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High Affinity Interaction between a Bivalve C-type Lectin and a Biantennary Complex-type N-Glycan Revealed by Crystallography and Microcalorimetry*

Open AccessPublished:August 07, 2008DOI:https://doi.org/10.1074/jbc.M804353200
      Codakine is an abundant 14-kDa mannose-binding C-type lectin isolated from the gills of the sea bivalve Codakia orbicularis. Binding studies using inhibition of hemagglutination indicated specificity for mannose and fucose monosaccharides. Further experiments using a glycan array demonstrated, however, a very fine specificity for N-linked biantennary complex-type glycans. An unusually high affinity was measured by titration microcalorimetry performed with a biantennary Asn-linked nonasaccharide. The crystal structure of the native lectin at 1.3Å resolution revealed a new type of disulfide-bridged homodimer. Each monomer displays three intramolecular disulfide bridges and contains only one calcium ion located in the canonical binding site that is occupied by a glycerol molecule. The structure of the complex between Asn-linked nonasaccharide and codakine has been solved at 1.7Å resolution. All residues could be located in the electron density map, except for the capping β1–4-linked galactosides. The α1–6-linked mannose binds to calcium by coordinating the O3 and O4 hydroxyl groups. The GlcNAc moiety of the α1,6 arm engages in several hydrogen bonds with the protein, whereas the GlcNAc on the other antenna is stacked against Trp108, forming an extended binding site. This is the first structural report for a bivalve lectin.
      Lectins are multivalent carbohydrate-binding proteins that play important roles in the social life of cells. A growing repertoire of lectins has been identified in invertebrates (
      • Vasta G.R.
      • Ahmed H.
      • Odom E.W.
      ), where these molecules are involved in self/nonself recognition (
      • Vasta G.R.
      • Ahmed H.
      • Fink N.E.
      • Elola M.T.
      • Marsh A.G.
      • Snowden A.
      • Odom E.W.
      ). For example, lectins play a role in aggregation mechanisms in corals and sponges (
      • Muller W.E.
      • Dorn A.
      • Uhlenbruck G.
      ) or in sperm-egg recognition in oysters (
      • Moy G.W.
      • Springer S.A.
      • Adams S.L.
      • Swanson W.J.
      • Vacquier V.D.
      ). Lectin mediation of symbiosis with algae or bacteria has been observed in coral (
      • Jimbo M.
      • Koike K.
      • Sakai R.
      • Muramoto K.
      • Kamiya H.
      ) and nematodes (
      • Bulgheresi S.
      • Schabussova I.
      • Chen T.
      • Mullin N.P.
      • Maizels R.M.
      • Ott J.A.
      ). Nevertheless, the most common function assessed for lectins in marine invertebrates is their role in innate immunity by specific binding of polysaccharide-coated pathogenic bacteria (
      • Iwanaga S.
      • Lee B.L.
      ,
      • Vasta G.R.
      ).
      Different lectins have been identified in bivalves and they most frequently belong to the C-type lectin family. Proteins from this group of calcium-dependent lectins have been reported in oysters (
      • Minamikawa M.
      • Hine M.
      • Russell S.
      • Huber P.
      • Duignan P.
      • Lumsden J.S.
      ,
      • Yamaura K.
      • Takahashi K.G.
      • Suzuki T.
      ), scallops (
      • Wang H.
      • Song L.
      • Li C.
      • Zhao J.
      • Zhang H.
      • Ni D.
      • Xu W.
      ), and clams (
      • Bulgakov A.A.
      • Park K.-I.
      • Choi K.-S.
      • Lim H.-K.
      • Cho M.
      ,
      • Gourdine J.-P.
      • Smith-Ravin E.J.
      ). C-type lectins are characterized by a carbohydrate recognition domain (CRD)
      The abbreviations used are: CRD, carbohydrate recognition domain; αMeMan, α-methyl-mannoside; ITC, isothermal titration microcalorimetry; nona-Asn, asparagine-linked complex type nonasaccharide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HA, hemagglutination; DC-SIGN, dendritic cell-specific intercellular adhesion.
      3The abbreviations used are: CRD, carbohydrate recognition domain; αMeMan, α-methyl-mannoside; ITC, isothermal titration microcalorimetry; nona-Asn, asparagine-linked complex type nonasaccharide; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HA, hemagglutination; DC-SIGN, dendritic cell-specific intercellular adhesion.
      with a conserved fold and the involvement of a calcium ion in carbohydrate binding (
      • Drickamer K.
      ). The crystal structure of mannose-binding protein was the first one to be described (
      • Weis W.I.
      • Kahn R.
      • Fourme R.
      • Drickamer K.
      • Hendrickson W.A.
      ). The CRD belongs to a larger family sharing a common fold and is referred to as the C-type lectin-like domain (
      • Drickamer K.
      ).
      Codakine is a 14-kDa C-type lectin purified from the gill of the tropical clam Codakia orbicularis (Linné 1758) by affinity chromatography on a mannose-agarose column (
      • Gourdine J.-P.
      • Smith-Ravin E.J.
      ). It forms homodimers and heterodimers with isoforms 1 (NCBI accession number AAX19697) and 2 (NCBI accession number ABQ40396) (
      • Gourdine J.-P.
      • Smith-Ravin E.J.
      ). A 19-amino acid peptide signal suggests that the lectin travels through a secretory pathway. The 129-amino acid sequence of the mature protein has significant sequence similarities to various fish lectins (33%). Sequence homologies have, moreover, been observed between codakine and mermaid nematodes (
      • Gourdine J.-P.
      • Markiv A.
      • Smith-Ravin J.
      ). These results point to the probable role for the gill-located codakine in either antibacterial protection or in the recognition of sulfur-oxidizing bacteria, symbionts that are needed for the survival of C. orbicularis in sandy anaerobic environments (
      • Berg C.J.
      • Alatalo P.
      ).
      In this paper, we present data on the specificity and affinity of codakine for various monosaccharides and oligosaccharides using inhibition of hemagglutination, glycan microarrays, and microcalorimetry. The native crystal structure displays a new covalent dimerization mode. The structure of the complex with a high affinity biantennary N-glycan displays a new oligosaccharide binding mode where both antennae are in contact with the protein.

      EXPERIMENTAL PROCEDURES

      Protein Purification—The previously described protein purification protocol (
      • Gourdine J.-P.
      • Smith-Ravin E.J.
      ) was used with minor modifications. Briefly, about 5 g of nitrogen frozen gill tissue of C. orbicularis was crushed with a pestle in 50 ml of buffer composed of 20 mm Tris-HCl, 100 mm NaCl, 100 μm CaCl2, pH 7.4 (T buffer). After a 10-min centrifugation at 10,000 rpm, the supernatant was dialyzed overnight at 4 °C against fresh T-buffer four times. Insoluble matter was pelleted by centrifugation as described above. The 0.25-μm filtered supernatant was loaded onto a mannose-agarose column pre-equilibrated with T-buffer. After washing with T-buffer containing 1 m NaCl, codakine was eluted by 0.1 m EDTA in T-buffer. The eluted fractions were pooled and dialyzed extensively during 2 days at 4 °C against T-buffer. The electrophoretic profile of eluted fractions was checked on 15% SDS-polyacrylamide gel (
      • Laemmli U.K.
      ). The molar extinction coefficient and optical density at 280 nm were used to determine the concentration of codakine.
      Hemagglutination Assays—Hemagglutination tests were performed using microtiter plates with U-bottom wells by the 2-fold serial dilution method. 25 μl of rabbit erythrocytes (2% in NaCl; Biomérieux) were mixed with serially diluted codakine in T-buffer (described above), starting with a concentration of 1 mg/ml. After a 30-min incubation at 37 °C, the plates were read. One unit of hemagglutination activity was defined as the highest dilution of lectin giving a complete hemagglutination (HA unit, μg/ml). For the inhibition tests, a dilution of codakine equivalent to four units of HA, corresponding to 15.6 μg/ml, was used. Inhibition of hemagglutination was assayed by 2-fold serial dilutions of the following sugars in T-buffer: d-mannose, d-galactose, d-glucose, N-acetylglucosamine, l-fucose, N-acetylneuraminic acid, and d-rhamnose, each starting at a concentration of 100 mm.
      Glycan Microarray Analysis—Purified codakine (1 mg/ml) was labeled with the Alexa Fluor® 488 protein labeling kit (Invitrogen) according to the instructions of the manufacturer. The purification of Alexa-labeled codakine was performed by mannose-agarose chromatography, as described above. Glycan microarray tests were carried out by the standard procedure of Core H of the Consortium for Functional Glycomics (
      • Blixt O.
      • Head S.
      • Mondala T.
      • Scanlan C.
      • Huflejt M.E.
      • Alvarez R.
      • Bryan M.C.
      • Fazio F.
      • Calarese D.
      • Stevens J.
      • Razi N.
      • Stevens D.J.
      • Skehel J.J.
      • van Die I.
      • Burton D.R.
      • Wilson I.A.
      • Cummings R.
      • Bovin N.
      • Wong C.H.
      • Paulson J.C.
      ).
      Preparation of Nonasaccharide-Asn 1—The protected compound Fmoc-1 was prepared from egg yolk by acid hydrolysis of the disialylated derivative of Fmoc-1 in analogy to the published procedures (
      • Kajihara Y.
      • Suzuki Y.
      • Yamamoto N.
      • Sasaki K.
      • Sakakibara T.
      • Juneja L.R.
      ,
      • Seko A.
      • Kotetsu M.
      • Nishizono M.
      • Enoki Y.
      • Ibrahim H.R.
      • Juneja L.R.
      • Kim M.
      • Yamamoto T.
      ). For Fmoc deprotection (Scheme 1), a 5-mg portion of Fmoc-1 was dissolved in H2O/MeOH (75 μl; 30:9), and 0.5 m NaOH (20 μl) was added in four portions. After shaking for 10 min (TLC; 2-propanol, 1 m ammonium acetate), the suspension was acidified to pH 4–5 with 10% acetic acid. Subsequently, the suspension was diluted with 0.1 m NH4HCO3 to a final volume of 200 μl. The suspension was cleared by centrifugation, and the supernatant was purified by gel filtration (Superdex 30 (16: 60); flow rate, 1 ml/min; eluent, 0.1 m NH4HCO3; detection, 214 and 260 nm). The peak eluting at 76 min was collected and lyophilized five times to yield 3.3 mg of nonasaccharide-Asn 1 (74.4%). The purity of 1 (
      • Kajihara Y.
      • Suzuki Y.
      • Yamamoto N.
      • Sasaki K.
      • Sakakibara T.
      • Juneja L.R.
      ) was confirmed by 360-MHz 1H NMR in D2O (Fig. S1).
      Titration Microcalorimetry—Thermodynamic parameters were estimated by isothermal titration calorimetry (ITC) using a Microcal (Northampton, MA) VP-ITC microcalorimeter. All experiments were carried out at 298 K. Ligands and lectins were prepared in T-buffer (described above). Titration of codakine binding was performed in a cell with a volume of 1.447 ml by 30 injections of 10 μl of ligand with 5-min intervals while stirring at 310 rpm. Blank titration with buffer was used as a reference. The experimental data were fitted to a theoretical titration curve using software supplied by Microcal with ΔH (enthalpy change), Ka (association constant), and n (number of binding sites/monomer) as adjustable parameters, from the classical relationship (
      • Wiseman T.
      • Williston S.
      • Brandts J.F.
      • Lin L.N.
      ). For each ligand, experiments were repeated two or three times.
      Crystallization and Data Collection—Native codakine was concentrated to 15 mg/ml in 20 mm Tris-Cl, pH 8, 5 mm CaCl2, and 5 mm α-methyl-mannoside on Vivaspin 5 kDa (Vivascience). All crystals were obtained by the hanging drop vapor diffusion method using 2-μl drops containing a 50:50 (v/v) mix of protein and reservoir solution at 20 °C. Initial crystallization screening was performed using the JCSG+ suite (Qiagen). After three months, crystals were obtained with a solution containing 0.2 m diammonium citrate, pH 5, 20% (w/v) polyethylene glycol 3350. A range of related conditions were used for co-crystallization of the complex between codakine (22 mg/ml in the same buffer) and nona-Asn (10 mm). One large cube-shaped crystal was obtained with 0.2 m lithium sulfate, 100 mm sodium acetate, pH 7.5, and 15% polyethylene glycol 4000 from a well with a broken lamella.
      Glycerol was added to 20% (v/v) to the crystallization solution as cryoprotectant prior to freezing crystals in a gaseous nitrogen stream at 100 K. Native and complex data were collected at the European Synchrotron Radiation Facility on an ADSC Q4R CCD detector (Quantum Corp.) on beamline ID14-1 and an ADSC Q315R detector on beamline ID29, respectively. Diffraction images were processed using MOSFLM (

      Leslie, A. G. W. (1992) Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, No. 26

      ). All further computing was performed using the CCP4 suite (
      • number4 C.c.p.
      ), unless otherwise stated. Data processing statistics are presented in Table 1.
      TABLE 1Data collection and refinement statistics
      NativeNona-Asn
      Data collection statistics
      Unit cell (Å)a = 82.91, b = 30.39, c = 67.09, β = 133.86a = 32.16, b = 100.19, c = 95.74
      BeamlineID14-1ID29
      SpacegroupC2C2221
      Wavelength (Å)0.9310.976
      Resolution limits (Å)33.11–1.30 (1.37–1.30)34.61–1.70 (1.74–1.70)
      Total observations87,473 (4,789)
      Values in parenthesis refer to the highest resolution shell
      96,802 (6,992)
      Unique reflections26,746 (2,230)17,204 (1,232)
      Completeness89.7 (52.5)98.6 (99.3)
      Multiplicity3.3 (2.1)5.6 (5.6)
      I〉/〈σI12.0 (2.1)5.3 (2.0)
      Rmerge
      Rmerge = Σ||I – 〈I〉|/|Σ〈I〉|
      (%)
      3.4 (32.3)8.3 (37.7)
      Wilson B-factor (Å2)10.419.70
      Refinement statistics
      Used reflections2538116302
      Rcryst
      Rcryst = (Σ||Fo – Fc||)/(Σ||Fo||)
      14.420.1
      Rfree16.125.8
      Root mean square bonds0.0190.018
      Root mean square angles1.7241.763
      Cruickshank's DPI0.050.11
      Protein atoms10571028
      Solvent atoms176121
      Other atoms26109
      B-Factors
      Overall12.323.6
      Protein atoms10.121.3
      Solvent atoms25.633.7
      Other atoms16.034.9
      Protein Data Bank code2VUV2VUZ
      a Values in parenthesis refer to the highest resolution shell
      b Rmerge = Σ||I – 〈I〉|/|Σ〈I〉|
      c Rcryst = (Σ||FoFc||)/(Σ||Fo||)
      Molecular Replacement and Structure Refinement—Attempts to solve the crystal structure of codakine by molecular replacement using the C-type lectin structures available in the Protein Data Bank were unsuccessful. A homology model was then constructed using FUGUE and ORCHESTRAR (Tripos Inc., St. Louis, MO). Among profile families identified by the FUGUE program, four templates with more than 25% identity with codakine were selected: human E-selectin (
      • Graves B.J.
      • Crowther R.L.
      • Chandran C.
      • Rumberger J.M.
      • Li S.
      • Huang K.S.
      • Presky D.H.
      • Familletti P.C.
      • Wolitzky B.A.
      • Burns D.K.
      ) (Protein Data Bank code 1ESL), human lithostathine (
      • Gerbaud V.
      • Pignol D.
      • Loret E.
      • Bertrand J.A.
      • Berland Y.
      • Fontecilla-Camps J.C.
      • Canselier J.P.
      • Gabas N.
      • Verdier J.M.
      ) (Protein Data Bank code 1QDD), human tetranectin (
      • Kastrup J.S.
      • Nielsen B.B.
      • Rasmussen H.
      • Holtet T.L.
      • Graversen J.H.
      • Etzerodt M.
      • Thogersen H.C.
      • Larsen I.K.
      ) (Protein Data Bank code 1TN3), and Hemitripterus americanus antifreeze protein (
      • Gronwald W.
      • Loewen M.C.
      • Lix B.
      • Daugulis A.J.
      • Sonnichsen F.D.
      • Davies P.L.
      • Sykes B.D.
      ) (Protein Data Bank code 2AFP). Structurally conserved regions were built by ORCHESTRAR, and loops were modeled by an ab initio approach. The conserved disulfide bridges and calcium ions were incorporated in the model that has been deposited in the Protein Model Data Base with accession number PM0074967.
      This model was then used for solving the codakine structure by molecular replacement with the program Phaser (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Win M.D.
      • Storoni L.C.
      • Read R.J.
      ). The program Acorn (
      • Foadi J.
      • Woolfson M.M.
      • Dodson E.J.
      • Wilson K.S.
      • Jia-xing Y.
      • Chao-de Z.
      ) was subsequently used to improve the electron density. A few correctly placed segments were chosen from the molecular replacement solution and used as starting coordinates for Acorn phasing. Among the smallest fragments tested, Acorn was able to phase the structure starting from the positions of eight sulfur atoms. The very high resolution (1.3 Å) of the native crystals allowed the program to calculate an excellent electron density map where ARpWarp (
      • Perrakis A.
      • Morris R.
      • Lamzin V.S.
      ) built the complete model. In the carbohydrate binding site, clear density could be observed for one calcium atom. Two glycerol molecules and one citrate molecule were also included in the model.
      The structure in complex with the nona-Asn was solved by molecular replacement with Molrep (
      • Vagin A.
      • Teplyakov A.
      ), using the native codakine structure as a search model. After the addition of a calcium ion and solvent molecules, clear residual electron density was visible for at least five sugar monomers. The oligosaccharide was docked manually into the electron density according to its chemical structure; no ambiguity at the glycosidic linkages was observed.
      For the refinement of each structure, 5% of the observations were immediately set aside for cross-validation analysis (
      • Brünger A.
      ) and were used to monitor various refinement strategies. Manual corrections of the models using Coot (
      • Emsley P.
      • Cowtan K.
      ) were interspersed with cycles of maximum likelihood refinement with REFMAC (
      • Murshudov G.N.
      • Vagin A.A.
      • Dodson E.J.
      ). The two models were validated with the WhatIf suite (
      • Vriend G.
      ) and deposited in the Protein Data Bank with accession numbers 2vuv and 2vuz for the native codakine and the nonasaccharide complexes, respectively. The refinement statistics are listed in Table 1.
      Miscellaneous Methods—Areas of buried surfaces were calculated with PISA (
      • Krissinel E.
      • Henrick K.
      ). All figures were drawn with the PyMOL Molecular Graphics System (DeLano Scientific LLC, San Carlos, CA) unless otherwise stated.

      RESULTS

      Overall Structure of Codakine—In the presence of α-methyl-mannoside (αMeMan), codakine crystallized in rock-shaped crystals of space group C2 that diffract to 1.3 Å resolution. The asymmetric unit contains one monomer, and a disulfide bond dimer is generated by the 2-fold axis. The monomer consists of a typical C-type lectin CRD with two parts: the lower part contains the two helices and three strands, and the upper part is composed of four strands and long loops (Fig. 1A) (
      • Drickamer K.
      ). As predicted previously by bioinformatics, the mature protein lacks the 19-amino acid signal peptide present in the nucleotide sequence. The first two β-sheets and the last one run antiparallel, and a salt bridge is observed linking the termini of the peptide chain. For the C-terminal amino acid, clear density for the backbone was found but not for the side chain. Apart from this, all amino acids could be modeled in the density map. Additional density was observed near Cγ of Pro91, which appears to be a 4-hydroxyproline.
      Figure thumbnail gr2
      FIGURE 1Crystal structure of codakine. A, graphical representation of the codakine dimer with disulfide bonds displayed as orange sticks. B, superimposition of the CRD of codakine (green) and DC-SIGN (blue). C, glycerol in the binding site with labeling of amino acids of interest. D, sequence alignment of codakine and DC-SIGN. Amino acids involved in monosaccharide binding are labeled with a blue star, and intramolecular disulfide bridges are shown with orange lines.
      Three intramolecular disulfide bonds are observed (Cys2–Cys13, Cys30–Cys124, Cys103–Cys116). The last two disulfide bridges are conserved in C-type lectins, linking the first helix to the last strand and one strand to a loop, respectively. The first bridge is present in long form C-type lectin-like domain (
      • Day A.J.
      ). One free cysteine, Cys53, is observed on the α2-helix. Cys44, the residue involved in the unique dimerization mode observed in this C-type lectin, is located at the beginning of the same helix. The dimer is further stabilized by hydrogen bonds from His51 and Glu55 to Asp11 and Gln8 on the other chain. Hydrophobic interactions were found for His51 stacking against Leu10 from the other chain and Leu10 against Phe9. Through the 2-fold symmetry, this creates a chain of contact between the 6 hydrophobic residues that extends to 20 Å; this results in a dimer interface area of about 400 Å2.
      C-type lectins usually contain two calcium ions in conserved binding sites referred to as site 1 and site 2 (
      • Drickamer K.
      ). In the present structure, only one calcium ion is present in site 2 that corresponds to the monosaccharide binding site. The calcium is coordinated to the acidic oxygen of Glu93, Glu101, and Asp113; to the oxygen atoms from amide Asn95 and Asn112; and to the main chain oxygen of Asp113. Analysis of the electron density indicates that αMeMan is not present in the site but is instead replaced by a well ordered glycerol molecule. Additional density was also observed and attributed to another glycerol molecule and a citrate molecule.
      Analysis of the glycerol located in the carbohydrate binding site indicates that two oxygen atoms establish the contacts classically observed for the carbohydrate ligand. These two oxygens complete the coordination sphere of the calcium. The first oxygen shows hydrogen bonds to the side chains of Asn95, Glu93, and Asn112, the second oxygen to the side chains of Glu101 and Asn112 and to the backbone oxygen of Asp113 (Fig. 1C).
      Affinity and Specificity for Monosaccharides—Hemagglutination inhibition assays were performed using rabbit erythrocytes. Codakine shows a hemagglutination activity for a minimum of 3.9 μg/ml (one unit of HA). Inhibition of interaction between the lectin and the red blood cells was assayed with a range of monosaccharides. The highest inhibition power (using 4 HA units of codakine) was observed for d-mannose and l-fucose) (25 mm). Glucose and GlcNAc have a weaker effect (100 mm), and no inhibition was observed for d-galactose, d-rhamnose, d-neuraminic acid even at the higher concentration. Codakine can therefore be confirmed as a mannose/fucose-specific lectin, as was predicted from the EPN peptide signature in the carbohydrate binding site (
      • Drickamer K.
      ).
      For a complete characterization of the interaction between codakine and the most efficient monosaccharides used in the hemagglutination assay, affinity and thermodynamic parameters were measured using ITC (Table 2). Mannose and GlcNAc have a rather low affinity with a dissociation constant of 0.27 and 0.46 mm, respectively. No affinity could be measured for galactose. The dissociation constant obtained for αMeMan is 52 μm, which is about 20 times more potent than the interaction with mannose, an increase frequently observed in lectin-carbohydrate interactions. The α-methyl-fucoside also appears to be a good inhibitor, as observed in hemagglutination assays. In all cases, the association is driven by enthalpy. The entropy term is weak and slightly favorable except for αMeMan. In this latter case, the strong enthalpy contribution (ΔH = -33.6 kJ/mol) is partly opposed by an entropy barrier (TΔS =-9.1 kJ/mol), as classically observed in protein-carbohydrate interactions (
      • Dam T.K.
      • Brewer C.F.
      ).
      TABLE 2Thermodynamic of the binding of codakine to carbohydrates S.D. values are in the range of 10%.
      SugarnKaKdΔGΔHTΔS
      × 104 m–1μmkJ/molkJ/molkJ/mol
      Mannose1
      Fixed value during fitting procedure
      0.37270–20.3–18.7–1.6
      GlcNAc1
      Fixed value during fitting procedure
      0.21465–19.0–14.6–4.4
      α–MeMan1
      Fixed value during fitting procedure
      1.9352–24.5–33.69.1
      α–Methyl-fucoside1
      Fixed value during fitting procedure
      1.6760–24.1–20.3–3.8
      Trimannoside0.91.2780–23.4–41.918.5
      Nona-Asn0.942320.432–36.2–45.38.9
      a Fixed value during fitting procedure
      Affinity and Specificity for Oligosaccharides—The specificity of the fluorescence-labeled lectin was evaluated by binding to the 320 oligosaccharides present on the glycan array available at the Consortium for Functional Glycomics (Fig. 2). Only 13 oligosaccharides are recognized with high affinity; they all represent complex-type biantennary N-glycans (complete data available as supplemental material). The shortest ligand is the core pentasaccharide, consisting of a chitobiose and a trimannoside with α1–3 and α1–6 linkage. Larger N-glycans elongated by the successive addition of GlcNAc, galactose, and sialic acid are also recognized by the lectin. Fucosylation at position 3 of the antennary GlcNAc moieties (Lewis x) is tolerated, but not fucosylation of the chitobiose core.
      Figure thumbnail gr3
      FIGURE 2Glycan array analysis of codakine as measured by fluorescence intensity. Monomer coding is as follows: black square; GlcNAc; grey circle; Man; white circle; Gal; grey triangle; Fuc; grey diamond; NeuAc.
      In order to characterize the thermodynamic and structural basis of this very narrow specificity, a biantennary nonasaccharide N-glycan linked to Asn (Scheme 1) was prepared from egg yolk. Codakine dissociation constants were measured for this compound and also for trimannoside in order to evaluate the influence of the internal chitobiose moiety on the high affinity (Fig. 3). The affinity for the trimannoside (Kd = 80 μm) is of the same order of magnitude as the one measured for αMeMan (Table 2). In contrast, the affinity for nona-Asn is submicromolar (Kd = 432 nm). To our knowledge, this is the highest affinity reported for a C-type lectin, excluding multivalency effects. The 200-fold increase observed for nona-Asn compared with the trimannoside could be due to either the terminal N-acetyllactosamine on each antenna or to the presence of internal chitobiose. Since the core pentasaccharide Man3GlcNAc2 is efficiently recognized in the glycan array, it could be concluded that the internal chitobiose has a crucial effect on the affinity.
      Figure thumbnail gr4
      FIGURE 3Titration calorimetry results of codakine with oligosaccharides. A, trimannoside (5 mm) in codakine (500 μm). B, nona-Asn (0.37 mm) in codakine (37 μm). Top, data from 30 automatic injections of 10 μl of oligosaccharide into the codakine-containing cell. Bottom, plot of the total heat released as a function of ligand concentration for the titration shown above (squares). The solid line represents the best least square fit for the obtained data.
      When analyzing the thermodynamic contributions, the binding of codakine to nona-Asn shows a higher enthalpy contribution (ΔH = -45.3 kJ/mol) than to trimannoside (ΔH = -41.9 kJ/mol). However, the difference in free energy (and therefore in affinity) is mainly due to the different entropy terms. Both oligosaccharides display an unfavorable entropy contribution, but the entropy for binding the trimannoside (TΔS =-18.5 kJ/mol) is much stronger than for the nonasaccharide (TΔS =-8.9 kJ/mol).
      Crystal Structure of Codakine·Nona-Asn—Cocrystallization of codakine with nona-Asn yielded one rock-shaped crystal diffracting to 1.7 Å in the C2221 space group. The overall shape of each monomer is similar to what is observed in the structure of codakine complexed with glycerol. The intermonomer disulfide bridge also occurs, albeit with a longer S–S bond distance (2.33 Å instead of 2.05 Å). Such a deviation from normality might be due to radiation damage. This variation, associated with rotational flexibility of this linkage, induces a slightly different orientation of the second monomer with respect to the first one (supplemental Fig. S2).
      Electron density could be readily identified for one calcium ion and 5 carbohydrate residues. After building the βGlcNAc12αMan13(βGlcNAc12αMan16) Man pentasaccharide, the two extra GlcNAc could be added at position 1 of the central βMan. The galactose residues capping the two antennae and the Asn moiety could not be located in the electron density (Fig. 4A).
      Figure thumbnail gr5
      FIGURE 4Crystal structure of codakine complexed with nona-Asn. A, representation of the final maximum likelihood weighted 2 mFo - DFo electron density map (contoured at 1.0σ, 0.34 e Å-3 for the nona-Asn oligosaccharide. B, hydrogen bond network between oligosaccharide and codakine amino acids. Intramolecular hydrogen bonds of the nonasaccharide are represented by blue dashed lines. C, comparison of oligosaccharide binding site in codakine (left) and DC-SIGN (right) (
      • Feinberg H.
      • Mitchell D.
      • Drickamer K.
      • Weis W.
      ). The star indicates the position of the calcium ion. Electrostatic surface was calculated with APBS (
      • Baker N.A.
      • Sept D.
      • Joseph S.
      • Holst M.J.
      • Mc Cammon J.A.
      ).
      The αMan of the 1–6 branch is located on the calcium ion in an orientation previously reported in DC-SIGN (
      • Feinberg H.
      • Mitchell D.
      • Drickamer K.
      • Weis W.
      ), with O3 and O4 coordinating the calcium ion. The OH3 group makes hydrogen bonds to the side chains of Glu93, Asn95, Glu101, and Asn112. The OH4 group binds to the Glu101 and Asn112 side chains and to the Asp113 main chain oxygen, and the O6 hydroxyl binds to the Arg115 side chain (Table 3). The GlcNAc residue of the 1–6 branch interacts with Glu93 via OH6, with Asn95 via the ring oxygen O5 and with Ser97 via the acetyl oxygen. The branching βMan establishes two hydrogen bonds between O4 and the Asn107 and Trp108 side chains. The αMan of the 1–3 branch is not in contact with the protein, but the antenna folds back, and the terminal GlcNAc is perfectly stacked onto Trp108, creating a strong hydrophobic interface. The two GlcNAc moieties of the chitobiose core do not make direct contact with the protein, but the one linked to βMan interacts with the Ser61 side chain via a water molecule and establishes two intramolecular hydrogen bonds with the ring oxygens of βMan and αMan in the 1–6 branch (Fig. 4B).
      TABLE 3List of contacts between nona-Asn and the proteins
      Atom 1Atom 2DistanceBuried protein surface
      Å2
      1–6 branch-Man7133
      Man7·O3Calcium2.48
      Glu93·OE22.82
      Glu93·OE13.19
      Asn95·ND22.78
      Asn95·OD13.03
      Asn112·OD13.33
      GlcNAc8·O53.16
      Man7·O4Calcium2.64
      Glu101·OE22.48
      Asp113·O2.85
      Asp112·ND22.98
      Asn112·OD13.37
      Man7·O6Arg115·NH13.19
      1–6 branch-GlcNAc8106
      GlcNAc8·O5Asn95·ND22.84
      GlcNAc8·O6Glu93·OE22.83
      GlcNAc8·O7Ser97·OG2.49
      1–3 branch-Man438
      Man4·O3GlcNAc5·O53.24
      1–3 branch-GlcNAc5116
      GlcNAc5Trp108Stacking
      GlcNAc5·O6Man3·O4Water-bridged
      Core-Man386
      Man3·O4Trp108·NE12.92
      Asn107·ND22.92
      Man3·O6Asn107·ND23.30
      Man3·O42.8
      Core-GlcNAc237
      GlcNAc2·O3Man3·O52.83
      GlcNAc2·O3Man7·O53.26
      Core-GlcNAc1
      GlcNAc1·O3GlcNAc2·O52.88
      The overall shape of the oligosaccharide is rather folded. The conformations of all of the glycosidic linkages lie in energy minima that were identified previously in energy maps (
      • Imberty A.
      • Delage M.M.
      • Bourne Y.
      • Cambillau C.
      • Pérez S.
      ,
      • Imberty A.
      • Gerber S.
      • Tran V.
      • Pérez S.
      ). In solution, the most flexible one would be the αMan1–6Man linkage that could generate several different overall shapes of the biantennary N-glycan. In the present case, the constraints created by the rather deep binding pocket result in a folded conformation, with the αMan of the 1–6 arm close to GlcNAc2 of the chitobiose core.

      DISCUSSION

      Comparison with Other C-type Lectins—The family of C-type lectins is characterized by low sequence similarities despite a well conserved fold. Indeed, structural comparison by secondary structure matching (
      • Krissinel E.
      • Henrick K.
      ) indicates high structural similarity with a range of vertebrate C-type lectin-like domains, such as the ones from scavenger receptor (
      • Feinberg H.
      • Taylor M.E.
      • Weis W.I.
      ) (0.97 Å for 122 amino acids), DC-SIGN (
      • Feinberg H.
      • Mitchell D.
      • Drickamer K.
      • Weis W.
      ) (1.29 Å for 123 amino acids), and langerin (
      • Chatwell L.
      • Holla A.
      • Kaufer B.B.
      • Skerra A.
      ) (1.09 Å for 115 amino acids). Superimposition with DC-SIGN (Fig. 1B) illustrates the high similarities in secondary structure and calcium site 2. Codakine lacks calcium site 1, due to differences in the loop connecting strands β3 and β4. The loop is shorter and lacks acidic amino acids, such as Glu324, in DC-SIGN. More importantly, Asp320 at the end of the DC-SIGN β3-strand is replaced by a lysine (Lys70) that extends into the groove. The positively charged side chain occupies the calcium location in site 1 and establishes the equivalent interactions with conserved amino acids (Asp102 and Asn96 side chains and Glu101 backbone oxygen). Langerin is the only other structure of CRDs lacking calcium in site 1, and it also contains a lysine residue occupying the place of calcium. However, in langerin, the connecting loop is longer, and the resulting larger groove acts as a secondary carbohydrate binding site (
      • Chatwell L.
      • Holla A.
      • Kaufer B.B.
      • Skerra A.
      ).
      Molecular Basis for the High Affinity toward Oligosaccharides—Codakine displays millimolar affinity for monosaccharides (Kd = 0.27 mm for mannose), which is in agreement with ITC data previously obtained for other C-type lectins, such as mannose-binding protein with mannoside (
      • Quesenberry M.S.
      • Lee R.T.
      • Lee Y.C.
      ) and tunicate lectin TC14 with fucose (
      • Poget S.F.
      • Legge G.B.
      • Proctor M.R.
      • Butler P.J.
      • Bycroft M.
      • Williams R.L.
      ). However, the strong affinity observed between codakine and the biantennary N-glycan (Kd = 0.432 μm) is unique when compared with other C-type lectin/oligosaccharide interactions. The dissociation constant for E-selectin interacting with Sialyl-Lewis X has been estimated at 120 μm by fluorescence polarization (
      • Jacob G.S.
      • Kirmaier C.
      • Abbas S.Z.
      • Howard S.C.
      • Steininger C.N.
      • Welply J.K.
      • Scudder P.
      ), whereas DC-SIGN interaction with Man9GlcNAc2 was measured with a Kd of 26 μm by competition assays (
      • Feinberg H.
      • Castelli R.
      • Drickamer K.
      • Seeberger P.H.
      • Weis W.I.
      ).
      Indeed, the high affinity appears to be due to the extended binding site with numerous contacts between the oligosaccharide and the protein side chain. The stacking between Trp108 and the GlcNAc of the 3 arm also seems to be very favorable. It results in a buried protein surface of 116 Å2, almost as large as the surface buried by the mannose at the main binding site (133 Å2). However, there are some discrepancies between this observation and the results derived from the glycan array data, since the core pentasaccharide Man3GlcNAc2, which lacks the GlcNAc moieties on the antennae, is also a high affinity ligand of codakine.
      When comparing the codakine·nona-Asn complex with the DC-SIGN·pentasaccharide βGlcNAc12αMan13(βGlcNAc12Man16)Man (
      • Feinberg H.
      • Mitchell D.
      • Drickamer K.
      • Weis W.
      ), only the βGlcNAc12Man disaccharide segments on the calcium sites are similar (Fig. 4C). In DC-SIGN, the rest of the pentasaccharide does not establish much contact with the protein, and the binding site appears to be more open. The deeper character of the codakine binding site is mainly due to the longer loop between α2-helix and β3-strand (Fig. 1D).
      The inner chitobiose core seems to play a crucial role for the high affinity binding to codakine. From the glycan array data, the presence of these residues of the core is strictly needed. However, no direct contact is observed between the two GlcNAc and the protein. Interestingly, for DC-SIGN, a similar effect is observed, since Man9GlcNAc2 has a 2–3-fold increase in affinity when compared with Man9. It was proposed that the inner GlcNAc residues restrict the conformation of nearby sugar groups, resulting in lower entropy of binding. This hypothesis is confirmed by our work, since the biantennary glycan with the chitobiose core has a significantly lower entropy barrier (-8 kJ/mol) than the trimannose compound (-18 kJ/mol). This is also correlated with the structural data, since the GlcNAc residue linked to βMan establishes one hydrogen bond to the αMan residue on the 1–6 arm, which should significantly stabilize this otherwise very flexible linkage.
      Structure-Function Relationship in Invertebrate Lectins—Codakine is the first mannose-specific invertebrate lectin to be structurally characterized. Crystal structures were previously obtained for tunicate (Polyandrocarpa misakiensis) lectin TC14 complexed with galactose (
      • Poget S.F.
      • Legge G.B.
      • Proctor M.R.
      • Butler P.J.
      • Bycroft M.
      • Williams R.L.
      ) and sea cucumber (Cucumaria echinata) CEL-I lectin complexed with GalNAc (
      • Sugawara H.
      • Kusunoki M.
      • Kurisu G.
      • Fujimoto T.
      • Aoyagi H.
      • Hatakeyama T.
      ). In all cases, the lectins adopt a dimeric arrangement allowing for opposite face presentation of carbohydrate binding sites that would be more appropriate for aggregating cells than for avidity binding (Fig. 5). All dimers are generated by 2-fold (or pseudo-2-fold) symmetry. TC14 is a noncovalent dimer with interaction through the α2-helix, whereas codakine and CEL-I dimerization involves an intermolecular disulfide bridge.
      Figure thumbnail gr6
      FIGURE 5Comparison of dimerization modes for invertebrate lectins. Calcium ions are represented as dark spheres, and intermolecular disulfide bridges, when present, are colored in black. A, codakine complexed with nona-Asn. B, tunicate (P. misakiensis) lectin TL14 complexed with galactose (
      • Poget S.F.
      • Legge G.B.
      • Proctor M.R.
      • Butler P.J.
      • Bycroft M.
      • Williams R.L.
      ). C, sea cucumber (C. echinata) CEL-I lectin complexed with N-acetyl-galactosamine (
      • Sugawara H.
      • Kusunoki M.
      • Kurisu G.
      • Fujimoto T.
      • Aoyagi H.
      • Hatakeyama T.
      ).
      The biological role and the natural ligands are not yet defined for the three lectins that have been crystallized. Invertebrate lectins are involved in innate immunity and are thought to bind to polysaccharides present at the surface of bacteria and parasites. Recently, the structural basis for the interaction between lung surfactant protein D, a C-type lectin, and heptose residues present in bacterial lipopolysaccharide was demonstrated (
      • Wang H.
      • Head J.
      • Kosma P.
      • Brade H.
      • Muller-Loennies S.
      • Sheikh S.
      • McDonald B.
      • Smith K.
      • Cafarella T.
      • Seaton B.
      • Crouch E.
      ). It would be of interest to determine which bacterial lipopolysaccharide fragments could mimic the N-glycan conformation observed in the present structure and which could be recognized by codakine.
      Additionally, codakine could play a role in the recognition of symbiotic sulfur-oxidizing bacteria (
      • Berg C.J.
      • Alatalo P.
      ). The presentation of opposite face binding sites resulting from dimeric association could result in the cross-linking of microorganisms to the invertebrate cell surface. GlcNAc-terminated biantennary glycans have been identified in invertebrates (
      • Gutternigg M.
      • Burgmayr S.
      • Poltl G.
      • Rudolf J.
      • Staudacher E.
      ), and codakine would therefore attach to the gill surface through this ligand. Further investigation of the polysaccharides present on the sulfur-oxidizing bacteria should provide important clues regarding the role of invertebrate lectins in symbiotic associations that are often of primary importance in marine organisms.

      Acknowledgments

      The glycan microarray analysis was provided by the Consortium for Functional Glycomics, funded by NIGMS, National Institutes of Health, Grant GM62116. We thank the European Synchrotron Radiation Facility, Grenoble, for access to synchrotron data collection facilities.

      Supplementary Material

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