Selective Binding of the Scavenger Receptor C-type Lectin to Lewisx Trisaccharide and Related Glycan Ligands
2005; Elsevier BV; Volume: 280; Issue: 24 Linguagem: Inglês
10.1074/jbc.m504197200
ISSN1083-351X
AutoresP.J. Coombs, Sarah A. Graham, Kurt Drickamer, Maureen E. Taylor,
Tópico(s)Complement system in diseases
ResumoThe scavenger receptor C-type lectin (SRCL) is an endothelial receptor that is similar in organization to type A scavenger receptors for modified low density lipoproteins but contains a C-type carbohydrate-recognition domain (CRD). Fragments of the receptor consisting of the entire extracellular domain and the CRD have been expressed and characterized. The extracellular domain is a trimer held together by collagen-like and coiled-coil domains adjacent to the CRD. The amino acid sequence of the CRD is very similar to the CRD of the asialoglycoprotein receptor and other galactose-specific receptors, but SRCL binds selectively to asialo-orosomucoid rather than generally to asialoglycoproteins. Screening of a glycan array and further quantitative binding studies indicate that this selectivity results from high affinity binding to glycans bearing the Lewisx trisaccharide. Thus, SRCL shares with the dendritic cell receptor DC-SIGN the ability to bind the Lewisx epitope. However, it does so in a fundamentally different way, making a primary binding interaction with the galactose moiety of the glycan rather than the fucose residue. SRCL shares with the asialoglycoprotein receptor the ability to mediate endocytosis and degradation of glycoprotein ligands. These studies suggest that SRCL might be involved in selective clearance of specific desialylated glycoproteins from circulation and/or interaction of cells bearing Lewisx-type structures with the vascular endothelium. The scavenger receptor C-type lectin (SRCL) is an endothelial receptor that is similar in organization to type A scavenger receptors for modified low density lipoproteins but contains a C-type carbohydrate-recognition domain (CRD). Fragments of the receptor consisting of the entire extracellular domain and the CRD have been expressed and characterized. The extracellular domain is a trimer held together by collagen-like and coiled-coil domains adjacent to the CRD. The amino acid sequence of the CRD is very similar to the CRD of the asialoglycoprotein receptor and other galactose-specific receptors, but SRCL binds selectively to asialo-orosomucoid rather than generally to asialoglycoproteins. Screening of a glycan array and further quantitative binding studies indicate that this selectivity results from high affinity binding to glycans bearing the Lewisx trisaccharide. Thus, SRCL shares with the dendritic cell receptor DC-SIGN the ability to bind the Lewisx epitope. However, it does so in a fundamentally different way, making a primary binding interaction with the galactose moiety of the glycan rather than the fucose residue. SRCL shares with the asialoglycoprotein receptor the ability to mediate endocytosis and degradation of glycoprotein ligands. These studies suggest that SRCL might be involved in selective clearance of specific desialylated glycoproteins from circulation and/or interaction of cells bearing Lewisx-type structures with the vascular endothelium. The scavenger receptor C-type lectin (SRCL) 1The abbreviations used are: SRCL, scavenger receptor C-type lectin; SR, scavenger receptor; CRD, carbohydrate-recognition domain; BSA, bovine serum albumin; LNFP, lacto-N-fucopentaose. 1The abbreviations used are: SRCL, scavenger receptor C-type lectin; SR, scavenger receptor; CRD, carbohydrate-recognition domain; BSA, bovine serum albumin; LNFP, lacto-N-fucopentaose. is also known as collectin-placenta 1 (CL-P1). Although originally cloned from liver and placenta, SRCL is expressed in many tissues (1Ohtani K. Suzuki Y. Eda S. Kawai T. Kase T. Keshi H. Sakai Y. Fukuoh A. Sakamoto T. Itabe H. Suzutani T. Ogaswara M. Yoshida I. Wakamiya N. J. Biol. Chem. 2001; 276: 44222-44228Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 2Nakamura K. Funakoshi H. Miyamoto K. Tokunaga F. Nakamura T. Biochem. Biophys. Res. Commun. 2001; 280: 1028-1035Crossref PubMed Scopus (91) Google Scholar). The receptor is found in endothelial cells from human umbilical vein and artery as well as in vascular endothelial cells of the heart, suggesting that it may be widely distributed in endothelia. The SRCL polypeptide contains an N-terminal intracellular domain, transmembrane anchor, and the extended extracellular domain (Fig. 1). The extracellular portion of the receptor is composed of a neck that contains coiled-coil and collagen-like regions and a C-terminal carbohydrate-recognition domain (CRD).The binding properties of SRCL have only been partially characterized. Binding to oxidized low density lipoprotein particles has been demonstrated and, by analogy to the type A scavenger receptors, this activity has been attributed to the collagen-like domain (1Ohtani K. Suzuki Y. Eda S. Kawai T. Kase T. Keshi H. Sakai Y. Fukuoh A. Sakamoto T. Itabe H. Suzutani T. Ogaswara M. Yoshida I. Wakamiya N. J. Biol. Chem. 2001; 276: 44222-44228Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Binding to Gram-negative bacteria has also been associated with the collagen-like domain, because a variant form of the protein lacking CRDs still shows binding activity for bacteria when expressed in Chinese hamster ovary cells (2Nakamura K. Funakoshi H. Miyamoto K. Tokunaga F. Nakamura T. Biochem. Biophys. Res. Commun. 2001; 280: 1028-1035Crossref PubMed Scopus (91) Google Scholar). Although the cytoplasmic domain of SRCL contains a potential endocytosis signal motif, conflicting data have been presented regarding the uptake of particulate ligands into cells (1Ohtani K. Suzuki Y. Eda S. Kawai T. Kase T. Keshi H. Sakai Y. Fukuoh A. Sakamoto T. Itabe H. Suzutani T. Ogaswara M. Yoshida I. Wakamiya N. J. Biol. Chem. 2001; 276: 44222-44228Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 2Nakamura K. Funakoshi H. Miyamoto K. Tokunaga F. Nakamura T. Biochem. Biophys. Res. Commun. 2001; 280: 1028-1035Crossref PubMed Scopus (91) Google Scholar).C-type CRDs fall broadly into two subgroups, those that bind galactose and related ligands and those that bind mannose, N-acetylglucosamine, and fucose. Mutagenesis of galactose-binding C-type CRDs, combined with studies in which the specificity of a mannose-binding CRD was converted to galactose binding, have led to the identification of a small set of residues that are essential for high affinity binding to galactose (3Drickamer K. Nature. 1992; 360: 183-186Crossref PubMed Scopus (443) Google Scholar, 4Iobst S.T. Drickamer K. J. Biol. Chem. 1994; 269: 15512-15519Abstract Full Text PDF PubMed Google Scholar). Examination of the sequence of SRCL reveals that all of these key residues are present. 2See www.imperial.ac.uk/research/animallectins for sequence alignments. 2See www.imperial.ac.uk/research/animallectins for sequence alignments. Thus, SRCL differs from DC-SIGN, the macrophage mannose receptor, serum mannose-binding protein, and other C-type lectins known to have roles in pathogen recognition, all of which fall into the second subgroup. Semiquantitative binding studies using cells expressing SRCL suggest that the receptor does bind sugar-containing ligands, with some preference for N-acetylgalactosamine (5Yoshida T. Tsuruta Y. Iwasaki M. Yamane S. Ochi T. Suzuki R. J. Biochem. (Tokyo). 2003; 133: 271-277Crossref PubMed Scopus (23) Google Scholar).The overall organization of SRCL shares features of both the type A scavenger receptors for modified low density lipoprotein and the collectins, which are soluble C-type lectins involved in pathogen recognition. The arrangement of domains in SRCL is most like the arrangement of the type A scavenger receptors, with the CRDs replacing cysteine-rich domains that are not related in sequence (6Peiser L. Mukhopadhyay S. Gordon S. Curr. Opin. Immunol. 2002; 14: 123-128Crossref PubMed Scopus (382) Google Scholar). Sequence comparisons reveal sequence similarities between the collagen-like and coiled-coil domains of SRCL and all three of the type A scavenger receptors, with particularly strong similarity to SR-A 3 (Fig. 1) (7Han H-J. Tokino T. Nakamura Y. Hum. Mol. Genet. 1998; 7: 1039-1046Crossref PubMed Scopus (84) Google Scholar). Although the collagen-like and coiled-coil domains would have similar elongated conformations in SRCL and the collectins, sequence comparisons provide no evidence for homology between SRCL and the collectins, and the domains are actually arranged in reverse order in the polypeptides. In addition, comparison of the CRD of SRCL with other C-type lectins shows that, in evolutionary terms, this domain falls within the family of type II transmembrane receptors that includes the asialoglycoprotein receptor, rather than forming part of the collectin cluster. Thus, it seems likely that the presence of similar domains in SRCL and the collectins represents independent gene shuffling events that have converged on a similar set of protein structural elements.To assess the biological functions of SRCL, both the sugar-binding characteristics and intracellular trafficking properties of the receptor need to be better defined. To this end, the results of this study demonstrated that the CRD of SRCL binds with unusually high selectivity to glycans containing the Lewisx epitope, that the CRDs are clustered in a trimer, and that the receptor is able to direct uptake and degradation of glycoprotein ligands.EXPERIMENTAL PROCEDURESMaterials—Oligosaccharides and LNFP III-BSA were purchased from Dextra Ltd. (Reading, UK). Galactose-BSA was obtained from E-Y Laboratories (San Mateo, CA). Radioisotopes were from Amersham Biosciences. Rabbit polyclonal antibodies to the CRD from SRCL were generated by Eurogentec (Seraing, Belgium) following their standard protocol using 2 mg of bacterially expressed protein as antigen.Cloning of SRCL cDNA—cDNA encoding human SRCL was amplified from human placental cDNA (Clontech) in two sections using the following pairs of primers: N-terminal section forward, 5′-AACCCACGGTCACGGTCACCATGAAAGACGACTTCGCAG-3′; N-terminal section reverse, 5′-TTGAGCTGACCATGCTTGGAGTCTACTAGCTTC-3′; C-terminal section forward, 5′-AAGTGGTCATCATGAACCTCAACAACCTGAACCTG-3′; C-terminal section reverse, 5′-TTGCGGCCGCTTATAATGCAGATGACAGTACTGTCTCCCTGTC-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. PCR products were cloned into the vector pCRIITOPO using the TOPO cloning kit (Invitrogen) and sequenced using an Applied Biosystems Prism 310 genetic analyzer. Segments of the cDNAs that were free of reverse transcription errors were combined using convenient restriction sites.Expression of the CRD from SRCL in Bacteria—The region of the cDNA coding for the CRD only (from residue 603 to the C terminus) was amplified using the following primers: forward primer, 5′-AAGGCCGGCCGAGGACAATAGCTGCCCGCCTCACTGGAAG-3′; reverse primer, 5′-TTGCGGCCGCTTATAATGCAGATGACAGTACTGTCTCCCTGTC-3′. PCR products were digested at the FseI and NotI sites in the primers and inserted into a modified pINIIIompA2 expression vector containing restriction sites for FseI and NotI downstream of the ompA signal sequence (8Grayeb J. Kimura H. Takahara M. Hsiung H. Masui Y. Inouye M. EMBO J. 1984; 3: 2437-2442Crossref PubMed Scopus (285) Google Scholar). The correct reading frame was generated by digesting the vector with FseI, followed by trimming of the 3′ extension with T4 polymerase and religation. The integrity of the final expression plasmid was verified by DNA sequencing. Luria-Bertani medium (1 liter), containing 50 μg/ml ampicillin, was inoculated with 30 ml of an overnight culture of Escherichia coli strain JA221 transformed with the expression plasmid. The culture was grown by shaking at 25–30 °C to an A550 of ∼0.8, and isopropyl-β-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, the cells were harvested by centrifugation at 4,000 rpm for 15 min in a Beckman JS-4.2 rotor. Bacterial pellets were resuspended in cold 10 mm Tris-Cl, 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 of 20 mm Tris-Cl, pH 7.8, 0.5 m NaCl, 20 mm CaCl2 (loading buffer). Lysed bacteria were centrifuged at 10,000 × g for 15 min, and the supernatant was recentrifuged at 100,000 × g for 1 h at 4°C. The supernatant was passed over a 10-ml column of galactose-Sepharose (9Fornstedt N. Porath J. FEBS Lett. 1975; 57: 187-191Crossref PubMed Scopus (211) Google Scholar) equilibrated in loading buffer. The column was washed with 10 × 2 ml of loading buffer and eluted with 10 × 2 ml of elution buffer (20 mm Tris-Cl, pH 7.8, 0.5 m NaCl, 2 mm EDTA). Fractions were analyzed by SDS-polyacrylamide gel electrophoresis. The CRD was identified by N-terminal sequencing on a Beckman LF3000 protein sequencer following transfer to polyvinylidene difluoride membranes (10Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar).Expression of the Extracellular Domain of SRCL in Chinese Hamster Ovary Cells—DNA coding for the extracellular domain of SRCL, without the cytoplasmic tail and the transmembrane region, was produced by amplifying the region coding for the N-terminal section of the protein using the forward primer 5′-AAAGTTGTAGAGAAATGGACAATGTCACAGGTGCC-3′ with the reverse primer described above and combining this section with the region coding for the C-terminal section amplified as above. The resulting DNA, coding for the entire extracellular domain of SRCL (starting at residue 60), was fused to codons specifying the dog preproinsulin signal sequence. The preproinsulin signal sequence has previously been shown to direct efficient secretion of other truncated receptors (11Taylor M.E. Bezouska K. Drickamer K. J. Biol. Chem. 1992; 267: 1719-1726Abstract Full Text PDF PubMed Google Scholar, 12Taylor M.E. Drickamer K. J. Biol. Chem. 1993; 268: 399-404Abstract Full Text PDF PubMed Google Scholar). The modified cDNA was inserted into the vector pED between the XbaI and EcoRI sites following the region encoding the adenovirus major late promoter and preceding the region encoding the dihydrofolate reductase gene (13Kaufman R.J. Davies M.V. Wasley L.C. Michnick D. Nuc. Acids Res. 1991; 19: 4485-4490Crossref PubMed Scopus (231) Google Scholar). The resulting plasmid was transfected into DXB11 Chinese hamster ovary cells (which are deficient in dihydrofolate reductase) using the calcium phosphate method (14Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6475) Google Scholar). Transfectants were selected by growth in minimal essential α medium without nucleosides and supplemented with 10% (v/v) dialyzed fetal calf serum. Amplification of protein expression was achieved by passaging cells into medium containing increasing concentrations (up to 0.5 μm) of methotrexate over several weeks. For protein production, cells were grown in medium containing 0.5 μm methotrexate. Once the cells reached confluence, the medium was changed and batches of medium were harvested every 2 days for 8 days. Tris-Cl, pH 7.8, CaCl2, and NaCl were added to the medium to give final concentrations of 20 and 20 mm and 0.5 m, respectively. Following centrifugation at 10,000 rpm in a Beckman JA14 rotor for 15 min, the medium was loaded onto a 10-ml column of galactose-Sepharose equilibrated in loading buffer. The column was washed with loading buffer before elution with elution buffer.Expression of Full-length SRCL in Fibroblasts—DNA coding for full-length SRCL was inserted into the retroviral expression vector pVcos (15Maddon P.J. Littman D.R. Godfrey M. Maddon D.E. Chess L. Axel R. Cell. 1985; 42: 93-104Abstract Full Text PDF PubMed Scopus (486) Google Scholar) 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 (16Southern P.J. Berg P. J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar). The final plasmid was transfected into ΨCre packaging cells to produce a pseudovirus that was used to infect Rat-6 fibroblasts, and fibroblast lines stably expressing SRCL were selected using G418 (17Stambach N.S. Taylor M.E. Glycobiology. 2003; 13: 401-410Crossref PubMed Scopus (149) Google Scholar). Cells were harvested by scraping, suspended in ∼10 volumes of 125 mm NaCl, 10 mm Tris-Cl, pH 7.5, and 0.5% Triton X-100, sonicated briefly, incubated 1 h at 4°C, and spun for 2 min at 18,000 × g. The supernatant was analyzed by SDS-polyacrylamide gel electrophoresis on a 12% gel, which was blotted onto nitrocellulose and probed with antibodies raised against the CRD (18Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5867) Google Scholar).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. Proteins were dialyzed extensively against 10 mm Tris-Cl, pH 7.8, 100 mm NaCl, 10 mm CaCl2. The extracellular domain of SRCL was analyzed at 5,000 and 8,000 rpm. For the CRD, rotor speeds of 10,000 and 21,000 rpm were used. Equilibrium distributions from three different loading concentrations (0.5, 0.25, and 0.125 mg/ml for the extracellular domain and 0.2, 0.1, and 0.05 mg/ml for the CRD) were analyzed simultaneously using the Nonlin curve fit program supplied with the instrument. Partial specific volumes for the SRCL fragments were determined from their amino acid composition and estimates of the extent of glycosylation (19Cohn E.J. Edsall J.T. Proteins, Amino Acids and Peptides as Ions and Dipolar Ions. Reinhold, New York1943: 370-381Google Scholar).Labeling of the Extracellular Domain of SRCL—For radio-iodination, the extracellular domain of SRCL, ∼100 μg of protein in 1.5 ml, was dialyzed against 150 mm NaCl, 25 mm HEPES, pH 7.8, 5 mm CaCl2 and reacted with 50 μCi of 125I-Bolton-Hunter reagent for 15 min at room temperature (20Bolton A.E. Hunter W.M. Biochem. J. 1973; 133: 529-539Crossref PubMed Scopus (2378) Google Scholar). Labeled protein was repurified by affinity chromatography on galactose-Sepharose. For fluorescent labeling, ∼250 μg of protein was dialyzed against water, lyophilized, dissolved in 0.5 ml of 0.5 m NaCl, 20 mm Na-bicine, 5 mm CaCl2, and reacted with 75 μg of fluorescein isothiocyanate that was dissolved at 1 mg/ml in dimethyl sulfoxide and added as five aliquots of 15 μl, followed by reaction overnight at 4 °C. EDTA was added to a concentration of 10 mm, and the labeled protein was repurified by gel filtration on a 10 × 300-mm Superdex 200 column (Amersham Biosciences) eluted with 0.1 m NaCl, 10 mm Tris-Cl, pH 7.8, 2.5 mm EDTA at a flow rate of 0.5 ml/min.Probing of Ligands—Glycoproteins were resolved on a 17.5% SDS-polyacrylamide gel and transferred to nitrocellulose. All of the glycoproteins were of bovine origin, except orosomucoid, which was from human serum. The blot was blocked at room temperature for 1 h with 2% bovine hemoglobin in wash buffer (125 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 25 mm CaCl2) and reacted for 1 h with the radio-iodinated extracellular domain of SRCL in the same solution at a concentration of ∼1 μg/ml. The blot was washed four times for 5 min each with cold wash buffer, and radioactivity was detected using a phosphorimaging device from Molecular Dynamics. Neoglycolipids, prepared by coupling oligosaccharides to phosphatidylethanolamine dipalmitate, were resolved by thin layer chromatography and fixed with isobutylmethacrylate (21Feizi T. Stoll M.S. Yuen C-T. Chai W. Lawson A.M. Methods Enzymol. 1994; 230: 484-519Crossref PubMed Scopus (118) Google Scholar). The chromatograms were then blocked and probed as for the glycoprotein blots. Fluorescein-labeled extracellular domain was used to probe the glycan array following the standard procedure of Core H of the Consortium for Functional Glycomics. 3www.functionalglycomics.org. Solid Phase Binding Assays—Polystyrene wells were coated overnight with either CRD or the extracellular domain of SRCL at a concentration of ∼50 μg/ml in loading buffer. Neoglycoprotein reporter ligands were created by iodination (22Greenwood F.C. Hunter W.M. Glover J.S. Biochem. J. 1963; 89: 114-123Crossref PubMed Scopus (6717) Google Scholar). Binding competition experiments and determination of the pH dependence of binding were performed as previously described (17Stambach N.S. Taylor M.E. Glycobiology. 2003; 13: 401-410Crossref PubMed Scopus (149) Google Scholar). Binding data were fitted using SigmaPlot software (Jandel Scientific).Molecular Modeling—The crystal structure of the galactose-binding derivative of mannose-binding protein with bound galactose was used as a starting point (23Kolatkar A. Weis W.I. J. Biol. Chem. 1996; 271: 6679-6685Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The structure of the Lewisx trisaccharide abstracted from the crystal structures of this ligand bound to DC-SIGN was used to model the ligand (24Guo Y. Feinberg H. Conroy E. Mitchell D.A. Alvarez R. Blixt O. Taylor M.E. Weis W.I. Drickamer K. Nat. Struct. Mol. Biol. 2004; 11: 591-598Crossref PubMed Scopus (479) Google Scholar), although similar results were obtained using coordinates derived from the crystal structure of sialyl-Lewisx bound to E-selectin and from the NMR structure of the free oligosaccharide. The galactose moiety of Lewisx was manually superimposed onto the galactose in the mannose-binding protein structure using Insight II modeling software.Endocytosis Experiments—Analysis of uptake and degradation of 125I-Gal-BSA or 125I-LNFP III-BSA by fibroblasts expressing SRCL was performed as described previously (25Mellow T.E. Halberg D. Drickamer K. J. Biol. Chem. 1988; 263: 5468-5473Abstract Full Text PDF PubMed Google Scholar).RESULTSExpression and Characterization of the SRCL Extracellular Domains—To initiate a study of the ligand-binding properties of SRCL, the C-terminal CRD was expressed in bacteria, whereas the entire extracellular domain was expressed in Chinese hamster ovary cells (Fig. 1). In the latter case, the insulin signal sequence was used to direct the protein to the endoplasmic reticulum so that it could be modified by hydroxylation and glycosylation before secretion into the medium. Both fragments of SRCL could be purified by affinity chromatography on galactose-Sepharose (Fig. 2). The CRD, on its own, binds weakly and washes off the affinity column even in the presence of Ca2+. The extracellular domain binds more effectively, and columns of sufficient capacity can be washed with Ca2+-containing buffer and eluted with EDTA. The extracellular domain fragment migrated as a broad band on SDS-polyacrylamide gels, suggesting that there is considerable heterogeneity in post-translational modification. The apparent molecular weight of 110–115 kDa deduced from the gels was consistent with the predicted presence of up to 13 N-linked glycans, 8 O-linked glycans, and mono- or disaccharide residues attached to hydroxylysine residues, all appended to a core polypeptide of 75 kDa.Fig. 2Gel electrophoresis of fractions from affinity chromatography of fragments of SRCL on galactose-Sepharose. Fractions from washing in the presence of Ca2+ (W) and elution with EDTA (E) are shown. Gels were stained with Coomassie Blue.View Large Image Figure ViewerDownload (PPT)Weak binding to highly substituted sugar resins is often characteristic of monomeric forms of C-type lectins. Therefore, the oligomeric states of the fragments were analyzed by equilibrium analytical ultracentrifugation (Fig. 3). The results demonstrated that the CRD is monomeric, but the entire extracellular domain formed a stable trimer. This result is consistent with the presence of the collagen-like domain, which would be expected to direct trimerization. In addition, the sequence of the N-terminal portion of the extracellular domain was predicted to form a 3-stranded coiled coil of α-helices, which would further stabilize the trimer. The relatively weak binding of both the CRD and the extracellular domain to galactose-Sepharose contrasts with other galactose-binding C-type lectins, such as the asialoglycoprotein receptor and the Kupffer cell receptor. Both monomeric and trimeric fragments of these receptors bind tightly to this resin and can only be eluted with EDTA, indicating that these receptors have inherently higher affinity for galactose. These results suggest that galactose on its own is not an optimal ligand for SRCL.Fig. 3Sedimentation equilibrium analytical ultracentrifugation of SRCL fragments. Data for three different loading concentrations, shown as symbols, were fitted globally, with the fitted curve shown as continuous lines. The deduced Mr of the CRD is 17,000 compared with the Mr of the CRD polypeptide calculated from the amino acid sequence, which is 16,441. The measured Mr of the extracellular domain is 317,000, which falls in the range of values calculated for a trimer with various possible degrees of glycosylation (313,000–322,000). Similar results were obtained at two different speeds for each sample. Results for 21,000 rpm for the CRD and 5,000 rpm for the extracellular domain are shown.View Large Image Figure ViewerDownload (PPT)Binding of SRCL to Lewisx and Related Structures—An initial screening for higher affinity ligands was undertaken by testing the binding of SRCL to a selection of glycoproteins. The glycoproteins were resolved by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and probed with the radiolabeled extracellular domain of SRCL (Fig. 4). Strong binding to asialo-orosomucoid is evident, but no binding to other desialylated glycoproteins was detected. Binding to glycoproteins bearing sialylated complex, high mannose, or sulfated N-linked glycans was also not detected.Fig. 4Binding of 125I-SRCL to glycoproteins. Aliquots of ∼2 μg of each glycoprotein were resolved by SDS-polyacrylamide gel electrophoresis on a 17.5% gel. Portions of the gel were stained with Coomassie Blue and blotted onto nitrocellulose for probing with 125I-SRCL. SBA, soybean agglutinin. RNase, ribonuclease. Samples in the first lane contain high mannose and hybrid glycans. Thyrotropin in the final lane contains sulfated glycans. Orosomucoid, fetuin, transferrin, and fibrinogen bear sialylated complex glycans.View Large Image Figure ViewerDownload (PPT)There are two relatively unusual features to the N-linked glycans on asialo-orosomucoid. First, many of the glycans have highly branched tri- and tetra-antennary structures. Second, many of these branches bear outer-arm fucose residues (26Treuheit M.J. Costello C.E. Halsall H.B. Biochem. J. 1992; 283: 105-112Crossref PubMed Scopus (147) Google Scholar). The contribution of these two features to the binding of SRCL to asialo-orosomucoid was assessed by testing the binding to highly branched glycans lacking fucose residues and glycans bearing single terminal fucose residues. The glycans were presented as neoglycolipids resolved by thin layer chromatography (Fig. 5). The radiolabeled extracellular domain of SRCL binds to the Lewisx- and Lewisa-containing ligands but not to the branched, unfucosylated oligosaccharides. Thus, SRCL shows selectivity for glycans bearing both terminal galactose and fucose residues.Fig. 5Binding of 125I-SRCL to neoglycolipids.A, chromatogram was developed with CHCl3/methanol/H2O 105:100:28. B, chromatogram was developed with CHCl3/methanol/H2O 11:9:2. Parallel chromatograms were stained with orcinol or fixed and probed with125I-SRCL. LNT, lacto-N-tetraose; LNnT, lacto-N-neotetraose. The boxes indicate Lewisx and Lewisa trisaccharide structures.View Large Image Figure ViewerDownload (PPT)A broader view of the binding selectivity of SRCL was obtained by probing a glycan array with fluorescently labeled extracellular domain. The array consists of over 150 different biotinylated oligosaccharides presented on streptavidin immobilized in polystyrene wells. The results reveal a striking selectivity for glycans containing terminal Lewisx trisaccharides, with no binding to sulfated or sialylated forms of these glycans (Fig. 6). Weaker binding was also observed for the isomeric Lewisa trisaccharide and to Lewisx, in which the galactose is substituted by N-acetylgalactosamine. In addition, very weak binding to blood group H substances was detected. The high degree of selectivity of SRCL is relatively unusual among C-type lectins.Fig. 6Binding of fluorescein-labeled SRCL to glycan array. Biotin glycosides were immobilized in streptavidin-coated wells. Binding and washing were conducted in the presence of 150 mm NaCl, 25 mm Tris-Cl, pH 7.5, and 2 mm CaCl2. The level of fluorescence was normalized to glycan 25 (Lewisx).View Large Image Figure ViewerDownload (PPT)Selectivity for Lewisx and Mechanism of Binding—Previous studies with the dendritic cell receptor DC-SIGN have revealed that this receptor, similar to SRCL, binds Lewisx and other glycans that bear terminal fucose and galactose residues, although it binds a broader range of such glycans. The primary binding site in the C-type CRD of DC-SIGN binds to the fucose moiety (24Guo Y. Feinberg H. Conroy E. Mitchell D.A. Alvarez R. Blixt O. Taylor M.E. Weis W.I. Drickamer K. Nat. Struct. Mol. Biol. 2004; 11: 591-598Crossref PubMed Scopus (479) Google Scholar), whereas the primary binding site in SRCL has the hallmarks of a galact
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