Characterization of carbohydrate recognition by langerin, a C-type lectin of Langerhans cells

Nicola S. Stambach and Maureen E. Taylor1

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


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Langerin is a type II transmembrane cell surface receptor found on Langerhans cells. The extracellular domain of langerin consists of a neck region containing a series of heptad repeats and a C-terminal C-type carbohydrate-recognition domain (CRD). A role for langerin in processing of glycoprotein antigens has been proposed, but until now there has been little study of the langerin protein. In this study, analytical ultracentrifugation and circular dichroism spectroscopy of recombinant soluble fragments of human langerin have been used to show that the extracellular region of this receptor exists as a stable trimer held together by a coiled coil of {alpha}-helices formed by the neck region. The langerin CRD shows specificity for mannose, GlcNAc, and fucose, but only the trimeric extracellular domain fragment binds to glycoprotein ligands. Langerin extracellular domain binds mammalian high mannose oligosaccharides, as well mannose-containing structures on yeast invertase but does not bind complex glycan structures. Full-length langerin stably expressed in rat fibroblast transfectants mediates efficient uptake and degradation of a mannosylated neoglycoprotein ligand. pH-dependent ligand release appears to involve interactions between the CRDs or between the CRDs and the neck region in the trimer. The results are consistent with a role for langerin in internalization of both self and nonself glycoprotein antigens.

Key words: carbohydrate recognition / C-type lectin / endocytosis / langerin


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Langerin (CD207) is a cell surface receptor unique to Langerhans cells, a subset of immature dendritic cells located in skin epidermis and mucosal tissues (Valladeau et al., 1999Go, 2000Go). Langerhans cells, like other dendritic cells, perform an essential function in the immune response with their ability to take up and process foreign and self antigens and then present them to T cells after migration to lymph nodes (Banchereau and Steinman, 1998Go). Langerin recycles between the plasma membrane and early endosomes and is associated with birbeck granules, subdomains of the endosomal recycling compartment that are specific to Langerhans cells (Valladeau et al., 1999Go, 2000Go; McDermott et al., 2002Go). Birbeck granules appear to form where langerin accumulates following internalization (McDermott et al., 2002Go). It has been proposed that langerin may play a role in antigen uptake by Langerhans cells, but up to now endocytosis has only been demonstrated using anti-langerin antibodies and no studies of ligand binding have been carried out (Valladeau et al., 1999Go, 2000Go; McDermott et al., 2002Go).

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., 2000Go, 2002Go; Takahara et al., 2002Go). Binding of mouse langerin to mannan-agarose has been demonstrated (Takahara et al., 2002Go), 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, 1992Go). 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., 1987Go; Mitchell et al., 2001Go). These receptors oligomerize through the formation of {alpha}-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., 2002Go).

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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Expression and physical characterization of langerin fragments
Two fragments of human langerin, one consisting of the CRD only, the other consisting of the whole extracellular region containing both the neck and the CRD, were expressed in bacteria (Figure 1). Consistent with the presence of the sequence EPN in the CRD of langerin (Figure 2), which is characteristic of mannose specificity, both the expressed CRD and the extracellular domain were found to bind to mannose-Sepharose (Figure 3). The CRD binds weakly, with protein appearing in the Ca2+-containing wash fractions as well as in the fractions eluted with ethylenediamine tetra-acetic acid (EDTA), but it was possible to purify the CRD on a long column. In contrast, langerin extracellular domain binds much more tightly to mannose-Sepharose and is only seen in eluted fractions. Weak binding of isolated CRDs to monosaccharide-Sepharose columns compared to stronger binding of larger fragments has been seen for other C-type lectins and is usually due to oligomerization of the larger fragments leading to multivalent binding (Mitchell et al., 2001Go; Bouyain et al., 2002Go).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Langerin expression constructs. The domain organization of full-length langerin is shown at the top, with the two soluble fragments produced by bacterial expression shown below. The starting amino acid residue of each expression construct is indicated, with numbering based on the residue numbers of full-length langerin.

 


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 2. Sequence comparisons of CRDs of mannose-binding C-type lectins. The langerin CRD is aligned with the CRDs of rat serum mannose-binding protein (MBP-A), the CRDs of human DC-SIGN and DC-SIGNR and CRD4 from the human mannose receptor (ManR). 1 and 2 denote residues involved in ligating two Ca2+ (Ca2+ 1 and Ca2+ 2) to MBP-A (Weis et al., 1992Go). Sugar is ligated at Ca2+ site 2, also known as the principal Ca2+ site.

 


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 3. Purification of langerin fragments by affinity chromatography on mannose-Sepharose. Langerin CRD and extracellular domain were expressed in 1 L bacterial culture. Langerin CRD was purified on a 10-ml column of mannose-Sepharose. The column was washed with 12x2-ml fractions (W) of Ca2+-containing buffer and eluted with 8x2-ml fractions (E) of EDTA-containing elution buffer (upper). Langerin extracellular domain was purified on a 2-ml column washed with 10 ml Ca2+-containing buffer and eluted with 8x1-ml of elution buffer (lower). Fractions were analyzed on 17.5% polyacrylamide gels that were stained with Coomassie blue.

 
The oligomeric state of the langerin fragments was investigated using analytical ultracentrifugation. Sedimentation equilibrium analysis of the langerin extracellular domain (Figure 4) shows that it exists as a single stable species with an apparent molecular mass (89.5 ± 12.3 kDa) corresponding to the theoretical value for a trimer (89.5 kDa). In contrast, the CRD is largely monomeric, although weak self-association is seen with increasing concentration. The results indicate that the neck region of langerin is essential for formation of trimers. The weak self-association of the CRD may represent contacts that would normally be formed between CRDs in the trimeric extracellular domain or could be artifactual due to exposure of hydrophobic regions that would be covered by the neck region in the trimer (Weis and Drickamer, 1994Go; Weis et al., 1991Go).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. Sedimentation equilibrium ultracentrifugation analysis of langerin fragments. Samples of langerin extracellular domain (0.5 mg/ml) or CRD (0.35 mg/ml) were loaded into the ultracentrifuge cells undiluted (circles), twofold diluted (squares), and fourfold diluted (triangles). Analyses of the extracellular domain at 14,000 rpm and the CRD at 25,000 rpm are shown. Absorbance was measured at 280 nm for the extracellular domain and 290 nm for the CRD. Molecular masses for dimers (59.7 kDa), trimers (89.5 kDa), and tetramers (119.4 kDa) of the extracellular domain calculated from the amino acid sequences are indicated, as are those for monomers (15.7 kDa) and dimers (31.3 kDa) of the CRD. The average apparent molecular mass obtained for the extracellular domain from analysis at 9000 rpm, 12,000 rpm, and 14,000 rpm is 89.5 ± 12.3 kDa. The average apparent molecular mass obtained for the CRD from analysis at 20,000 rpm and 25,000 rpm is 17.6 ± 0.9 kDa.

 
The secondary structure of the neck region of langerin was investigated using circular dichroism spectroscopy. The spectrum for the neck region only was calculated as a difference spectrum using spectra obtained for the extracellular domain and for the CRD (Figure 5). The difference spectrum shows the characteristics of an {alpha}-helical conformation with minima at 208 and 223 nm and a maximum at 193 nm. The results are consistent with formation of a trimeric coiled coil of {alpha}-helices in the neck region of langerin.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5. Determination of the secondary structure of the neck region of langerin by circular dichroism. The circular dichroism spectrum of the neck region of langerin (C) was calculated as the difference between the spectrum of the extracellular domain (A) and the spectrum of the CRD (B). The mean residue ellipticity at 220 nm for the 128-amino acid neck region in the difference spectrum is approximately 30x103 deg cm2/dmol.

 
Sugar-binding specificity of langerin CRD
The sugar-binding properties of langerin CRD were investigated using a solid phase assay in which monosaccharides or oligosaccharides compete for binding of 125I-Man–bovine serum albumin (BSA) to immobilized CRD (Table I). Mannose, GlcNAc, and fucose are approximately equally effective as inhibitors of 125I-Man-BSA binding, showing inhibition constants of 2–3 mM. The weak inhibition seen with galactose is likely to be due to interaction with the anomeric hydroxyl of the free sugar because {alpha}-methylgalactoside does not inhibit binding of 125I-Man-BSA. This nonphysiological binding of galactose has also been seen with other mannose-binding C-type lectins (Mitchell et al., 2001Go; Ng et al., 1996Go; East et al., 2002Go). The results indicate that langerin, like other C-type lectins, discriminates between monosaccharides through recognition of C-3 and C-4 hydroxyl groups. Like other mannose-binding C-type lectins, langerin binds preferentially to sugars with equatorial C-3 and C-4 hydroxyl groups. Binding appears to be affected by substituents at the 2 position, because N-acetylmannosamine is approximately 2.5-fold less effective an inhibitor than mannose and GlcNAc, suggesting that the N-acetyl group in the axial position interferes with binding. Glucose is also less effective an inhibitor than GlcNAc, suggesting that interactions with the equatorial N-acetyl group might contribute to binding of GlcNAc. Overall, langerin shows specificity for the same range of monosaccharides as other mannose-binding C-type lectins such as the mannose receptor, DC-SIGN, and serum mannose-binding protein (MBP) (Taylor et al., 1992Go; Mitchell et al., 2001Go; Drickamer, 1992Go).


View this table:
[in this window]
[in a new window]
 
Table I. Sugar binding by langerin CRD

 
Interestingly, the Man9GlcNAc2 oligosaccharide is a 10-fold more effective inhibitor on a molar basis of 125I-Man-BSA binding to langerin CRD than is mannose (Figure 6 and Table I). This is a greater enhancement of binding than would be expected if it were simply due to the increased concentration (threefold) of terminal mannose residues. Based on sequence similarity to other C-type CRDs (Drickamer, 1992Go) and the specificity studies described, each langerin CRD is predicted to ligate a single mannose residue to a Ca2+ through hydroxyl groups 3 and 4. However, the inhibition results with the Man9GlcNAc2 oligosaccharide suggest that there may be additional interactions between the CRD and other parts of the oligosaccharide.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6. Inhibition of 125I-Man-BSA binding to langerin CRD by mannose and Man9GlcNAc2. Data were obtained using the competition assay. Experimental values (symbols) are shown together with the theoretical curves (lines) fitted to the data.

 
Glycoprotein binding by langerin
Langerin binding to oligosaccharides was further investigated using radioiodinated CRD or extracellular domain to probe a glycoprotein blot containing a range of oligosaccharide structures. Using this method, binding of langerin CRD to glycoproteins was not detected, consistent with the low affinity of the monomeric CRD for sugars. However, langerin extracellular domain binds to glycoproteins containing high mannose structures, confirming that oligomerization of langerin is essential for high affinity binding to oligosaccharides (Figure 7). The extracellular domain binds strongly to yeast invertase, which is heavily glycosylated with mannose-containing oligosaccharides. Langerin extracellular domain also binds to bovine ribonuclease B and soybean agglutinin, each of which contains a single glycosylation site occupied by a high mannose oligosaccharide, as well as to ovalbumin, which contains some high mannose oligosaccharides in addition to hybrid structures. Weak binding to horseradish peroxidase is detected, consistent with the low proportion of high mannose structures found on this glycoprotein. Langerin does not bind to glycoproteins containing complex oligosaccharides, even when the oligosaccharides have been desialylated. The results with glycoproteins were confirmed by probing neoglycolipids prepared by coupling oligosaccharides released from glycoproteins to phosphatidylethanolamine dipalmitate (Mizuochi et al., 1989Go). Langerin extracellular domain binds to Man5–Man9 oligosaccharides released from bovine ribonuclease B, but not to asialo bi-, tri-, or tetraantennary complex oligosaccharides (data not shown).



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 7. Binding of langerin extracellular domain to glycoproteins. Glycoproteins (2.5 µg of each) were resolved by SDS–PAGE on a 17.5% gel, blotted onto nitrocellulose, and incubated with radioiodinated langerin extracellular domain.

 
Endocytosis of a neoglycoprotein by langerin
To investigate whether langerin is capable of endocytosing mannose-containing ligands, rat-6 fibroblast cell lines stably transfected with full-length langerin were produced. Fibroblasts were chosen as recipient cells because previous studies have shown that transfection of langerin into fibroblast cell lines induces formation of birbeck granules, suggesting that trafficking of langerin in these cells will be similar to that in Langerhans cells (Valladeau et al., 2000Go, 2002Go). In addition, Rat-6 fibroblast transfectants have been used to study endocytosis by other C-type lectins, including the chicken hepatic lectin and the mannose receptor (Mellow et al., 1988Go; Taylor et al., 1990Go). Transfectants expressing langerin were tested for their ability to take up and degrade 125I-Man-BSA. Radioiodinated Man-BSA was used as the test ligand because this neoglycoprotein binds to the langerin CRD in the solid phase binding assay and has been used extensively for analysis of endocytosis by the mannose receptor (Taylor et al., 1990Go, 1992Go). The langerin expressing cells efficiently endocytose 125I-Man-BSA (Figure 8). Cell-associated radioactivity increases rapidly on warming and reaches a plateau. After a short lag period, degraded ligand, measured as trichloroacetic acid–soluble radioactivity, is released into the medium. The results are very similar to those seen with rat-6 cells expressing the mannose receptor (Taylor et al., 1990Go, 1992Go; data not shown).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8. Uptake and degradation of 125I-Man-BSA by transfected cells expressing langerin. Cells (2.5 x 106/35-mm plate) were preincubated with 125I-Man-BSA for 30 min at 4°C. Following incubation at 37°C for the times indicated, radioactivity associated with cells (open symbols) and acid-soluble fragments in the medium (closed symbols) were analyzed as described (Mellow et al., 1988Go). Circles, processing of 125I-Man-BSA present at a concentration of 1 µg/ml; triangles, values at 5 h when a 50-fold excess of unlabeled Man-BSA was present throughout the experiment.

 
The fact that 125I-Man-BSA internalized following binding to langerin at the cell surface is subsequently degraded indicates that the ligand must be released from langerin in an internal compartment and targeted to the lysosomes. Other endocytic C-type lectins release ligand in the acidic environment of the endosomes. For the chicken hepatic lectin, the mannose receptor and the asialoglycoprotein receptor, pH-dependent ligand release is associated with a decrease in affinity for Ca2+ at low pH, so that bound Ca2+ and therefore bound sugar are released from the C-type CRDs (Loeb and Drickamer, 1988Go; Mullin et al., 1994Go; Feinberg et al., 2000Go; Wragg and Drickamer, 1999Go). However, the exact mechanism for pH-related change in Ca2+ affinity seems to be different in each of these receptors. pH dependence of 125I-Man-BSA binding to langerin CRD and langerin extracellular domain is shown in Figure 9. For the extracellular domain, little variation of 125I-Man-BSA binding is seen with changing pH when the assays are carried out at 20 mM CaCl2 (Figure 9A), but a sharp decrease in binding is seen below pH 5.5 when the assays are carried out at 1 mM CaCl2. These results suggest that, as for several other endocytic C-type lectins, release of ligand by langerin at endosomal pH is associated with decreased affinity for Ca2+.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 9. pH dependence of Man-BSA binding by langerin. pH dependence of (A) langerin extracellular domain or (B) langerin CRD binding to 125I-Man-BSA was determined using the solid phase binding assay. The buffers used were sodium acetate (pH 4.0–5.0), 2-(N-morpholino)ethanesulfonic acid (pH 5.0–7.0), and Tris-HCl (pH 7.0–8.5) all at a final concentration of 25 mM with 0.15 M NaCl and either 20 mM CaCl2 or 1 mM CaCl2. Results are presented as mean ± SD of two experiments for the extracellular domain and three experiments for the CRD.

 
Interestingly, the CRD alone shows a different profile for pH dependence of binding to 125I-Man-BSA (Figure 9B) when compared with the extracellular domain. Also, the CRD shows similar pH dependence of 125I-Man-BSA binding at 20 mM CaCl2 and at 1 mM CaCl2, indicating little change in Ca2+ affinity with change in pH. Thus it appears that interactions between the CRDs in the trimeric fragment or between the CRDs and the neck region are required to cause pH-dependent reduction of Ca2+ affinity, leading to ligand release. Analytical ultracentrifugation of the langerin extracellular domain at low pH indicates that it exists as a stable trimer even at pH 4.0 (apparent molecular mass 86.4 ± 11 kDa); thus pH-dependent loss of ligand binding activity is not due to dissociation of the trimer.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Biochemical characterization and carbohydrate-binding studies of langerin have provided evidence for a role of langerin in uptake of glycoprotein antigens. Langerin is seen to share many features with other C-type lectins, but differences are likely to reflect its distinct biological function and ligands.

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., 2001Go), MBP (Drickamer, 1992Go), the mannose receptor (Taylor et al., 1992Go), and Endo180 (East et al., 2002Go). 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., 1992Go). 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., 2001Go). MBP binds only the terminal residue, with the rest of the oligosaccharide pointing away from the CRD (Weis et al., 1992Go). 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., 2001Go), but is not seen with the CRD of MBP (Mitchell et al., 2001Go; Lee et al., 1992Go).

The finding that the neck region of langerin mediates oligomerization and forms a coiled-coil of {alpha}-helices is consistent with the presence of 17 typical heptad repeats in this region (http://ctld.glycob.ox.ac.uk) (Lupas, 1996Go). 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, 1996Go). 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, 1996Go). 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., 1990Go). 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., 2001Go).

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., 2002Go), 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., 1990Go).

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, 1998Go). 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., 1994Go; Feinberg et al., 2000Go; Wragg and Drickamer, 1999Go). 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., 1997Go).

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., 2002Go). 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., 2002Go). 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., 2002Go). Thus langerin is likely to be the major receptor involved in endocytosis of glycoprotein antigens by Langerhans cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Restriction enzymes and other DNA modifying enzymes were obtained from New England Biolabs (Beverly, MA). Oligonucleotides were obtained from Invitrogen (Carlsbad, CA). Monosaccharides and glycoproteins were from Sigma-Aldrich (St. Louis, MO). Na125I, Bolton and Hunter reagent, and isopropyl-ß-D-thiogalactoside were from Amersham Pharmacia (Little Chalfont, U.K.). Man-BSA was purchased from E-Y Laboratories (San Mateo, CA) and iodinated by the chloramine-T method (Greenwood et al., 1963Go). Immulon 4 96-well microtiter plates were obtained from Dynex Technologies (Southampton, UK). Mannose-Sepharose was prepared by the divinyl sulfone method (Fornstedt and Porath, 1975Go). Man9-GlcNAc2 oligosaccharide prepared by hydrazinolysis of soybean agglutinin (Ashford et al., 1987Go) was a gift from Dan Mitchell and Brian Matthews (Glycobiology Institute, Oxford University). Oligosaccharides from bovine ribonuclease B and asialo tetraantennary complex oligosaccharides were obtained from Glyko (Novato, CA). Asialo bi- and triantennary oligosaccharides were from Dextra Laboratories (Reading, UK).

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., 1984Go). 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 25–30°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 25–30°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 sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and langerin CRD was identified by N-terminal sequencing on a Beckman LF3000 protein sequencer following transfer to polyvinylidene difluoride membranes (Matsudaira, 1987Go).

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 SDS–PAGE. 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, 1943Go).

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, 1967Go).

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% SDS–polyacrylamide 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., 1989Go). 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., 1985Go) 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, 1982Go). The final plasmid was transfected into {psi}Cre cells (Danos and Mulligan, 1988Go) using the calcium phosphate method (Wigler et al., 1979Go). 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., 1988Go), except that specificity was measured using an excess of unlabeled ligand.


    Acknowledgements
 
We thank Russell Wallis and Dan Mitchell for help with analytical ultracentrifugation, Oliver Holt for assistance with neoglycolipid overlays, and Kurt Drickamer for help with circular dichroism spectroscopy and comments on the manuscript. This work was supported by Grant 041845 from the Wellcome Trust. The analytical ultracentrifugation facility is supported by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust.

1 To whom correspondence should be addressed; e-mail: mt{at}glycob.ox.ac.uk Back


    Abbreviations
 
BSA, bovine serum albumin; CRD, carbohydrate-recognition domain; EDTA, ethylenediamine tetra-acetic acid; MBP, serum mannose-binding protein; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Ashford, D.A., Dwek, R.A., Welply, J.K., Amatayakul, S., Homans, S.W., Lis, H., Taylor, G.N., Sharon, N., and Rademacher, T.W. (1987) The ß1-2-D-xylose and {alpha}1-3-L-fucose substituted lectins from Erythrina cristagalli lectin. Isolation, characterisation and comparison with other legume lectins. Eur. J. Biochem., 166, 311–320.[Abstract]

Banchereau, J. and Steinman, R.M. (1998) Dendritic cells and the control of immunity. Nature, 392, 245–252.[CrossRef][ISI][Medline]

Bouyain, S., Silk, N J., Fabini, G., and Drickamer, K. (2002) An endogenous Drosophila receptor for glycans bearing {alpha}1,3-linked core fucose residues. J. Biol. Chem., 277, 22566–22572.[Abstract/Free Full Text]

Cohn, E.J. and Edsall, J.T. (1943) Proteins, amino acids and peptides as ions and dipolar ions. Reinhold, New York, pp. 370–381.

Danos, O. and Mulligan, R.C. (1988) Safe and efficient generation of recombinant retroviruses with amphotrophic and ecotrophic host range. Proc. Natl. Acad. Sci. USA, 85, 6460–6464.[Abstract]

Drickamer, K. (1992) Engineering galactose-binding activity into a C-type mannose-binding protein. Nature, 360, 183–186.[CrossRef][ISI][Medline]

East, L., Rushton, S., Taylor, M.E., and Isacke, C.M. (2002) Characterization of sugar binding by the mannose receptor family member, Endo-180. J. Biol. Chem., 277, 50469–50475.[Abstract/Free Full Text]

Engering, A., Geijtenbeek, T.B.H., van Vliet, S.J., Wijers, M., van Liempt, E., Demaurex, N., Lanzavecchia, A., Fransen, J., Figdor, C.G., Piguet, V., and van Kooyk, Y. (2002) The dendritic cell-specific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells. J. Immunol., 168, 2118–2126.[Abstract/Free Full Text]

Feinberg, H., Park-Snyder, S., Kolatkar, A.R., Heise, C.T.,Taylor, M.E., and Weis, W.I. (2000) Structure of a C-type carbohydrate-recognition domain from the macrophage mannose receptor. J. Biol. Chem., 275, 21539–21548.[Abstract/Free Full Text]

Feinberg, H., Mitchell, D.A., Drickamer, K., and Weis, W.I. (2001) Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR. Science, 94, 2163–2166.[CrossRef]

Fornstedt, N. and Porath, J. (1975) Characterization studies on a new lectin found in seeds of Vicia ervilia. FEBS Lett., 57, 187–191.[CrossRef][ISI][Medline]

Ghrayeb, J., Kimura, K., Takahara, M., Hsuing, H., Masui, Y., and Inouye, M. (1984) Secretion cloning vectors in Escherichia coli. EMBO J., 3, 2437–2442.[Abstract]

Greenwood, F.C., Hunter, W.M., and Glover, J.S. (1963) The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem. J., 89, 114–123.[ISI]

Halberg, D.F., Wager, R.E., Farrell, D.C., Hildreth, J. IV, Quesenberry, M.S., Loeb, J.A., Holland, E.C., and Drickamer, K. (1987) Major and minor forms of the rat liver asialoglycoprotein receptor are independent galactose-binding proteins. Primary structure and glycosylation heterogeneity of minor receptor forms. J. Biol. Chem., 262, 9828–9838.[Abstract/Free Full Text]

Hirs, C.H.W. (1967) Detection of peptides by chemical methods. Methods Enzymol., 11, 325–329.

Lee, R.T., Ichikawa, Y., Kawasaki, T., Drickamer, K., and Lee, Y.C. (1992) Multivalent ligand binding by serum mannose-binding protein. Arch. Biochem. Biophys., 299, 129–136.[ISI][Medline]

Lee, S.J., Evers, S., Roeder, D., Parlow, A.F., Risteli, J., Risteli, L., Lee, Y.C., Feizi, T., Langen, H., and Nussenzweig, M.C. (2002) Mannose receptor-mediated regulation of serum glycoprotein homeostasis. Science, 295, 1898–1901.[Abstract/Free Full Text]

Loeb, J.A. and Drickamer, K. (1988) Conformational changes in the chicken receptor for endocytosis of glycoproteins. Modulation of ligand-binding by Ca2+ and pH. J. Biol. Chem., 263, 9752–9760.[Abstract/Free Full Text]

Lupas, A. (1996) Coiled coils: new structures and new functions. Trends Biochem. Sci., 21, 375–382.[CrossRef][ISI][Medline]

Maddon, P.J., Littman, D.R., Godfrey, M., Maddon, D.E., Chess, L., and Axel, R. (1985) The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family. Cell, 42, 93–104.[ISI][Medline]

Matsudaira, P. (1987) Sequence from picomole quantities of protein electroblotted ont polvinylidene difuoride membranes. J. Biol. Chem., 262, 10035–10038.[Abstract/Free Full Text]

McDermott, R., Ziylan, U., Spehner, D., Bausinger, H., Lipsker, D., Mommaas, M., Cazenave, J.-P., Raposo, G., Goud, B., de la Salle, H., and others. (2002) Birbeck granules are subdomains of endosomal recycling compartment in human epidermal langerhans cells, which form where langerin accumulates. Mol. Biol. Cell, 13, 317–335.[Abstract/Free Full Text]

Mellow, T.E., Halberg, D., and Drickamer, K. (1988) Endocytosis of N-acetylglucosamine-containing glycoproteins by rat fibroblasts expressing a single species of chicken liver glycoprotein receptor. J. Biol. Chem., 263, 5468–5473.[Abstract/Free Full Text]

Mitchell, D.A., Fadden, A.J., and Drickamer, K. (2001) A novel mechanism of carbohydrate-recognition by the C-type lectins DC-SIGN and DC-SIGN-R. Subunit organization and binding to multivalent ligands. J. Biol. Chem., 276, 28939–28945.[Abstract/Free Full Text]

Mizuochi, T., Loveless, W.R., Lawson, A.M., Chai, W., Lachmann, P.J., Childs, R.A., Thiel, S., and Feizi, T. (1989) A library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins reveals that conglutinin binds to certain complex-type as well as high mannose-type oligosaccharide chains. J. Biol. Chem., 264, 13834–13839.[Abstract/Free Full Text]

Mullin, N.P., Hall, K.T., and Taylor, M.E. (1994) Characterization of ligand binding to a carbohydrate-recognition domain of the macrophage mannose receptor. J. Biol. Chem., 269, 28405–28413.[Abstract/Free Full Text]

Ng, K.K., Drickamer, K., and Weis, W.I. (1996) Structural analysis of monosaccharide recognition by rat liver mannose-binding protein. J. Biol. Chem., 274, 663–674.[CrossRef]

Rice, K., Weisz, O.A., Barthel, T., Lee, R.T., and Lee, Y.C. (1990) Defined geometry of binding between triantennary glycopeptide and the asialoglycoprotein receptor of rat hepatocytes. J. Biol. Chem., 265, 18429–18434.[Abstract/Free Full Text]

Southern, P.J. and Berg, P. (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet., 1, 327–341.[Medline]

Suzuki, K., Doi, T., Imanishi, T., Kodama, T., and Tanaka, T. (1997) The conformation of the alpha-helical coiled coil domain of the macrophage scavenger receptor is pH dependent. Biochemistry, 36, 15140–15146.[CrossRef][ISI][Medline]

Takahara, K., Omatsu, Y., Yashima, Y., Maeda, Y., Tanaka, S., Iyoda, T., Clusen, B., Matsubara, M., Letterio, J., Steinman, R.M., Matsuda, Y., and Inaba, K. (2002) Identification and expression of mouse langerin (CD207) in dendritic cells. Int. Immunol., 14, 433–444.[Abstract/Free Full Text]

Taylor, M.E., Conary, J.T., Lennarz, M.R., Stahl, P.D., and Drickamer, K. (1990) Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. J. Biol. Chem., 265, 12156–12162.[Abstract/Free Full Text]

Taylor, M.E., Bezouska, K., and Drickamer, K. (1992) Contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor. J. Biol. Chem., 267, 1719–1726.[Abstract/Free Full Text]

Turville, S.G., Cameron, P.U., Handley, A., Lin, G., Pohlmann, S., Doms, R.W., and Cunningham, A.L. (2002) Diversity of receptors binding HIV on dendritic cell subsets. Nature Immunol., 3, 975–983.[CrossRef][ISI][Medline]

Valladeau, J., Duvert-Frances, V., Pin, J.-J., Dezutter-Dambuyant, C., Vincent, C., Massacrier, C., Vincent, J., Yoneda, K., Banchereau, J., Caux, C., and others. (1999) The monoclonal antibody DCGM4 recognizes langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface. Eur. J. Immunol., 29, 2695–2704.[CrossRef][ISI][Medline]

Valladeau, J., Ravel, O., Dezutter-Dambuyant, C., Moore, K., Kleijmeer, M., Liu, Y., Duvert-Frances, V., Vincent, C., Schmitt, D., Davoust, J., and others. (2000) Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces formation of birbeck granules. Immunity, 12, 71–81.[ISI][Medline]

Valladeau, J., Clair-Moninot, V., Dezutter-Dambuyant, C., Pin, J.-J., Kissenpfennig, A., Mattéi, M.-G., Ait-Yahia, S., Bates, E.E.M., Malissen, B., Koch, F., and others. (2002) Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissue. J. Immunol., 168, 782–792.[Abstract/Free Full Text]

Weis, W.I. and Drickamer, K. (1994) Trimeric structure of a C-type mannose-binding protein. Structure, 2, 1227–1240.[ISI][Medline]

Weis, W.I. and Drickamer, K. (1996) Structural basis of lectin-carbohydrate recognition. Annu. Rev. Biochem., 65, 441–473.[CrossRef][ISI][Medline]

Weis, W.I., Kahn, R., Fourme, R., Drickamer, K., and Hendrickson, W.A. (1991) Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing. Science, 254, 1608–1615.[ISI][Medline]

Weis, W.I., Drickamer, K., and Hendrickson, W.A. (1992) Structure of a C-type mannose-binding protein complexed with an oligosaccharide. Nature, 360, 127–134.[CrossRef][ISI][Medline]

Wigler, M., Sweet, R., Sim, G.K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R. (1979) Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell, 16, 777–785.[ISI][Medline]

Wragg, S. and Drickamer, K. (1999) Identification of amino acid residues that determine pH dependence of ligand binding to the asialoglycoprotein receptor. J. Biol. Chem., 274, 35400–35406.[Abstract/Free Full Text]