bleeding time assay, DSS did not augment bleeding risk. ConclusionThese findings indicated that DSS effectively inhibited platelet activation by depressing the collection of ROS and the release of platelet mtDNA without arousing hemorrhage risk. DSS might represent a promising candidate drug for thrombosis and cardiovascular disease therapeutics.

Drug-like Inhibitors of DC-SIGN Based on a Quinolone Scaffold.

DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) is a pattern recognition receptor expressed on immune cells and involved in the recognition of carbohydrate signatures present on various pathogens, including HIV, Ebola, and SARS-CoV-2. Therefore, developing inhibitors blocking the carbohydrate-binding site of DC-SIGN could generate a valuable tool to investigate the role of this receptor in several infectious diseases. Herein, we performed a fragment-based ligand design using 4-quinolone as a scaffold. We synthesized a library of 61 compounds, performed a screening against DC-SIGN using an STD reporter assay, and validated these data using protein-based 1H-15N HSQC NMR. Based on the structure-activity relationship data, we demonstrate that ethoxycarbonyl or dimethylaminocarbonyl in position 2 or 3 is favorable for the DC-SIGN binding activity, especially in combination with fluorine, ethoxycarbonyl, or dimethylaminocarbonyl in position 7 or 8. Furthermore, we demonstrate that these quinolones can allosterically modulate the carbohydrate binding site, which offers an alternative approach toward this challenging protein target.

Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage

Abnormal cell surface glycosylation plays a major role in disease processes such as immune evasion. However, the underlying role of glycans is yet to be fully understood. Binding information obtained from glycan arrays can provide critical starting points for downstream applications such as the development of carbohydrate‐based inhibitors, vaccines, and other therapeutics. However, it is challenging to use powerful techniques like DNA deep sequencing to analyze glycan recognition due to the lack of 1:1 correspondence between DNA and glycan structures. Therefore, we have developed Liquid Glycan Array (LiGA), a technology that allows for genetic encoding of glycans. LiGA provides a 1:1 correspondence between the glycan displayed in multiple copies on a bacteriophage carrier and the phage genetic material. LiGA is generated by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido‐modified glycans. The display of glycans on each phage virion can be controlled from 30‐1500 copies to probe the critical variables in glycan recognition: valency and density. A simple pulldown of the LiGA along with lectins followed by deep sequencing of the DNA in the bound phage decodes the recognized glycans. LiGA is target agnostic and measures binding profile of lectins expressed on intact cells, such as hCD22 (Siglec‐2) and DC‐SIGN (Dendritic Cell‐Specific Intercellular adhesion molecule‐3‐Grabbing Non‐integrin), and in live mice (Nat. Chem. Bio. 17, 806–816, 2021). From a mixture of 50‐100 multivalent glycan probes, LiGA identifies the glycan‐phage conjugates with optimal valency and density for binding to antibodies and lectins on cells in vitro and in vivo.Sialic acid‐binding immunoglobulin‐type lectins (Siglecs) expressed on the surface of immune cells are exploited by cancer to evade immune response. We applied LiGA to study the binding specificity of Siglec‐7, a cell surface receptor that cancer cells use to evade immune response from natural killer (NK) cells. Additionally, we explored the roles of valency and density in ganglioside interaction with Siglec‐1 using a cell‐based assay. Building on these successes, we plan to use LiGA to identify the valency and affinity required by trans‐ glycan to overcome the cis‐ masking on the surface of immune cells.

Interaction of Host Pattern Recognition Receptors (PRRs) with Mycobacterium Tuberculosis and Ayurvedic Management of Tuberculosis: A Systemic Approach.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb), infects the lungs' alveolar surfaces through aerosol droplets. At this stage, the disease progression may have many consequences, determined primarily by the reactions of the human immune system. However, one approach will be to more actively integrate the immune system, especially the pattern recognition receptor (PRR) systems of the host, which notices pathogen-associated molecular patterns (PAMPs) of Mtb. Several types of PRRs are involved in the detection of Mtb, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), Dendritic cell (DC) -specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), Mannose receptor (MR), and NOD-like receptors (NLRs) related to inflammasome activation. In this study, we focus on reviewing the Mtb pathophysiology and interaction of host PPRs with Mtb as well as adverse drug effects of anti-tuberculosis drugs (ATDs) and systematic TB treatment via Ayurvedic medicine.