Interactions of LSECtin and DC-SIGN/DC-SIGNR with viral ligands: Differential pH dependence, internalization and virion binding

The calcium-dependent lectins DC-SIGN and DC-SIGNR (collectively termed DC-SIGN/R) bind to high-mannose carbohydrates on a variety of viruses. In contrast, the related lectin LSECtin does not recognize mannose-rich glycans and interacts with a more restricted spectrum of viruses. Here, we analyzed whether these lectins differ in their mode of ligand engagement. LSECtin and DC-SIGNR, which we found to be co-expressed by liver, lymph node and bone marrow sinusoidal endothelial cells, bound to soluble Ebola virus glycoprotein (EBOV-GP) with comparable affinities. Similarly, LSECtin, DC-SIGN and the Langerhans cell-specific lectin Langerin readily bound to soluble human immunodeficiency virus type-1 (HIV-1) GP. However, only DC-SIGN captured HIV-1 particles, indicating that binding to soluble GP is not necessarily predictive of binding to virion-associated GP. Capture of EBOV-GP by LSECtin triggered ligand internalization, suggesting that LSECtin like DC-SIGN might function as an antigen uptake receptor. However, the intracellular fate of lectin-ligand complexes might differ. Thus, exposure to low-pH medium, which mimics the acidic luminal environment in endosomes/lysosomes, released ligand bound to DC-SIGN/R but had no effect on LSECtin interactions with ligand. Our results reveal important differences between pathogen capture by DC-SIGN/R and LSECtin and hint towards different biological functions of these lectins.     

中国汉族人群DC-SIGN和DC-SIGNR基因遗传多态性

目的了解中国汉族人群DC-SIGN和DC-SIGNR基因颈区重复序列的遗传多态性分布，获得相应位点的汉族人群的遗传学数据。方法应用PCR技术、琼脂糖凝胶电泳结合测序对DC-SIGN和DC-SIGNR基因的颈区重复序列分型，计算DC-SIGNR的多态信息含量。结果DC-SIGN多态性低，颈区绝大多数为等位基因7重复，该等位基因频率为0．9808，但亦检出少量的等位基因4、5、6、8等变异，而美国白人只含有等位基因6、8变异；DC-SIGNR存在高度多态性，多态信息含量为0．5312，存在4、5、6．7、8、9等位基因，检出16种基因型。6／5、7／4、7／5、7／6、7／7、9／5、9．／7、9／9基因型和5、6、7、9等位基因频率在中国汉族人群和美国白人中的分布构成差异有统计学意义（P〈0．01），与美国白人比较，中国汉族人群似乎存在更多的插入突变。结论中国汉人的DC-SIGN和DC-SIGNR基因型分布和基因频率与美国白人相比其差异有统计学意义，并有其独特的群体遗传学特征。     

DC-SIGN和DC-SIGNR基因多态性分析与HIV-1易感性关系研究

目的通过分析DC-SIGN和DC-SIGNR的基因多态性分布及基因频率在健康人群、HIV-1感染者和HIV-1长期暴露不感染人群（ES）的差别,探讨这两个基因与HIV-1长期暴露不感染的关系。方法纳入三组研究对象,包括健康对照组（160例）,HIV-1感染者组（267例）,ES组（37例）。扩增DC-SIGN和DC-SINGR基因的颈区重复序列进行分析。结果三组人群DC-SIGN基因型以7/7为主,等位基因呈低度多态性。DC-SIGNR基因型以7/7为主,等位基因呈高度多态性,出现了较多的非7/7基因型,其中等位基因6在三组间以及在HIV-1感染者和ES之间分布的差异均有统计学意义（P〈0.05）。结论 DC-SIGN基因多态性变异很低,对人群HIV-1易感性的研究意义不大;DC-SIGNR基因呈高度多态性,其低频率等位基因6可能是人群对HIV-1不易感的保护因素。     

The protease allergen Der p 1 cleaves cell surface DC-SIGN and DC-SIGNR: experimental analysis of in silico substrate identification and implications in allergic responses

Background The cysteine protease Der p 1 from the house dust mite Dermatophagoides pteronyssinus is one of the most potent allergens known. An attractive mechanism for a component of Der p 1 allergenicity lies in its ability to cleave key regulatory molecules from leucocyte surfaces, subverting cellular function and driving abnormal immunoglobulin E (IgE) responses. Objective Although CD23, CD25 and CD40 have already been identified as major Der p 1 targets, other significant substrates may also exist. Methods To investigate this, knowledge of the proteolytic properties of Der p 1 was used to perform in silico digestion of human dendritic cell surface proteins, using the prediction of protease specificity (PoPS) bioinformatics tool, in conjunction with cellular in vitro analysis and cleavage site determination. Results Targets identified included DC‐SIGN and DC‐SIGNR, two C‐type lectins implicated mostly in pathogen trafficking. Treatment of positively expressing cells with Der p 1 led to loss of detectable surface DC‐SIGN and DC‐SIGNR. Digestion of purified soluble recombinant DC‐SIGN and DC‐SIGNR, followed by N‐terminal sequencing and MALDI mass spectrometry, indicated in each case one major cleavage site and several minor sites, the former correlating well with Der p 1 enzymology and the folded state of the substrate proteins. Loss of DC‐SIGN from the cell surface led to reduced binding of intracellular adhesion molecule‐3, an endogenous DC‐SIGN ligand expressed on naïve T cells which is thought to be involved in T‐helper type 1 cytokine signalling. Conclusion These data provide evidence of lectin involvement in the initiation of the allergic response and the value of using genome‐wide in silico digestion tools.     

LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus

Cellular attachment factors like the C-type lectins DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) can augment viral infection and might promote viral dissemination in and between hosts. The lectin LSECtin is encoded in the same chromosomal locus as DC-SIGN/R and is coexpressed with DC-SIGNR on sinusoidal endothelial cells in liver and lymphnodes. Here, we show that LSECtin enhances infection driven by filovirus glycoproteins (GP) and the S protein of SARS coronavirus, but does not interact with human immunodeficiency virus type-1 and hepatitis C virus envelope proteins. Ligand binding to LSECtin was inhibited by EGTA but not by mannan, suggesting that LSECtin unlike DC-SIGN/R does not recognize high-mannose glycans on viral GPs. Finally, we demonstrate that LSECtin is N-linked glycosylated and that glycosylation is required for cell surface expression. In summary, we identified LSECtin as an attachment factor that in conjunction with DC-SIGNR might concentrate viral pathogens in liver and lymph nodes.     

Role of Homozygous DC-SIGNR 5/5 Tandem Repeat Polymorphism in HIV-1 Exposed Seronegative North Indian Individuals

Despite multiple sexual exposures to HIV-1 virus, some individuals remain HIV-1 seronegative. Although several genetic factors have been related to HIV-1 resistance, the homozygosity for a mutation in CCR5 gene (the 32-bp deletion, i.e., CCR5-Delta32 allele) is presently considered the most relevant one. The C-type lectins, DC-SIGN (present on dendritic cells and macrophages) and DC-SIGNR (present on endothelial cells in liver and lymph nodes) efficiently bind and transmit HIV-1 to susceptible cell in trans, thereby augmenting the infection. A potential association of the DC-SIGN and DC-SIGNR neck domain repeat polymorphism and risk of HIV-1 infection is currently under debate. To determine the influence of host genetic factors on HIV-1 resistance, we conducted genetic risk association study in HIV-1-exposed seronegative (n = 47) individuals, HIV-1 seronegative (n = 262) healthy control, and HIV-1-infected seropositive patients (n = 168) for polymorphism in neck domain of DC-SIGN and DC-SIGNR genes. The DC-SIGN and DC-SIGNR genotypes were identified by polymerase chain reaction method in DNA extracted from peripheral blood and confirmed by sequencing. Fisher exact or χ ² test was used for static analysis. DC-SIGN genotype and allele distribution was fairly similar in HIV-1-exposed seronegative, HIV-1 seropositive, and HIV-1 seronegative control. There was no statistical significance in the differences in the distribution of DC-SIGN genotypes. A total of 13 genotypes were found in DC-SIGNR neck repeat region polymorphism. Among all the genotypes, only 5/5 homozygous showed significant reduced risk of HIV-1 infection in HIV-1-exposed seronegative individuals (p = 0.009). A unique genotype 8/5 heterozygous was also found in HIV-1 seropositive individual, which is not reported elsewhere.     

Distinct usage of three C-type lectins by Japanese encephalitis virus: DC-SIGN,DC-SIGNR,and LSECtin

Infection with West Nile virus and dengue virus, two mosquito-borne flaviviruses, is enhanced by two calcium-dependent lectins: dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and its related molecule (DC-SIGNR). The present study examined the relationship between Japanese encephalitis virus (JEV) infection and three lectins: DC-SIGN, DC-SIGNR, and liver sinusoidal endothelial cell lectin (LSECtin). Expression of DC-SIGNR resulted in robust JEV proliferation in a lymphoid cell line, Daudi cells, which was otherwise non-permissive to infection. DC-SIGN expression caused moderate JEV proliferation, with effects that varied according to the cells in which JEV was prepared. LSECtin expression had comparatively minor, but consistent, effects, in all cell types used in JEV preparation. While DC-SIGN/DC-SIGNR-mediated JEV infection was inhibited by yeast mannan, LSECtin-mediated infection was inhibited by N-acetylglucosamine β1-2 mannose. Although involvement of DC-SIGN/DC-SIGNR in infection seems to be a common characteristic, this is the first report on usage of LSECtin in mosquito-borne flavivirus infection.     

DC-SIGN and DC-SIGNR: helping hands for HIV

The ability of dendritic cells (DCs) to promote HIV infection of T cells efficiently has been linked to the C-type lectin DC-specific intercellular adhesion molecule (ICAM)-grabbing nonintegrin (DC-SIGN). DC-SIGN and its homolog DC-SIGN-related (DC-SIGNR) capture and transmit human and simian immunodeficiency viruses to a wide variety of receptor-positive cells. The expression patterns of DC-SIGN and DC-SIGNR suggest that they might play roles in both horizontal and vertical transmission, as well as dissemination of virus within the host. In vivo experiments are required to prove these hypotheses and might pave the way for the development of new inhibitors of viral entry.     

DC-SIGNR基因多态性与人类免疫缺陷病毒-1易感性的关联研究

目的 探索DC-SIGNR基因多态性对中国汉族人群人类免疫缺陷病毒-1(human immunodeficiency virus-1,HIV-1)病毒易感性的影响.方法 用聚合酶链反应、琼脂糖凝胶电泳结合测序对345例HIV-1血清阳性感染者及468例H1V-1血清阴性高危人群的DC-SIGNR基因绞链区重复序列分型,然后用卡方检验检测DC-SIGNR基因各亚型在不同人群组中出现的频率.结果 共发现DC-SIGNR基因14个基因型和5种等位基因,其中等位基因7出现的频率最高(67.1%),明显高于美国白人(67.1% vs.46.0%,P<0.01).HIV-1感染组7/7基因型的出现频率明显低于高危人群组(38.55% vs.48.29%,P=0.0057),而HIV-1感染组的9/5基因型的出现频率明显高于高危人群组(4.35% vs.1.07%,P=0.0029).结论 中国人群DC-SIGNR基因多态性分布有着独特的遗传学特征,中国人群中DC-SIGNR基因型9/5可能增加对HIV-1的易感性.     

Five mouse homologues of the human dendritic cell C-type lectin, DC-SIGN

DC-SIGN, a human C-type lectin, is expressed on the surface of dendritic cells (DC), while a closely related human gene, DC-SIGNR or L-SIGN, is found on sinusoidal endothelial cells of liver and lymph node. Both DC-SIGN and DC-SIGNR/L-SIGN can bind ICAM-3 and HIV gp120, and transmit HIV to susceptible cells in trans. Here, we report the cloning of five mouse genes homologous to human DC-SIGN and DC-SIGNR/L-SIGN. Only one gene, named mouse DC-SIGN, is highly expressed in DC, and is not found in a panel of mouse macrophage and lymphocyte cell lines. The other four genes, named mouse SIGNR1 (SIGN-Related gene 1), SIGNR2, SIGNR3 and SIGNR4, are expressed at lower levels in various cells according to RT-PCR and Northern blot analyses on RNA. All the genes of mouse DC-SIGN and SIGNRs map to adjacent regions of chromosome 8 A1.2-1.3. However, like human DC-SIGN, only the mouse DC-SIGN gene is closely juxtaposed to the CD23 gene, while the other four SIGNR genes are located close to each other in a neighboring region. mRNAs of mouse DC-SIGN and three SIGNR genes encode type II transmembrane proteins (DC-SIGN, 238 amino acids; SIGNR1, 325 amino acids; SIGNR3, 237 amino acids; SIGNR4, 208 amino acids), but the SIGNR2 gene only encodes a carbohydrate recognition domain (CRD) without a cytosolic domain and a transmembrane domain (SIGNR2, 178 amino acids). Amino acid sequence similarities between the CRD of human DC-SIGN and the mouse homologues are 67% for DC-SIGN, 69% for SIGNR1, 65% for SIGNR2, 68% for SIGNR3 and 70% for SIGNR4 respectively. However, the membrane proximal neck domains in the mouse genes are much shorter than their counterparts in human DC-SIGN and DC-SIGNR/L-SIGN. This family of mouse C-type lectins is therefore complex, but only one of the new genes, DC-SIGN, is juxtaposed to CD23 and is expressed at high levels in DC.     

可溶性DC-SIGN的表达、纯化及其生物学活性分析

目的　在大肠杆菌中表达并纯化DC SIGN融合蛋白并对该融合蛋白的抗原特异性和生物学活性进行分析。方法　以重组质粒pcDNA3 .1 DC SIGN为模板 ,进行PCR扩增出带KpnⅠ和SacⅠ酶切位点的人DC SIGN凝集素cDNA ,经相应酶切后插入原核表达载体pET 3 2a( ) ,转化大肠杆菌AD494(DE3 ) ,经IPTG诱导表达DC SIGN融合蛋白 ,用Ni2 NTA树脂对融合蛋白进行纯化 ,以Westernblot试验进行鉴定 ,通过HIV与DC SIGN的亲和实验研究融合蛋白的生物学活性。结果　酶切鉴定证实DC SIGN凝集素基因已插入原核表达载体pET 3 2a( )。重组表达质粒pET 3 2a( ) DL在大肠杆菌AD494(DE3 )中成功表达了DC SIGN融合蛋白 ,其相对分子质量 (Mr)约为 3 5× 10 3。Ni2 NTA树脂纯化后 ,融合蛋白的纯度可达 90 %以上。Westernblot试验显示DC SIGN融合蛋白与鼠抗人DC SIGN抗体有特异性免疫反应。结论　在大肠杆菌中表达DC SIGN融合蛋白 ,用亲和层析的方法对其进行初步纯化 ;HIV与DC SIGN的亲和实验表明 ,可溶性DC SIGN融合蛋白能抑制R5和X4HIV与DC SIGN受体结合     

Identification of the mycobacterial carbohydrate structure that binds the C-type lectins DC-SIGN, L-SIGN and SIGNR1

Mycobacterium tuberculosis represents a worldwide health risk and although macrophages are primarily infected, dendritic cells (DC) are important in inducing cellular immune responses against M. tuberculosis. Recent studies have demonstrated that M. tuberculosis targets the DC-specific C-type lectin DC-SIGN to inhibit the immuno-stimulatory function of DC through the interaction of the mycobacterial mannosylated lipoarabinomannan (ManLAM) to DC-SIGN, which prevents DC maturation and induces the immuno-suppressive cytokine IL-10. This may contribute to survival and persistence of M. tuberculosis. Here, we have identified the specific pathogen-derived carbohydrate structure on ManLAM that is recognized by DC-SIGN. We have synthesized the mannose-cap oligosaccharides man-ara, (man)2-ara and (man)3-ara, and demonstrate that these neoglycoconjugates are specifically bound by DC-SIGN. Moreover, we demonstrate that the human and murine DC-SIGN homologue L-SIGN and SIGNR1, respectively, also interact with mycobacteria through ManLAM. Both homologues have the highest affinity for the (man)3-ara structure, similar to DC-SIGN. This study provides information about the specific carbohydrate structures on pathogens that are recognized by DC-SIGN, and may provide strategies to develop vaccines against these pathogens. Moreover, the identification of SIGNR1 as a receptor for ManLAM will enable in vivo studies to investigate the role of DC-SIGN in M. tuberculosis pathogenesis.     

树突状细胞特异表面分子DC-SIGN的研究进展

DC-SIGN是一种特异表达于树突状细胞(DC)表面的Ⅱ型跨膜蛋白,在机体生理和病理免疫调节中发挥着重要作用,可以与ICAM-3结合,从而介导DC与T细胞的相互作用,其CRD区可以与HIV-1、HCV、结核杆菌等多种病原体表面糖蛋白结合,从而促进病原体感染。最近的研究表明DC-SIGN与肿瘤免疫、免疫逃避也密切相关。     

Domains of macaque DC-SIGN essential for capture and transfer of simian immunodeficiency virus

The C-type lectin DC-SIGN mediates the capture and transfer of simian immunodeficiency virus (SIV) from macaque dendritic cells (DCs) to permissive T-cells. To further identify the determinants in macaque DC-SIGN required for capture and transfer of virus, we created mutants containing deletions or point mutations in the extracellular domains, and tested their ability to capture and transmit SIV. We found that SIV bound to the carbohydrate recognition domain (CRD) of macaque DC-SIGN via the envelope protein. In addition, deleting the C-terminal half of the CRD, or mutating amino acids within this region that contact Ca(2+) or mannose, disrupted virion capture activity. However, an N-terminal CRD deletion mutant was capable of binding SIV, indicating that this region was not necessary for binding. Finally, deletion of the neck domain also reduced the capacity for macaque DC-SIGN to capture SIV. Interestingly, ICAM-3, the cellular ligand for DC-SIGN, did not bind to any of the DC-SIGN mutants, including mutants with amino acid changes in the N-terminal region of the CRD. These data suggest that the binding sites for SIV and ICAM-3 may be distinct but overlapping. Together, the data demonstrate the importance of both the neck and the CRD of macaque DC-SIGN for efficient capture of SIV and binding to ICAM-3.     

AP-1和ETS-1位点缺失的DC-SIGN启动子荧光素酶报告质粒的构建及其活性研究

目的 研究AP-1和ETS-1转录因子结合位点(TFBS)缺失对树突状细胞特异性胞间黏附分子-3结合非整合素(DC-SIGN)启动子活性的影响,探讨DC-SIGN表达的机制.方法 从人外周血中提取全基因组DNA,设计DC-SIGN启动子序列上下游引物、转录因子结合位点两侧的引物及其相应的连接引物,利用PCR方法,通过不同的引物组合扩增出转录因子结合位点两侧的片段,再用连接引物连接两片段,得到转录因子结合位点缺失的DC-SIGN启动子序列,通过双酶切、连接的方法,将得到的DC-SIGN启动子序列定向克隆入荧光素酶报告质粒,将上述质粒通过脂质体转染Hacat和293细胞,48 h后检测荧光素酶的活性.结果 体外扩增得到的转录因子结合位点缺失的DC-SIGN启动子序列以及重组的荧光素酶报告质粒经双酶切、基因测序鉴定正确,荧光素酶活性检测结果显示AP-1位点缺失使DC-SIGN启动子活性下降20%(293细胞)和10%(Hacat细胞),带增强子的DC-SIGN启动子活性下降了40%～50%;ETS-1位点缺失的普通型和带增强子型DC-SIGN启动子的活性几乎消失.结论 ETS-1转录因子结合位点在DC-SIGN启动子活性表达中起重要作用,AP-1转录因子结合位点作用不大.     

DC-SIGN荧光融合蛋白的构建、表达和生物学功能初探

目的 构建绿色荧光蛋白（EGFP）标记的DC-SIGN分子（DC-SIGN-EGFP融合蛋白）,并在哺乳动物细胞中获得功能性表达。方法 PCR方法获得DC-SIGN分子和EGFP分子的cDNA,分别克隆入真核表达载体pEGFP-N1和真核表达载体pCI,使EGFP荧光蛋白分别位于DC-SIGN蛋白的C末端和N末端。在转染COS-7细胞后,在激光共聚焦显微镜和流式细胞仪检测重组分子的表达及融合蛋白在细胞定位情况。激光共聚焦显微镜检测融合蛋白的功能情况。结果 获得了预期的重组表达质粒,经DNA测序证实开放阅读框正确,转染COS-7细胞后绿色荧光蛋白位于细胞膜,仅EGFP位于DC-SIGNN-末端的融合蛋白可被抗DC-SIGN抗体结合。EGFP位于DC-SIGN N-末端的融合蛋白可有效摄取DC-SIGN受体的高亲和力配体le（x）寡糖。结论 所构建的DC-SIGN-EGFP融合蛋白在细胞内表达后具有细胞表面受体分子的特征性分布,可被抗DC-SIGN抗体检测及摄取特异性配体,为进一步深入研究DC-SIGN分子功能提供了良好的模型。     

Expression of DC-SIGN and DC-SIGNR on human sinusoidal endothelium: a role for capturing hepatitis C virus particles

Hepatic sinusoidal endothelial cells are unique among endothelial cells in their ability to internalize and process a diverse range of antigens. DC-SIGNR, a type 2 C-type lectin expressed on liver sinusoids, has been shown to bind with high affinity to hepatitis C virus (HCV) E2 glycoprotein. DC-SIGN is a closely related homologue reported to be expressed only on dendritic cells and a subset of macrophages and has similar binding affinity to HCV E2 glycoprotein. These receptors function as adhesion and antigen presentation molecules. We report distinct patterns of DC-SIGNR and DC-SIGN expression in human liver tissue and show for the first time that both C-type lectins are expressed on sinusoidal endothelial cells. We confirmed that these receptors are functional by demonstrating their ability to bind HCV E2 glycoproteins. Although these lectins on primary sinusoidal cells support HCV E2 binding, they are unable to support HCV entry. These data support a model where DC-SIGN and DC-SIGNR on sinusoidal endothelium provide a mechanism for high affinity binding of circulating HCV within the liver sinusoids allowing subsequent transfer of the virus to underlying hepatocytes, in a manner analogous to DC-SIGN presentation of human immunodeficiency virus on dendritic cells.     

Role of the C-type lectins DC-SIGN and L-SIGN in Leishmania interaction with host phagocytes

Leishmaniasis is a parasitic disease that courses with cutaneous or visceral clinical manifestations. The amastigote stage of the parasite infects phagocytes and modulates the effector function of the host cells. Our group has described that the interaction between Leishmania and immature monocyte-derived dendritic cells (DCs) takes place through dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin that specifically recognizes fungal, viral and bacterial pathogens. The DC-SIGN-mediated recognition of Leishmania amastigotes does not induce DC maturation, and the DC-SIGN ligand/s on Leishmania parasites is/are still unknown. We have also found that the DC-SIGN-related molecule L-SIGN, specifically expressed in lymph node and liver sinusoidal endothelial cells, acts as a receptor for L. infantum, the parasite responsible for visceral leishmaniasis, but does not recognize L. pifanoi, which causes the cutaneous form of the disease. Therefore, DC-SIGN and L-SIGN differ in their ability to interact with Leishmania species responsible for either visceral or cutaneous leishmaniasis. A deeper knowledge of the parasite-C-type lectin interaction may be helpful for the design of new DC-based therapeutic vaccines against Leishmania infections.