Functional comparison of the mouse DC-SIGN, SIGNR1, SIGNR3 and Langerin, C-type lectins
Kazuhiko Takahara,
Yusuke Yashima,
Yoshiki Omatsu,
Hideo Yoshida,
Yukino Kimura,
Young-Sun Kang1,
Ralph M. Steinman1,
Chae Gyu Park1 and
Kayo Inaba
Laboratory of Immunobiology, Department of Animal Development and Physiology, Division of Systemic Life Science, Graduate School of Biostudies, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan 1 Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
K. Takahara and Y. Yashima contributed equally to this work.
Correspondence to: K. Inaba; E-mail: kayo{at}lif.kyoto-u.ac.jp
Transmitting editor: H. Karasuyama
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Abstract
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The mouse (m) DC-SIGN family consists of several homologous type II transmembrane proteins located in close proximity on chromosome 8 and having a single carboxyl terminal carbohydrate recognition domain. We first used transfected non-macrophage cell lines to compare the polysaccharide and microbial uptake capacities of three of these lectinsDC-SIGN, SIGNR1 and SIGNR3to another homologue mLangerin. Each molecule shares a potential mannose-recognition EPN-motif in its carbohydrate recognition domain. Using an anti-Tag antibody to follow Tag-labeled transfectants, we found that each molecule could be internalized, although the rates differed. However, mDC-SIGN was unable to take up FITCdextran, FITCovalbumin, zymosan or heat-killed Candida albicans. The other three lectins showed distinct carbohydrate recognition properties, assessed by blocking FITCdextran uptake at 37°C and by mannan binding activity at 4°C. Furthermore, only SIGNR1 was efficient in mediating the capture by transfected cells of Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium, while none of the lectins tested were competent to capture Gram-positive bacteria, Staphylococcus aureus. Interestingly, transfectants with SIGNR1 lacking the cytoplasmic domain were capable of binding FITCzymosan in a manner that was abolished by EDTA or mannan, but not laminarin. In addition, resident peritoneal CD11b+ cells expressing SIGNR1 bound zymosan at 4°C in concert with a laminarin-sensitive receptor. Therefore these homologous C-type lectins have distinct recognition patters for microbes despite similarities in the carbohydrate recognition domains.
Keywords: dendritic cells, macrophage, Gram-negative bacteria, phagocytosis
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Introduction
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C-type lectins are expressed in most cell types including macrophages and dendritic cells (DCs), which must internalize various glycoproteins and microbes for the purposes of clearance and antigen presentation to T lymphocytes (1). Human (h) DC-SIGN (CD209) is of special interest because it is involved in the interaction of certain DCs with several different pathogens including several viruses [HIV-1 (2), HCV (3), dengue virus (4,5), CMV (6), ebola virus (7)] and other microbes [mycobacteria (8,9), Leishmania (10) and candida species (11)]. This type II transmembrane protein has a single C-type lectin domain and is expressed on immature monocyte-derived DCs.
Previously, we have cloned five mouse hDC-SIGN homologues that form a gene cluster in an
210 kb region on chromosome 8 (12). Only one of these named mDC-SIGN was expressed in a restricted fashion in DCs, while the others had a different tissue distribution and were termed SIGNR1, SIGNR2, SIGNR3 and SIGNR4 for SIGN-related proteins. Despite similarities in the carbohydrate recognition domains (CRDs) of the mouse homologues to hDC-SIGN, the membrane proximal neck domains were much shorter.
It will be important to begin to identify the natural ligands for this new family of C-type lectins. We and Geijtenbeek et al. have recently reported that SIGNR1 is primarily expressed on a subset of macrophages in spleen marginal zone and lymph node medulla, and that SIGNR1 is involved in the uptake of fluorescence-labeled dextran (FITCdextran) and capsular polysaccharides of Streptococcus pneumoniae (1315). SIGNR1 dependent uptake was observed for both splenic marginal zone macrophages in vivo and by cell lines transfected with the SIGNR1 gene. Except for SIGNR4, the mouse DC-SIGN family has an EPN motif that is postulated to recognize mannose residues (16,17) (SIGNR4 contains a QPN motif).
We now report on the recognition of polysaccharides and microbes as well as endocytic capabilities of mDC-SIGN and SIGNRs using transfected non-macrophage cell lines, so that we could distinguish the function of the SIGN family from the many other C-type lectins expressed by macrophages and DCs. We were also interested in comparing mDC-SIGN, SIGNR1 and SIGNR3 with mLangerin, another homologous C-type lectin that has an EPN motif in its CRD but is expressed on a distinct subset of DCs (12,18). We further show that SIGNR1 is expressed by resident peritoneal CD11b+ cells and recognizes zymosan in cooperation with laminarin-sensitive receptor. Our results reveal differences in polysaccharide recognition, endocytic capacities and microbe capture among these lectins.
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Methods
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Cells and cultures
Human embryonic kidney HEK293T cells, African green monkey kidney COS-7 cells, mouse fibroblast NIH3T3 cells and Chinese hamster ovary CHO cells were maintained in DMEM containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. For CHO transfectants, 1 mg/ml of G418 (Sigma-Aldrich, Irvine, CA) was added to the medium.
Vector constructions and transient expression of lectins
The coding sequences of mDC-SIGN, SIGNR1, SIGNR3 (12) and mLangerin (18,19) were amplified with KlenTaq DNA polymerase (Sigma-Aldrich), and then cloned into pEF6/V5-His (Invitrogen, Carlsbad, CA) and into pcDNA4/HisMax (Invitrogen) to add V5 and Xpress Tag sequence at C-terminus and N-terminus, respectively, according to the manufacturers protocol. Cloned cDNAs were transfected with Lipofectamine 2000 reagent (Invitrogen) into HEK293T and COS-7 or with TransFast transfection reagent (Promega, Madison, WI) into NIH3T3. Following 24 h culture, cells were collected, seeded either into tissue culture plates or onto poly L-lysine (Sigma-Aldrich)-coated cover slips placed in culture vessels at an appropriate cell density, and cultured for another 24 h. Transfection efficiencies were over 80% for HEK293T cells, 2530% for NIH3T3 cells and 3545% for COS-7 cells, as assessed by staining with anti-Tag mAbs.
Kinetic and microscopic analyses of internalization of C-type lectins
Single cell suspensions of HEK293T cell transfectants were incubated with 1 µg/ml of anti-V5 monoclonal antibody (mAb) (mouse IgG2a; Invitrogen) for 30 min on ice, washed with cold PBS and then incubated for various periods at 37°C to allow internalization. At the end of the incubation, the cells were stained with biotindonkey F(ab')2 anti-mouse IgG antibody (Jackson Immuno Research, West Grove, PA) and phycoerythrin-labeled (PE-) streptavidin (Jackson Immuno Research), and then acquired with a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA). The relative amounts of remaining Ab on the cell surface were analyzed with Flowjo (Tree Star, Inc., San Carlos, CA) or CELLQuest (Becton Dickinson). In order to facilitate internalization, transfectants were first labeled with anti-V5 mAb and biotindonkey F(ab')2 anti-mouse IgG antibody before the incubation at 37°C. Cells incubated at 4°C served as controls.
To detect the internalized lectins by immunostaining, NIH3T3 transfectants on cover slips were first treated with 1 µg/ml of anti-V5 antibody at 4°C for 30 min. After extensive washes, the cells were incubated at 37°C for 60 min, fixed with ice-cold 3.7% formalin in phosphate-buffered saline (PBS) followed by permeabilization by 0.01% saponinPBS supplemented with 1% FCS. Then, cells were stained with biotindonkey F(ab')2 anti-mouse IgG antibody + Texas Redstreptavidin (Dupont NEN, Boston, MA) in combination with rat anti-transferrin receptor (TIB219) or LAMP-2 (ABL-93) mAb + FITC-labeled donkey F(ab')2 anti-rat IgG (Jackson Immuno Research) antibody. After mounting with 1% propyl gallate (Wako, Osaka, Japan) in 50% glycerol, internalization of lectins was analyzed with a deconvolution microscope (BX51-FL: Olympus, Tokyo, Japan) using imaging software (Openlab: Improvision, Coventry, UK). Images were stored digitally and exported to Adobe Photoshop 7.0 for reproduction.
Capture of FITCdextran and FITCovalbumin (OVA) by transfectants
HEK293T cell transfectants expressing lectins with N-terminal Xpress Tag were cultured in the presence of neutral FITCdextran (
-1,6-glucan) with molecular weight of 4.3 (FD-4), 40 (FD-40S), 250 (FD-250S), 500 (FD-500S) or 2000 kDa (FD-2000S) (all from Sigma-Aldrich) at 16 µg/ml for 1 h or 1.6 µg/ml for 12 h. FITCOVA (Sigma-Aldrich) containing mainly (Man)nGlcNAcGlcNAc-Asn was also added to the culture at 12.5 µg/ml for 12 h. Controls were HEK293T cells transfected with pcDNA4/HisMax. After washing with PBS containing 5 mM EDTA to detach surface bound materials, cells were analyzed with flow cytometry. For inhibition assays, transfectants were incubated with graded doses of ß-glucan (from barley: G-6513), mannan (from Saccharomyces cerevisiae: M-7504), N-acetylglucosamine (GlcNAc: A-8625), N-acetylgalactosamine (GalNAc: A-2795) or Fucose (F-2252) (all from Sigma-Aldrich) for 15 min at room temperature prior to the addition of 16 µg/ml of FITCdextran (molecular weight 2000 kDa) for 1 h at 37°C. Blocking efficacies were indicated as percent inhibition according to the formula: (% FITC+ cells in the control group without blocking agents that in experimental groups) / that in control group x 100. To examine affinity of lectin molecules for 2000 kDa FITCdextran, the transfectants were preincubated with graded doses of mannan for 20 min at room temperature prior to the addition of 16 µg/ml FITCdextran and then cultured for 1 h at 37°C.
Binding assay using mannan agarose
Binding of the lectins to mannan agarose was tested as described previously (18). Briefly, cell lysates in lysis buffer (50 mM TrisHCl pH 7.6, 150 mM NaCl, 25 mM CaCl2, 0.5% Triton X-100, 1 mM PMSF and 20 µg/ml aprotinin) were prepared from HEK293T cells transiently transfected with the pcDNA4/HisMax constructs and then incubated with mannan agarose beads (Sigma-Aldrich: M9917) for 12 h at 4°C. Then, the agarose beads were washed with binding buffer (lysis buffer including 0.05% Triton X-100) five times with or without 25 mM EDTA. Proteins bound to the agarose beads were separated on 12% SDSPAGE under denaturing conditions and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The membrane was probed with HRP-labeled anti-Xpress antibody (Invitrogen) for Langerin, mDC-SIGN and SIGNR3 or rabbit anti-SIGNR1 Ab (13) followed by HRP-anti-rabbit IgG, then visualized by LumiGLO chemiluminescent substrate (Cell Signaling Technology, Beverly, MA) according to the manufacturers protocols.
Uptake of microbes including zymosan
COS-7 transfectants with pcDNA4/HisMax constructs on cover slips were cultured with Texas Redzymosan A (four particles/cell; Molecular Probes, Eugene, OR) or Texas RedE. coli (20 particles/cell; Molecular Probes) for 12 h in CO2 incubator. Salmonella typhimurium (strain GIFU5001) obtained from Dr M. Mitsuyama (Kyoto University) was labeled with FITC after heat treatment at 60°C for 30 min as described (20), and added to the cell culture at 20 particles/cell for 6 h. Candida albicans at the yeast stage was killed at 80°C for 30 min and added to the cell culture at two particles/cell for 4 h. Bound and internalized C. albicans was visualized by staining with FITCanti-C. albicans antibody (Biogenesis, Kingston, NH). Following extensive washes, cells were fixed-permeabilized and stained with anti-Xpress mAb (mouse IgG1; Invitrogen) followed sequentially by biotindonkey F(ab')2 anti-mouse IgG antibody (Jackson Immuno Research) and FITC- or Texas Redstreptavidin to visualize transfectants expressing lectins. After mounting with 1% propyl gallate in 50% glycerol, cells were analyzed with fluorescence microscopy. In order to determine the internalization of FITCzymosan A (Molecular Probes), cells capturing zymosan were treated with 0.05% trypan blue with 13 mM potassium dihydrogen citrate in saline at pH 4.5 for a few minutes before observations by fluorescence microscopy (21). Inhibition assays were performed by preincubating COS-7 transfectants with ß-glucan, 2000 kDa dextran (both at 100 µg/ml) or mannan (at 100, 500 or 1000 µg/ml) for 15 min at 4°C, and then continuing the culture by adding Texas Redzymosan for another 3 h. The number of captured zymosan particles was enumerated under the fluorescence microscope after visualizing cells expressing lectins by staining with Abs as described above. At least 200 FITC+ transfectants were examined, and cells capturing more than two particles were taken as positive. The index was calculated as the average number of captured zymosan particles per FITC+ cell.
Zymosan-binding assay of CHO-SIGNR1 transfectants and peritoneal cells
For binding assays with transfectants at 4°C, CHO cells that stably expressed whole SIGNR1 or SIGNR1 lacking 47 N-terminal amino acids including putative adopter protein-binding sites (called #48) (13) were used, because cell membrane of the transient transfected cells was fragile for binding assay. The stable transfectants were mixed at 2 x 105/well with 8 x 105/well FITCzymosan in 100 µl of Hanks-balanced saline solution (HBSS) containing 1% FCS and 0.1% NaN3 in V-bottomed 96-well plates and centrifuged at 250 g for 5 min. After incubation for 4 h on ice, the cells were suspended and analyzed with flow cytometry. For inhibition assay, 1 mg/ml of mannan, laminarin (Sigma-Aldrich: #L-9634) or 20 mM EDTA was added and incubated for 1 h prior to the addition of FITCzymosan.
Resident peritoneal cells were obtained by injecting ice-cold PBS containing 5 mM EDTA. After washing, cells were preincubated with anti-CD16/32 mAb for 30 min for Fc-block and stained with PECD11b (clone M1/70; BD PharMingen, San Diego, CA) and biotinER-TR9 (BMA Biomedicals, Augst, Switzerland) (22) followed by streptavidinCy-Chrome (BD PharMingen) to detect SIGNR1 expression. BiotinDX5 (CD49b; BD PharMingen) was used as an isotype-matched control. For binding assays, bulk resident peritoneal cells were first blocked for Fc-binding and stained with PE-labeled anti-CD11b. Then the cells were mixed with FITCzymosan at four particles/cells and spun in V-bottomed 96-well plates as above. Inhibition assay was also performed by preincubating the cells in the presence of 1 mg/ml of mannan or laminarin, 25 mg/ml of ER-TR9 (Bachem Bioscience, King of Prussia, PA) or rat IgM (clone R4-22; BD PharMingen) on ice for 1 h before the assay.
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Results
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Internalization by antibody cross-linking
We first studied the internalization of mouse DC-SIGN, SIGNR1, SIGNR3 and Langerin molecules using HEK293T transfectants with C-terminus Tag. As shown in Fig. 1(A), nearly 90% of cells expressed lectins at comparable level. Ligation of the lectins with the antibody resulted in the reduction of the cell surface lectins, demonstrating the internalization. However, the efficiency was greatest for SIGNR3 and Langerin, which were taken up more rapidly than mDC-SIGN and SIGNR1 (Fig. 1B, left panel). When secondary Ab was added to facilitate cross-linking, more rapid and efficient internalization was observed for all lectins, with Langerin being a little more efficient than SIGNR3 (Fig. 1B, right panel).

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Fig. 1. Internalization of mouse DC-SIGN homologues and Langerin by antibody cross-linking. (A) HEK293T cells were transfected with V5 C-terminal tagged-lectins. Cell surface expression of the lectins was checked using anti-V5 tag antibody. (B) The transfectants were treated with (left panel) anti-V5 antibody or (right panel) anti-V5 antibody followed by biotindonkey F(ab')2 anti-mouse IgG antibody. Following washing, cells were incubated for the indicated time points at 37°C, and the antibodies remaining on the cell surface were visualized by staining with biotindonkey F(ab')2 anti-mouse IgG antibody and PEstreptavidin (left panel) or PEstreptavidin (right panel). Internalization is given by the percentage of MFI in experimental groups relative to control samples at 4°C. The MFIs in the control groups of (left panel) and (right panel) were 86 and 92 for SIGNR1, 62 and 55 for SIGNR3, 86 and 82 for mDC-SIGN and 46 and 43 for Langerin, respectively. (C) NIH3T3 transfectants expressing C-terminal tagged-lectins were treated with anti-V5 antibody. After 60 min incubation at 37°C, the anti-V5 antibodies were visualized with biotindonkey F(ab')2 anti-mouse IgG antibody followed by Texas Redstreptavidin. To localize the internalized antibodies, the transfectants were also stained with anti-transferrin receptor (TfR) or anti-LAMP-2.
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By deconvolution microscopy, the internalized anti-Tag mAbs in NIH3T3 transfectants were observed in numerous vesicles, of which some were double labeled with anti-transferrin receptor mAb to mark early endosomes (Fig. 1C, left panels). Consistent with the results of Fig. 1(B), relatively large amounts of SIGNR1 and mDC-SIGN remained on the cell surface (Fig. 1C), even after 1 h incubation. All of the lectins were localized in LAMP-2 negative non-lysosomal compartments (Fig. 1C, right panels), as reported for hDC-SIGN (23,24).
Different sugar recognition by the lectins
We next examined the endocytosis of FITCdextran and FITCOVA using HEK293T cell transfectants expressing the N-terminal-tagged lectins, since both dextran and OVA bind to macrophage mannose receptors via an EPN motif in one of the CRDs (16,17). Total expression levels of the lectins in transfectants were tested by intracellular staining with anti-tag antibody (Fig. 2A). As shown in Fig. 2(B), none of the transfectants showed uptake for small 4.3 kDa FITCdextran. mDC-SIGN was also unable to endocytose any of the larger FITCdextrans, even though mDC-SIGN was internalized following cross-linking with the monoclonal antibody (Fig. 1B). Likewise, mDC-SIGN failed to take up FITCOVA. In contrast, SIGNR3 and Langerin took up 40 kDa FITCdextran, the form that is often used for endocytosis assays. However, SIGNR1 endocytosed only the larger FITC dextrans, 250 kDa or more. Similar results were also obtained when the transfectants were incubated at 1/10 of concentration of 40 or 2000 kDa FITCdextran but for 12 h (data not shown). Cells transfected with control plasmid were negative for the uptake of any size of FITCdextran. FITCOVA, a glycosylated protein, was also taken up by SIGNR1, SIGNR3 and Langerin, but not by mDC-SIGN (Fig. 2B, bottom-right panel).

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Fig. 2. Comparison of internalization capacities for FITCdextran and FITCOVA as well as binding affinity for mannan. (A) Expression of SIGNR1, SIGNR3, mDC-SIGN and Langerin in transfectants were checked using anti-Xpress tag antibody. Open histogram indicates a control staining of transfectants with the parental plasmid. (B) The transfectants were incubated with various molecular weights of FITCdextran for 1 h or FITCOVA for 12 h (bottom right panel). Open histograms indicate background uptake by transfected cells with the parental plasmid. The results shown are representative of five independent experiments. (C) Inhibition of FITCdextran (molecular weight 2000 kDa) uptake by soluble carbohydrates in HEK293T cell transfectants. Cells were incubated with 1100x higher concentrations of soluble ß-glucan, mannan, GalNAc and GlcNAc for 15 min before incubation with 16 µg/ml FITCdextran for 1 h. Inhibition is indicated as the percent decrease of FITC+ cells in experimental groups compared to respective controls without blocking sugars. FITC+ cells in control groups were 72% for SIGNR1, 74% for SIGNR3 and 32% for Langerin. A representative result from four independent experiments is shown. (D) Titration of the inhibitory effect by mannan. The FITCdextran uptake was inhibited by graded amounts of mannan. The relative amount shown as 1 indicates 16 µg/ml mannan. (E) Mannan binding properties of the lectins. Cell lysates of the HEK293T transfectants were incubated with mannanagarose. Bound protein was detected by western blotting using anti-Xpress or anti-SIGNR1 antibody with/without EDTA.
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The effects of sugar inhibitors on FITCdextran uptake were also investigated (Fig. 2C). Mannan completely blocked FITCdextran uptake via any the lectins tested, even at the same concentration as FITCdextran. In contrast, excess amounts of ß-glucan and GalNAc were unable to inhibit endocytosis of FITCdextran. A large dose (
1.6 mg/ml) of fucose substantially reduced their FITCdextran uptake, while GlcNAc was only inhibitory for SIGNR1 (57.8 ± 1.7%) and Langerin (43.6 ± 2.6%), but not for SIGNR3. These results for Langerin were comparable to competition data using radio-labeled mannose-bound BSA by Stambach et al. (25). In addition, the monosaccharide specificity for SIGNR1 and Langerin was similar to those for other C-type lectins, such as mannose receptor, serum mannose-binding protein and hDC-SIGN (2628). Thus, these results indicate that SIGNR3 has distinct specificity from SIGNR1 and Langerin. The oligosaccharide sequences recognized by these three lectins have been investigated in detail (C. Galustian, C. G. Park, W. Chai, M. Kiso, S. A. Bruening, Y. S. Kang, R. M. Steinman and T. Feizi, submitted for publication).
More precise analysis was performed for the inhibition by mannan of FITCdextran uptake via SIGNR1, SIGNR3 and Langerin (Fig. 2D). FITCdextran was most efficiently blocked by mannan in the case of Langerin, followed by SIGNR1 and SIGNR3. To extend these data, lectin binding assays for mannanagarose were carried out using cell lysates of the transfectants. We found that Langerin and SIGNR1, but not SIGNR3, bound mannan in a Ca2+-dependent manner (Fig. 2E). Although SIGNR1 appeared to capture mannan less efficiently than Langerin, this result was not conclusive since the amounts of lectins were not adjusted.
SIGNR1, SIGNR3 and Langerin capture zymosan via its mannan moiety
Mannan is the major constituent of zymosan and is recognized by mannose-binding receptors (29,30). Thus the recognition and uptake of FITCzymosan was studied. For this experiment, we used COS-7 cells because their large size permits easier observation of zymosan binding/internalization under the microscope. Similar to the data with 2000 kDa FITCdextran and FITCOVA, transfectants of SIGNR1, SIGNR3 and Langerin were capable of capturing a large number of zymosan particles (Fig. 3A) and heat-killed C. albicans (Fig. 3B). For DC-SIGN, only a small number of the transfectants, if any, could bind a few yeast particles (data not shown). The internalization function of SIGNR1 and SIGNR3 was also confirmed using trypan blue quenching method for FITCzymosan (Fig. 3C). This treatment reduced the percentages of FITC+ zymosan-bearing SIGNR1- and SIGNR3-transfected cells from 7585% to 1530%. Control transfectants with the parental plasmid were negative for capture of zymosan. Therefore, SIGNR1 and SIGNR3 can internalize zymosan, although uptake was moderate in our transfectants.

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Fig. 3. Capture of yeast and yeast-like particles zymosan. COS-7 transfectants of N-terminal tagged-SIGNR1, SIGNR3, DC-SIGN and Langerin were incubated with Texas Redzymosan (A) for 12 h and C. albicans (B) for 4 h. Lectins expressed by the transfectants were visualized with anti-Xpress Tag antibody. Internalization of FITCzymosan was confirmed by quenching fluorescence using treatment with trypan blue. (C) Intracellular zymosan particles stayed brightly positive for FITC, whereas extracellular ones largely lost fluorescence by trypan blue (arrows). (D) COS-7 transfectants of SIGNR1, SIGNR3 and Langerin were preincubated with ß-glucan, dextran or mannan before adding zymosan particles. After staining with anti-Xpress mouse IgG antibody followed by biotindonkey F(ab')2 anti-mouse IgG antibody + FITCstreptavidin, cells capturing more than two zymosan particles were regarded as positive cells, and only the numbers of particles in positive cells were counted. Index denotes the number of particles per cell in more than 200 transfectants. Left and middle panels show results with the inhibitors at 100 µg/ml. Right panel indicates that a higher amount of mannan is required for inhibition in the case of SIGNR1 transfectants. Result shown is representative of three separate experiments.
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The blocking of zymosan uptake was also tested using mannan and ß-glucan as inhibitors, since a large part of the yeast cell wall is composed of mannan, ß-glucan and chitin (29). Although mannan at 100 µg/ml completely abrogated FITCdextran uptake via SIGNR1 (Fig. 2C), an only slight inhibition of zymosan uptake was detected at the same dose of mannan (Fig. 3D, left and middle panels). However, a large excess of mannan (500 and 1000 µg/ml) significantly reduced zymosan capture by SIGNR1 (Fig. 3D, right panel). In contrast to SIGNR1, mannan was still a potent inhibitor for zymosan uptake by SIGNR3 and Langerin. ß-glucan had no effect on SIGNR1 and SIGNR3, but substantially inhibited the activity of Langerin. On the other hand, dextran significantly inhibited SIGNR3- and Langerin-mediated capture of zymosan.
Capture of Gram-negative bacterial by SIGNR1, but not the other lectins tested
In striking contrast to the data with FITCzymosan, Gram-negative bacteria, such as E. coli and S. typhimurium, were captured, but only by SIGNR1 transfectants, and not by the other C-type lectin transfectants (Fig. 4A and B). On the other hand, the Gram-positive bacterium Staphylococcus aureus was not captured by any type of transfectants examined here (data not shown).

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Fig. 4. Capture of Gram-negative bacteria. COS-7 transfectants were incubated with Texas RedE. coli (A) for 12 h or FTICS. typhimurium (B) for 6 h. Lectins expressed by the transfectants were visualized as in Fig. 3.
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Zymosan capture by SIGNR1-tranfectant is mediated via the recognition for mannan moiety
Because of the broad specificity of SIGNR1 for microbes, we attempted to detect direct binding of zymosan by SIGNR1 transfectants at 4°C. For this purpose, CHO cells stably expressing intact or truncated SIGNR1 (#48) were utilized. As shown in Fig. 5(A), both transfectants effectively and comparably bound FITCzymosan (7278%), and the treatment with mannan and EDTA, but not laminarin, resulted in a complete block of zymosan binding to control levels (
6%).

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Fig. 5. Direct binding of stably SIGNR1-transfected CHO cells and of resident peritoneal cells expressing SIGNR1. (A) SIGNR1 itself bound FITCzymosan in a mannan and EDTA-sensitive manner. CHO-SIGNR1 and CHO-#48 cells lacking the cytoplasmic tail of SIGNR1 were incubated with FITCzymosan on ice with/without inhibitors described. Reduction of FITCzymosan capture by inhibitors is depicted in the right panel. (B) Expression of SIGNR1 on resident peritoneal cells was analyzed by flow cytometry. A majority of CD11b+ cells were stained with ER-TR9, anti-SIGNR1 mAb (left and center panels). Open histogram represents control staining with DX-5 antibody. The CD11b+ cells were capable of binding FITCzymosan at 4°C (right panel). (C) Cooperation in zymosan binding by SIGNR1 and laminarin-sensitive receptor. Peritoneal cells were pretreated with inhibitors represented for 1 h. Binding (%) indicates the percentage of FITC+ cells in CD11b+ cells.
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Expression of SIGNR1 and zymosan binding by a subpopulation of resident peritoneal cells
As reported previously, SIGNR1 protein is expressed by macrophages in spleen and lymph nodes (13). SIGNR1+ marginal zone macrophages play a role in capturing Streptococcus pneumoniae (15). Although we are able to obtain such SIGNR1+ macrophages from spleen, it is difficult to prepare a large number of SIGNR1+ macrophages using conventional methods. We therefore sought another source of SIGNR1+ cells using monoclonal antibody ER-TR9 (22), and found that 75% or more of CD11b+ cells in the resident peritoneal cavity express SIGNR1 (Fig. 5B, left and center panels). This made it possible to examine a role of SIGNR1 zymosan recognition by direct binding assays at 4°C. About 45% of CD11b+ peritoneal cells were able to bind FITCzymosan (Fig. 5B, right panel), and this binding was significantly inhibited by SIGNR1-specific ER-TR9 mAb (Fig. 5C). An excess of mannan was effective, although both ER-TR9 and mannan did not completely inhibit, as in Fig. 5(A). Since macrophages are known to express other C-type lectins with different carbohydrate specificity, such as Dectin-1 recognizing ß-glucan on zymosan, laminarin, a known ligand for Dectin-1 was used as an inhibitor (31). Laminarin itself blocked zymosan binding to SIGNR1+ peritoneal cells, and the blocking was augmented by co-addition of mannan (Fig. 5C). Similar results were also obtained using heat-killed C. albicans (data not shown). Therefore, it is likely that SIGNR1 and a laminarin-sensitive receptor, possibly Dectin-1, on a population of peritoneal cells cooperate to bind zymosan by recognizing different carbohydrate moieties.
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Discussion
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In the present study, we demonstrate the distinctive uptake of polymerized dextran, glycosylated protein and microbes, as well as the binding capacity to mannan and zymosan, by C-type lectins in the mouse DC-SIGN family (DC-SIGN, SIGNR1 and SIGNR 3) and Langerin, of which all share an EPN-motif in their CRD. Results are summarized in Table 1.
For the presentation of exogenous antigens, it is necessary to internalize and deliver them to LAMP-2-positive late endosome/lysosome compartments, where processed antigens are loaded onto MHC class II molecules. These four lectins on the cell surface of transfectants could each be internalized by cross-linking with specific antibody in HEK293 T cell and NIH3T3 transfectants. However, the internalized antibodies were detected in cytoplasmic compartments lacking LAMP-2 in NIH3T3 transfectants. As for SIGNR1, Geijtenbeek et al. (14) recently suggested a rapid sorting of FITCdextran captured by SIGNR1 into lysosome-like compartments using an erythroleukemic cell line K562, although they did not double label with a lysosomal marker. In contrast, we find that internalized FITCdextran is delivered to compartments lacking LAMP-1/CD107a, pan-lysosomal maker, using stably transfected RAW264.7 SIGN-R1 transfectants (13). These discrepancies may depend on cell types tested. In human DCs, however, Langerin is also reported to deliver bound mAb to non-lysosomal compartments (32), while DC-SIGN sorts captured mycobacteria and specific mAb into lysosomal compartments (9). Therefore, it is also possible that adaptor proteins for each lectin are involved in internalization and intracellular sorting.
Our results show that the efficacy of uptake of bound mAb varies amongst the lectins. The results of Fig. 1(B) are consistent with those of Fig. 1(C) where DC-SIGN and SIGNR1 are less efficient for internalization than SIGNR3 and Langerin. These distinctions may reflect differences in the cytoplasmic tails. It has been shown that mDC-SIGN and SIGNR1 have a single dileucine motif (DEELL and DDDELM, respectively), which binds adaptor proteins for the clathrin-coated pit, while SIGNR3 has both a dileucine motif and tyrosine internalization motif (DEECLM and YSDI) (12). Langerin has a distinct type of proline-rich sequence (PREPPP) that may bind a signal protein containing a src homology 3 (SH3) domain (33), leading to a rapid internalization by cross-linking with anti-Langerin mAb (32). Examination of internalization efficiency of these lectins using transfectants of other cell types remains to be conducted. Regarding uptake of polymerized FITCdextran, mDC-SIGN was found to be inactive even for high molecular weight forms (although mDC-SIGN was capable of internalizing a ligated mAb as mentioned). Furthermore, none of the microbes tested were captured by mDC-SIGN. In contrast, SIGNR3 and Langerin were capable of efficiently taking up FITCdextran even with a small molecular weight. SIGNR1, however, could only take up highly polymerized dextran. Moreover, uptake of high molecular weight FITCdextran was blocked by mannan with an efficacy order of Langerin > SIGNR1 > SIGNR3, and direct binding to mannanagarose was only detected with Langerin and SIGNR1, but not DC-SIGN and SIGNR3, in a Ca2+-dependent manner. These observations and the results showing distinctive blocking efficiency with free monosaccharides for FITCdextran uptake, indicate that the lectins tested here have characteristic sugar recognition abilities, even though they share a common EPN motif in their single CRD domains.
SIGNR1, SIGNR3 as well as Langerin could also capture zymosan and C. albicans. However, capture via Langerin was much more efficiently inhibited not only by mannan but also by ß-glucan and polymerized dextran, than that via SIGNR1 and SIGNR3. Furthermore, Langerin-mediated capture was the lowest among these three lectins, in terms of the number of zymosan particles captured per transfectant. On the other hand, the capture via SIGNR1 was not much affected by mannan, even though CRDs of SIGNR1 and SIGNR3 are homologous; 69.9% in amino acid sequence. This might be ascribed to the difference in their avidity for mannan, but this possibility is unlikely, since mannan was more potent in blocking the uptake of FITCdextran by SIGNR1 than that by SIGNR3. Another possibility is that SIGNR1 and SIGNR3 recognize different target moieties on zymosan particles, since dextran was more effective in inhibiting the capture of zymosan by SIGNR3. Alternatively, SIGNR1 might recognize a particular spatial arrangement or pattern of target sugars on the zymosan particles. It has been reported that the binding specificities of hDC-SIGN and L-SIGN might be generated by the spatial arrangement of target sugars in high mannose oligosaccharides (27,34). We also demonstrated that SIGNR1 molecules primarily exist in aggregated form in situ (13). Furthermore, the potency to bind zymosan through the recognition of mannan was also shown in SIGNR1-transfected CHO cells. This binding was dependent on Ca2+, but did not require the cytoplasmic tail. Therefore, SIGNR1 may have a higher avidity to multivalent/repetitive target sugars spaced on zymosan.
Interestingly, SIGNR1 was able to capture Gram-negative bacteria, such as E. coli and S. typhimurium, but not Gram-positive bacteria; S. aureus in the present study. However, we previously reported that capsular polysaccharide of Streptococcus pneumoniae, Gram-positive bacteria, can be recognized by SIGNR1 (15). This discrepancy has yet to be solved, but may be ascribed to the loss or damage of capsule of fluorescence-labeled S. aureus available on the market.
At present, we cannot exclude the possibility that polysaccharide and microbial uptake could be somehow influenced by intracellular tag. However, SIGNR1, SIGNR3 and Langerin with intracellular tag are competent for uptake of dextran and OVA, and their endocytic capacity is dependent on ligand and its structure. Therefore, we suspect the effect of the expression tag on endocytic activity.
Recently, Dectin-1, a ß-glucan receptor, has been reported to collaborate with Toll-like receptor (TLR) 2 to recognize microbes for inflammatory response (35,36). Therefore, it will be of interest to elucidate the involvement of other pattern recognition receptors, such as TLRs, for the recognition of Gram-negative bacteria in collaboration with SIGNR1. In addition, results showing cooperative recognition of yeast by SIGNR1 and laminarin-sensitive receptor (possibly Dectin-1) on peritoneal cells may lead us to anticipate more complicated regulation in pattern recognition systems in situ. During revision of this manuscript, similar results demonstrating participation of both SIGNR1 and Dectin-1 for recognizing zymosan were reported by Taylor et al. (37). The different properties of mouse DC-SIGN, SIGNR1, SIGNR3 and Langerin indicate that distinct microbial components are recognized by lectins expressed by cells of the innate immune system.
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Acknowledgements
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This work was supported in part by Grant-in-Aid for Scientific Research (14370075 and 15590336), a Grant-in-Aid for Scientific Research on Priority Area (13140202) from the Ministry of Education, Science, Sports and Culture of Japan and by grant AI13013 and AI40045 from the NIH to R.M.S. K.I. is also supported by The Naito Foundation and Uehara Memorial Foundation, and Y.O. and Y.K. are supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology.
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Abbreviations
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CRDcarbohydrate recognition domain
DCdendritic cell
DC-SIGNDC-specific ICAM-3 grabbing non-integrin
LAMPlysosomal-associated membrane protein
SIGNRSIGN-related gene
TLRToll-like receptor
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