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J. Biol. Chem., Vol. 277, Issue 25, 22566-22572, June 21, 2002

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An Endogenous Drosophila Receptor for Glycans Bearing 1,3-Linked Core Fucose Residues^*

Samuel Bouyain^§, Nicholas J. Silk, Gustáv Fabini^¶, and Kurt Drickamer

From the  Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom and the ^¶ Glycobiology Division, Institut für Chemie der Universität für Bodenkultur, Muthgasse 18, A-1190 Wien, Austria

Received for publication, March 25, 2002, and in revised form, April 17, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The genome of Drosophila melanogaster encodes several proteins that are predicted to contain Ca²⁺-dependent, C-type carbohydrate-recognition domains. The CG2958 gene encodes a protein containing 359 amino acid residues. Analysis of the CG2958 sequence suggests that it consists of an N-terminal domain found in other Drosophila proteins, a middle segment that is unique, and a C-terminal C-type carbohydrate-recognition domain. Expression studies show that the full-length protein is a tetramer formed by noncovalent association of disulfide-linked dimers that are linked through cysteine residues in the N-terminal domain. The expressed protein binds to immobilized yeast invertase through the C-terminal carbohydrate-recognition domain. Competition binding studies using monosaccharides demonstrate that CG2958 interacts specifically with fucose and mannose. Fucose binds ~5-fold better than mannose. Blotting studies reveal that the best glycoprotein ligands are those that contain N-linked glycans bearing 1,3-linked fucose residues. Binding is enhanced by the additional presence of 1,6-linked fucose. It has previously been proposed that labeling of the Drosophila neural system by anti-horseradish peroxidase antibodies is a result of the presence of difucosylated N-linked glycans. CG2958 is a potential endogenous receptor for such neural-specific carbohydrate epitopes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animal lectins provide a mechanism for recognition of protein- and lipid-linked glycans. Recognition of endogenous glycoconjugates at the cell surface, in the extracellular matrix, and in serum can lead to intercellular adhesion and signaling as well as uptake and degradation. Animal lectins are diverse in structure, but they usually contain modular carbohydrate-recognition domains (CRDs)¹ (1, 2). The C-type CRDs are the largest and most diverse class of CRDs. These domains share a common fold and show Ca²⁺-dependent sugar binding activity, although their selectivity for different carbohydrate ligands varies. Binding of sugars involves formation of a ternary complex between the protein, a bound Ca²⁺, and the sugar (3). Selectivity for particular sugar ligands is determined in large part by the disposition of Ca²⁺-ligating residues in the protein, but can also reflect additional interactions with nearby regions of the protein surface. The C-type CRDs are a subset of a larger family of protein modules, the C-type lectin-like domains (CTLDs) (4). Although the CTLDs share a common fold, many do not bind Ca²⁺ and they often interact with ligands others than sugars.

Profile analysis provides a powerful method for identifying CTLDs in proteins identified by genome sequence analysis. Using information about the structures and sugar binding activities of known C-type CRDs, it is possible to identify CTLDs that are likely to display carbohydrate binding activity. This approach has been applied to the complete genomic sequences of model organisms such as Caenorhabditis elegans (5) and Drosophila melanogaster (2) as well as to the human genome (see ctld.glycob.ox.ac.uk for a current update). The screen of the Drosophila genome revealed 32 genes that encode proteins containing CTLDs, of which only 6 have appropriate residues at the key positions required for Ca²⁺ and sugar binding in the C-type CRDs. Among these potential CRDs, the domain encoded in the CG2958 gene (also designated Lectin-24DB) shows a particularly strong resemblance to mammalian CRDs that bind mannose, fucose, and N-acetylglucosamine. The Ca²⁺-ligating residues are in the same configuration as those in serum mannose-binding protein and other C-type lectins that bind sugars with hydroxyl groups in the orientation corresponding to the 3- and 4-hydroxyl groups of mannose (6). In addition, the CTLD of CG2958 contains a cluster of basic residues (Arg-Lys-Lys at positions 347-349) that can form a secondary binding site that enhances binding of C-type CRDs to anionic oligosaccharides (7).

To probe a possible role of CG2958 in sugar-mediated recognition, the protein has been expressed and its carbohydrate-binding properties have been examined. CG2958 appears to be an endogenous receptor for 1,3-linked fucose residues linked to the core GlcNAc residue of N-linked oligosaccharides in Drosophila.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Restriction enzymes were purchased from New England Biolabs. Both the Advantage 2 polymerase mix and the adult Drosophila Matchmaker cDNA library were supplied by CLONTECH. Synthetic oligonucleotides were obtained from Invitrogen. Yeast invertase, glycans, and glycoproteins used in binding assays were obtained from Sigma. Man-BSA, purchased from E-Y Laboratories, had an average density of 30 mol of mannose/mol of protein. Na¹²⁵I, Bolton-Hunter reagent, and nitrocellulose were purchased from Amersham Biosciences. Immulon 4 polystyrene wells were from Dynex Technologies. Affinity resins were prepared by the method of Fornstedt and Porath (8). RNase B oligosaccharides were purchased from Oxford GlycoSciences, and oligosaccharides from soybean agglutinin were a kind gift of Daniel Mitchell and Brian Matthews (Glycobiology Institute, University of Oxford, Oxford, United Kingdom).

Cloning and Mutagenesis-- cDNA sequences encoding the CRD of CG2958 were amplified with forward primer 5'-aaggccggccaccaaggtcttctggccgaaatttgagcga-3' and reverse primer 5'-ttgcggccgcttaaacttctttgtcggtttgacaaataac-3'. For amplification of the CRD and neck region, the alternative forward primer 5'-aaggccggcctccttggaggaatcagcgcagaaggttcca-3' was used. The 5' end of the cDNA was amplified using forward primer 5'-aaggccggcccgagcggaatccacagaaaattcccgatcc-3' and reverse primer 5'-ttgcggccgccattttcattagcctcgcatcgagttgagc-3'. The primers include restriction sites for FseI or 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 modified to contain the restriction sites FseI and NotI downstream of the ompA signal sequence (9). Portions of cDNAs without reverse transcription errors were recombined using convenient restriction sites. The resulting plasmids were used to transform Escherichia coli strain JA221. To avoid 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 the vector with FseI followed by trimming of the 3' extensions with T4 DNA polymerase. Mutagenesis was performed by substituting double-stranded synthetic oligonucleotides for restriction fragments in the expression plasmid.

Expression and Purification of Proteins-- An aliquot of an overnight culture of transformed bacteria (20 ml) was used to inoculate 1 liter of Luria-Bertini medium containing 50 µg/ml ampicillin, and the culture was grown at 25 °C and 200 rpm. Expression of proteins was induced, when the A_550 nm reached 0.8, by adding isopropyl--D-thiogalactopyranoside to a final concentration of 40 µM and CaCl₂ to a final concentration of 100 mM. Cells were grown for another 16-18 h.

Harvested cells were suspended in loading buffer (150 mM NaCl, 25 mM CaCl₂, and 25 mM Tris-Cl, pH 7.8) and sonicated with the large probe of a Branson 250 sonifier at full power for a total of 10 min. The lysate was spun for 15 min in a Beckman JA14 rotor at 11,000 × g and for another 60 min at 100,000 × g in a Beckman Ti55.2 rotor. The supernatant was applied to a 1-ml column of invertase-Sepharose equilibrated with 5 ml of loading buffer. The column was washed with loading buffer, and proteins were recovered by elution with five aliquots of 0.5 ml of eluting buffer (150 mM NaCl, 2.5 mM EDTA, and 25 mM Tris-Cl, pH 7.8). In most cases, the expressed proteins were further purified on a C4 reverse phase column (4.6 × 50 mm) eluted with an acetonitrile gradient increasing from 10 to 60% at a rate of 1.25%/min in the presence of 0.1% trifluoroacetic acid. Fractions were concentrated for 30 min to remove acetonitrile and then lyophilized.

Binding Assays-- Aliquots (50 µl) of the neck and CRD fragment of CG2958 (0.1 mg/ml) in loading buffer were pipetted into Immulon 4 wells, and the plate was incubated overnight at 4 °C. Protein solution was removed, and wells were filled with 5% BSA in loading buffer. After 2 h at 4 °C, the blocking solution was discarded and wells were washed three times with cold HEPES-buffered saline (136 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl₂, 0.5 mM MgCl₂, 19 mM Na-HEPES, pH 7.5). Aliquots (100 µl) of inhibitor solutions at various concentrations in HEPES-buffered saline containing 1 mg/ml BSA and ¹²⁵I-Man-BSA (~0.5 µg/ml) were added to the protein-coated wells. After 2 h at 4 °C, wells were washed three times with cold HEPES-buffered saline and counted in a Wallac Wizard  counter.

Results were fitted to a simple competition binding equation (10), in which K_I is the concentration of inhibitor that gives 50% inhibition of ¹²⁵I-Man-BSA binding, using the SigmaPlot program from Jandel Scientific. Results reported as averages ± standard deviations are from at least two experiments, each performed in duplicate. Assays showing little or no inhibition or with inhibitors available in limited quantities were performed only once, with duplicate samples for each data point.

Blotting and Overlay Procedures-- For iodination, 20 µl (0.1 mCi) of Bolton-Hunter reagent (11) was dried with argon, CG2958 (100 µg in 200 µl of 100 mM NaCl, 25 mM CaCl₂, and 25 mM Na-HEPES, pH 7.8) was added, and the reaction was allowed to proceed at room temperature for 10 min. After addition of 800 µl of loading buffer, the labeled protein was recovered on invertase-Sepharose as described above. Glycoprotein samples (10 µg) were run on an SDS-polyacrylamide gel under reducing conditions, and the gel was transferred onto nitrocellulose (12). The membrane was blocked with 2% hemoglobin in HEPES-buffered saline for 1 h at room temperature and incubated for 90 min with a solution of ¹²⁵I-CG2958 in HEPES-buffered saline in the presence of 2% hemoglobin. Following three washes with cold HEPES-buffered saline for 5, 10, and 10 min, radioactivity was detected using a PhosphorImager (Molecular Dynamics).

For dot blots, nitrocellulose membranes were soaked in gel transfer buffer and clamped into a 96-well dot blot apparatus. Protein samples prepared in 100 µl of gel transfer buffer were filtered through the membrane under vacuum, followed by another 100 µl of gel transfer buffer. The membrane was removed from the apparatus and blocked and incubated with ¹²⁵I-CG2958 as for the gel blot. Neoglycolipids were prepared and resolved by thin layer chromatography following published procedures (13). The chromatograms were blocked and with ¹²⁵I-CG2958 as described for the gel blots.

Analytical Methods-- Polyacrylamide gel electrophoresis was performed by the method of Laemmli, using gels containing 17.5% acrylamide (14). Samples were prepared by heating at 100 °C for 5 min in sample buffer, either in the presence or absence of 1% 2-mercaptoethanol. Equilibrium analytical ultracentrifugation was carried out as described previously in a Beckman XLA-70 centrifuge (15). Protein was analyzed at three different loading concentrations at 12,000 rpm and 20 °C. Equilibrium distributions were fitted globally to a single species model, using the software supplied with the centrifuge. The partial specific volume of CG2958 was calculated as 0.730 ml/g from the amino acid composition (16).

Computational Methods-- The SwissProt data base (17) was accessed through the European Bioinformatics Institute web site, and sequence comparisons were performed using Fast A with the default parameters. Molecular modeling was performed with the Insight II software (Biosym), starting with coordinates for the CRD of mannose-binding protein modified to display selectin-like binding characteristics (Protein Data Bank entry 2kmb).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sequence and Expression of CG2958-- The deduced amino acid sequence of CG2958 is shown in Fig. 1. The CTLD was previously identified using profile scanning algorithms, and the extent of the signal sequence was predicted using hydropathy plots combined with the sequence preference of signal peptidase (18, 19). The domain organization of the remainder of the protein was investigated by screening the SwissProt data base of protein sequences with the portion of CG2958 between the signal sequence and the CTLD. The results revealed that several proteins in Drosophila contain sequences similar to residues 21-90 of CG2958. Within this N-terminal domain, two cysteine residues are conserved in most of these proteins. No related sequences in other organisms were detected. The remaining portion of CG2958, between the N-terminal domain and the CTLD, was rescreened against the protein sequence data base, but no significant sequence similarity was detected.

View larger version (58K): [in this window] [in a new window]   Fig. 1.   Comparison of the sequence of Drosophila protein CG2958 with other proteins sharing related domains. The N-terminal portion of the deduced amino acid sequence of CG2958 is compared with other Drosophila proteins that contain homologous sequences. The C-terminal CTLD is compared with C-type CRDs from human E-selectin and rat serum mannose-binding protein. Elements of secondary structure are -helix (), -strand (), and loop (L). Ca2+-binding residues are designated by the numbers 1 and 2. Conserved cysteine residues throughout the sequences and conserved amino acids in the N-terminal domain are shaded. The stretches of three basic amino acid residues in CG2958 and E-selectin are underlined, and residues that have been mutated are indicated with asterisks.

A CG2958 cDNA was created using the polymerase chain reaction. Sequencing of multiple independent clones revealed a consistent discrepancy at the codon for amino acid 134, which encodes lysine in the Drosophila genome sequence (codon AAA) and arginine in the cDNAs (codon AGA). This difference may reflect a strain difference or a polymorphism in the Drosophila population. Based on the sequence analysis, vectors were created from the amplified cDNA to express the entire CG2958 polypeptide, a fragment lacking the N-terminal domain, and a smaller fragment consisting only of the CTLD. In each case, a bacterial signal sequence was fused to the open reading frame to allow export into the periplasm of E. coli. Induction of protein synthesis in the presence of Ca²⁺ allows correct folding of many CTLDs under such conditions (10). In the case of CG2958, sonication of the bacteria in Ca²⁺-containing buffer and passage over a column containing immobilized yeast invertase resulted in binding of the expressed fragments, which could be eluted with EDTA (Fig. 2). The intact protein and the CRD + neck fragment were retained more effectively on the column than was the fragment consisting only of the CRD.

View larger version (79K): [in this window] [in a new window]   Fig. 2.   SDS-polyacrylamide gel electrophoresis of intact CG2958 and C-terminal fragments purified by affinity chromatography on invertase-Sepharose. Protein was purified from 500 ml of bacterial culture. The CRD fragment was purified on a 2-ml column, and the intermediate and full-length proteins were purified on 1-ml columns. W, wash fractions in Ca2+-containing loading buffer; E, elution fractions with EDTA-containing elution buffer. Aliquots (25 µl) from the Ca2+-containing wash fractions and the EDTA-containing elution fractions were examined by SDS-polyacrylamide gel electrophoresis (17.5% gel) followed by staining with Coomassie Blue.

Weak binding of isolated CRDs to affinity columns and stronger binding of larger lectin fragments often reflects multivalent binding by oligomers of the larger fragments. Portions of the lectin polypeptide outside the CRDs are often necessary to stabilize such oligomers (4). The oligomeric states of the CG2958 fragments were investigated by gel electrophoresis and sedimentation analysis (Fig. 3). When analyzed by SDS-polyacrylamide gel electrophoresis, the intact protein runs as a covalent dimer, whereas the fragments that lack the N-terminal domain run as monomers. Thus, CG2958 consists of covalent dimers of polypeptides that are held together by disulfide bonds between cysteine residues in the N-terminal domain. Sedimentation analysis gives a molecular mass of 156,800 Da, which corresponds to a tetramer with a calculated molecular mass of 156,900 Da. These results demonstrate that CG2958 consists of a dimer of covalent dimers, in which each polypeptide comprises an N-terminal oligomerization domain, an intervening neck, and a C-terminal CRD. Although formation of the covalent oligomer requires the N-terminal domain, the relatively tight binding of the neck + CRD fragment to the invertase-Sepharose column suggests that noncovalent oligomerization can be mediated by the neck region.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Analysis of the oligomeric state of CG2958. Left, SDS-polyacrylamide gel electrophoresis (12.5% gel) of expressed portions of CG2958 analyzed in the presence and absence of reducing agent. Right, sedimentation equilibrium ultracentrifugation of intact CG2958. Scans of sectors with loading concentrations of 30 µg/ml (top), 15 µg/ml (middle), and 8 µg/ml (bottom) are presented. The protein distribution and the results of fitting are shown as circles and lines, respectively. Deviations of the experimental data from the fitted curves are shown at the top of each panel.

Analysis of Sugar Binding Activity-- The observation that CG2958 binds to immobilized yeast invertase in a Ca²⁺-dependent manner suggested that it interacts with the extensive mannose-containing glycans attached to this protein. To obtain direct evidence for carbohydrate-dependent binding, the fragment representing the head plus neck region of CG2958 was immobilized on polystyrene wells. This fragment was chosen because it binds tightly to the affinity resin and is produced in high yield. Radiolabeled Man-BSA was found to bind to the immobilized protein and was therefore used as a reporter ligand to test the binding of potential sugar ligands in competition assays. These assays were first used to establish relative affinities of CG2958 for various monosaccharides (Table I).

The inhibition data reveal that, among the neutral monosaccharides tested, the most effective inhibitors of Man-BSA binding are L-fucose and the - and -methyl fucosides. Among the D-hexoses tested, only mannose showed inhibition in the low millimolar range. Weak inhibition observed with free galactose is probably caused by interaction with the anomeric hydroxyl group of the free sugar, as has been previously observed (20), because -methyl-D-galactoside does not inhibit binding. The selective binding of mannose and -methyl-D-mannoside suggests that, as with other C-type lectins, the relative orientation of the 3- and 4-hydroxyl groups is important for binding to CG2958. The assays also reveal that binding is sensitive to the orientation and nature of the 2-substituent, because glucose, GlcNAc, N-acetylmannosamine, and 2-deoxyglucose are all poor inhibitors. Thus, the data suggest that the 2-, 3-, and 4-hydroxyl groups of mannose may interact with the binding site of CG2958.

In the light of the selectin-like aspects of the CG2958 sequence, various potential anionic ligands were tested. In the inhibition assay, only sialic acid showed any inhibition at the concentrations tested. The measured K_I indicates that it binds to CG2958 with higher affinity than glucose, but the affinity is still 7-fold lower than the affinity for mannose. Polymers containing glucuronic acid as well as various sulfated sugars were also tested, but none of these have enhanced affinity compared with glucuronic acid. Finally, the result using fucoidan as an inhibitor indicates that sulfated fucose shows decreased rather than increased affinity compared with fucose. Direct incubation with radioiodinated sialyl Lewis^x-BSA also failed to demonstrate any binding (data not shown).

Possible Orientations of Monosaccharides in the Binding Site of CG2958-- The high selectivity for fucose and the submillimolar K_I are unusual in C-type CRDs, so it was of interest to determine what structural features of the CRD might enhance binding to this monosaccharide. In previous studies, the sequence Lys-Lys-Lys has been inserted into the CRD from mannose-binding protein in the positions corresponding to the Arg-Lys-Lys sequence in CG2958 (21). The crystal structure of this modified CRD complexed with the fucose-containing sialyl-Lewis^x tetrasaccharide (22) was used as a starting point to model the binding site of CG2958. There are four orientations in which pairs of hydroxyl groups of fucose can be superimposed on the hydroxyl groups that interact with Ca²⁺ in the binding site (Fig. 4). Two of these structures involve interactions between Ca²⁺ and the 2- and 3-hydroxyl groups (orientations A and B) and two involve interaction with the 3- and 4-hydroxyl groups (orientations C and D). In orientation D, there are clashes with the modeled protein surface, so this orientation can be dismissed. In orientation C, the fucose residue approaches the side chains of residues that correspond to Asn³²⁷ and Arg³⁴⁷ of CG2958 in the model. Binding of the 2- and 3-hydroxyl groups, as in the original crystal structure, projects the sugar mostly away from the protein surface, but in orientation B there is a potential hydrogen bond between O1 of the sugar and Arg³⁴⁷.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Possible orientations of fucose in the binding site of a C-type CRD. The model is based on the arrangement of fucose in the binding site of the CRD from mannose-binding protein that has been modified to include three lysine residue analogous to those found in E-selectin (22). Side chains of residues near the binding site have been changed to correspond to the residues found in CG2958: Asn327 and Arg347. Four possible orientations of fucose, indicated at the bottom of the figure, allow superposition of hydroxyl groups of fucose on O2 and O3 of fucose in the A orientation seen in the crystal structure. Orientation A is shown in white and orientation C in gray. Atoms are shaded black for carbon, gray for nitrogen, and white for oxygen.

Because interactions of residues Asn³²⁷ or Arg³⁴⁷ with the O1 and O2 substituents of fucose could contribute to selective binding to this sugar, their role in preferential binding to fucose was tested by mutagenesis. The effects of changing these residues to alanine or lysine on the relative binding affinities for mannose and fucose were examined using the competition binding assay (Table II). Changing Asn³²⁷ to alanine has the most severe effect on the relative affinity for fucose, causing a nearly 3-fold reduction in the K_I for fucose compared with mannose. There is a marginal effect when residue Arg³⁴⁷ is modified. The only orientation in which significant interactions with Asn³²⁷ would be predicted is orientation C, suggesting that this is the most likely way that fucose interacts with CG2958. This orientation is similar to the orientation observed in crystals of E- and P-selectin with bound sialyl-Lewis^x ligand.

Oligosaccharide Ligands for CG2958-- To screen for potential oligosaccharide ligands for CG2958, the expressed protein was radioiodinated and used to probe a glycoprotein blot containing a range of glycan structures (Fig. 5). As a positive control, yeast invertase was included on the blot. The strong binding observed confirms that the radioiodinated material retains binding activity. No interaction with glycoproteins bearing complex N-linked glycans was detected. Binding to high mannose oligosaccharides is suggested by the interaction with RNase B, which contains a range of oligosaccharides from Man₃GlcNAc₂ to Man₉GlcNAc₂ (23). In contrast, no binding to soybean agglutinin was detected, although this glycoprotein bears Man₉GlcNAc₂ oligosaccharides (24). To determine whether CG2958 binds preferentially to the smaller oligosaccharides that are more abundantly expressed on RNase B, the oligosaccharides from RNase B and soybean agglutinin were released and presented to CG2958 in the form of neoglycolipids on a thin layer chromatogram (Fig. 5). In this format, binding to all of the mannose-containing structures, including Man₉GlcNAc₂, was observed at levels proportional to their abundance (25). These results suggest that binding of CG2958 to oligosaccharides may be influenced by the proteins to which they are conjugated.

View larger version (59K): [in this window] [in a new window]   Fig. 5.   Binding of glycoconjugates by CG2958. Left, glycoproteins (10 µg each) were resolved by SDS-polyacrylamide gel electrophoresis (17.5% gel), blotted onto nitrocellulose, and incubated with radioiodinated CG2958. Right, neoglycolipids made from oligosaccharides derived from bovine ribonuclease B and soybean agglutinin were resolved by thin layer chromatography and stained with radioiodinated CG2958.

The other glycoprotein ligand detected in the blotting procedure is horseradish peroxidase. As a plant glycoprotein, horseradish peroxidase bears glycans containing sugars, such as xylose, and linkages, including fucose residues 1,3-linked to the inner core GlcNAc residue, that are not found in mammalian glycoproteins (26, 27). Xylose is not found in N-linked glycans on Drosophila glycoproteins, but the core 1,3-fucose residue is present (28). The most striking finding from the comparison of monosaccharide affinities is the relatively high selectivity for fucose, suggesting that the binding to horseradish peroxidase might be a result of the presence of core fucose. Goat immunoglobulin G, which bears core 1,6-linked fucose (29), did not react, suggesting that the binding might be specific for 1,3-linked fucose.

The possibility that CG2958 binds to core 1,3-linked fucose residues was investigated using various modified human serum transferrin preparations, in an approach previously used for studying binding of anti-horseradish peroxidase antibody (28). After removal of the terminal sialic acid and galactose residues, fucose was added enzymatically in vitro to the inner GlcNAc residue in either 1,3- or 1,6-linkage. The nonreducing terminal GlcNAc residues were then removed to expose the modified Man₃GlcNAc₂ core. In preliminary studies, these proteins were run on SDS-polyacrylamide gels, blotted onto nitrocellulose, and probed with radioiodinated CG2958. Only the proteins bearing structures modified with 1,3-linked or 1,3- and 1,6-linked core fucose were bound by the labeled protein (data not shown). Similar modified proteins in which the nonreducing terminal GlcNAc residues were retained were also tested, but no binding was observed. Binding to the fucosylated Man₃GlcNAc₂ core structures was quantified by performing a dot blot assay and comparing the binding to that obtained with dilutions of horseradish peroxidase (Fig. 6). Binding to 1-µg aliquots of the modified transferrin samples exceeds binding to comparable amounts of horseradish peroxidase, even though here are only two N-glycosylation sites in transferrin and seven in horseradish peroxidase and the in vitro fucosylation of transferrin is incomplete. The results confirm that binding is dependent on the presence of 1,3-linked fucose, and they suggest that addition of 1,6-linked fucose enhances the binding. The enhancement of CG2958 binding to modified transferrin by 1,6-fucosylation is consistent with the findings that all 1,3-fucosylated N-glycans found in adult flies are difucosylated and that 1,6-fucosylated N-glycans are the preferred substrates for the 1,3-fucosyltransferase.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Binding of CG2958 to glycoproteins detected by dot blot assay. Samples consisted of various concentrations of horseradish peroxidase (A) or 1 µg of each of the modified transferrin preparations (B). Following incubation with radioiodinated CG2958, binding was quantified using a PhosphorImager. Averages of duplicate samples, corrected for background binding, are shown. In all cases, the results for duplicates were within 5% of each other.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results presented here demonstrate that Drosophila protein CG2958 is a fucose-binding lectin that interacts specifically with 1,3-linked fucose attached to the core of N-linked glycans. This binding can occur with oligosaccharides that are attached to proteins, so CG2958 is a potential endogenous receptor for Drosophila glycoproteins that bear core 1,3-fucosylated glycans. CG2958 is predicted to be secreted, and the full-length, expressed protein is soluble in the absence of detergents. Its tetrameric structure indicates that it would have the ability to interact with multiple N-linked glycans simultaneously. Depending on the geometry of the tetramer, such multivalent binding could lead to high affinity attachment to cell surfaces or to cross-linking of glycoproteins.

CG2958 was initially considered a potential selectin ortholog, largely based on the presence of a cluster of three basic residues adjacent to the predicted primary sugar-binding site in both the selectins and CG2958. In previous studies, it has been shown that such a cluster of residues can form a secondary, ionic strength-sensitive subsite in C-type CRDs, facilitating the binding of sialyl-Lewis^x and other anionic oligosaccharide ligands (7). Additionally, like the selectin CRDs, the CRD in CG2958 appears to contain a single Ca²⁺-binding site, because several of the acidic amino acid residues that usually form the secondary site are not conserved in CG2958 (Fig. 1). The present studies suggest that CG2958 and the selectins share the ability to bind fucose-containing ligands, but their oligosaccharide-binding characteristics are very different. CG2958 does not bind with high affinity to any of the anionic oligosaccharide or polysaccharide ligands tested. Thus, there seems to be little basis for suggesting that CG2958 might function in a selectin-like fashion in Drosophila.

Despite the differences between the CRDs in the selectins and CG2958, it is interesting that these proteins do share the ability to bind to fucose attached to N-acetylglucosamine in 1,3 linkage. The modeling and mutagenesis results suggest a possible orientation of fucose-containing ligands in the binding site of the CRD in CG2958, in which the anomeric hydroxyl group would be near to one of the loops of the polypeptide adjacent to the conserved Ca²⁺ that forms the nucleus of the sugar-binding site (Fig. 4). The portions of the E- and P-selectin CRDs that correspond to the loop containing Asn³²⁷ in CG2958 form part of the extended sialyl-Lewis^x-binding sites (30). Differences in sequence between these loops in the selectins and CG2958 preclude exactly analogous contacts, but the results suggest that the loop may be a common determinant of ligand-binding specificity beyond the simple monosaccharide-binding site. Although the modeling studies are suggestive, further structural analysis will clearly be necessary.

The pattern of staining of Drosophila embryos with anti-horseradish peroxidase antibodies suggests a role for core 1,3-linked fucose in the nervous system (31-33). The ability of CG2958 to serve as an endogenous receptor for glycoproteins bearing core 1,3-linked fucose further suggests that this protein might also be expressed in the nervous system. The Berkeley Drosophila Genome Project (34) has reported expressed sequence tags for the mRNA encoding CG2958 in cDNA libraries from head, brain, and sensory organs in the larval-early pupal stage suggestive of expression throughout development (identifiers HL05328 and LP02926).

Structural data for glycoconjugates in invertebrates, including Drosophila, are limited (35, 36). However, the information that is available suggests that there are substantial differences between the glycans in invertebrates and vertebrates. Particularly striking differences are observed in the terminal sugars present on glycans. Although sialic acid and galactose are common terminal elaborations of vertebrate glycans, these sugars are rare, if present at all, in invertebrates. In contrast, fucose appears to be much more abundant in invertebrates. These data suggest that cell surface carbohydrates have evolved somewhat differently in different animal lineages. The results reported here suggest that endogenous receptors for cell surface glycans have evolved in parallel to recognize a distinct complement of sugar structures.

	ACKNOWLEDGEMENTS

We thank Dawn Torgersen for help with protein production and binding assays, Russell Wallis for assistance with the analytical ultracentrifugation, and Iain Wilson and Maureen Taylor for comments on the manuscript.

	FOOTNOTES

^* This work was supported in part by Grant 041845 from the Wellcome Trust, Grant 43/B13743 from the Biotechnology and Biological Sciences Research Council (BBSRC), and Grant P13810-GEN from the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (to Iain B. H. Wilson). The analytical ultracentrifugation facility is supported by the Wellcome Trust and the BBSRC.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported by a studentship from the Wellcome Trust.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, United Kingdom. Tel.: 44-1865-275727; Fax: 44-1865-275339; E-mail: kd@glycob.ox.ac.uk.

Published, JBC Papers in Press, April 17, 2002, DOI 10.1074/jbc.M202825200

	ABBREVIATIONS

The abbreviations used are: CRD, carbohydrate-recognition domain; CTLD, C-type lectin-like domain; BSA, bovine serum albumin; Man-BSA, mannosylated-bovine serum albumin.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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