Anda di halaman 1dari 30

Mol Immunol. 2010 March; 47(6): 12161225. PMCID: PMC2837148 doi: 10.1016/j.molimm.2009.12.016 Copyright 2010 Elsevier Ltd.

. C-type lectin Langerin is a -glucan receptor on human Langerhans cells that recognizes opportunistic and pathogenic fungi Marein A.W.P. de Jong,ab Lianne E.M. Vriend,b Bart Theelen,c Maureen E. Taylor,d Donna Fluitsma,b Teun Boekhout,c and Teunis B.H. Geijtenbeeka a Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands b Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands c CBS Fungal Diversity Center, Utrecht, The Netherlands d Division of Molecular Biosciences, Department of Life Sciences, Imperial College, London, UK Teunis B.H. Geijtenbeek: Corresponding author. Tel.: +31 205666309. Email: Received December 4, 2009; Revised December 22, 2009; Accepted December 23, 2009. This document may be redistributed and reused, subject to certain conditions. This document was posted here by permission of the publisher. At the time of the deposit, it included all changes made during peer review, copy editing, and publishing. The U. S. National Library of Medicine is responsible for all links within the document and for incorporating any publisher-supplied amendments or retractions issued subsequently. The published journal article, guaranteed to be such by Elsevier, is available for free, on ScienceDirect, at: This article has been cited by other articles in PMC.

Other Sections

Abstract Langerhans cells (LCs) lining the stratified epithelia and mucosal tissues are the first antigen presenting cells to encounter invading pathogens, such as viruses, bacteria and fungi. Fungal infections form a health threat especially in immuno-compromised individuals. LCs express C-type lectin Langerin that has specificity for mannose, fucose and GlcNAc structures. Little is known about the role of human Langerin in fungal infections. Our data show that Langerin interacts with both mannan and -glucan structures, common cell-wall carbohydrate structures of fungi. We have screened a large panel of fungi for recognition by human Langerin and, strikingly, we observed strong binding of Langerin to a variety of Candida and Saccharomyces species and Malassezia furfur, but very weak binding was observed to Cryptococcus gattii and Cryptococcus neoformans. Notably, Langerin is the primary fungal receptor on LCs, since the interaction of LCs with the different fungi was blocked by antibodies against Langerin. Langerin recognizes both mannose and -glucans present on fungal cell walls and our data demonstrate that Langerin is the major fungal pathogen receptor on human LCs that recognizes pathogenic and commensal fungi. Together these data may provide more insight in the role of LCs in fungal infections. Keywords: -Glucan, C-type lectin, Fungi, Langerhans cells, Langerin, Yeast

Other Sections



Langerin (CD207) is a C-type lectin expressed in human exclusively by Langerhans cells (LCs), which constitute a subset of dendritic cells that are located in epithelium of mucosal tissues and epidermis (Fithian et al., 1981; Patterson et al., 2002). LCs play a key role in the induction of immune responses against invading pathogens by capturing and processing foreign antigens and migrating to draining lymph nodes to present processed antigens to T cells (Banchereau and Steinman, 1998). As a C-type lectin, Langerin is thought to play a role in pathogen recognition by facilitating pathogen uptake and processing for antigen presentation (Hunger et al., 2004). Langerin is a type II transmembrane protein that contains one calcium-dependent carbohydrate recognition domain with a short cytoplasmic tail with a proline rich motif (Valladeau et al., 2003). Langerin forms a trimer on the cell surface and upon crosslinking with either a cell-bound or a soluble ligand, Langerin induces the formation of Birbeck granules, which are LC-specific intracellular organelles that appear as tennis-racket like structures with a size of <1 m in diameter that are thought to be part of the endosomal recycling pathway (Valladeau et al., 2000). Although Langerin is characterized as a pathogen recognition receptor, only few pathogens have been demonstrated to interact with Langerin. Both HIV-1 (de Witte et al., 2007; Turville et al., 2001) and Mycobacteria leprae (Hunger et al., 2004) have been identified as pathogens that interact with human Langerin, whereas murine Langerin was shown to bind to Candida albicans (Takahara et al., 2002, 2004). Langerin is important in protecting against HIV-1 transmission, since HIV-1 captured by Langerin is rapidly internalized into Birbeck granules for degradation, thereby preventing HIV-1 infection of LCs (de Witte et al., 2007; Turville et al., 2001). Based on the carbohydrate recognition specificity for mannose, fucose and Nacetyl-glucosamine monosaccharides (GlcNAc) (Stambach and Taylor, 2003), it is likely that Langerin has a broader specificity for pathogens than has been identified so far. Fungal species are ubiquitous residents of human skin and many can cause invasive infections especially in immuno-compromised individuals. They can be acquired from the environment, such as Cryptococcus neoformans, or are part of the normal skin microbiota, such as Candida albicans (Richardson, 2005) or Malassezia species (Batra et al., 2005; Gupta et al., 2004). The close contact of the fungi with the epidermis and epithelial barrier suggests that LCs might be involved in fungal defense. Therefore we have investigated the interaction of a panel of fungi with Langerin on LCs. Fungi, such as Candida species ubiquitously express -glucans and mannosylated carbohydrate structures in their cell wall (Chaffin et al., 1998; Gantner et al., 2005). Here we demonstrate that Langerin not only recognizes mannose, fucose and GlcNAc structures, but also -glucan structures. To our knowledge, thus far dectin-1 is the only C-type lectin in human that has been associated with -glucan binding (Brown et al., 2003; Brown and Gordon, 2001). Here, we demonstrate that aside from dectin-1, Langerin also interacts with -glucans, which can possibly mediate recognition of fungi and bacteria to initiate an antimicrobial response. In addition, we demonstrate that Langerin is a receptor for Candida species, Saccharomyces species and Malassezia furfur, but only weakly interacts with Cryptococcus gattii and Cryptococcus neoformans. Our data further show that Langerin is the main C-type lectin receptor for fungi on primary human LCs even though these also express dectin-1. Thus, we have demonstrated that Langerin on LCs is a pathogen recognition receptor for -glucans and a broad range of fungi. Increased knowledge of the pathogen recognition profile by Langerin on LCs can contribute to the understanding of the pathogenicity of fungi.

Other Sections



2.1. Langerin recognizes Candida, Malassezia and Saccharomyces species A large panel of pathogenic fungi was screened for binding to recombinant Langerin in an ELISA system (Table 1). This recombinant Langerin contains the whole extracellular region including the carbohydrate recognition domain and the coiledcoil stalk that allows oligomerization (Stambach and Taylor, 2003). To determine the specificity of the binding, polysaccharide mannan was used to inhibit the C-type lectin domain. Langerin interacted strongly with all Saccharomyces species, Malassezia furfur and Candida species, including C. albicans, C. dubliensis, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, C. tropicalis, C. lusitaniae, C. nivariensis, C. orthopsilosis and C. metapsilosis (Fig. 1A and B). In contrast, no or low binding was observed for Cr. neoformans varieties and Cr. gattii of different serotypes (Fig. 1B). The inclusion of an acapsular Cr. neoformans (CBS 7935) demonstrated that the lack of binding is not due to the capsule. Table 1 Yeast strains and CBS numbers.

Fig. 1 Langerin recognizes Candida species, M. furfur and Saccharomyces species. (A and B) Yeast strains were coated onto ELISA plates (107/ml) and binding to recombinant Langerin was determined. Polysaccharide mannan was used to determine the specificity of (more ...) To investigate the interaction between these fungi and Langerin in depth, a selection was made and a titration was performed. To determine the specificity of the Langerin binding ELISA, we investigated the ability of mannan and anti-Langerin to block the interaction of Langerin with coated ligands. Both inhibitors prevented Langerin binding to different concentrations of mannan and C. albicans, although mannan is slightly more efficient in blocking Langerin compared to anti-Langerin (Fig. 1C and D). In addition, both life and heatkilled C. albicans were compared and similar binding patterns were observed, demonstrating that heat-treatment did not alter the binding to Langerin (Fig. 1E). Langerin efficiently interacted with all Candida species tested, namely C. albicans, C. glabrata, C. guilliermondii, C. krusei, C. lusitaniae, and C. nivariensis, and Saccharomyces cerevisiae at a concentration of 106 and 107/ml, whereas most binding was lost at 105/ml or lower (Figs. 1D and 2A, B and E). No difference was observed between virulent and nonvirulent strains of S. cerevisiae (Fig. 2E). Cryptococcus species only weakly interacted with Langerin at high concentrations which was abrogated after dilution (Fig. 2C and D). These data demonstrate that Langerin is a fungal receptor that interacts with Malassezia furfur, Saccharomyces and Candida species but not with Cryptococcus species. Fig. 2 Titration of a selection of fungi. Yeast strains from Candida species (A and B), Cryptococcus neoformans (C), Cryptococcus gattii (D), and Saccharomyces cerevisiae (E) were coated onto ELISA plates at different concentrations. Mannan was used to determine (more ...)

2.2. Langerin binds to -glucans Langerin has been shown to interact strongly with mannose, fucose and GlcNAc structures (Fig. 3A) (Stambach and Taylor, 2003). In addition, Langerin interacts with both Lewis B and Lewis Y through terminal fucose structures, whereas it does not interact with internal fucose present in Lewis A and Lewis X (de Jong et al., 2009). In general, fungal cell walls contain a large amount of mannose structures, chitin, as well as (13) and (16)-glucans at the inner-layer and budding scars (Chaffin et al., 1998). Mannan is a major component of the mannose structures on the cell wall and also a well-known ligand for Langerin (Stambach and Taylor, 2003; Chaffin et al., 1998). Therefore we investigated whether Langerin also interacts with other fungal-derived structures. Strikingly, Langerin strongly interacted with curdlan, laminarin and zymosan (Fig. 3B) and this binding was inhibited by both mannan and laminarin (3CF). Both curdlan and laminarin contain large amount of -glucans, while zymosan is a ghost cell derivative of S. cerevisiae, containing both mannose and -glucans. It has previously been demonstrated that -glucans partially inhibit zymosan binding to mouse Langerin, which suggests a role for Langerin in -glucan binding (Takahara et al., 2004). Here, we demonstrate that Langerin directly interacts with -glucans. These data demonstrate that Langerin recognizes also -glucans, which could facilitate recognition of fungi. Fig. 3 Langerin is a receptor for -glucans. (A) Langerin specifically interacts with mannose, fucose and GlcNAc structures. Carbohydrate binding to Langerin was determined by recombinant Langerin ELISA. Specificity was determined by mannan, anti-Langerin (more ...) 2.3. Cellular Langerin interacts with Candida species and Zymosan but not with Cryptococcus species Next, we investigated whether a selection of this panel also interacted with cellular Langerin. Fluorescently labelled C. albicans, C. krusei, Cr. neoformans, Cr. gattii, and zymosan were incubated with a Langerin transduced cell-line, or a mock transduced cell-line. We selected the Jurkat T cell cell-line since it does not express dectin-1, which has been identified as a primary receptor for -glucans (Fig. 4A). C. albicans, C. krusei and zymosan strongly interacted with Langerin transduced cells, which was completely inhibited by mannan, whereas no binding was observed to mock-transduced cells (Fig. 4B). In concordance with the Langerin binding ELISA, Cr. neoformans and Cr. gattii did neither interact with the Langerin transduced cells, nor with the mock-transduced cells. These data strongly suggest that Langerin recognizes Candida species but not Cryptococcus species. Fig. 4 Candida species and zymosan interact with cellular Langerin. (A) Langerin and dectin-1 expression on Jurkat cell-line transduced with Langerin. Thin line represents isotype control, filled histogram represents specific staining. (B) Langerin expressing (more ...)

2.4. Langerin is the primary receptor for fungi on primary Langerhans cells Opportunistic fungal infections often enter through the skin or mucosal tissues, where LCs are the predominant antigen presenting cells. Therefore, we investigated whether primary human LCs interact with different fungi. CD1a+ LCs isolated from human epidermis express high levels of Langerin, low levels of dectin-1 and no DC-SIGN (Fig. 5A). Both DC-SIGN

and dectin-1 have been shown to bind fungi through mannan and -glucan structures, respectively (Cambi et al., 2008; Brown et al., 2003). Primary LCs were incubated with fluorescently labelled C. albicans, C. krusei, Cr. neoformans, Cr. gattii, and zymosan and binding was measured by flow cytometry. Strikingly, LCs strongly interacted with C. albicans, C. krusei, and zymosan, but not with Cr. neoformans or Cr. gattii (Fig. 5B). The binding to C. albicans, C. krusei, and zymosan was primarily mediated via Langerin, since both mannan and specific antibodies against Langerin inhibited the binding to LCs (Fig. 5B). The blocking antibody against dectin-1 only weakly interfered with fungi binding to LCs, strongly suggesting that the contribution of dectin-1 on LCs is minor. Cr. neoformans and Cr. gattii failed to interact with LCs even at very high concentrations (data not shown). We observed a background binding of approximately 10%, which could be due to other receptors, such as complement receptor 3 expressed in low levels by LCs (Depanfilis et al., 1990; Netea et al., 2008). Fig. 5 Langerin is the primary receptor for fungi on LCs. (A) CD1a, Langerin, dectin-1 and DC-SIGN expression on primary immature LCs. Thin line represents isotype control, filled histogram represents specific staining. (B) Binding of different fungi to human (more ...)

Next, we have used electron microscopy to investigate the uptake of C. albicans by LCs. LCs are able to phagocytose C. albicans (Fig. 6A), but we also observed C. albicans at the cell surface (Fig. 6). Notably, Langerin was regularly localized at the interface of the cell surface and C. albicans, either when phagocytosed or cell-bound (Fig. 6BD). Irregular and detached cell wall was due to the EM preparation procedure. Thus, on LCs Langerin is the primary receptor for fungi such as C. albicans and the interaction can lead to phagocytosis of the fungi particles. Fig. 6 Electron microscopic analysis of Candida albicans interaction with LCs. (A) Electron microscopic picture of LCs which has internalized C. albicans (C). Arrow is directed towards C. albicans. (BD) Gold labelling of Langerin demonstrates Langerin (more ...)

Other Sections

Fig. 6 Electron microscopic analysis of Candida albicans interaction with LCs. (A) Electron microscopic picture of LCs which has internalized C. albicans (C). Arrow is directed towards C. albicans. (BD) Gold labelling of Langerin demonstrates Langerin localization at the binding site of LCs and C. albicans. Arrows are directed towards Langerin gold labelling. Picture (B) is a magnification of (A), and (D) is a magnification of (C).

3. Discussion Opportunistic fungal infections are a well-known problem, especially in immunocompromised patients, such as HIV-1 infected people and transplantation patients. With an increase in the survival of these patients, there is also an increasingly large number of patients that suffer from opportunistic fungal infections (McNeil et al., 2001). Candida species, especially C. albicans, and Cryptococcus species are the most common causes of invasive fungal infections in immuno-compromised patients (Pappas et al., 2001, 2003). These fungi can colonize the skin and mucosal tissues that contain LCs and this dendritic cell subset might be crucial in the defense against fungi. Our data show that Langerin is the major receptor on primary LCs for Candida species, Saccharomyces species, and Malassezia furfur. Notably, we did not observe any interaction of LCs with Cryptococcus species, suggesting that this pathogenic fungus is evading immune recognition by LCs. Although C. albicans is still the leading cause of candidiasis, infections with variants such as C. parapsilosis and C. glabrata are increasingly common (Almirante et al., 2005; Arendrup

et al., 2008). The cell wall of Candida species consists of mannose structures (mannan), chitins (GlcNAc structures) and -1,3- and -1,6-glucose polymers (-glucans). The mannan components are present on the outer cell layer, whereas chitins and -glucans are the structural components of the wall and exposed at budding scars (Karkowska-Kuleta et al., 2009). The C-type lectins dectin-1 and DC-SIGN are known fungal receptors that recognize fungi through -glucan and mannan structures, respectively (Cambi et al., 2008). Here we demonstrate that Langerin is also a fungal receptor that interacts with a large variety of Candida species including C. albicans, C. parapsilosis, C. glabrata, C. krusei, C. nivariensis, C. lusitaniae and more. This suggests that Candida species express conserved structures that interact with Langerin. Strikingly, we observed that Langerin does not only recognizes mannose structures on fungi but also 1,3-glucan structures. Currently, dectin-1 is the main C-type lectin that specifically interacts with -glucans and interaction of dectin-1 on macrophages and dendritic cells with C. albicans can modulate immune responses (Brown, 2006). Here, we demonstrate that Langerin also has affinity for -glucans and that on human primary LCs, Langerin accounts for the majority of the interaction of Candida species. Together these data show that Langerin shares carbohydrate specificities with both DC-SIGN and dectin-1 (van Kooyk and Geijtenbeek, 2003; Brown, 2006). The signalling pathways for dectin-1 and DC-SIGN have been largely identified and dectin-1 plays a central role in the defense against fungal infections (Brown, 2006; Geijtenbeek and Gringhuis, 2009). Since dectin-1 appears to play a minor role in the binding of C. albicans to LCs, and Langerin interaction is more prominent, it is of great interest to investigate the immunological consequences of Langerin binding to fungi. Using electron microscopy we observed co-localization of Langerin with C. albicans. We have previously demonstrated that interaction of viruses such as HIV-1 and Langerin can lead to internalization in Langerin positive Birbeck granules (de Witte et al., 2007). Due to the large size of C. albicans it seems unlikely that C. albicans can be internalized efficiently into Birbeck granules. However, our data demonstrate that LCs phagocytose fungi albeit not very efficiently. Questions remain whether Langerin is able to capture and degrade fungal pathogens in a similar manner as it is capable of degrading HIV-1. Noteworthy, we did observe that C. albicans but not Cryptococcus species could prevent the binding of HIV-1 to Langerin (data not shown), suggesting this could interfere with the protective barrier provides by Langerin on LCs and facilitate HIV-1 infection of LCs (de Witte et al., 2007). However, this remains to be further determined. S. cerevisiae is one of the most well-studied yeast and shows high similarity to C. albicans cell-wall architecture (Karkowska-Kuleta et al., 2009). Indeed, similar to C. albicans, S. cerevisiae strongly interacted with Langerin. In addition, we observed comparable binding the non-virulent genome-sequenced strain of S. cerevisiae and a virulent strain. These data suggest that Langerin binding does not account for the differences in virulence. Malassezia species are present as normal skin flora, however they are also associated with several skin diseases such as seborrehic dermatisis, atopic dermatitis, pityriasis versicolor and Malassezia folliculitis (Batra et al., 2005; Gupta et al., 2004). Most Malassezia species are lipid dependent (Xu et al., 2007), while one species, M. pachydermatis, is lipophilic and their cell wall is relatively thick (Mittag, 1995). Recently, it was demonstrated that Malassezia species have low mannose content in the cell wall, but strikingly Malassezia species reacted with anti--1,3-glucan antibody (Shibata et al., 2009). Here, we demonstrate that Langerin interacts with M. furfur and this suggests that the interaction is mediated via -glucan structures. M. furfur and M. pachydermatis recently been identified as an activating ligand for C-type lectin Mincle (Yamasaki et al., 2009). Notably, we did not observe binding of Langerin to M. pachydermatis (data not shown). No difference in galactomannan spectra was observed by Shibata et al. (2009), however M. pachydermatis is a single non-lipid dependent

species, which might account for the difference in binding to Langerin because of differences in cell-wall composition. Cryptococcus species Cr. neoformans, which has a predilection for the central nervous system and is highly prevalent in HIV-1 infected patients, and Cr. gattii, which can cause infections in immune-competent patients, did not interact with Langerin. Although it has been described that the capsule of Cr. neoformans determines its virulence and allows immune escape by circumventing recognition by antigen presenting cells (Vecchiarelli et al., 2003; Kozubowski et al., 2009), binding by Langerin was not rescued using an acapsular variant. These data suggest that underneath the capsule, no carbohydrate structures are present that are recognized by Langerin. It remains to be determined whether acapsular Cr. neoformans can elicit proper immune response in LCs. In this screening of opportunistic and pathogenic fungi interaction with Langerin and LCs we demonstrate that Langerin is a receptor for Candida species, Saccharomyces species and Malassezia furfur and we have shown that Langerin is a -glucan receptor. This knowledge strongly suggests that Langerin is an important fungal receptor on LCs. It remains to be determined whether the binding is mediated via mannose structures or -glucans present on fungi. Moreover, future studies are necessary to further examine the immunological consequences of these fungal interactions with Langerin on LCs and how this can contribute to the pathogenicity of fungal infections.

Other Sections

4. Materials and methods 4.1. Antibodies and reagents The following antibodies and reagents were used: CD1a-FITC, Langerin-PE, DCGM4 (antiLangerin) (all Immunotech), goat anti-mouse-FITC (Zymed Laboratories Inc.), isotype control IgG1 and IgG2b, anti dectin-1 (259931), and polyclonal anti-Langerin (all R&D systems), AZN-D1 (anti-DC-SIGN) (Geijtenbeek et al., 2000), 10E2 (anti-Langerin) (de Witte et al., 2007). Mannan from S. cerevisiae, curdlan from Alcaligenes faecalis, laminarin from Laminaria digitata, zymosan from S. cerevisiae (all SigmaAldrich), and zymosanFITC (Molecular probes). Biotinylated polyacrylamide (PAA)-coupled glycoconjucates Mannose, Fucose, GlcNAc (N-acetyl-glucoseamine), GalNAc (N-acetyl-galactoseamine), Lewis (Le)X (Gal1-4(Fuc1-3)GlcNAc), LeY (Fuc1-2Gal1-4(Fuc1-3)GlcNAc), LeA (Gal1-4(Fuc1-3)GlcNAc), LeB (Fuc1-2Gal1-4(Fuc1-3)GlcNAc) were obtained from Lectinity. The following buffers were used: TSM buffer: Tris buffer (20 mM TrisHCl, pH 7, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2) (TSM), TSA buffer: TSM supplemented with BSA. PBA buffer: PBS supplemented with 0.5% BSA and 0.02% Azide. 4.2. Yeast preparation Yeast strains were obtained from CBS Fungal Biodiversity Centre ( and included some new hybrid strains (Bovers et al., 2006, 2008). Yeast strains were cultured for 3 days at 25 C on YPGA medium followed by inoculation in Sabouraud dextrose broth and incubated at 25 C for 3 days, while shaking. Malassezia strains were cultured on solid mLNA medium for 3 days at 30 C. Yeast strains were harvested and heat inactivated for 1 h at 56 C. Optical density measurement at 600 nm was used to determine the concentration. Yeast strains were washed extensively in PBS before storage at 20 C at a concentration of 5 108/ml until use. 4.3. Cell-lines and LC isolation Jurkat and Jurkat-Langerin transduced cells were produced as described previously and were cultured in RPMI medium with 10% FCS (Valladeau et al., 2000; de Witte et al., 2007).

To isolate human LCs, human tissue was obtained from healthy donors undergoing corrective breast or abdominal surgery after informed consent in accordance with institutional guidelines and used within 3 h after surgery. The skin is cut in 0.3 mm slices containing the dermis and epidermis and incubated overnight at 4 C in dispase II (1 mg/ml, Roche diagnostics) in Iscoves Modified Dulbecco's medium (IMDM) and gentamycin (10 g/ml) overnight. The epidermis was mechanically separated and cultured in IMDM, 10% FCS, gentamycin (10 g/ml), GM-CSF (800 U/ml) and IL-4 (800 U/ml). After 3 days the cells were harvested and layered on a ficoll density gradient to obtaining a 90% pure LC population as determined by flow cytometry and used immediately (de Witte et al., 2007). 4.4. Langerin binding ELISA Different fungi (104107/ml), carbohydrate structures (2 g/ml) or -glucans were coated onto ELISA plates overnight at room temperature. Non-specific binding was blocked by incubating the plate with 2% TSA buffer for 1 h at 37 C. Recombinant human Langerin (Stambach and Taylor, 2003) (2 g/ml in 2% TSA) was added for 1 h at 37 C. Unbound Langerin was washed away and binding was determined using an anti-Langerin antibody (DCGM4, Beckman Coulter Inc.) followed by peroxidise-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch). Specificity was determined in the presence of mannan (1 mg/ml) or anti-Langerin monoclonal antibody 10E2 (10 g/ml) (de Witte et al., 2007). Efficient coating of the different fungi was confirmed with plant-lectin binding ConA (Canavalia ensiformis). 4.5. Facs analysis 50,000 cells (Jurkat, Jurkat-Langerin or LCs) were washed in PBA and incubated with specific antibodies (5 g/ml) or isotype controls for 30 min at 4 C and followed by an incubation with FITC-labelled secondary antibodies for 30 min at 4 C, or cells were incubated with directly labelled antibodies for 30 min at 4 C. Cells were washed and binding was measured using flow cytometry. 4.6. Fitc labelling Yeast strains were incubated with Fluorescein, 5-isothiocyanate isomer I (FITC) (Sigma) (1 mg/ml in DMSO) for 1 h at room temperature. Yeast strains were washed extensively in PBS to eliminate unbound FITC. The solution was resuspended at a concentration of 1 108/ml and stored at 4 C in the dark until use. 4.7. Cell binding assay 50,000 cells (Jurkat, Jurkat-Langerin or LCs) were washed in 0.5% TSA and pre-incubated with mannan, blocking antibodies against Langerin or dectin-1, or isotype controls for 15 min at 37 C. Different strains of FITC-labelled yeast were added for 45 min at 37 C. Cells were washed and binding was measured using flow cytometry. Forward-site scatter gating was used to exclude unbound yeast. 4.8. Electron microscopy LCs (1 106) were incubated with 1 107 C. albicans for 4 h. Cells were fixed and sections were prepared according to standard protocol (de Witte et al., 2007). Sections were stained for Langerin as described previously using polyclonal Langerin antibody (R&D) (de Witte et al., 2007). Acknowledgements This work was supported by the Dutch Scientific Organization (NWO) Grant 91204025 (MdJ) and the Wellcome Trust Grant 075565 (MET). We would like to thank Boerhaave Kliniek and Dr. A. Knottenbelt (Flevoziekenhuis) for their valuable support.

Other Sections


Almirante B., Rodriguez D., Park B.J., Cuenca-Estrella M., Planes A.M., Almela M., Mensa J., Sanchez F., Ayats J., Gimenez M., Saballs P., Fridkin S.K., Morgan J., Rodriguez-Tudela J.L., Warnock D.W., Pahissa A. Epidemiology and predictors of mortality in cases of Candida bloodstream infection: results from population-based surveillance, barcelona, Spain, from 2002 to 2003. J. Clin. Microbiol. 2005;43:18291835. [PMC free article] [PubMed] Arendrup M.C., Fuursted K., Gahrn-Hansen B., Schonheyder H.C., Knudsen J.D., Jensen I.M., Bruun B., Christensen J.J., Johansen H.K. Semi-national surveillance of fungaemia in Denmark 20042006: increasing incidence of fungaemia and numbers of isolates with reduced azole susceptibility. Clin. Microbiol. Infect. 2008;14:487494. [PubMed] Banchereau J., Steinman R.M. Dendritic cells and the control of immunity. Nature. 1998;392:245252. [PubMed] Batra R., Boekhout T., Gueho E., Cabanes F.J., Dawson T.L., Gupta A.K. Malassezia Baillon, emerging clinical yeasts. FEMS Yeast Res. 2005;5:11011113. [PubMed] Bovers M., Hagen F., Kuramae E.E., Diaz M.R., Spanjaard L., Dromer F., Hoogveld H.L., Boekhout T. Unique hybrids between the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii. FEMS Yeast Res. 2006;6:599607. [PubMed] Bovers M., Hagen F., Kuramae E.E., Hoogveld H.L., Dromer F., St-Germain G., Boekhout T. AIDS patient death caused by novel Cryptococcus neoformansC. gattii hybrid. Emerg. Infect. Dis. 2008;14:11051108. [PMC free article] [PubMed] Brown G.D. Dectin-1: a signalling non-TLR pattern-recognition receptor. Nat. Rev. Immunol. 2006;6:3343. [PubMed] Brown G.D., Gordon S. Immune recognition. A new receptor for beta-glucans. Nature. 2001;413:3637. [PubMed] Brown G.D., Herre J., Williams D.L., Willment J.A., Marshall A.S., Gordon S. Dectin-1 mediates the biological effects of beta-glucans. J. Exp. Med. 2003;197:11191124. [PMC free article] [PubMed] Cambi A., Netea M.G., Mora-Montes H.M., Gow N.A., Hato S.V., Lowman D.W., Kullberg B.J., Torensma R., Williams D.L., Figdor C.G. Dendritic cell interaction with Candida albicans critically depends on N-linked mannan. J. Biol. Chem. 2008;283:2059020599. [PMC free article] [PubMed] Chaffin W.L., Lopez-Ribot J.L., Casanova M., Gozalbo D., Martinez J.P. Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol. Mol. Biol. Rev. 1998;62:130180. [PMC free article] [PubMed] de Jong M.A., de Witte L., Santegoets S.J., Fluitsma D., Taylor M.E., de Gruijl T.D., Geijtenbeek T.B. Mutz-3-derived Langerhans cells are a model to study HIV-1 transmission and potential inhibitors. J. Leukoc. Biol. 2009;87 de Witte L., Nabatov A., Pion M., Fluitsma D., de Jong M.A., de Gruijl T.D., Piguet V., van Kooyk Y., Geijtenbeek T.B. Langerin is a natural barrier to HIV-1 transmission by Langerhans cells. Nat. Med. 2007;13:367371. [PubMed] Depanfilis G., Soligo D., Manara G.C., Ferrari C., Torresani C., Zucchi A. Human normalresting epidermal langerhans cells do express the type-3 complement receptor. Br. J. Dermatol. 1990;122:127136. Fithian E., Kung P., Goldstein G., Rubenfeld M., Fenoglio C., Edelson R. Reactivity of Langerhans cells with hybridoma antibody. Proc. Natl. Acad. Sci. U.S.A. 1981;78:2541 2544. [PMC free article] [PubMed] Gantner B.N., Simmons R.M., Underhill D.M. Dectin-1 mediates macrophage recognition of Candida albicans yeast but not filaments. EMBO J. 2005;24:12771286. [PMC free article] [PubMed] Geijtenbeek T.B., Gringhuis S.I. Signalling through C-type lectin receptors: shaping immune responses. Nat. Rev. Immunol. 2009;9:465479. [PubMed]

Geijtenbeek T.B., Torensma R., van Vliet S.J., van Duijnhoven G.C., Adema G.J., van Kooyk Y., Figdor C.G. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell. 2000;100:575585. [PubMed] Gupta A.K., Batra R., Bluhm R., Boekhout T., Dawson T.L. Skin diseases associated with Malassezia species. J. Am. Acad. Dermatol. 2004;51:785798. [PubMed] Hunger R.E., Sieling P.A., Ochoa M.T., Sugaya M., Burdick A.E., Rea T.H., Brennan P.J., Belisle J.T., Blauvelt A., Porcelli S.A., Modlin R.L. Langerhans cells utilize CD1a and Langerin to efficiently present nonpeptide antigens to T cells. J. Clin. Invest. 2004;113:701 708. [PMC free article] [PubMed]

The Role of Sebaceous Gland Activity and Scalp Microfloral Metabolism in the Etiology of Seborrheic Dermatitis and Dandruff
Byung In Ro* and Thomas L Dawson 1. Department of Dermatology, College of Medicine, Chung Ang University, Seoul, Korea 2. Beauty Care Technology Division, The Procter & Gamble Company, Cincinnati, Ohio, USA Correspondence: Thomas L. Dawson Jr, 11810 East Miami River Road, Cincinnati, Ohio, 45252, USA. Email: Received 20 September 2004; Accepted 21 October 2004. Top of page

Most common scalp flaking disorders show a strong correlation with sebaceous gland (SG) activity. Early SG activity in the neonate results in microfloral colonization and cradle cap. After maternal hormonal control subsides, there is little SG activity until puberty, when the SG turns on under sex hormone control. When the SG activity increases, the present but low Malassezia population has a new food source and proliferates, resulting in the scalp itching and flaking common to greater than 50% of adults. Dry scalp flaking, dandruff, and seborrheic dermatitis are chronic scalp manifestations of similar etiology differing only in severity. The common etiology is a convergence of three factors: (1) SG secretions, (2) microfloral metabolism, and (3) individual susceptibility. Dandruff and seborrheic dermatitis (D/SD) are more than superficial stratum corneum disorders, including alteration of the epidermis with hyperproliferation, excess lipids, interdigitation of the corneal envelope, and parakeratosis. The pathogenic role of Malassezia in D/SD has recently been elucidated, and is focused on their lipid metabolism. Malassezia restricta and M. globosa require lipids. They degrade sebum, free fatty acids from triglycerides, consume specific saturated fatty acids, and leave behind the unsaturates. Penetration of the modified sebaceous secretions results in inflammation, irritation, and scalp flaking. Keywords: dandruff, microflora, sebaceous gland, seborrheic dermatitis, sebum Abbreviations: D/SD, dandruff and seborrheic dermatitis; SG, sebaceous gland Top of page

Sebaceous Gland (SG) Activity

Human SG are found over the entire skin surface (except the palms of the hands and soles of the feet), but sebum secretion is highest on the scalp, face, chest, and back (Strauss and Pochi, 1968a). Sebum is produced under hormonal control, with SG active at birth under the control of maternal androgens. They quickly reduce in size and sebum production until the onset of puberty. As puberty begins the SG again activate, this time under the control of circulating androgens. The sebum secretion rate increases throughout the teens, remains steady through the 20s and 30s, then lessens with age (Strauss et al, 1983;Dawber, 1997). Throughout the active period of sebum secretion, the secretion rate is higher in males than in females. In males, the rate remains higher longer, into the 50s and 60s, but in females, the secretion rate drops quickly after menopause (Strauss and Pochi, 1968b). Common scalp flaking disorders all show a strong temporal correlation with sebaceous activity, following the pattern of early cradle cap, low incidence until puberty, increasing incidence through the teens, second and third decades, then declining (Dawber, 1997;Gupta et al, 2003,2004a,b). The primary functions of sebum have historically been controversial, but are recently being elucidated. Sebum is involved in development of epidermal structure and maintenance of the epidermal permeability barrier (Pilgram et al, 2001), carrying anti-oxidants to the skin surface (Theile et al, 1999 ), protection from microbial colonization, generation of body odor, and pheromone generation (Kligman, 1963). It has also recently come to light that sebum is directly involved in skin-specific hormonal signaling, epidermal differentiation, and protection of the skin from ultraviolet irradiation (Thiboutot et al, 2003;Zouboulis, 2003). Top of page

Composition of Human Sebum

When secreted human sebum is a complex mixture of triglycerides, fatty acids, wax esters, sterol esters, cholesterol, cholesterol esters, and squalene Figure 1 (Strauss et al, 1983). As the sebum is secreted, it consists primarily of triglycerides and esters, which are broken down by commensal microbes into diglycerides, monoglycerides, and the constituent free fatty acids. Human sebum contains both saturated and unsaturated fatty acids, with a preponderance of unsaturates. The fatty acid chain lengths of human sebum vary considerably, but are predominantly 16 and 18 carbons (stearic, C18:0, oleic, C18:1 9, linoleic, C18:2 9 12, palmitic, 16:0, sapienic, 16:1 6, and palmitoleic, C16:1 9, Figure 1). The role of specific fatty acids of human sebum becomes apparent when we examine the metabolism of Malassezia.
Figure 1.

Relative composition of human sebum. Samples of human sebum were collected and analyzed by gas chromatography. Peaks were identified by comparison to known standards. Identifications confirmed by GC-mass spectrometry. Full figure and legend (48K)

Top of page

Role of Malassezia
Over 100 y ago, Malassez implicated the yeast Pityrosporum in the etiology of dandruff (Malassez, 1874). Although there has been much debate regarding whether the yeast is actually a causative agent (Leyden et al, 1976;Shuster, 1984) there is now general agreement (Pierard Franchimont et al, 2000;Gupta and Kohil, 2004 ). Early SG activity in the neonate allows initial Malassezia colonization and is likely an initiating factor for cradle cap. The Malassezia population then drops dramatically, only to re-appear as SG activity increases at the onset of puberty (Gupta and Kohil, 2004). As the SG begins increased activity, the present but low Malassezia population has a new food source and proliferates (Gupta et al, 2001;Gupta and Kohil, 2004). Malassezia, however, have a very specific taste for individual fatty acids (Gueho et al, 1996,1998). The Malassezia lipases are non-specific and degrade any available triglycerides Figure 2. The saturated fatty acids are consumed, and the abundant unsaturates are left on the skin (Figure 2 and Figure 3).
Figure 2.

Triglyceride degradation and increased free fatty acids after incubation of artificial sebum by Malassezia globosa. Lipid composition analyzed as in Figure 1, but following incubation of M. globosa for 24 hours with defined lipid matrix. Full figure and legend (30K)

Figure 3.

Triglyceride and fatty acid composition of sebum extracted from human scalp. Lipid profile analyzed as in Figure 1, samples collected from a dandruff sufferer with high Malassezia counts before (red, primarily free fatty acids) or after (blue, both triglycerides and free fatty acids) treatment with a commercial antifungal shampoo. Full figure and legend (25K)

Recently, novel molecular methods have overcome the difficulties presented by culture of Malassezia, and the specific Malassezia species present on human scalp have been elucidated (Gupta et al, 2000;Gaitanis et al, 2002;Gemmer et al, 2002;Sugita and Nishikawa, 2003;Sugita et al, 2003). Malassezia nomenclature has evolved over the last century, but the genus now consists of 10 distinct species: M. globosa, M. restricta, M. furfur, M. sympodialis, M. slooffiae, M. obtusa, M. nana, M. dermatis, M. japonica, and the sole non-lipid-dependent species, M. pachydermatis. All except M. pachydermatis can be found on human skin, but the most common species on human scalp are M. restricta and M. globosa (Gemmer et al, 2002). Further molecular investigation will undoubtedly produce more distinct genetic entities, but detailed biochemical and physiological experiments will be needed to define the actual species. Top of page

Etiologic Mechanism of Dandruff and Seborrheic Dermatitis (D/SD)

D/SD are chronic clinical scalp conditions affecting greater than 50% of the population, the primary symptom of which is visibly excessive scalp scaling. Seborrheic dermatitis is a more severe disorder which can include increased desquamation of facial areas other than the scalp and visible inflammation. Although dandruff is not a life-threatening disease, its presence can lead to loss of self-esteem and a negative social image (Hay and Graham-Brown, 1997). D/SD are characterized by itching and visible dry or oily flakes, induced by excess turnover of scalp cells (Dawber, 1997). D/SD are more than just superficial disorders of the stratum corneum, including alteration of the epidermis with hyperproliferation, excess intercellular and intracellular lipids, interdigitation of the corneal envelope, and parakeratosis (McOsker and Hannon, 1967;Warner et al 2001 ,). Although Malassezia are not numerically correlated to D/SD, recent evidence strongly supports their causal role (Gupta and Kohil, 2004). This evidence includes the effectiveness of multiple chemical entities whose sole common mechanism of action is antifungal activity, as well as the very distinct numerical correlation of reduction in severity with reduction of Malassezia numbers (Shuster, 1984). Combination of several recent lines of investigation points out a novel mechanism for the etiology of D/SD. M. restrica and M. globosa require lipids as food source (Guillot and Gueho, 1995;Gueho et al, 1996;Guillot et al, 1996), and are perfectly adapted for life on the human scalp. The Malassezia degrade sebum, freeing multiple fatty acids from triglycerides Figure 2. They consume the very specific saturated fatty acids necessary for their proliferation, leaving behind the unsaturated fatty acids Figure 3. Experimentally, it can be shown that the changes in sebum composition over time are a direct result of Malassezia metabolism. Table I illustrates the effect of removing scalp microflora with an antimicrobial shampoo (removal of microorganisms verified by molecular analysis, data not shown). The sebum composition changes back to near normal levels of triglyercides and free fatty acids.
Table I - Relative composition of human sebum.

Full table

Top of page

Individual Susceptibility
Penetration of the modified sebaceous secretions into the stratum corneum breaks down the skin barrier function, resulting in inflammation, irritation, and the resultant scalp flaking. Recent data shows that the penetration and inflammation response to the fatty acids are different between dandruff and non-dandruff sufferers.1 Additionally, immunodeficiency, such as AIDS, allows excess Malassezia proliferation, resulting in severe D/SD. Physical factors, nutritional disorders, drugs, and neurotransmitter abnormalities are additional aggravating factors. Top of page

The common etiology of D/SD is therefore a convergence of three factors: (1) SG secretions, which provide the substrate for Malassezia growth; (2) Malassezia metabolism of the sebaceous secretions, releasing irritating unsaturated fatty acids; and (3) individual susceptibility to the penetration of the fatty acids and the resultant inflammation. Top of page

1 DeAngelis Y, Leland M, Gemmer C, et al: The three etiologic facets of dandruff and seborrheic dermatitis: Malassezia fungi, sebaceous lipids, and individual sensitivity. Intercontinental Meeting of Hair Research Societies, Conference poster, 2004. Top of page

1. Dawber, R: Diseases of the Hair and Scalp. London: Blackwell Science, p 499504, 1997 2. Gaitanis, G, Velegraki, A, Frangoulis, E, et al: Identification of Malassezia species from patient skin scales by PCR-RFLP. Clin Microbiol Infect 8: 162173, 2002 3. Gemmer, CM, DeAngelis, YM, Theelen, B, Boekhout, T, Dawson, TL: Fast, noninvasive method for molecular detection and differentiation of Malassezia yeast species on human skin and application for the method to dandruff microbiology. J Clin Microbiol 40: 33503357, 2002 | Article | PubMed | ISI | ChemPort | 4. Gueho, E, Boekhout, T, Ashbee, HR, Guillot, J, Van Belkum, A, Faergemann, J: The role of Malassezia species in the ecology of human skin and as pathogens. Med Mycol 36(Suppl):220229, 1998 | PubMed | 5. Gueho, E, Midgley, G, Guillot, J: The genus Malassezia with description of four new species. Antonie Van Leeuwenhoek 69: 337355, 1996 | PubMed | ISI | ChemPort |

6. Guillot, J, Gueho, E: The diversity of Malassezia yeasts confirmed by rRNA sequence and nuclear DNA comparisons. Antonie van Leeuwenhoek 67: 297314, 1995 7. Guillot, J, Gueho, E, Lesourd, M, Midgley, G, Chevrier, B, Dupont, B: Identification of Malassezia species. J Mycol Med 6: 103110, 1996

Dual Specificity of Langerin to Sulfated and Mannosylated Glycans via a Single C-type Carbohydrate Recognition Domain* Hiroaki Tateno, Koji Ohnishi, Rikio Yabe, Norihito Hayatsu, Takashi Sato, Motohiro Takeya, Hisashi Narimatsu, and Jun Hirabayashi1 From the Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, Central 2, 1-1-1 Umezono, Ibaraki 305-8568 and the Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan 1 To whom correspondence should be addressed: AIST, Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan., Tel.: Phone: 81-29-861-3124; Fax: 81-29-861-3125; E-mail: Received July 7, 2009; Revised November 5, 2009 This article has been cited by other articles in PMC.

Other Sections

Abstract Langerin is categorized as a C-type lectin selectively expressed in Langerhans cells, playing roles in the first line of defense against pathogens and in Birbeck granule formation. Although these functions are thought to be exerted through glycan-binding activity of the Ctype carbohydrate recognition domain, sugar-binding properties of Langerin have not been fully elucidated in relation to its biological functions. Here, we investigated the glycanbinding specificity of Langerin using comprehensive glycoconjugate microarray, quantitative frontal affinity chromatography, and conventional cell biological analyses. Langerin showed outstanding affinity to galactose-6-sulfated oligosaccharides, including keratan sulfate, while it preserved binding activity to mannose, as a common feature of the C-type lectins with an EPN motif. By a mutagenesis study, Lys-299 and Lys-313 were found to form extended binding sites for sulfated glycans. Consistent with the former observation, the sulfated Langerin ligands were found to be expressed in brain and spleen, where the transcript of keratan sulfate 6-O-sulfotransferase is expressed. Moreover, such sulfated ligands were upregulated in glioblastoma relative to normal brain tissues, and Langerin-expressing cells were localized in malignant brain tissues. Langerin also recognized pathogenic fungi, such as Candida and Malassezia, expressing heavily mannosylated glycans. These observations provide strong evidence that Langerin mediates diverse functions on Langerhans cells through dual recognition of sulfated as well as mannosylated glycans by its uniquely evolved C-type carbohydrate-recognition domain. Keywords: Carbohydrate/Function, Glycoproteins, Glycoproteins/Carbohydrates, Glycosylation, Immunology/Innate Immunity, Receptors/Oligosaccharides

Other Sections


Langerhans cells (LCs)2 are a subset of dendritic cells resident in an immature state in skin epidermis and mucosal epithelium. They have been considered to play a sentinel role through their specialized function in antigen capture and migratory capacity to secondary lymphoid tissue to initiate specific immunity (1). LCs are characterized by the presence of a cytoplasmic organelle, Birbeck granules (BGs), and the cell surface LC-specific receptor, Langerin (CD207) (2, 3). Langerin was first identified as a molecule specifically expressed in LCs, which is recognized by LC-specific mAb DCGM4 (2). Langerin belongs to group II C-type lectins, most of which have been reported to recognize glycan ligands in a Ca2+-dependent manner. Langerin is a 37.5-kDa (328 amino acids) type II transmembrane receptor consisting of an N-terminal short cytoplasmic domain, a transmembrane domain, a neck region, and a C-terminal C-type carbohydrate-recognition domain (CRD) (2). The cytoplasmic domain contains a proline-rich motif (WPREPPP) as a potential signal transduction site. The CRD contains an EPN (GluPro-Asn) motif characteristic of C-type lectins with mannose (Man) specificity (4). Mannanbinding protein and dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) are representative members of this Man-specific subfamily. Langerin functions as an endocytic receptor, which has been believed to be involved in selfdefense against invading pathogens. Langerin is expressed on the cell surface and has the ability to uptake (5), process, and present antigens to T cells through the major histocompatibility complex I and II pathways (6), although it lacks a typical tyrosine-based internalization motif (2). Recently, Langerin was reported to have activity to prevent human immunodeficiency virus-1 (HIV-1) transmission (7). Indeed, Langerin binds to HIV-1 through gp120 with mannosylated glycans and uptakes into the BGs, resulting in HIV-1 degradation and viral clearance. Langerin also functions as an essential component of the induction of BG formation as proved by the following findings: (a) inside the cell, Langerin exists mainly in the BGs (2); (b) transient transfection of Langerin cDNA into the fibroblastic mouse cell line induces BG formation (1); and (c) disruption of the Langerin gene abolishes the BGs in LCs (8). Interestingly, the C-type CRD of Langerin is essential for the BG formation, because the activity is abrogated by eliminating the CRD (2). In agreement with this observation, calcium deprivation results in unzippering of the BGs and transformation into vesicles (3). A recent report also demonstrated that antibody binding to the Langerin CRD induces global rearrangements of LC morphology (9). Undoubtedly, the Langerin CRD is essential for its biological functions in LCs. However, its sugar-binding specificity in relation to diverse functions in LCs remains to be elucidated. In terms of sugar-binding specificity, two studies have been reported. Stambach et al. analyzed the glycan-binding specificity of Langerin by solid phase and glycoprotein blotting assays, showing that the Langerin CRD binds to Man, fucose, and N-acetylglucosamine (GlcNAc) similar to other C-type lectins with the EPN motif, and that the oligomeric structure formed via the neck region is required for sufficient binding (10). More recently, Galustian et al. reported the glycan-binding specificity of Langerin using carbohydrate microarray (11). In contrast to the report by Stambach et al., Langerin was shown to bind to sulfated Lex-related glycans, whereas only weak binding was observed for high mannose-type N-glycans. Therefore, current understanding about sugar-binding specificity of Langerin has been rather confused. More systematic analysis is necessary to elucidate sugar-binding properties of Langerin in relation to its biological functions on LCs. Here, we analyzed glycan-binding specificity of Langerin using glycoconjugate microarray (12), frontal affinity chromatography (FAC) (13, 14), and cell biological assays. We demonstrate that Langerin is a unique receptor with dual specificity to sulfated and

mannosylated glycans via a single C-type CRD. The binding mechanism and putative functions of the dual specificity of Langerin are also discussed in this study.

Other Sections

EXPERIMENTAL PROCEDURES Materials FITC-labeled rabbit anti-donkey IgG (H+L), Cy3-labeled donkey anti-human Fc, peroxidase-labeled goat anti-human IgG (H+L), Cy3-labeled donkey anti-mouse IgG (H+L), and Cy3-labeled streptavidin were purchased from Jackson ImmunoResearch (West Grove, PA). FITC-labeled rat anti-CD3 and anti-CD19 mAbs were purchased from BD Biosciences. Mouse anti-human Langerin mAb (12D6, Novocastora) was purchased from Mitsubishi Chemical Medience Corp. (Tokyo, Japan). N-Acetyllactosamine (LacNAc)-polyacrylamide (PAA)-biotin, 6-sulfo-N-acetylglucosamine (GlcNAc)-PAA- biotin, 6-sulfo-LacNAc-PAAbiotin, and 6-sulfo-LacNAc-PAA-biotin probes were purchased from Glycotech (Gaithersburg, MD). Anti-highly sulfated keratan sulfate mAb (5D4) was purchased from Seikagaku Co. (Tokyo, Japan). Arthrobacter ureafaciens sialidase was purchased from Roche Diagnostics (Tokyo, Japan). Tissue microarrays of astrocytic tumors were purchased from Cybrdi, Inc. (Frederick, MD). Tissue specimens of gliomas were also obtained from the Kumamoto University Hospital. Marathon Ready cDNAs of various human tissues were purchased from Takara (Shiga, Japan). The human Langerin (#4692155), KS6ST (#4814499), and GlcNAc6ST (#4562846) cDNAs were purchased from Funakoshi Co., Ltd. (Tokyo, Japan). Protein A-Sepharose 4 Fast Flow was purchased from GE Amersham Biosciences. Pyridylaminated (PA) bovine corneal KS (KS-I) and shark cartilaginous KS (KS-II) were generous gifts from Seikagaku Co. Fungi Saccharomyces cerevisiae (Institute of Food Microbiology number = 49922), Candida albicans (40009), Candida glabrata (40018), Candida guilliermondii (46828), Candida kefyr (46842), Candida krusei (46839), Candida parapsilosis (52607), Candida tropicalis (40065), Malassezia furfur (52635), Malassezia pachydermatis (48586), and Cryptococcus neoformans (B4500) were obtained from the National BioResource Project. S. cerevisiae, Candida species, and C. neoformans were cultured in YPD medium (10% Tryptone, 5% dried yeast extract, 10% glucose, pH 5.8). Malassezia species were cultured in Malassezia medium (10% Tryptone, 10% dried yeast extract, 20% glucose, 0.5% Tween 80). Langerin-Fc Fusion Protein The CRD region of human Langerin (193328 amino acids) was amplified by PCR using specific primer sets (forward and reverse, 5CTCGAGATGACTGTGGAGAAGGAGGCCCCTGATGCG-3 and 5GATATCCGGTTCTGATGGGACATAGGGTCGCTTACA-3), and ligated into a pSecTag/FRT/V5-His vector (Invitrogen Japan K.K., Tokyo, Japan). The Fc region of human IgG1 was then inserted into the C terminus of the Langerin CRD sequence via AgeI and PmeI sites. Site-directed mutagenesis of Langerin-Fc was also performed using a Genetailor sitedirected mutagenesis system (Invitrogen). The primers used for construction of Langerin-Fc mutants were as follows: for E285A, 5-GAGGTTCTGGATTCCAGGTGCGCCCAACAAT3 (sense) and 5-CACCTGGAATCCAGAACCTCGCACTTTGGA-3 (antisense); for N287A, 5-TCTGGATTCCAGGTGAGCCCGCCAATGCTGGG-3 (sense) and 5GGGCTCACCTGGAATCCAGAACCTCGCACT-3 (antisense); for K299A, 5ATGAACACTGTGGCAATATAGCGGCTCCCTCAC-3 (sense) and 5TATATTGCCACAGTGTTCATTGTTCCCAGC-3 (antisense); and for K313A, 5GGAATGATGCCCCATGTGACGCAACGTTTCTTT-3 (sense) and 5-

GTCACATGGGGCATCATTCCAGGCCTGAAG-3 (antisense). The expression constructs were transiently transfected into HEK293T cells cultured in Opti-MEM (Invitrogen) supplemented with 5% low IgG fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen) by Lipofectamine LTX (Invitrogen) followed by the manufacturer's procedure. Fusion proteins secreted into medium were purified by affinity chromatography on Protein A-Sepharose 4 Fast Flow (Amersham Biosciences). Preparation of CHO Cells Stably Expressing Full-length Langerin The full-length human Langerin (CD207) gene (1328 amino acids) was amplified by PCR using specific primer sets (forward and reverse, 5CTCGAGATGACTGTGGAGAAGGAGGCCCCTGATGCG-3 and 5GATATCCGGTTCTGATGGGACATAGGGTCGCTTACA-3) and ligated into a pcDNA5/FRT/V5-His vector (Invitrogen). The expression vector was transfected into an FlpIn CHO cell line (Invitrogen) by Lipofectamine LTX. CHO cells stably expressing the fulllength Langerin were selected with 0.5 mg/ml hygromycin B (Invitrogen). Preparation of Sulfotransferase Expression Vectors The full-length cDNA of KS6ST (CHST1, keratan sulfate Gal-6-sulfotransferase), GlcNAc6ST (CHST2, carbohydrate N-acetylglucosamine-6-O-sulfotransferase 2), and IGlcNAc6ST (CHST5, intestinal N-acetylglucosamine 6-O-sulfotransferase) were ligated into pcDNA3.1 (+) or () vectors (Invitrogen) via EcoRV sites. Glycoconjugate Microarray The sugar-binding specificity of Langerin was analyzed by glycoconjugate microarray as described previously (12). Briefly, Langerin-Fc chimera (1 g/ml) was precomplexed with Cy3-labeled donkey anti-human Fc (1.4 g/ml) in TBS-T (10 mm Tris-HCl, pH 7.4, containing 150 mm NaCl (TBS), 1% (v/v) Triton X-100) containing 1 mm CaCl2, and then applied onto a glycoconjugate microarray (version 4.2). As negative control experiments, binding was also analyzed in TBS-T containing 10 mm EDTA. After incubation at 20 C overnight, binding was detected by the evanescent-field fluorescence-assisted scanner, SCprofiler (GP Biosciences Ltd., Yokohama, Japan) under Cy3 mode. Culture supernatants containing Langerin-Fc were precomplexed with Cy3-labeled anti-human Fc in the presence or absence (control) of inhibitors and applied onto the glycoconjugate microarray. 50 mm of Man and Glc, and 5 g/ml 6-sulfo-LacNAc-PAA (6-sulfo-LacNAc), 6-sulfo-LacNAc-PAA (6-sulfo-LacNAc), and LacNAc-PAA (LacNAc), and 10 mm EDTA were used as inhibitors. Glycans used for glycoconjugate microarray are shown in supplemental Fig. 1 and supplemental Table 1. Binding of Langerin-Fc to CHO Cells Transfected with Sulfotransferase cDNA CHO cells (1 105) were cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen) in 6-well plates at 37 C. After 2 days, sulfotransferase expression vectors (2.5 g) were transfected into CHO cells using Lipofectamine LTX (6.25 l, Invitrogen) in accordance with the manufacturer's procedure. Cells were recovered, resuspended in TSA buffer (TBS containing 10 mg/ml bovine serum albumin, 2 mm CaCl2, and 2 mm MgCl2), and incubated with or without 25 milliunits of A. ureafaciens sialidase at 37 C for 30 min. After washing with TSA buffer, cells were incubated with 20 g/ml Langerin-Fc chimera precomplexed with 20 g/ml Cy3-labeled donkey anti-human Fc on ice for 1 h. As negative controls, cells were also incubated with Langerin-Fc chimera in EDTA buffer (TBS containing 10 mg/ml bovine serum albumin and 5 mm EDTA). Flow cytometry data were acquired on a FACSCanto-II cytometer (BD Biosciences) and analyzed using FACSDiva and FlowJo software. Probe Binding Assay

Langerin-CHO cells (2 105) were cultured in 6-well plates for 2 days at 37 C. After washing with TSA buffer, cells were incubated with glycoside-PAA probes (10 g/ml) precomplexed with Cy3-streptavidin (10 g/ml) on ice for 1 h. Cells were then recovered with trypsin, and flow cytometry data were acquired on a FACSCanto-II cytometer and analyzed using FACSDiva and FlowJo. As a negative control, probe binding was also analyzed in the presence of EDTA. Probe Internalization Assay Langerin-CHO cells (2 105) were cultured in 6-well plates for 2 days at 37 C. After washing with TSA buffer, cells were incubated with glycoside-PAA probes (20 g/ml) precomplexed with Cy3-streptavidin (20 g/ml) in TSA buffer at 37 C for 1 h to trigger internalization. Cells were then recovered with trypsin, and the internalized fluorescence was detected by flow cytometry. Flow cytometry data were acquired on a FACSCanto-II cytometer and analyzed using FACSDiva and FlowJo. Negative control experiments were also performed using EDTA buffer. FAC FAC was performed as described previously (13, 14). Briefly, Langerin-Fc fusion proteins were immobilized on Protein A-Sepharose 4 Fast Flow at a concentration of 12 mg/ml, and the resulting lectin-immobilized gel was packed into a miniature column (inner diameter, 2 mm; length, 10 mm, bed volume, 31.4 l, Shimadzu) and connected to an automated FAC system (FAC-1). A panel of 142 PA-glycans was successively injected into the columns by the auto-sampling system, and elution of the PA-glycans was detected by fluorescence (excitation, 310 nm; emission, 380 nm). The elution front of each PA-glycan relative to that of an appropriate control (lactose-PA), referred to as V V0, was then determined. Glycans used for FAC are shown in supplemental Fig. 2. To determine Kd values, Bt (effective ligand content) was assumed to be 50% of the immobilized Langerin-Fc. Real-time PCR Real-time PCR was performed using an ABI Prism 7700 Sequence Detection System (Invitrogen) as previously described (15). Standard curves for the KS6ST gene and the human glyceraldehyde-3-phosphate dehydrogenase gene, as an endogenous control, were generated by serial dilution of each plasmid DNA. Data are shown as the relative amount of KS6ST transcript normalized by the amount of glyceraldehyde-3-phosphate dehydrogenase transcript in the same cDNA. Binding of Langerin-Fc to Mouse Splenocytes Mouse spleen was ground between two frosted glass slides and passed through a cottonplugged Pasteur pipette. Erythrocytes were lysed with BD Pharmingen Cell Lysis Buffer (BD Biosciences). Splenocytes (1 106) were incubated with or without 25 milliunits of A. ureafaciens sialidase (Roche Diagnostics K.K.) in TSA buffer at 37 C for 30 min. After washing with TSA buffer, cells were then incubated with Langerin-Fc fusion proteins (20 g/ml) precomplexed with Cy3-labeled donkey anti-human Fc (20 g/ml) on ice for 1 h. Cells were also stained with FITC-labeled rat anti-CD3 and anti-CD19 mAbs. Flow cytometry data were acquired on a FACSCanto-II cytometer and analyzed using FACSDiva and FlowJo. Tissue Staining Brain tissue paraffin-embedded sections were obtained from Cybrdi, Inc. and the Kumamoto University Hospital (Kumamoto, Japan). After deparaffinization with xylene and rehydration with ethanol, tissue sections were immersed in methanol containing 0.3% hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity. For Langerin immunostaining, tissue sections were pretreated with microwave to unmask antigen and then incubated with antiLangerin mAb (12D6). Tissue sections were then stained using Histofine Simple Stain MAX-PO (M) secondary antibody (Nichirei Biosciences, Inc., Tokyo, Japan). The

immunoreactions were visualized using a diaminobenzidine substrate kit (Vector Labs, Burlingame, CA) (brown) and counterstained with hematoxylin (blue). Tissue sections were also stained with Langerin-Fc (20 g/ml) precomplexed with peroxidase-conjugated goat anti-human-IgG (10 g/ml). Binding of Langerin-Fc to Live Fungi Langerin-Fc fusion proteins (0.5 g/ml) were precomplexed with Cy3-labeled anti-human Fc (0.5 g/ml) in TSA buffer at room temperature for 30 min and then incubated with 1 105 of live fungi on ice for 1 h. After washing with TSA buffer, flow cytometry data were acquired on a FACSCanto-II cytometer and analyzed using FACSDiva and FlowJo. Langerin-Fc was also incubated with fungi in EDTA buffer.

Other Sections

RESULTS Glycoconjugate Microarray Screening of Langerin-Fc Chimera Langerin is a type II transmembrane protein containing a single extracellular C-type CRD and a cytoplasmic domain. To examine its glycan-binding specificity, the soluble LangerinFc chimera was generated by cloning the CRD into the pSecTag/FRT/V5-His vector followed by insertion of the Fc portion of human IgG1 at the C terminus of the Langerin CRD. Using the Langerin-Fc chimera, potential glycan ligands were first screened using the recently developed glycoconjugate microarray comprising 98 immobilized compounds (supplemental Fig. 1 and supplemental Table 1) (12). Langerin-Fc chimera was precomplexed with Cy3labeled donkey anti-human Fc antibody and then incubated with glycoconjugate microarray. Binding was detected using the evanescent-field fluorescence-assisted scanner. At a low gain (Fig. 1A), Langerin-Fc exhibited highly selective binding to 6-sulfo-LacNAc (38, [6SO4]Gal14GlcNAc), whereas no binding was observed for either its position isomer, 6sulfo-LacNAc (37, Gal1-4[6-SO4]GlcNAc), or unsulfated form (35, Gal1-4GlcNAc). A weak, but significant signal was also observed for 6-sulfated GlcNAc (48, [6-SO4]GlcNAc), suggesting that sulfate at the C-6 of the non-reducing end sugar might be important for Langerin recognition. On the other hand, at a high gain (Fig. 1B), extensive binding to a certain set of glycans with Man (shown in green), GlcNAc (shown in blue), and fucose (shown in red) was observed, in agreement with the previous report (10): that is, Langerin-Fc bound to heavily mannosylated glycans such as Man (54) and Man polymers (55), yeast invertase (57), and mannan from S. cerevisiae and C. albicans (93 and 94). Notably, no binding was observed for a typical endogenous glycoprotein, thyroglobulin (28 and 46) expressing high mannose-type N-glycans, indicating that mannose multivalency and geometry, characteristic of exogenous organisms, might be important for Langerin recognition. A similar observation has been reported for mannan-binding protein (16). Langerin-Fc also exhibited binding to agalacto-AGP (50) containing highly branched agalactosylated N-glycans as well as fucosylated glycans (1 and 2), but to a much lesser extent. Binding signals were abolished in the presence of EDTA (Fig. 1C), indicating that the results are due to specific interactions via the C-type CRD. To further clarify whether the binding of Langerin to sulfated and mannosylated glycans is mediated by the same binding site, we then performed inhibition assay. As shown in Fig. 1D, binding of Langerin-Fc to both 6-sulfo-LacNAc and yeast invertase was abrogated in the presence of either 50 mm Man or 5 g/ml 6-sulfo-LacNAc-PAA, but no inhibitory effect was observed for 5 g/ml 6sulfo-LacNAc-PAA and LacNAc-PAA, consistent with the results obtained in Fig. 1 (A and B). These results indicate that dual specificity of Langerin to sulfated and mannosylated glycans share the same ligand binding site. FIGURE 1.

Analysis of the glycan-binding specificity of Langerin-Fc chimera by glycoconjugate microarray. Langerin-Fc fusion proteins were precomplexed with Cy3-labeled anti-human Fc in TBS-T containing 1 mm CaCl2 at room temperature for 15 min and directly (more ...)

Lys-299 and Lys-313 Form Extended Binding Sites for the Recognition of Sulfated Glycans Structurally, the Langerin CRD exhibits some differences from other known C-type CRDs having the EPN motif, such as DC-SIGN (9): two lysine residues, Lys-299 and Lys-313, are present in the Langerin CRD, but not in the DC-SIGN CRD (supplemental Fig. 3) (9, 17). The presence of such positively charged amino acids in the CRD is likely involved in the formation of salt bridges with negatively charged sulfated ligands (9, 17). Expression plasmids of WT and mutant forms of Langerin-Fc were transfected into HEK293T cells, and the resulting culture supernatants were analyzed by glycoconjugate microarray with the aid of a Cy3-labeled secondary antibody. The expression of WT and mutant forms of Langerin-Fc in the culture supernatants was confirmed by Western blotting using peroxidase-labeled goat anti-human IgG (H+L) (Fig. 2A). As shown in Fig. 2B, binding of Langerin-Fc to both invertase and 6-sulfo-LacNAc was abolished by mutation of either Glu-285 or Asn-287, which are components of the EPN motif. Interestingly, binding of Langerin-Fc to 6-sulfoLacNAc was also canceled by mutation of either Lys-299 or Lys-313, whereas binding of Langerin-Fc to 6-sulfo-GlcNAc was abolished by mutation of Lys-313, but not Lys-299. No effect of either mutation was observed on the binding to invertase. These results demonstrate that Lys-299 and Lys-313 form extended binding sites for the recognition of sulfated glycans. Binding mechanism of Langerin to 6-sulfo-LacNAc and 6-sulfo-GlcNAc might be slightly different. FIGURE 2. Lys-299 and Lys 313 are extended binding sites for the recognition of sulfated glycans. A, WT and mutant forms of Langerin-Fc expression vectors were transfected into HEK293T cells, and 1 ml of the resulting culture supernatants was incubated with 10 (more ...)

KS6ST Generates Langerin Ligands Having shown that Langerin-Fc chimera recognize 6-sulfo-LacNAc as a preferred glycan ligand, we then examined whether KS6ST, which has activity to transfer sulfate to the C-6 of Gal of LacNAc on glycoproteins (18), is responsible for the generation of a Langerin ligand. CHO cells were transfected with the KS6ST expression plasmid with or without sialidase pretreatment, and binding of Langerin-Fc was analyzed by flow cytometry. As shown in Fig. 3, Langerin-Fc bound to sialidase-treated CHO cells transfected with KS6ST (bold line), whereas only weak binding was observed before sialidase pretreatment (thin line). This observation indicates that Langerin recognizes 6-sulfo-LacNAc but not its sialylated form. Combined with the results obtained by glycoconjugate microarray, it is likely that Langerin recognizes 6-sulfo-LacNAc on glycoproteins expressed at the cell surface, in which synthesis KS6ST is involved. FIGURE 3. KS6ST generates sulfated Langerin ligands. Untreated (thin line) and sialidase-treated CHO cells (bold line) transfected with or without a KS6ST

(CHST1, keratan sulfate Gal-6-sulfotransferase) expression vector was incubated with Langerin-Fc chimera precomplexed (more ...) Langerin Mediates Binding to and Uptake of 6-Sulfo- LacNAc To assess the binding of Langerin to 6-sulfo-LacNAc under more physiological conditions, we examined whether the full-length Langerin expressed at the cell surface also binds to this structure. For this purpose, CHO cells stably expressing Langerin (Langerin-CHO) were first established (Fig. 4A), and binding of biotin-labeled PAA probes precomplexed with Cy3streptavidin to Langerin-CHO cells was detected by flow cytometry (Fig. 4B). The 6-sulfoLacNAc-PAA-biotin probe bound to Langerin-CHO cells (Fig. 4B, bold line), whereas no binding was observed with either 6-sulfo-LacNAc-PAA-biotin (Fig. 4B, thin line) or LacNAc-PAA-biotin probes (Fig. 4B, gray). Therefore, Gal-6-sulfate is considered to be essential for Langerin binding, in agreement with the results obtained by glycoconjugate microarray. Binding of the 6-sulfo-LacNAc-PAA-biotin probe to Langerin-CHO cells was cancelled in the presence of EDTA, indicating that Langerin binding is dependent on the Ctype CRD. FIGURE 4. Langerin expressed at the cell surface mediates binding to and uptake of 6-sulfated LacNAc. A, expression of the full-length Langerin on CHO cells. Langerin-CHO cells were stained with goat anti-Langerin IgG followed by FITC-labeled anti-goat (more ...)

One of the roles of Langerin expressed on LCs is to bind antigens and internalize them into the BGs. Therefore, we next investigated whether Langerin could endocytose bound 6-sulfoLacNAc-PAA-biotin probes into intracellular compartments. For this purpose, LangerinCHO cells were incubated with the biotin-labeled 6-sulfo-LacNAc-PAA-biotin probe precomplexed with Cy3-streptavidin via a biotin tag at 37 C for 60 min. After this internalization process, residual cell surface probes were removed by 5 mm EDTA, and internalized Cy3-labeled probes were detected by flow cytometry. As shown in Fig. 4C, internalization of the 6-sulfo-LacNAc-PAA-biotin probe was detected after 60 min of incubation. On the other hand, apparently no internalization was observed with either 6-sulfoLacNAc-PAA-biotin or LacNAc-PAA-biotin used as negative controls. These results demonstrate that 6-sulfo-LacNAc serves as a glycan ligand for Langerin. Langerin Recognizes Keratan Sulfate Keratan sulfate (KS) is a Gal-6-sulfated oligosaccharide consisting of a linear polymer of LacNAc, (Gal14GlcNAc13)n that is sulfated to a varying degree on the C-6 of either GlcNAc or Gal residues. Therefore, it is possible that KS serves as an endogenous ligand for Langerin. To prove this, binding of Langerin to KS was first investigated by FAC using two types of natural KS polymers, bovine corneal KS (KS-I) and shark cartilaginous KS (KS-II). KS-II is more extensively sulfated than KS-I (19). In Fig. 5A, Langerin showed much stronger affinity to highly sulfated KS-II (Kd = 3.3 105 m) than KS-I (Kd = 103 m). Similarly, no binding signal was detected for KS-I (91) by glycoconjugate microarray (Fig. 1). Weak, but significant binding was also observed for a KS-related glycan, 922 ([6S]Gal1-

4GlcNAc13[6S]Gal14GlcNAc, Kd = 7 102 m). On the other hand, Langerin showed no binding to other glycans (for the list of standard 142 glycans, see supplemental Fig. 2) representing N-glycans, O-glycans, glycolipid-type glycans, and milk oligosaccharides, including 3-sulfolactose (918). Binding affinity to KS-II was found to be 100-fold higher than to high mannose-type N-glycans (Kd > 103) under the current experimental condition. It seems that Langerin requires multivalency for the recognition of mannosylated glycans. FIGURE 5. Langerin recognizes KS. A, binding of Langerin-Fc chimera to KS analyzed by FAC. Dotted lines represent PA-labeled lactose used as negative controls. Solid lines represent PA-labeled corneal (left panel) and cartilaginous KS (right panel). B, CHO cells (more ...) We then investigated whether Langerin binds to highly sulfated KS expressed at the cell surface. For this purpose, we generated KS-expressing cells by double transfection with both KS6ST and GlcNAc6ST into CHO cells. In the KS synthesis, KS6ST has activity to transfer sulfate to the C-6 of Gal, whereas GlcNAc6ST acts to transfer sulfate to the C-6 of GlcNAc. As shown in Fig. 5B, left panel, double transfected-CHO cells were stained with 5D4 mAb, which is specific for highly sulfated KS. Therefore, the double- transfected cells are considered to produce KS on cell surfaces. Langerin-Fc chimera exhibited binding to the double-transfected CHO cells even without sialidase treatment (Fig. 5B, middle panel). Upon sialidase treatment, however, much enhanced binding was observed, consistent with the previous experiment showing sialylation inhibits Langerin binding (Fig. 3). Although Langerin-Fc bound to both 5D4-positive and -negative CHO cells, the lectin gave stronger binding on 5D4-positive CHO cells (Fig. 5B, right panel). Similar results were obtained by cotransfection with KS6ST and I-GlcNAc6ST (CHST5, intestinal N-acetylglucosamine 6-Osulfotransferase) (data not shown) (20). Therefore, it is strongly suggested that cells expressing highly sulfated KS serve as target ligands for Langerin. Quantitative Real-time PCR Analysis of the KS6ST Transcript in Human Tissues Having shown that KS6ST is responsible for transferring sulfate to the C-6 of Gal recognized by Langerin, we performed quantitative real-time PCR analysis to see which human tissues express KS6ST mRNA. Previously, KS6ST mRNA was reported to be expressed in brain (21) and spleen (18) analyzed by Northern blotting. Consistently, the expression of KS6ST mRNA was high in brain and spleen (Fig. 6), and expression was also detected in lung, pancreas, and thyroid tissues. FIGURE 6. Quantitative real-time PCR analysis of the KS6ST transcript in human tissues. Data are shown relative to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript. Langerin Ligands Are Expressed in Mouse Spleen Because spleen was one of the dominant tissues expressing KS6ST mRNA, we analyzed expression of Langerin ligands using mouse splenocytes and Langerin-Fc chimera as a probe. As shown in Fig. 7A, Langerin-Fc chimera bound to splenocytes even without sialidase pretreatment. Approximately 50% of splenocytes were estimated to be positive for Langerin binding. However, the binding was strongly increased with the sialidase pretreatment, again confirming that the sialylation event is inhibitory to some degree on Langerin binding to sulfated ligands. Langerin-Fc bound to both CD3+ T and CD19+ B cells even without sialidase treatment, and the binding was enhanced after sialidase treatment, indicating that sulfated Langerin ligands are expressed on both intact CD3+ T and CD19+ B cells (Fig. 7B). FIGURE 7. Binding of Langerin-Fc to mouse splenocytes. A, mouse splenocytes were

incubated with Langerin-Fc chimera precomplexed with Cy3-labeled antihuman Fc with or without sialidase pretreatment. B, mouse splenocytes were double stained with FITC-labeled (more ...)

Brain Glioblastoma Is a Candidate Target for Langerin Recently, an LN229 glioblastoma cell line was shown to express 5D4-positive highly sulfated KS (22). If our assumption that highly sulfated KS is an endogenous ligand for Langerin is the case, such glioblastoma cells should be targets for Langerin recognition. Thus, we examined the binding ability of Langerin to KS-expressing glioblastoma cells by flow cytometry. As shown in Fig. 8A, left panel, LN229 cells were stained with 5D4 mAb, confirming that highly sulfated KS is indeed expressed on the cells (22). Langerin-Fc chimera bound to LN229 cells with sialidase pretreatment, whereas little or no binding was observed for untreated LN229 cells (Fig. 8A, right panel) consistent with the previous observations (Fig. 3). The results provide the possibility that glioblastoma cells are target cells for Langerin in vivo. FIGURE 8. Glioblastoma is a candidate target for Langerin. A, LN229 glioblastoma cells were stained with 5D4 followed by incubation with Cy3-labeled donkey anti-mouse IgG (left panel) and Langerin-Fc chimera precomplexed with Cy3-labeled anti-human Fc (more ...)

KS6ST mRNA as well as its product, KS, is expressed in brain (19, 21) (Fig. 6). Recently, the expression of 5D4-positive highly sulfated KS was reported to be up-regulated depending on the malignancy of astrocytic tumors (23). Because Langerin bound to LN229 glioblastoma cells (Fig. 8A), we hypothesized that glioma cells could be an endogenous target for Langerin. Brain tissue sections were incubated with Langerin-Fc chimera, and the binding was observed by microscopy. As shown in Fig. 8B, left panel, normal brain tissues were weakly, but significantly, stained with Langerin-Fc. On the other hand, greatly enhanced staining was observed for grade IV astrocytic tumors (Fig. 8B, right panel), where prominent staining was detected at the cell surface of tumor cells. Because the staining was inhibited by EDTA, the binding is attributed to the C-type CRD (data not shown). These results indicate that Langerin ligands are expressed at a basal level even in normal brain tissues, whereas their expression is dramatically up-regulated in malignancy. We then examined the expression of Langerin in normal brain and glioma tissues using antihuman Langerin mAb (12D6) (Fig. 8C). From a histopathological viewpoint, glioma are classified into grades I to IV based on the degree of malignancy. Glioma includes astrocytoma, oligodendrocytoma, ependymoma, and oligoastrocytoma. Among them, astrocytoma is the most common. Langerin-expressing cells were detected in two clinical cases, including astrocytoma and malignant ependymoma, but not in normal brain tissues. In grade IV astrocytoma (glioblastoma), Langerin-expressing cells were diffusely localized in vascular-rich stromal tissues (Fig. 8C, right panel). Morphologically, however, Langerinexpressing cells are not tumor cells, but are probably dendritic cell-like cells infiltrated from blood vessels. Langerin Recognizes Live Pathogenic Fungi So far, we have described sulfated glycans as the most possible Langerin ligands. However, Langerin-Fc chimera also bound to heavily mannosylated ligands, such as yeast mannan and

invertase analyzed by glycoconjugate microarray (Fig. 1). In this regard, LCs basically residing in skin are believed to be involved in sensing and capturing invading pathogens for efficient antigen presentation to nave T cells present in draining lymph node. To investigate this point from a glycobiological viewpoint, we extended analysis by examining whether Langerin recognizes live pathogenic fungi, such as Candida, Malassezia, and Cryptococcus, by flow cytometry. As shown in Fig. 9, Langerin-Fc chimera precomplexed with Cy3-labeled donkey anti-human Fc strongly bound to fungi in a Ca2+-dependent manner, which include both pathogenic (Candida and Malassezia) and non-pathogenic (Saccharomyces) species. On the other hand, no significant binding was observed for pathogenic Cryptococcus neoformans. These results indicate that Langerin recognizes extensive categories of fungi, consistent with an intrinsic role of LCs in anti-fungal immunity. FIGURE 9. Binding of Langerin-Fc to live pathogenic fungi. Langerin-Fc fusion proteins (0.5 g/ml) were precomplexed with Cy3-labeled anti-human Fc (0.5 g/ml) in TSA buffer at room temperature for 30 min and then incubated with 1 (more ...) Other Sections

DISCUSSION There are lines of evidence that sulfation of cell surface glycoproteins plays critical roles in cell-cell communications. GlcNAc 6-sulfate is involved in several recognition phenomena, including selectin-mediated lymphocyte homing (24), activation of CD44-mediated cellular interaction (25), and dendritic cell function (26). GlcNAc 6-sulfate even serves as a ligand for siglecs, such as CD22 (Siglec-2) (27, 28) and Siglec-9 (29). In contrast, the functional importance of Gal 6-sulfate is much less understood, although Gal-6-sulfated sialyl LewisX was shown to be a candidate ligand for Siglec-8 (30) and its mouse paralog, Siglec-F (29, 31). In 2004, Galustian et al. (11) reported binding activity of Langerin to sulfated LewisX glycans, although the importance of sulfation on Langerin recognition has not been fully elucidated. In this study, we have obtained clear evidence that Langerin recognizes Gal-6sulfated lactosamine (6-sulfo-LacNAc) and polylactosamine (KS), although Langerin is a Ctype lectin with the consensus EPN motif predicting basic specificity to Man. Sialylation clearly inhibited Langerin binding to Gal-6-sulfated glycans unlike the results obtained by Galustian et al. showing that Langerin binds to both sialylated and non-sialylated Gal-6sulfated glycans (11). The two lysine residues, Lys-299 and Lys-313, were found to be involved in the recognition of sulfated ligands. These positively charged amino acids in the CRD are likely involved in the formation of salt bridges with negatively charged sulfated ligands. Further studies are required to understand the more precise binding mechanism of Langerin to its sulfated ligands. Langerin exhibited strong binding to KS. KS was first identified in bovine cornea in 1953 (32) and was later shown to be expressed in cartilage, bone, and the central nervous system (19). In fact, KS is expressed on a subpopulation of microglia (33) and spinal cord (34). Interestingly, KS synthesis is up-regulated in the lesions on central nervous system injury (35). Recently, 5D4-positive highly sulfated KS was reported to be up-regulated depending on the malignancy of astrocytic tumors (22). Therefore, Gal-6 sulfation is considered to be closely involved in brain biology. In this report, we showed that Langerin ligands are strongly expressed in malignant astrocytic tumors. However, Langerin also binds to heavily mannosylated glycans such as mannan but not to glycoproteins containing human-type high mannose-type N-glycans such as thyroglobulin (Fig. 1). Increased expression of heavily

mannosylated glycans in glioblastoma has not been reported. Therefore, it is reasonable to speculate that Langerin binds to glioblastoma tissues via Gal-6-sulfated glycans. Consistently, Langerin bound to the KS-expressing LN229 glioblastoma cell line after sialidase treatment, most probably via Gal-6-sulfated glycans. In LN229, Gal-6-sulfated glycans were sialylated. However, glycans expressed on tissues are much more heterogeneous. Therefore, it is not surprising that non-sialylated Gal-6-sulfated glycans are expressed in the glioblastoma tissues. Coincidently, Langerin-expressing cells were found to be localized in glioma, but not in normal brain tissues. Therefore, LCs could interact with glioma cells via the Langerin C-type CRD. The consequences of the interactions between LCs and astrocytic tumors were not studied in this report, but it could induce anti-tumor immunity or immune tolerance. Further studies are essential to understand the functions of Gal-6-sulfated glycans in the brain. Generation of antibody specific for Gal-6 sulfation would be helpful for this purpose. The interactions between C-type lectins and tumor-associated glycans have previously been described. Mannan-binding protein recognizes human colorectal carcinoma cells through clusters of tandem repeats of the Lewisb/Lewisa epitopes, resulting in anti-tumor immunity (36, 37). DC-SIGN was also shown to recognize colorectal cancer cells through the recognition of LewisX and LewisY antigens present on carcinoembryonic antigen, whereas normal colon tissues with low levels of LewisX and LewisY antigens do not interact with DCSIGN (38, 39). Macrophage galactose-binding lectin was reported to recognize Tn antigens present on MUC1, which are tumor-associated glycosylation in colon carcinoma (40). Therefore, it is possible that Langerin may be a novel member of C-type lectins, which recognize tumor-specific glycosylation and is involved in anti-tumor immunity. Langerin also exhibited binding to heavily mannosylated glycans such as mannan, which is a major structural component of fungal cell walls. Previously, Takahara et al. (41) reported that Langerin mediates the uptake of C. albicans. In this study, we have confirmed the binding ability of Langerin to live pathogenic fungi. Langerin was found to recognize a series of Candida species as well as Malassezia furfur in a Ca2+-dependent manner, whereas no binding was observed for C. neoformans. Indeed, there is a striking difference between the structures of mannosylated glycans in their cell walls, e.g. between C. albicans (42) and C. neoformans (43): C. albicans expresses N- and O-linked mannoproteins, whereas C. neoformans expresses glucuronoxylomannan as a major component. Together with the observation that Langerin exhibits no binding to thyroglobulin expressing high mannose-type N-glycans, mannose multivalency, geometry, and/or spatial distribution might be important factors for Langerin recognition (44). In this study, Langerin was shown to bind to sulfated as well as mannosylated glycans. We have also demonstrated its potential functions on glioma biology and anti-fungal immunity to be exerted through the C-type CRD. The mannose receptor expressed on macrophages, a member of the C-type lectin, is known to bind to both sulfated and mannosylated glycans. In this case, however, such dual specificities are attributed to distinct CRDs, of which the evolutionary origins are different: the former is performed by an R-type CRD, whereas the latter is performed by consecutive C-type CRDs present in a single polypeptide. In this context, Langerin is extremely unique among C-type lectins, recognizing both sulfated and mannosylated glycans via a single C-type CRD. Such specificity of Langerin should be directly involved in the functions of LCs. Supplementary Material Supplemental Data Click here to view. Acknowledgments We thank M. Fukumura, Y. Kubo, and K. Kiyohara for technical assistance.

This work was supported in part by the New Energy and Industrial Technology Development Organization, grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan (to H. T.), and the National BioResource Project in Japan. The on-line version of this article (available at contains supplemental Figs. 13 and Table 1.

The abbreviations used are: Langerhans cell Chinese hamster ovary carbohydrate recognition domain


DC-SIGN dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin FAC Gal KS KS-I KS-II LacNAc Man PA BG mAb HIV-1 FITC PAA frontal affinity chromatography d-galactose keratan sulfate bovine corneal KS shark cartilaginous KS N-acetyllactosamine d-mannose pyridylamino Birbeck granule monoclonal antibody human immunodeficiency virus, type 1 fluorescein isothiocyanate polyacrylamide.

Other Sections

REFERENCES 1. Romani N., Holzmann S., Tripp C. H., Koch F., Stoitzner P. (2003) APMIS 111, 725740. [PubMed] 2. Valladeau J., Ravel O., Dezutter-Dambuyant C., Moore K., Kleijmeer M., Liu Y., DuvertFrances V., Vincent C., Schmitt D., Davoust J., Caux C., Lebecque S., Saeland S. (2000) Immunity 12, 7181. [PubMed]

3. Valladeau J., Dezutter-Dambuyant C., Saeland S. (2003) Immunol. Res. 28, 93107. [PubMed] 4. Weis W. I., Taylor M. E., Drickamer K. (1998) Immunol. Rev. 163, 1934. [PubMed] 5. McDermott R., Bausinger H., Fricker D., Spehner D., Proamer F., Lipsker D., Cazenave J. P., Goud B., De La Salle H., Salamero J., Hanau D. (2004) J. Invest. Dermatol. 123, 7277. [PubMed] 6. Idoyaga J., Suda N., Suda K., Park C. G., Steinman R. M. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 15241529. [PMC free article] [PubMed] 7. de Witte L., Nabatov A., Pion M., Fluitsma D., de Jong M. A., de Gruijl T., Piguet V., van Kooyk Y., Geijtenbeek T. B. (2007) Nat. Med. 13, 367371. [PubMed] 8. Kissenpfennig A., At-Yahia S., Clair-Moninot V., Stssel H., Badell E., Bordat Y., Pooley J. L., Lang T., Prina E., Coste I., Gresser O., Renno T., Winter N., Milon G., Shortman K., Romani N., Lebecque S., Malissen B., Saeland S., Douillard P. (2005) Mol. Cell Biol. 25, 8899. [PMC free article] [PubMed] 9. Thpaut M., Valladeau J., Nurisso A., Kahn R., Arnou B., Vivs C., Saeland S., Ebel C., Monnier C., Dezutter-Dambuyant C., Imberty A., Fieschi F. (2009) Biochemistry 18, 2684 2698. 10. Stambach N. S., Taylor M. E. (2003) Glycobiology 13, 401410. [PubMed] 11. Galustian C., Park C. G., Chai W., Kiso M., Bruening S. A., Kang Y. S., Steinman R. M., Feizi T. (2004) Int. Immunol. 16, 853866. [PubMed] 12. Tateno H., Mori A., Uchiyama N., Yabe R., Iwaki J., Shikanai T., Angata T., Narimatsu H., Hirabayashi J. (2008) Glycobiology 18, 789798. [PubMed] 13. Tateno H., Nakamura-Tsuruta S., Hirabayashi J. (2007) Nat. Protoc. 2, 25292537. [PubMed] 14. Nakamura-Tsuruta S., Kominami J., Kamei M., Koyama Y., Suzuki T., Isemura M., Hirabayashi J. (2006) J. Biochem. 140, 285291. [PubMed] 15. Sato T., Sato M., Kiyohara K., Sogabe M., Shikanai T., Kikuchi N., Togayachi A., Ishida H., Ito H., Kameyama A., Gotoh M., Narimatsu H. (2006) Glycobiology 16, 11941206. [PubMed] 16. Weis W. I., Drickamer K. (1996) Annu. Rev. Biochem. 65, 441473. [PubMed] 17. Chatwell L., Holla A., Kaufer B. B., Skerra A. (2008) Mol. Immunol. 45, 19811994. [PubMed] 18. Torii T., Fukuta M., Habuchi O. (2000) Glycobiology 10, 203211. [PubMed] 19. Funderburgh J. L. (2000) Glycobiology 10, 951958. [PubMed]