Transmembrane C-type Lectin Research Articles

Characterization of the transmembrane C-type lectin reveals a novel phagocytic receptor for hemocytes of Tegillarca granosa

C-type lectins (CTLs), a superfamily of calcium-dependent carbohydrate binding proteins, which remain uncharacterized in bivalve mollusks such as blood clam Tegillarca granosa, plays a pivotal role in non-self-recognition and pathogen clearance within the innate immune system. In this study, we identified a CTL as a potential marker gene for immune cells in T. granosa using single-cell RNA-sequence (scRNA-seq). Structural predictions confirmed TgCTL as a functional protein, being characterized by a classic C-type lectin domain (CTLD) and a stable transmembrane structure. Furthermore, the Vibrio parahaemolyticus challenge experiment indicated that the TgCTL gene can significantly respond to pathogen invasion as a marker of immune response and can effectively eliminate pathogens. Results of RNA interference (RNAi) showed that TgCTL knockdown led to a significant decrease in hemocytes phagocytic activity (P < 0.05). Taken together, the results of this study revealed that TgCTL, a marker for immune response, could participate in immune defense against pathogens by enhancing phagocytosis of hemocytes, providing new insights into the antimicrobial immune response of T. granosa.

Characterization of four spliced isoforms of a transmembrane C-type lectin from Procambarus clarkii and their function in facilitating WSSV infection

C-type lectin (CTL) is an important pattern recognition receptor that play vital functions in the innate immunity. Many soluble CTLs in crustacean participate in the inhibition or promotion of white spot syndrome virus (WSSV) infection. However, whether transmembrane CTLs participate in WSSV infection in crustacean remains unknown. In the present study, four spliced isoforms of a transmembrane CTL (designated as PcTlec) from Procambarus clarkii were identified for the first time. The genome structure of PcTlec contains eight exons, six known introns, and one unknown intron. PcTlec-isoform1 is produced by intron retention, whereas PcTlec-isoform3 and PcTlec-isoform4 are produced by exon skipping. All of them contain the transmembrane domain and characteristic carbohydrate recognition domain (CRD). Four PcTlec isoforms were mainly expressed in the hepatopancreas, stomach, and intestine. After WSSV challenge, the expression levels of PcTlec-isoform1-4 in the intestine were upregulated. The knockdown of the region shared by four PcTlec isoforms evidently decreased the expression of WSSV envelope protein VP28 and the copies of viral particles. A recombinant protein (rPcTlec-CRD) containing the CRD that was shared by four PcTlec isoforms was acquired by procaryotic expression system. The injection of purified rPcTlec-CRD protein evidently increased the VP28 expression and WSSV copies during viral infection. Moreover, rPcTlec-CRD could directly bind to WSSV and interact with VP28 protein. These findings indicate that new-found transmembrane CTL isoforms in P. clarkii may act as viral receptors that facilitate WSSV infection. This study contributes to the recognition and understanding of the functions of transmembrane CTLs in crustacean in the infection of host by WSSV.

Perspective on automated in vivo drug screening using the chodl mutant zebrafish line.

Perspective on automated in vivo drug screening using the chodl mutant zebrafish line.

Molecular characterization and cellular localization of a transmembrane C-type lectin receptor in hemocytes from the scallop Argopecten purpuratus

Recognition of potential pathogens is a crucial function of the immune system of every organism. In this context, great efforts have been made in the last years to characterize the diversity of pattern recognition receptors (PRRs) present in cultured aquatic species, such as bivalve mollusks. Among the great diversity of PRRs described so far, C-type lectin receptors (CLRs) comprise a large family of innate immune receptors that can recognize different carbohydrates present on the cell surface of microorganisms. CLRs can be found extracellularly in soluble form, or transmembrane, located on immune cell surfaces. To date, almost only soluble CLRs have been described in bivalves and so far, no transmembrane CLRs are identified in pectinids. In the present study, a sequence showing structural homologies to transmembrane CLRs (tmCLR) was characterized from scallop Argopecten purpuratus hemocytes, named ApCLR. The cDNA sequence displays 948 nucleotides, coding for a deduced protein of 316 amino acids with a molecular weight of 35.24 kDa. The protein sequence showed characteristic domains of a tmCLR, such as a signal peptide, an extracellular N-terminal carbohydrate recognition domain (CRD), one transmembrane domain and a cytoplasmic tail. Phylogenetic analysis revealed that ApCLR groups with soluble CLRs from scallops but also with transmembrane CLRs from vertebrates, such as chondrolectins and layilins. Gene expression analysis showed that ApCLR was constitutively present in gill, mantle, digestive gland, gonad and hemocyte tissues from non-immune challenged scallops and it was most strongly expressed in the gill and mantle tissues. Furthermore, ApCLR was significantly upregulated in scallop hemocytes at 6 h after scallop injection with heat-killed Vibrio splendidus. At the protein level, ApCLR is located at the plasma membrane of hemocytes, observed as punctate signals by immunofluorescence and confocal analysis. Results obtained in this study reveal the first transmembrane CLR in scallops. Also, results showed that ApCLR is present at the surface of hemocytes and increase its transcript availability during the immune challenge, suggesting a potential role in the recognition of microorganisms.

Transmembrane C-type lectin receptors in immunity

Transmembrane C-type lectin receptors in immunity

ISSUE HIGHLIGHTS - July 2020.

ISSUE HIGHLIGHTS - July 2020.

Evaluation of CD304 expression in pediatric Blineage acute lymphoblastic leukemia

BACKGROUND: Acute lymphoblastic leukemia is the most common form of cancer (25–30%) and predominant subtype of leukemia (75–80%) in children. However, childhood ALL has considerable phenotypic and genotypic heterogeneity, which is of diagnostic and prognostic importance. CD304 is a transmembrane glycoprotein C-type lectin found on plasmacytoid dendritic cells, It acts as a receptor for class III semaphorins mediating neuronal guidance and axonal growth.Aim of the study: to study expression of CD304 in de novo newly diagnosed pediatric B- lineage ALL patients and to evaluate its association with hematological and clinical parameters.Patients and methods: This cross sectional study was conducted on 30 pediatric patients with de novo newly diagnosed BALL randomly selected in relation to sex included (16 males and 14females) with their age ranging 1-14 year.RESULTS: This study revealed that CD304 was detected in (56.67%) of newly diagnosed B-ALL patients and (43.33%) of them were negative for CD304 expression. It was detected that CD304 was not associated with age, gender, clinical presentations, other prognostic hematological parameters (Hb, WBC count, platelate count), blast percentage, NCI risk, B-ALL subtype, positive PAS and response to induction therapy.CONCLUSIONS: CD304 expression was independent on the hematological and physical characteristics of patients, since the majority of positive CD304B-ALL cases were in high risk groups of NCI criteria and 60% of non- responder were positive CD304. PAS stain was detected in standard risk groups of NCI criteria, common B-ALL subtype and response to induction therapy.

Characterization and Functional Analysis of Two Transmembrane C-Type Lectins in Obscure Puffer (Takifugu obscurus).

C-type lectins (CTLs) have received widespread attention in animal immune responses. In the present study, two CTLs (ToCTL1 and ToCTL2) were identified from obscure puffer Takifugu obscurus. The open reading frames of ToCTL1 and ToCTL2 were 687 and 1,380 bp, respectively. The predicted ToCTL1 and ToCTL2 proteins contained a single transmembrane region and one typical carbohydrate recognition domain (CRD). Quantitative real-time polymerase chain reaction detected ToCTL1 and ToCTL2 transcripts in all examined tissues, with high levels in the intestine and kidney, and their expression levels were remarkably altered upon Vibrio harveyi and Aeromonas hydrophila infection. The recombinant proteins ToCTL1-CRD and ToCTL2-CRD agglutinated the Gram-negative and Gram-positive bacteria in a Ca2+-dependent manner. rToCTL1-CRD and rToCTL2-CRD exhibited evident binding activities against seven kinds of bacteria and polysaccharides (lipopolysaccharide and peptidoglycan) in a Ca2+-independent manner. Moreover, rToCTL1-CRD and rToCTL2-CRD could inhibit the growth of four types of bacteria in vitro. These findings collectively demonstrated that ToCTL1 and ToCTL2 could be involved in host defense against bacterial infection in T. obscurus.

Dominant role of splenic marginal zone lipid rafts in the classical complement pathway against S. pneumoniae

Lipid rafts (LRs) play crucial roles in complex physiological processes, modulating innate and acquired immune responses to pathogens. The transmembrane C-type lectins human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and its mouse homolog SIGN-R1 are distributed in LRs and expressed on splenic marginal zone (MZ) macrophages. The DC-SIGN-C1q or SIGN-R1-C1q complex could mediate the immunoglobulin (Ig)-independent classical complement pathway against Streptococcus pneumoniae. Precise roles of LRs during this complement pathway are unknown. Here we show that LRs are indispensable for accelerating the DC-SIGN- or SIGN-R1-mediated classical complement pathway against S. pneumoniae, thus facilitating rapid clearance of the pathogen. The trimolecular complex of SIGN-R1-C1q-C4 was exclusively enriched in LRs of splenic MZ macrophages and their localization was essential for activating C3 catabolism and enhancing pneumococcal clearance, which were abolished in SIGN-R1-knockout mice. However, DC-SIGN replacement on splenic MZ macrophage’s LRs of SIGN-R1-depleted mice reversed these defects. Disruption of LRs dramatically reduced pneumococcal uptake and decomposition. Additionally, DC- SIGN, C1q, C4, and C3 were obviously distributed in splenic LRs of cadavers. Therefore, LRs on splenic SIGN-R1+ or DC-SIGN+ macrophages could provide spatially confined and optimal bidirectional platforms, not only for usual intracellular events, for example recognition and phagocytosis of pathogens, but also an unusual extracellular event such as the complement system. These findings improve our understanding of the orchestrated roles of the spleen, unraveling a new innate immune system initiated from splenic MZ LRs, and yielding answers to several long-standing problems, including the need to understand the profound role of LRs in innate immunity, the need to identify how such a small portion of splenic SIGN-R1+ macrophages (<0.05% of splenic macrophages) effectively resist S. pneumoniae, and the need to understand how LRs can promote the protective function of DC-SIGN against S. pneumoniae in the human spleen.

Macrophage galactose-type lectin 1 (MGL-1) regulates neutrophil mobilization during pulmonary Gram negative pneumonia

Abstract Macrophage galactose-type lectin 1 (MGL-1) is a type 2 transmembrane C-type lectin receptor. We recently reported that MGL1-mediated responses are required to mitigate disease severity in Klebsiella pneumoniae (KPn) induced murine pneumosepsis. Here we investigated the role of MGL-1 in neutrophil development and homeostasis during KPn infection. We found that MGL-1−/− mice exhibit severe neutrophilia in their lungs upon KPn infection. Adaptive transfer experiments showed that MGL-1 deficiency on neutrophils significantly increased their capacity to infiltrate the lungs. Analysis of neutrophil numbers and their progenitors in various compartments showed that increased mobilization of mature neutrophils from bone marrow storage pools, but not the progenitor proliferation, accounted for the increased systemic neutrophil counts. This was correlated with elevated levels of neutrophil mobilizing factors G-CSF and MCP-2 in MGL-1−/− mice. Collectively, our findings demonstrate that MGL-1 has a profound role in neutrophil homeostasis during acute inflammatory condition.

A transmembrane C-type lectin receptor mediates LECT2 effects on head kidney-derived monocytes/macrophages in a teleost, Plecoglossus altivelis

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine involved in many diseases in which immune dysfunction is present. Ayu LECT2 (PaLECT2), which interacts with a C-type lectin receptor (PaCLR), was shown to activate ayu head kidney-derived monocytes/macrophages (MO/MΦ) to improve the outcomes of fish upon bacterial infections. However, it is not known if PaCLR mediates PaLECT2 effects on ayu MO/MΦ. In this study, we determined the role of PaCLR in signal transduction of PaLECT2 on ayu MO/MΦ. We expressed the PaCLR ectodomain in Escherichia coli and produced a refolded recombinant protein (rPaCLR) that was then used to produce the anti-PaCLR IgG (anti-PaCLR) for neutralization. Addition of the refolded PaLECT2 mature peptide (rPaLECT2m) to ayu MO/MΦ cultures, increased cytokine expression, induced chemotaxis, and enhanced phagocytosis and bactericidal activity of these cells were observed. When we added anti-PaCLR to block the ectodomain of PaCLR, these effects were significantly inhibited. Based on our previous works and the data presented here, we conclude that PaCLR mediates the immunomodulatory effects of PaLECT2 on ayu MO/MΦ, thus defining a mechanism by which LECT2 protects fish against pathogens.

Expression of CD69 molecule in Rheumatoid Arthritis Iraqi patients

Background: Rheumatoid arthritis (RA) is an autoimmune disease, where the normal joint tissues attacked by body’s immune system, causing their inflammation. Cluster of Differentiation 69 (CD69) is a human transmembrane C-Type lectin protein encoded by the CD69 gene. It’s expression was induced by activation (in vivo and in vitro) of T lymphocytes and Natural Killer (NK) Cells. As CD69 early activation has been implicated in the pathogenesis of some inflammatory diseases, its expression on peripheral blood T-lymphocytes must be evaluated.Objective: To evaluate the expression of CD69 on peripheral blood T-lymphocytes in RA Iraqi patients. Patients and methods: This study carried out between March 2014 and May 2014. The study involved 40 patients diagnosed with RA attending rheumatology outpatient’s clinic at Baghdad medical city teaching hospital diagnosed clinically and classified according to the 2010 American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) classification criteria for rheumatoid arthritis, compared to 40 apparently healthy individuals as a control group. Erythrocyte sedimentation rate (ESR) was measured by Westergren method. Flow cytometric immunophenotype analysis applied on T-lymphocytes for measuring the fluorescence intensity of CD3 and CD69 expression on those cells.Results: This study showed that the mean of ESR in RA patients was (66.8) while in healthy control it is only (20.4). And the mean of CD3 was higher in healthy individuals (41.88) than in RA patients (35.32), while the mean of CD69 was significantly higher among RA patients (11.09) than that of healthy controls (3.58). Also a moderately strong positive linear correlation was found between CD69 expression and RA severity.Conclusions: the expression of CD69 was up-regulated in freshly isolated peripheral blood lymphocytes from RA patients in comparison to healthy individuals.

Molecular characterization of a transmembrane C-type lectin receptor gene from ayu (Plecoglossus altivelis) and its effect on the recognition of different bacteria by monocytes/macrophages

C-type lectin receptors (CTLRs) play vital roles in immune responses as pattern-recognition receptors (PRRs). In this study, we identified a novel C-type lectin receptor (PaCTLRC) gene from ayu, Plecoglossus altivelis. Predicted PaCTLRC is a single transmembrane receptor with a typical carbohydrate recognition domain (CRD) at its C-terminus. Sequence comparison and phylogenetic tree analysis showed that PaCTLRC was most closely related to Atlantic salmon (Salmo salar) CLRC, but was significantly different from two other ayu CTLRs, aCLR and PaCD209L. PaCTLRC transcript was detected in all tested tissues and cells, with high levels in the liver; and its expression was significantly altered upon Vibrio anguillarum infection. Refolded recombinant PaCTLRC (rPaCTLRC) agglutinated three types of Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus and Streptococcus iniae) and four types of Gram-negative bacteria (Aeromonas hydrophila, Escherichia coli, V. anguillarum and Vibrio parahaemolyticus) in a Ca2+-dependent manner in vitro, and Gram-positive bacteria were shown to be biologically relevant ligands for PaCTLRC. rPaCTLRC bound to d-mannose, d-galactose, l-fucose, N-acetyl-d-glucosamine (GlcNAc), lipopolysaccharide (LPS) and peptidoglycan (PGN), exhibiting a relative binding strength to d-mannose and PGN. d-Mannose, l-fucose, GlcNAc, LPS and PGN could inhibit the agglutinating activity of rPaCTLRC, while d-galactose did not functioned. PaCTLRC neutralization using anti-PaCTLRC IgG resulted in the inhibition of phagocytosis by ayu monocytes/macrophages (MO/MΦ) of S. aureus but not of E. coli, and produced a consistently higher survival rate of S. aureus than that of E. coli. d-Mannose, LPS and PGN treatment had no significant influence on the phagocytosis of ayu MO/MΦ. These results suggest that PaCTLRC may serve as a Gram-positive bacteria-preferred PRR which is involved in pathogen recognition and signal transduction in ayu MO/MΦ.

Characterization of functional mannose receptor in a continuous hybridoma cell line

BackgroundThe mannose receptor is the best described member of the type I transmembrane C-type lectins; however much remains unanswered about the biology of the receptor. One difficulty has been the inability to consistently express high levels of a functional full length mannose receptor cDNA in mammalian cells. Another difficulty has been the lack of a human macrophage cell line expressing a fully functional receptor. Commonly used human macrophage cell lines such as U937, THP-1, Mono-Mac and HL60 do not express the mannose receptor. We have developed a macrophage hybridoma cell line (43MR cells) created by fusion of U937 cells with primary human monocyte-derived macrophages, resulting in a non-adherent cell line expressing several properties of primary macrophages. The purpose of this study was to identify and select mannose receptor-expressing cells using fluorescence-activated cell sorting and to characterize the expression and function of the receptor.ResultsIn the current study we show that the mannose receptor found on this novel cell has endocytic characteristics consistent with and similar to the mannose receptor found on the surface of monocyte-derived human macrophages and rat bone marrow-derived macrophages. In addition, we demonstrate that these cells engage and internalize pathogen particles such as S. aureus and C. albicans. We further establish the transfectability of these cells via the introduction of a plasmid expressing influenza A hemagglutinin.ConclusionsThe 43MR cell line represents the first naturally expressed MR-positive cell line derived from a human macrophage background. This cell line provides an important cell model for other researchers for the study of human MR biology and host-pathogen interactions.

Targeting DC-SIGN via its neck region leads to prolonged antigen residence in early endosomes, delayed lysosomal degradation, and cross-presentation

AbstractTargeting antigens to dendritic cell (DC)–specific receptors, such as DC-SIGN, induces potent T cell-mediated immune responses. DC-SIGN is a transmembrane C-type lectin receptor with a long extracellular neck region and a carbohydrate recognition domain (CRD). Thus far, only antibodies binding the CRD have been used to target antigens to DC-SIGN. We evaluated the endocytic pathway triggered by antineck antibodies as well as their intracellular routing and ability to induce CD8+ T-cell activation. In contrast to anti-CRD antibodies, antineck antibodies induced a clathrin-independent mode of DC-SIGN internalization, as demonstrated by the lack of colocalization with clathrin and the observation that silencing clathrin did not affect antibody internalization in human DCs. Interestingly, we observed that anti-neck and anti-CRD antibodies were differentially routed within DCs. Whereas anti-CRD antibodies were mainly routed to late endosomal compartments, anti-neck antibodies remained associated with early endosomal compartments positive for EEA-1 and MHC class I for up to 2 hours after internalization. Finally, cross-presentation of protein antigen conjugated to antineck antibodies was approximately 1000-fold more effective than nonconjugated antigen. Our studies demonstrate that anti-neck antibodies trigger a distinct mode of DC-SIGN internalization that shows potential for targeted vaccination strategies.

Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Non-integrin (DC-SIGN) Recognizes a Novel Ligand, Mac-2-binding Protein, Characteristically Expressed on Human Colorectal Carcinomas

Dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a type II transmembrane C-type lectin expressed on DCs such as myeloid DCs and monocyte-derived DCs (MoDCs). Recently, we have reported that DC-SIGN interacts with carcinoembryonic antigen (CEA) expressed on colorectal carcinoma cells. CEA is one of the most widely used tumor markers for gastrointestinal cancers such as colorectal cancer. On the other hand, other groups have reported that the level of Mac-2-binding protein (Mac-2BP) increases in patients with pancreatic, breast, and lung cancers, virus infections such as human immunodeficiency virus and hepatitis C virus, and autoimmune diseases. Here, we first identified Mac-2BP expressed on several colorectal carcinoma cell lines as a novel DC-SIGN ligand through affinity chromatography and mass spectrometry. Interestingly, we found that DC-SIGN selectively recognizes Mac-2BP derived from some colorectal carcinomas but not from the other ones. Furthermore, we found that the α1-3,4-fucose moieties of Le glycans expressed on DC-SIGN-binding Mac-2BP were important for recognition. DC-SIGN-dependent cellular interactions between immature MoDCs and colorectal carcinoma cells significantly inhibited MoDC functional maturation, suggesting that Mac-2BP may provide a tolerogenic microenvironment for colorectal carcinoma cells through DC-SIGN-dependent recognition. Importantly, Mac-2BP was detected as a predominant DC-SIGN ligand expressed on some primary colorectal cancer tissues from certain parts of patients in comparison with CEA from other parts, suggesting that DC-SIGN-binding Mac-2BP bearing tumor-associated Le glycans may become a novel potential colorectal cancer biomarker for some patients instead of CEA.

Increased Expression of Macrophage-Inducible C-type Lectin in Adipose Tissue of Obese Mice and Humans

OBJECTIVEWe have provided evidence that saturated fatty acids, which are released from adipocytes via macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for the Toll-like receptor (TLR) 4 complex in macrophages, thereby aggravating obesity-induced adipose tissue inflammation. The aim of this study was to identify the molecule(s) activated in adipose tissue macrophages in obesity.RESEARCH DESIGN AND METHODSWe performed a cDNA microarray analysis of coculture of 3T3-L1 adipocytes and RAW264 macrophages. Cultured adipocytes and macrophages and the adipose tissue of obese mice and humans were used to examine mRNA and protein expression.RESULTSWe found that macrophage-inducible C-type lectin (Mincle; also called Clec4e and Clecsf9), a type II transmembrane C-type lectin, is induced selectively in macrophages during the interaction between adipocytes and macrophages. Treatment with palmitate, a major saturated fatty acid released from 3T3-L1 adipocytes, induced Mincle mRNA expression in macrophages at least partly through the TLR4/nuclear factor (NF)-κB pathway. Mincle mRNA expression was increased in parallel with macrophage markers in the adipose tissue of obese mice and humans. The obesity-induced increase in Mincle mRNA expression was markedly attenuated in C3H/HeJ mice with defective TLR4 signaling relative to control C3H/HeN mice. Notably, Mincle mRNA was expressed in bone-marrow cell (BMC)-derived proinflammatory M1 macrophages rather than in BMC-derived anti-inflammatory M2 macrophages in vitro.CONCLUSIONSOur data suggest that Mincle is induced in adipose tissue macrophages in obesity at least partly through the saturated fatty acid/TLR4/NF-κB pathway, thereby suggesting its pathophysiologic role in obesity-induced adipose tissue inflammation.

Secreted M-Ficolin Anchors onto Monocyte Transmembrane G Protein-Coupled Receptor 43 and Cross Talks with Plasma C-Reactive Protein to Mediate Immune Signaling and Regulate Host Defense

Although transmembrane C-type lectins (CLs) are known to initiate immune signaling, the participation and mechanism of action of soluble CLs have remained enigmatic. In this study, we found that M-ficolin, a conserved soluble CL of monocyte origin, overcomes its lack of membrane-anchor domain by docking constitutively onto a monocyte transmembrane receptor, G protein-coupled receptor 43 (GPCR43), to form a pathogen sensor-cum-signal transducer. On encountering microbial invaders, the M-ficolin-GPCR43 complex activates the NF-κB cascade to upregulate IL-8 production. We showed that mild acidosis at the local site of infection induces conformational changes in the M-ficolin molecule, which provokes a strong interaction between the C-reactive protein (CRP) and the M-ficolin-GPCR43 complex. The collaboration among CRP-M-ficolin-GPCR43 under acidosis curtails IL-8 production thus preventing immune overactivation. Therefore, we propose that a soluble CL may become membrane-associated through interaction with a transmembrane protein, whereupon infection collaborates with other plasma protein to transduce the infection signal and regulate host defense. Our finding implies a possible mechanism whereby the host might expand its repertoire of immune recognition-cum-regulation tactics by promiscuous protein networking. Furthermore, our identification of the pH-sensitive interfaces of M-ficolin-CRP provides a powerful template for future design of potential immunomodulators.

Mutation in the DC-SIGN cytoplasmic triacidic cluster motif markedly attenuates receptor activity for phagocytosis and endocytosis of mannose-containing ligands by human myeloid cells

The transmembrane C-type lectin, dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), has three conserved cytoplasmic tail motifs: the tyrosine (Y)-based, dileucine (LL), and triacidic cluster (EEE), which are believed to regulate ligand binding, uptake, and trafficking. We mutated each of these motifs by alanine substitution and tested their roles in phagocytosis and receptor-mediated endocytosis of the highly mannosylated ligands, Mycobacterium tuberculosis mannose-capped lipoarabinomannan (ManLAM) and HIV-1 surface glycoprotein gp120, respectively, in transfected human myeloid K-562 cells. Compared with wild-type and other mutants, the EEE mutant of DC-SIGN showed a reduced cell-surface expression, near abolishment in the phagocytosis of ManLAM-coated beads (90.5+/-0.4%), and a marked reduction in the endocytosis of soluble gp120 (79.3+/-0.7%). Although, the Y mutant of DC-SIGN did not exhibit any effect on phagocytosis and intracellular trafficking to the phagolysosome, the LL mutant caused the majority of the receptor and/or ligands to remain bound to the cell surface, indicating a role for the LL motif as an internalization signal. The majority of the EEE mutant protein was found to be retained by the intracellular trans-Golgi network and not by the late endosomal/lysosomal compartment of transfected K-562 cells. Collectively, our data indicate a dual role for the EEE motif as a sorting signal in the secretory pathway and a lysosomal targeting signal in the endocytic pathway.

DC-SIGN and L-SIGN: the SIGNs for infection

Two closely related trans-membrane C-type lectins dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin (DC-SIGN or CD209) and liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN also known as DC-SIGNR, CD209L or CLEC4M) directly recognize a wide range of micro-organisms of major impact on public health. Both genes have long been considered to share similar overall structure and ligand-binding characteristics. This review presents more recent biochemical and structural studies, which show that they have distinct ligand-binding properties and different physiological functions. Of importance in both these genes is the presence of an extra-cellular domain consisting of an extended neck region encoded by tandem repeats that support the carbohydrate-recognition domain, which plays a crucial role in influencing the pathogen-binding properties of these receptors. The notable difference between these two genes is in this extra-cellular domain. Whilst the tandem-neck-repeat region remains relatively constant size for DC-SIGN, there is considerable polymorphism for L-SIGN. Homo-oligomerization of the neck region of L-SIGN has been shown to be important for high-affinity ligand binding, and heterozygous expression of the polymorphic variants of L-SIGN in which neck lengths differ could thus affect ligand-binding affinity. Functional studies on the effect of this tandem-neck-repeat region on pathogen-binding, as well as genetic association studies for various infectious diseases and among different populations, are discussed. Worldwide demographic data of the tandem-neck-repeat region showing distinct differences in the neck-region allele and genotype distribution among different ethnic groups are presented. These findings support the neck region as an excellent candidate acting as a functional target for selective pressures exerted by pathogens.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-008-0350-2) contains supplementary material, which is available to authorized users.