Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
Received on December 13, 2002; revised on January 6, 2003; accepted on January 6, 2003
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Abstract |
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Key words: carbohydrate recognition / C-type lectin / endocytosis / langerin
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Introduction |
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Langerin is orientated as a type II transmembrane protein with an extracellular region consisting of a neck and a C-terminal C-type carbohydrate-recognition domain (CRD) (Valladeau et al., 2000, 2002
; Takahara et al., 2002
). Binding of mouse langerin to mannan-agarose has been demonstrated (Takahara et al., 2002
), consistent with the presence in the langerin CRD of residues shown to be necessary for Ca2+-dependent binding of mannose and related sugars to C-type CRDs (Drickamer, 1992
). The domain organization and transmembrane orientation of langerin is similar to that of several other endocytic C-type lectins, including the hepatic asialoglycoprotein receptor and another dendritic cell receptor, DC-SIGN (Halberg et al., 1987
; Mitchell et al., 2001
). These receptors oligomerize through the formation of
-helical coiled coils in the neck region, and this oligomerization is important for binding of carbohydrate ligands. Preliminary studies indicate that detergent solubilized mouse langerin in cell lysates can be cross-linked to form higher oligomers, but the precise nature of the oligomeric structures has not yet been determined (Takahara et al., 2002
).
This article presents biochemical characterization and carbohydrate-binding studies of recombinant fragments of human langerin, as well as analysis of endocytosis of a neoglycoprotein ligand by langerin. The results provide evidence that langerin has a role in internalization of glycoprotein antigens.
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Results |
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Discussion |
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The CRD of langerin shows specificity for monosaccharides similar to several other mannose-binding C-type lectins, including DC-SIGN and DC-SIGNR (Mitchell et al., 2001), MBP (Drickamer, 1992
), the mannose receptor (Taylor et al., 1992
), and Endo180 (East et al., 2002
). The langerin CRD contains all five residues shown to ligate Ca2+ and sugar in other C-type CRDs (Figure 2). Thus, it is likely that the mechanism of sugar binding by the langerin CRD involves ligation of two equatorial hydroxyl groups of a monosaccharide by two pairs of glutamic acid and asparagine residues at the conserved principal Ca2+ site as is seen in the crystal structures of MBP, DC-SIGN, and DC-SIGNR (Weis et al., 1992
). However, the CRDs of DC-SIGN and DC-SIGNR differ from that of MBP in their ability to bind to an internal mannose residue at the principal Ca2+ site and to form contacts with additional residues in a high mannose oligosaccharide (Feinberg et al., 2001
). MBP binds only the terminal residue, with the rest of the oligosaccharide pointing away from the CRD (Weis et al., 1992
). It is not possible to say whether the CRD of langerin binds a terminal mannose residue or an internal one at the principal Ca2+ site. However, the inhibition studies with Man9GlcNAc2 and mannose showing enhanced binding of Man9GlcNAc2 suggest that as in the CRDs of DC-SIGN and DC-SIGNR, the langerin CRD may contact more than one monosaccharide in the oligosaccharide. Enhancement of Man9GlcNAc2 binding compared to mannose is also seen for the CRDs of DC-SIGN and DC-SIGNR, although to a much greater extent than demonstrated here for the langerin CRD (Mitchell et al., 2001
), but is not seen with the CRD of MBP (Mitchell et al., 2001
; Lee et al., 1992
).
The finding that the neck region of langerin mediates oligomerization and forms a coiled-coil of -helices is consistent with the presence of 17 typical heptad repeats in this region (http://ctld.glycob.ox.ac.uk) (Lupas, 1996
). Oligomerization of polypeptides containing a single CRD is a common feature of many C-type lectins and is important for determining specificity as well as affinity for oligosaccharides. Typically, C-type CRDs bind a single monosaccharide residue with mM affinity and must be clustered to allow multivalent high-affinity binding to oligosaccharides (Weis and Drickamer, 1996
). Trimerization of langerin is clearly essential for binding of oligosaccharides because binding of the CRD to glycoproteins could not be detected in the solution-phase blotting assays.
Two other C-type lectins that form trimers are the asialoglycoprotein receptor and MBP. In each case, trimerization is essential for high affinity binding to oligosaccharides. However, the arrangements of the CRDs in the trimers confer very different specificities on each of these proteins. The mannose-binding CRDs of MBP are spaced too far apart to allow binding to more than one terminal residue of a mammalian high mannose oligosaccharide, but they are appropriately spaced to bind mannose residues arrayed on the surfaces of pathogens (Weis and Drickamer, 1996). Thus MBP is unable to bind mammalian high mannose oligosaccharides, so inappropriate complement fixation is prevented. In contrast, the geometric arrangement of three galactose-binding CRDs in the asialoglycoprotein receptor allows high-affinity binding to a desialylated triantennary complex oligosaccharide (Rice et al., 1990
). Langerin differs from MBP in being able to bind mammalian high mannose structures. Thus it is likely that the CRDs in the langerin trimer must be clustered more closely together than those in MBP. The arrangement of CRDs in langerin must also be different from that in DC-SIGN and DC-SIGNR because these two proteins both form tetramers rather than trimers, an arrangement that is believed to be important for binding multiple high mannose structures (Mitchell et al., 2001
).
The data presented herein showing that langerin can bind mammalian high mannose structures as well as yeast invertase, together with the earlier finding that langerin can bind to yeast mannan immobilized on agarose (Takahara et al., 2002), indicate that langerin can bind endogenous glycoproteins as well as glycoconjugates of microorganisms. The specificity of the CRD for GlcNAc as well as mannose indicates that langerin will probably bind glycoconjugates found on Gram-negative bacteria, as well as the mannose-containing structures of yeast and other fungi. The ability to bind both endogenous and exogenous glycoconjugates is a feature that langerin shares with the mannose receptor. However, the arrangement of CRDs allowing multivalent binding in the mannose receptor is very different from that of langerin and most other C-type lectins because it contains multiple C-type CRDs in a single polypeptide (Taylor et al., 1990
).
The demonstration that langerin is able to mediate endocytosis and subsequent degradation of a glycoconjugate ligand provides evidence consistent with a role for this protein in uptake of glycoprotein antigens. Although langerin is unusual among endocytic receptors in inducing formation of birbeck granules, the kinetics of uptake and degradation of ligand by langerin are very similar to those for other endocytic C-type lectins, suggesting that ligand is targeted to lysosomes. Capture of antigens followed by antigen processing and presentation of peptides in complex with MHC class II molecules to T cells is the major function of Langerhans cells. Many soluble antigens are taken up by fluid-phase pinocytosis, but it is clear that receptor-mediated endocytosis is also important for antigen uptake (Banchereau and Steinman, 1998). Binding and endocytosis of glycoconjugates by langerin would enhance processing and presentation of this class of antigens. Because langerin can bind oligosaccharides found on mammalian proteins as well as those found on microorganisms, it is likely to play a role in uptake and processing of both self and nonself antigens. Release of ligand by langerin at low pH is consistent with ligand release in an endosomal compartment. As for several other endocytic C-type lectins, pH-dependent release of glycoconjugates from langerin appears to be due to a pH-dependent change in Ca2+ affinity, leading to loss of Ca2+ binding and therefore loss of sugar binding (Loeb and Drickamer, 1987; Mullin et al., 1994
; Feinberg et al., 2000
; Wragg and Drickamer, 1999
). Each receptor seems to have a different mechanism for bringing about this pH-dependent loss of Ca2+; in langerin, interactions between the CRDs in the trimer may be required. Alternatively, it is possible that a pH-dependent conformational change in the coiled-coil neck region near the ligand binding domains contributes to loss of ligand binding by langerin, as is thought to be the case in the macrophage scavenger receptor (Suzuki et al., 1997
).
It is interesting that dendritic cells express several different mannose-binding endocytic C-type lectins. Langerin, DC-SIGN, and the mannose receptor are all potentially capable of playing a role in internalization of glycoprotein antigens by dendritic cells. However, although there is likely to be some overlap in ligand specificity of these three proteins, differences in the ways that the CRDs interact with monosaccharides and in the arrangement of multiple CRDs suggest that each of these proteins may have a distinct subset of glycoconjugate ligands. In addition, the expression patterns of the three proteins are different. The mannose receptor, though present on some populations of dendritic cells, is also expressed on macrophages, including Kupffer cells in the liver, as well as liver endothelial cells. Although the mannose receptor may play a role in the immune response by mediating endocytosis of glycoconjugates of pathogens, its major function appears to be in clearance of proteins bearing high mannose oligosaccharides, such as lysosomal enzymes that are released as part of the inflammatory response (Lee et al., 2002). DC-SIGN has a more restricted expression pattern, mainly confined to subsets of dendritic cells, and analysis of uptake, processing, and presentation of a DC-SIGN antibody by dendritic cells suggests that this receptor can enhance uptake and presentation of antigens (Engering et al., 2002
). In contrast, expression of langerin appears to be restricted to Langerhans cells, and neither the mannose receptor nor DC-SIGN is expressed on this subset of dendritic cells (Turville et al., 2002
). Thus langerin is likely to be the major receptor involved in endocytosis of glycoprotein antigens by Langerhans cells.
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Materials and methods |
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Cloning of langerin cDNA
cDNA encoding human langerin was amplified from human lung cDNA (Marathon Ready cDNA, Clontech, Oxford, UK) with forward primer 5'-aaggccggccaa- gggtgagcactcaggatgactgtggaga-3' and reverse primer 5'-ttgcggccgctcacggttctgatgggacatagggtcgctt-3'. Following denaturation at 95°C for 1 min, 40 cycles of 95°C for 30 s, and 68°C for 1 min were carried out. Polymerase chain reaction (PCR) products were cloned into the vector pCR II-TOPO using the TOPO cloning kit (Invitrogen) and sequenced using an ABI prism 310 Genetic Analyzer. Portions of cDNAs free of reverse transcription errors were combined using convenient restriction sites.
Expression and purification of soluble langerin fragments
The region of the cDNA coding for the CRD only was amplified using the forward primer 5'-aaggccggcccaggtggtttctcaaggctggaagtacttc-3' and the reverse primer already described. The primers include restriction sites for FseI or NotI. PCR products were digested with FseI and NotI and inserted into a modified pINIIIompA2 expression vector containing restriction sites for FseI and NotI downstream of the ompA signal sequence (Ghrayeb et al., 1984). The correct reading frame was generated by digesting the vector with FseI, followed by trimming of the 3' extension with T4 polymerase. The integrity of the final expression plasmid was verified by DNA sequencing. Luria-Bertani medium (1 L), containing 50 µg/ml ampicillin, was inoculated with 30 ml of an overnight culture of Escherichia coli strain JA221 transformed with the langerin CRD expression plasmid. The culture was grown with shaking at 2530°C to an A550 of approximately 1, and isopropyl-ß-D-thiogalactoside and CaCl2 were added to final concentrations of 50 µM and 100 mM, respectively. After growth for a further 18 h at 2530°C, cells were harvested by centrifugation at 4000 rpm for 15 min in a Beckman JS-4.2 rotor (Beckman, High Wycombe, UK). Bacterial pellets were resuspended in cold 10 mM Tris-HCl, pH 7.8, followed by centrifugation at 12,000 rpm for 15 min at 4°C in a Beckman JA14 rotor. Bacteria were sonicated in 30 ml 25 mM Tris-HCl, pH 7.8, 0.15 M NaCl, 25 mM CaCl2 (loading buffer). Lysed bacteria were centrifuged at 10,000xg for 15 min, and the supernatant was recentrifuged at 100,000xg for 1 h at 4°C. The supernatant was passed over a 10-ml column of mannose-Sepharose equilibrated in loading buffer. The column was washed with 24 ml loading buffer and eluted with 10x2 ml elution buffer (25 mM Tris-HCl, pH 7.8, 0.15 M NaCl, 2 mM EDTA). Fractions were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) and langerin CRD was identified by N-terminal sequencing on a Beckman LF3000 protein sequencer following transfer to polyvinylidene difluoride membranes (Matsudaira, 1987
).
The DNA coding for the langerin extracellular domain was cloned into the pT5T expression vector using appropriate restriction sites and a synthetic oligonucleotide designed to bridge the end of the coding sequence and the BamHI site of the vector. The resulting plasmid was transformed into E. coli strain BL21/DE3. Luria-Bertani medium (1 L), containing 50 µg/ml ampicillin, was inoculated with 25 ml of an overnight culture of bacteria containing the langerin extracellular domain expression plasmid. The culture was grown with shaking at 37°C to an A550 of approximately 0.5, and protein expression was induced by addition of isopropyl-ß-D-thiogalactoside to a final concentration of 100 mg/L. After growth for a further 3 h at 37°C, cells were harvested by centrifugation at 4000 rpm for 15 min in a Beckman JS-4.2 rotor. Bacterial pellets were resuspended in cold 10 mM Tris-HCl, pH 7.8, followed by centrifugation at 12,000 rpm for 15 min at 4°C in a Beckman JA14 rotor. Bacteria were lysed by sonication (4 bursts of 30 s duration) in 25 ml 10 mM Tris-HCl, pH 7.8, and inclusion bodies were isolated by centrifugation at 10,000xg for 15 min at 4°C. The pellet was solubilized by brief sonication in 20 ml 6 M guanidine-HCl containing 100 mM Tris-HCl, pH 7.0, and 0.01 % ß-mercaptoethanol and incubated at 4°C for 30 min. The mixture was centrifuged at 100,000xg for 30 min at 4°C, and the supernatant was diluted threefold with cold loading buffer by slow addition with stirring. The diluted mixture was dialyzed against 3x2 L of loading buffer. After dialysis, insoluble precipitate was removed by centrifugation at 100,000xg for 1 h at 4°C, and the supernatant was loaded onto a 2-ml column of mannose-Sepharose equilibrated in loading buffer. The column was washed with 10 ml loading buffer and eluted with 8x1 ml elution buffer. Fractions were analyzed by SDSPAGE. The identity of the langerin extracellular domain was confirmed by N-terminal sequencing.
Analytical ultracentrifugation
Equilibrium sedimentation analysis was carried out in a Beckman Optima XL-A analytical ultracentrifuge equipped with absorbance optics using an An60Ti rotor at 20°C. Analysis of langerin extracellular domain was performed at 9000 rpm, 12,000 rpm, and 14,000 rpm. For the CRD, rotor speeds of 20,000 and 25,000 were used. Equilibrium distributions from three different loading concentrations were analyzed simultaneously using the Nonlin curve fit program supplied with the instrument. Partial specific volumes for the proteins were determined from their amino acid composition (Cohn and Edsall, 1943).
Circular dichroism spectroscopy
Circular dichroism spectra were measured on a Jasco J600 spectrophotometer (Jasco, Great Dunmow, UK) using 200-µl samples in a 1 mm quartz cuvette at 20°C. Five scans from 190 to 250 nm were carried out on each sample using a band width of 1 nm and a scan rate of 20 nm/min. Protein concentrations were determined using the alkaline ninhydrin assay (Hirs, 1967).
Sugar competition assays
Plastic microtiter plates with removable wells (Immulon 4) were coated with langerin CRD or extracellular domain (50 µl/well of 100 µg/ml solutions in loading buffer). Following incubation overnight at 4°C, the wells were washed three times with cold loading buffer, filled with 5% (w/v) BSA in loading buffer and incubated for 2 h at 4°C. After washing the wells three times with cold loading buffer, aliquots (100 µl) of a range of concentrations of monosaccharide or oligosaccharide in loading buffer containing 125I-Man-BSA (1 µg/ml) and 5% BSA were added to the wells in duplicate. Following incubation at 4°C for 2 h, the wells were washed four times with cold loading buffer and counted on a gamma counter. Values for Ki (the inhibitor concentration that gives 50% inhibition of 125I-Man-BSA binding) for each inhibitor were determined by fitting the data to the following equation for simple competitive inhibition: fraction of maximal binding = KI/(KI + [Inhibitor]).
pH dependence assays
pH dependence of 125I-Man-BSA binding to langerin CRD and langerin extracellular domain was determined as described for the sugar competition assays, except that 125I-Man-BSA was incubated with buffers of different pH rather than with inhibitors.
Glycoprotein blotting and neoglycolipid overlays
For iodination, 20 µl (0.1 mCi) of Bolton Hunter reagent was dried with argon and langerin CRD or langerin extracellular domain (100 µg in 200 µl of 25 mM HEPES, pH 7.8, 100 mM NaCl, 25 mM CaCl2) was added. After incubation for 10 min at room temperature, the reaction was stopped by addition of 800 µl loading buffer, and labeled protein was isolated on a 2-ml column of mannose-Sepharose. Glycoprotein samples were run on 17.5% SDSpolyacrylamide gels and transferred to nitrocellulose. The nitrocellulose membrane was blocked with 2% hemoglobin in loading buffer for 1 h at room temperature and incubated with 125I-labeled langerin CRD or extracellular domain in loading buffer containing 2% hemoglobin for 90 min. Following four washes for 5 min with cold loading buffer, radioactivity was detected using a phosphorimager. Neoglycolipids were prepared and resolved following published procedures (Mizuochi et al., 1989). The chromatograms were blocked and incubated with 125I-labeled langerin extracellular domain as described.
Expression of full-length langerin in fibroblasts
DNA coding for full-length langerin was inserted into the retroviral expression vector pVcos (Maddon et al., 1985) at the EcoRI site. The neomycin-resistance gene, under the control of the herpes virus thymidine kinase promoter, was inserted into the resulting vector at the unique ClaI site (Southern and Berg, 1982
). The final plasmid was transfected into
Cre cells (Danos and Mulligan, 1988
) using the calcium phosphate method (Wigler et al., 1979
). Following incubation of the cells with the calcium phosphate-DNA coprecipitate for 4 h at 37°C, cells were grown for 48 h in Dulbecco's modified Eagle's medium containing 10% calf serum. Medium containing pseudovirus was collected, filtered through a 0.45-µm filter, and used to infect rat 6 cells for 2 h in the presence of 8 µg/ml polybrene. Infected cells were grown overnight in Dulbecco's modified Eagle's medium containing 10% calf serum before initiating selection by inclusion of 400 µg/ml G418 in the medium. After approximately 2 weeks, colonies were isolated by trypsinization within cloning cylinders. Langerin expression was detected by western blotting of cell lysates using a rabbit polyclonal antibody raised against bacterially expressed langerin CRD (prepared by Eurogentec, Seraing, Belgium). Analysis of uptake and degradation of 125I-Man-BSA by fibroblasts expressing langerin was performed as described previously for cells expressing the chicken hepatic lectin (Mellow et al., 1988
), except that specificity was measured using an excess of unlabeled ligand.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: mt{at}glycob.ox.ac.uk
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Abbreviations |
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References |
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