The Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX21 3QU, United Kingdom
Received on December 19, 2002; revised on March 10, 2003; accepted on March 12, 2003
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Abstract |
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Key words: clearance / glycoprotein / Kupffer cell / lectin / receptor
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Introduction |
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The existence of an additional hepatic system for recognition of glycoproteins was postulated based on the uptake of synthetic neoglycoproteins with exposed fucose residues (Lehrman et al., 1986b). A novel receptor, designated the Kupffer cell receptor (KCR), was extracted from rat liver; it was suggested that this protein serves as a fucose receptor (Haltiwanger et al., 1986
; Lehrman et al., 1986a
; Lehrman and Hill, 1986
). The KCR was shown to be present on the surface of Kupffer cells and is a type II transmembrane protein, consisting of an N-terminal cytoplasmic tail, a transmembrane region, a long neck region, and a C-terminal C-type CRD (Hoyle and Hill, 1988
).
Based on their ligand-binding properties, C-type CRDs can be divided into two groups: those that bind mannose, GlcNAc, and fucose and those that bind galactose and GalNAc (Drickamer, 1999; Weis et al., 1998
). CRDs in these two groups differ in a few key residues around the ligand-binding site. Ligand binding to the C-type CRDs involves interaction of two adjacent hydroxyl groups with Ca2+. Thus the primary determinant of binding selectivity is usually the orientation of the 3- and 4-hydroxyl groups of the sugar, although fucose presents a special case because it is an L sugar. In general, C-type lectins that bind fucose well do not bind galactose well and vice versa. The KCR, having been described as a fucose receptor, might appear to be a member of the former group, but studies on the purified protein suggest that it actually binds fucose relatively poorly compared to galactose (Lehrman et al., 1986a
). Sequence alignments also indicate that the KCR would be predicted to be a galactose/GalNAc-binding protein. Because most of the previous work on the ligand-binding properties of the KCR was carried out on protein purified from liver, the possibility of contamination by the mannose receptor or the asialoglycoprotein receptor has been a persistent concern in the interpretation of these studies. Nevertheless, the fact that the receptor was purified from rat liver by affinity chromatography on immobilized fucose suggested that it must have significant fucose-binding character.
The aims of this article were to produce a recombinant extracellular fragment of the KCR free from any other lectins and to facilitate a more definitive analysis of its sugar-binding properties and its structural organization. Overlapping binding specificities of the KCR and the asialoglycoprotein receptor and the absence of a functional gene for the KCR in humans have important implications for the role of multiple hepatic lectins in clearance of glycoproteins and particles from circulation.
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Results |
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Of particular interest is the ability of the receptor to interact with fucose and related sugars. Fucose competes only threefold less effectively than galactose. Binding of fucose to the asialoglycoprotein receptor is 15-fold weaker than binding of galactose (Iobst and Drickamer, 1994), so fucose competes for binding to the KCR relatively well. However, there is a marked difference in the affinity of the KCR for the methyl glycosides of fucose. The very weak binding of
-methyl fucoside suggests that most mammalian fucose-containing oligosaccharides would not be good ligands for the KCR.
Interactions with glycoproteins and oligosaccharides
To assess the types of endogenous glycans that might serve as ligands for the KCR, the trimeric KCR-B fragment was radiolabeled and used to probe blots of glycoproteins that bear different types of N- and O-linked glycans (Figure 7). The major glycoproteins detected in this way are desialylated 1-acid glycoprotein and fetuin. The absence of reactivity with the native proteins before treatment with neuraminidase is consistent with the critical role of terminal galactose residues in ligand binding. However, asialotransferrin and asialofibrinogen show little or no reactivity. The glycans attached to
1-acid glycoprotein and fetuin are predominantly tetra- and triantennary complex structures, and those attached to transferrin and fibrinogen are mostly biantennary (Green et al., 1988
; Shiyan and Bovin, 1997
). There are five glycosylation sites in
1-acid glycoprotein, three in fetuin, and one in transferrin. Thus the different reactivities of these glycoproteins could reflect differences in the structures of the attached glycans or their number and spacing.
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Discussion |
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Because the recombinant KCR fragments are free of contamination from other liver lectins, such as the asialoglycoprotein receptor and the mannose receptor, they also provide an opportunity to document unambiguously the sugar-binding specificity of this receptor. For the monosaccharides tested, essentially the same rank order of binding is obtained as was determined in earlier studies (Lehrman et al., 1986a). These studies demonstrate that the order of affinities is similar to that seen for the hepatic asialoglycoprotein receptor, although the range of affinities compared to galactose is much more compressed. Thus, GalNAc inhibits binding to the KCR approximately 6-fold better than does galactose, whereas the affinity of the asialoglycoprotein receptor for GalNAc is 60-fold higher than the affinity for galactose (Iobst and Drickamer, 1996
). Similarly, the affinity of the KCR for
-methyl mannoside is about 6-fold lower than the affinity for
-methyl galactoside, and the affinity difference is approximately 40-fold for the asialoglycoprotein receptor (Iobst and Drickamer, 1994
). These results suggest that the binding site does not exclude mannose and related sugars very effectively.
A high specificity ratio for galactose over mannose is associated with precise positioning of a specificity-determining tryptophan residue in the asialoglycoprotein receptor. The tryptophan residue is held in position by an adjacent glycine-rich loop (Iobst and Drickamer, 1994; Kolatkar and Weis, 1996
). Although a tryptophan residue is found at an equivalent position in the KCR, the adjacent sequence differs significantly from the glycine-rich loop of the asialoglycoprotein receptor (Figure 1). Studies using mutant forms of the C-type CRD from mannose-binding protein suggest that changes in the glycine-rich loop can lead to failure to exclude mannose and related sugars from a galactose-binding site (Iobst and Drickamer, 1994
).
Perhaps the most surprising result from the monosaccharide inhibition studies is the very poor interaction with methyl fucosides. The fact that both - and ß-methyl fucosides inhibit less effectively than free fucose suggests that some of the apparent affinity for fucose results from interaction involving the 1- and 2-hydroxyl groups in the way that other free sugars have been shown to interact with C-type CRDs (Ng et al., 1996
). Fucose in mammalian glycans is found almost exclusively in
linkage, so the very poor inhibition by
-methyl fucoside suggests that such glycans would be poor ligands for the receptor. Although this result is consistent with previous studies with neoglycoproteins (Lehrman et al., 1986a
), the monosaccharide inhibition studies reported herein provide a quantitative comparison. The neoglycolipid binding and inhibition studies employing Lewisx structures are consistent with the primary binding site on glycoproteins, such as asialo
1-acid glycoprotein, being terminal galactose residues with little if any contribution by fucose.
Previous studies have documented the ability of the KCR to mediate clearance of glycoprotein from circulation, and the loss of ligand-binding activity observed at acidic pH is consistent with a role in receptor-mediated endocytosis (Lehrman et al., 1986a,b
). The pH dependence of ligand binding is similar to that of the asialoglycoprotein receptor, which correlates with the presence of a key histidine residue that mediates loss of binding activity to the asialoglycoprotein receptor at low pH (Figure 1) (Feinberg et al., 2000
; Wragg and Drickamer, 1999
).
The extended neck of the receptor and the preference for galactose-containing ligands are consistent with the suggestion that the KCR is the galactose particle receptor, which participates in binding and internalization of ligands over 12 nm in size (Kuiper et al., 1994). However, the KCR clearly has high affinity for endogenous glycoproteins from which sialic acid is removed, suggesting that it might have a function parallel to the asialoglycoprotein receptor in clearance of desialylated glycoproteins. This possibility has important implications for understanding the phenotype of asialoglycoprotein receptordeficient mice. Such mice fail to show rapid clearance of asialoglycoproteins from circulation, but no long-term accumulation of desialylated serum glycoproteins is observed. A slower uptake of such glycoproteins mediated by the KCR might provide enough residual clearance capacity to keep circulating levels of asialoglycoproteins low while not being evident in short-term clearance studies.
The observed binding to thyrotropin suggests that the KCR may also bind terminal GalNAc residues on the hormones, which have become exposed due to loss of sulfate. Thus, the KCR could serve as a back-up for clearance of hormones that usually bear the 4-sulfo-GalNAc marker, which leads to endocytosis by the mannose receptor (Fiete et al., 1998). The preferential binding to GalNAc is consistent with previous studies in which adhesion to glycolipids was measured because it was observed that the best binding is obtained with a glycolipid bearing a terminal GalNAcß1-3Gal structure (Tiemeyer et al., 1992
).
Analysis of the human genome sequence reveals a partial gene closely related to the gene for the KCR in rats and mice encoded on chromosome 2, region 2p13.3, which is near to the gene for another C-type lectin, langerin (Figure 1). The fragment consists of three exons encoding a CRD and one exon encoding a sequence homologous to the C-terminal portion of the neck of the rat KCR. Extensive searches of the region 3' to the gene fragment fail to detect any additional exons, suggesting that this is a pseudogene. No cDNAs have been reported for this gene in any of the expressed sequence tag libraries. Attempts to amplify a cDNA from a liver library using primers that lie within the portion of the gene that is present fail to yield any detectable amplification product, providing further evidence that this gene is not functional (Fadden and Drickamer, unpublished data). Thus there appears to be no human ortholog of the KCR. The blotting studies using desialylated rat and human serum show that similar ligands are present in both species, so it seems unlikely that the KCR interacts with a set of ligands found uniquely in rodents or specifically absent from humans. If the KCR recognizes ligands that are also recognized by the asialoglycoprotein receptor, its role may have become redundant.
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Materials and methods |
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Cloning of the extracellular domain of the KCR
The cDNA sequence encoding the extracellular domain of the KCR was amplified using the primers 5'-aaggccggccagacagaagcagaatgaggatcatcctgtc-3' (forward) and 5-ttgcggccgctcagctctggtccgttctggccacagacca-3' (reverse). The CRD-encoding region was amplified with the alternative forward primer 5'-aaggccggcccaggttctccagctgatcatgcaggactgg-3'. These primers include restriction sites for FseI and NotI. Following denaturation at 95°C for 1 min, 40 cycles of 95°C for 30 s and 68°C for 1 min were executed. Fragments were digested with FseI and NotI and inserted into a pINIIIompA2 expression vector (Grayeb et al., 1984) modified to contain the restriction sites FseI and NotI downstream of the ompA signal sequence. In each case, a mistake-free clone was generated using restriction fragments from two different clones. The resulting plasmids were used to transform E. coli strain JA221. To avoid mutations due to possible toxicity of the expressed fragments, the FseI site was introduced in a way that interrupts the reading frame. The correct reading frame was then generated by digesting with FseI followed by trimming of the 3' extensions with T4 DNA polymerase. The sequence of all plasmids was confirmed by DNA sequencing.
Expression and purification of soluble KCR fragments
Protein expression was carried out in LB medium containing 50 µg/ml ampicillin. An overnight culture (200 ml) was diluted into 6 L of medium and incubated at 30°C with shaking until an A550 of 0.8 was reached. Following addition of 10 µg/L of isopropyl-ß-thiogalactoside and CaCl2 to a final concentration of 100 mM, cultures were incubated for a further 20 h at 30°C with shaking. Cells were harvested by centrifugation at 4000xg for 15 min at 4°C. The cell pellet was suspended in 300 ml loading buffer (25 mM Tris-Cl, pH 7.8, 125 mM NaCl, 25 mM CaCl2) and lysed by sonication (6x30-s bursts). Debris was removed by centrifugation at 10,000xg for 15 min at 4°C and then 137,000xg for 45 min at 4°C in a Beckman (Palo Alto, CA) 45Ti rotor. The clarified extract was loaded onto a 5-ml column of galactose-Sepharose equilibrated with loading buffer. The column was washed with five aliquots of 2-ml loading buffer and eluted with five aliquots of 2-ml elution buffer (25 mM Tris-Cl, pH 7.8, 125 mM NaCl, 2.5 mM EDTA). Fractions were analyzed by SDSpolyacrylamide gel electrophoresis (PAGE).
Protein from the affinity column was dialyzed against 50 mM Tris-Cl, pH 7.8, containing 50 mM NaCl and loaded onto a 1-ml Mono-Q column equilibrated with 50 mM Tris-Cl, pH 7.8, and eluted with a gradient from 0 to 500 mM NaCl over 20 min at a flow rate of 1 ml/min. Absorbance was measured at 280 nm, and 1-ml fractions were collected and analyzed by SDSPAGE.
Analytical methods
Analytical ultracentrifugation was carried out in a Beckman XL-A analytical ultracentrifuge using an An60Ti rotor. Proteins were dialyzed against 10 mM Tris-Cl, pH 7.8, 150 mM NaCl, 5 mM CaCl2. Experiments were performed at 6500, 8000 and 48,000 rpm at 20°C. Data were collected at either 233 nm or 280 nm, with the wavelength adjusted so that the initial absorbance was approximately 0.5. A baseline scan at 360 nm was used to correct for optical imperfections. The equilibrium distributions from different loading concentrations were analyzed simultaneously using the software supplied with the centrifuge.
Proteins were sequenced on a Beckman LF-3000 sequencer following blotting onto polyvinylidine difluoride membranes (Matsudaira, 1987). Circular dichroism spectra were measured on a Jasco J600 spectropolarimeter (Great Dunmow, Cambidgeshire, UK) using 200-µl samples in a 1-mm quartz cuvette at room temperature. Five scans were carried out on each sample from with a bandwidth of 2 nm and a scan rate of 20 nm/min. Protein concentrations were determined by alkaline ninhydrin assay (Hirs, 1967
).
Protein cross-linking
The KCR fragments purified by anion exchange chromatography were dialyzed against 100 mM sodium HEPES, pH 7.5, 150 mM NaCl. CaCl2 was added to some samples to a final concentration of 10 mM. Aliquots (25 µl) of each sample were treated with bis(sulfosuccinimidyl) suberate for 60 min at room temperature. Samples were diluted 1:1 with double-strength gel sample buffer, heated to 100°C for 5 min, and run on SDSpolyacrylamide gels.
Solid phase binding assay
Wells of microtiter plates were coated by incubating with 50 µl KCR-B fragment in loading buffer overnight at 4°C. Wells were washed twice with loading buffer and blocked with 5% BSA in loading buffer for 2 h at 4°C. The wells were washed twice again and incubated with 125I-galactose-BSA and competing sugar in loading buffer containing 5% BSA. After incubation at 4°C for 2 h, the wells were washed three times, dried, and counted in a -counter. Experiments were performed in duplicate and the data averaged. A nonlinear least squares fitting program (SigmaPlot, Jandel Scientific, San Rafael, CA) was used to fit the data to the following equation:
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Glycoprotein and neoglycolipid binding assays
The KCR-B fragment was dialyzed into 25 mM sodium-HEPES, pH 7.5, 100 mM NaCl, 25 mM CaCl2. Approximately 100 µg protein in 200 µl was reacted with 0.1 mCi of Bolton-Hunter reagent (Bolton and Hunter, 1973) for 10 min at room temperature. The iodinated protein was mixed with 1 ml loading buffer, repurified on a 1-ml column of galactose-Sepharose, and diluted into 50 ml loading buffer containing 2% bovine hemoglobin.
Serum was dialyzed against water and diluted eightfold prior to digestion with neuraminidase. Aliquots (40 µl) were treated with 50 U neuraminidase for 3 h at 37°C using buffer supplied by the manufacturer. Following addition of an equal volume of double-strength sample buffer, samples were heated to 100°C for 5 min. Aliquots containing the equivalent of 0.25 µl serum were resolved on 17.5% SDSpolyacrylamide gels and blotted onto nitrocellulose. The membrane was blocked with 2% hemoglobin in loading buffer for 60 min at room temperature and incubated with the radio-iodinated KCR-B fragment for 90 min at room temperature, followed by four 5-min washes with cold loading buffer. Radioactivity was detected using a phosphorimager from Molecular Dynamics (Chesham, Buckinghamshire, UK).
Neoglycolipids were prepared by conjugation of oligosaccharides to phosphatidylethanolamine dipalmitate in the presence of sodium cyanoborohydride (Mizuochi et al., 1989). High performance thin-layer chromatograms of the neoglycolipids were developed with chloroform: methanol:water (105:100:28, v/v). After fixation for 30 s in 1 mg/ml poly(isobutylmethacrylate) in hexane, chromatograms were blocked and incubated with iodinated KCR-B following the same procedures as for the glycoprotein gel blots. Oligosaccharides were released from
1-acid glycoprotein by hydrazinolysis (Patel and Parekh, 1994
). Sialic acid was released by treatment with 50 mM H2SO4 for 60 min at 80°C. Published procedures were used for repurification of released oligosaccharides on Dowex 50 resin and of neoglycolipids on C8 cartridges (Feizi et al., 1994
).
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: kd{at}glycob.ox.ac.uk
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Abbreviations |
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References |
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