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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

How a Plant Lectin Recognizes High Mannose Oligosaccharides^1,[C],[OA]

Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel, Pleinlaan 2, B–1050 Brussel, Belgium (A.G.-P., L.B., L.W., R.L.); Department of Molecular and Cellular Interactions, VIB, Pleinlaan 2, B–1050 Brussel, Belgium (A.G.-P., L.B., L.W., R.L.); and Centre de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique (affiliated with Joseph Fourier), BP53, 38041 Grenoble cedex 09, France (A.I.)

	ABSTRACT

TOP ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The crystal structure of Pterocarpus angolensis seed lectin is presented in complex with a series of high mannose (Man) oligosaccharides ranging from Man-5 to Man-9. Despite that several of the nine Man residues of Man-9 have the potential to bind in the monosaccharide-binding site, all oligomannoses are bound in the same unique way, employing the tetrasaccharide sequence Man(1–2)Man(1–6)[Man(1–3)]Man(1–. Isothermal titration calorimetry titration experiments using Man-5, Man-9, and the Man-9-containing glycoprotein soybean (Glycine max) agglutinin as ligands confirm the monovalence of Man-9 and show a 4-times higher affinity for Man-9 when it is presented to P. angolensis seed lectin in a glycoprotein context.

Despite the wealth of data obtained from x-ray crystallography, relatively few structures of lectins in complex with large oligosaccharides are available. Only 12 out of the more than 500 structures of lectins and carbohydrate-binding domains currently present in the Protein Data Bank (Berman et al., 2002) have a bound ligand corresponding to at least a pentasaccharide. These can be placed into three groups. The first one concerns lectins in complex with a fucosylated or nonfucosylated complex-type biantennary oligosaccharide or with the pentasaccharide GlcNAc(1–2)Man(1–3)[GlcNAc(1–2)Man(1–6)]Man derived thereof. They include the legume lectins Lathyrus ochrus lectin, concanavalin A (con A) and Pterocarpus angolensis seed lectin (PAL; Bourne et al., 1994b; Moothoo and Naismith, 1998; Buts et al., 2006), bovine galectin 1 (Bourne et al., 1994a), and DC-SIGN, a C-type lectin (Feinberg et al., 2001). The second group concerns sialylated pentasaccharides recognized by cholera toxin (Merritt et al., 1994) and influenza virus hemagglutinin (Ha et al., 2003; Gamblin et al., 2004). When looking at the high Man family of glycans, the only crystal structure of a lectin in complex with a complete Man-9 entity is that of the anti-HIV protein cyanovirin (Botos et al., 2002). A number of other structures of complexes with pentasaccharides derived from Man-9 are also available for rat Man-binding protein (Weis et al., 1992), cyanovirin-N (Botos et al., 2002), snowdrop lectin (Wright and Hester, 1996), and artocarpin (Jeyaprakash et al., 2004).

Detailed thermodynamic data for carbohydrate binding are equally limited to mono-, di-, or trisaccharides in most cases (Dam and Brewer, 2002). Exceptions are the interactions of high Man oligosaccharides with con A (Mandal et al., 1994), cyanovirin-N (Shenoy et al., 2002), and the human antibody 2G12 (Wang et al., 2004), of the pentasaccharide GlcNAc(1–2)Man(1–3)[GlcNAc(1–2)Man(1–6)]Man with PAL, con A, and other Diocleinae lectins (Mandal et al., 1994; Dam et al., 1998; Buts et al., 2006) and of bivalent pentasaccharides and sialofetuin with different galectins (Ahmad et al., 2004; Dam et al., 2005). Only for con A, PAL, and cyanovirin-N is at least one corresponding crystal structure available.

The Man-binding lectin from the seeds of P. angolensis (PAL) has been previously studied in detail by x-ray crystallography and a variety of biophysical techniques. This resulted in a clear picture of how this lectin recognizes mono-, di-, and trisaccharides (Loris et al., 2004) as well as complex-type oligosaccharides (Buts et al., 2006) and how its structure, stability, and activity respond to demetalization (Garcia-Pino et al., 2006). Here we report the crystal structures of PAL in complex with a series of high Man ligands ranging from Man-5 to Man-9. To our knowledge, not only is this the first time that the structure of a full-length high Man ligand is determined in complex with a plant protein, it is only the second complex for any lectin and the first time the whole range of naturally occurring high Mans are probed systematically.

	RESULTS AND DISCUSSION

TOP ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Overall Structure

The crystal structures of PAL in complex with six high Man-type glycans were determined at high resolution (Table I ). The crystals contain a dimer of PAL in their asymmetric unit, the two subunits of which will be termed A and B. The overall structure of the PAL dimer has previously been described in detail (Loris et al., 2003, 2004) and remains unaltered in the complexes presented in this article. These two subunits are chemically identical and show only minor conformational differences that are irrelevant for carbohydrate binding. Nevertheless, they present different crystal packing environments to the carbohydrate-binding site.

The carbohydrate-binding site of subunit B on the other hand faces a large solvent channel and can easily accommodate large oligosaccharide ligands without having to disturb either the crystal packing or the ligand conformation. Interactions and ligand conformations observed in this binding site are therefore assumed to reflect the dominant situation in solution. Further interpretation of the results presented in this article will be based upon the situation observed in subunit B unless otherwise stated.

Recognition Sequence for the Lectin

The chemical structures of the different high Man oligosaccharides used in this study and the fragments that could be built in the respective electron density maps are shown in Figure 1 . The soaking experiments resulted in all cases in occupation of the carbohydrate-binding site of the B-subunit with four to five carbohydrate residues resulting from the high Man oligosaccharides. The carbohydrate sequence that is systematically observed is Man^D3(1–2)Man^B(1–6)[Man^A(1–3)]Man⁴'(1–, corresponding essentially to the D3 arm and part of the D2 arm of the high Man chains (Fig. 2 ). Man^B occupies the monosaccharide-binding site (nomenclature of individual monosaccharides according to Koles et al., 2004).

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Oligosaccharides used in this work. The Man residues that show clear electron density and interact with the lectin are highlighted in blue for each carbohydrate. The monosaccharide nomenclature used (Koles et al., 2004) is indicated for Man-9.

View larger version (57K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Different electron density maps for all of the oligosaccharides used in this study as observed in the binding site of monomer B. All maps were calculated by removing the sugar residues from the final coordinates and applying one round of slow-cool refinement to remove potential bias of Man-9 and drawn at a level of 3 . The atomic model is superimposed. A, Man-5. B, Man-6. C, Man-7D1. D, Man-7D3. E, Man-8D1D3. F, Man-9. [See online article for color version of this figure.]

Interactions between Carbohydrate and Protein

Man(1–2)Man(1–6)[Man(1–3)]Man(1– interacts with the protein via a network of van der Waals contacts and hydrogen bonds that is illustrated in Figures 3 and 4 and Table II . This interaction network builds on those observed earlier for the smaller constituents Man(1–2)Man and Man(1–3)[Man(1–6)]Man. The monosaccharide-binding site dominates over all subsites in both the number of hydrogen bonds formed and the amount of surface buried upon complexation, a feature seen in the overall majority of protein:carbohydrate complexes.

View larger version (31K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Details of interactions between Man-9 and PAL. Residues of the binding site of PAL are colored according to atom type and selected residues are labeled. The pentasaccharide moiety of Man-9 that is visible in the structure is shown in green, except for the Man residue in the monosaccharide-binding site that is shown in black. Water molecules are drawn as light blue spheres and hydrogen bonds are indicated by dotted lines.

View larger version (14K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Two-dimensional schematic of the hydrogen bond network between Man-9 and PAL. Different Man residues are labeled according to the nomenclature of Koles et al. (2004).

A unique and identical binding mode is observed for all high Man oligosaccharides. This comes as a surprise as one would envisage several of the Man residues of Man-9 to be able to occupy the monosaccharide-binding site. Based upon the affinities for different mono-, di-, and trimannoses (Buts et al., 2006) one would expect a very poor selectivity resulting in sliding of the oligosaccharide over the binding surface of the lectin. To look for possible explanations for this apparent super-selectivity, we applied a modeling approach to identify possible factors hindering alternative binding modes.

All possible alternatives of Man residues that can be imagined to occupy the monosaccharide-binding site are summarized in Figure 5 . The branched Mans Man³ and Man⁴' are sterically excluded from the monosaccharide-binding site because of substitutions on O3 and O6 and in the case of Man³ also because of its -linkage.

View larger version (8K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Potential multivalency of Man-9. Each of the five Man residues that can theoretically bind in the monosaccharide-binding site of PAL are highlighted in red. In the crystal structures only ManB is observed to bind in the monosaccharide-binding site. Theoretically, each of the three arms could interact with a different lectin molecule via ManD1, ManD2, ManD3, and ManC. The other four Mans are excluded from the monosaccharide-binding site for steric reasons. Even with the binding mode observed via ManC as shown in Figures 1 to 3, the D3 arm remains free to interact with a second lectin molecule. Still, only a 1:1 stoichiometry is observed in solution and a unique binding mode in the crystal.

Finally, Man^A and Man⁴ remain as potential alternative candidates to occupy the monosaccharide-binding site. This would correspond to the binding of the trisaccharide Man(1–3)[Man(1–6)]Man in its reverse orientation, Man(1–3)Man occupying the monosaccharide-binding site and the +1 subsite. The isolated disaccharide Man(1–3)Man is known to bind to PAL in this manner (Loris et al., 2004). Therefore, we modeled Man(1–3)[Man(1–6)]Man in the PAL-binding site based upon the coordinates of the Man(1–3)Man complex and allowed the (1–6) linkage to vary. The (1–6) linkage is highly flexible due to its additional torsion angle. With in the gauche-trans conformation (+60°), the third Man (Man^B or Man⁴', respectively) points directly toward the solvent, making any further substitution possible (although without generating additional specific contacts). However, steric hindrance is expected for the chitobiose stem, especially when Man⁴ is located in the monosaccharide-binding site because of the -linkage in the Man³(1–4)GlcNAc² moiety. Therefore, Man^A and Man⁴ are probably excluded from the monosaccharide-binding site.

In conclusion, four binding modes different from the one observed in our crystal structures (Fig. 5) as well as multivalent behavior of the oligosaccharides remain theoretically possible. Although we predict that the affinities for these alternative binding modes would be slightly lower (probably similar to that of a monosaccharide), the binding mode observed in the crystal is not expected to be all dominating. Our earlier work also showed that isolated Man(1–3)[Man(1–6)]Man and Man(1–2)Man each bind in a single binding mode (Loris et al., 2004) while two are expected. For Man(1–2)Man, the 5-times higher binding constant (compared to Man) could still explain the observation of an apparent single-binding mode in the crystal structure. For Man(1–3)[Man(1–6)]Man, this argument is already much weaker as this trisaccharide binds only twice as strong as Man (Buts et al., 2006). However, in the case of Man-9 with a total of at least five possible binding modes, it is expected that the binding mode observed in our crystal structures would constitute well below 50% of the population, creating a contradiction between experiment and theory.

In the case of Man-7D1, it is even more remarkable that Man^B and not Man^C is not found in the monosaccharide-binding site. This would result in the recognition of at least Man^D1(1–2)Man^C though the –1 and M subsites. Assuming additivity of the subsite interactions, this binding mode should occur with an affinity of at least 5 times that of Man. The binding mode observed in the crystal with Man^B occupying the monosaccharide-binding site and Man⁴' and Man^A on the +1 and +2 subsites corresponds to the situation of the trisaccharide Man(1–3)[Man(1–6)]Man that binds with only twice the affinity of Man. The selection of the binding mode of Man-7D1 remains therefore unexplained.

Thermodynamics of Oligomannose Binding

To confirm the results obtained by crystallography, the solution binding of PAL to Man-5, Man-9, and soybean (Glycine max) agglutinin (a homotetrameric protein containing one Man-9 high Man oligosaccharide chain on each subunit) were measured by isothermal titration calorimetry (ITC). Examples of the experimental data are shown in Figure 6 and results are summarized in Table IV . Of interest is that binding of PAL to Man-9 and soybean agglutinin (SBA) is not exclusively enthalpy driven, but also shows a favorable entropy term. This seems to be a key feature for the better carbohydrate ligands of PAL as it was previously also observed for GlcNAc(1–2)Man (Buts et al., 2006). In the case of con A, a favorable entropy contribution to the binding of Man(1–2)Man was attributed to a sliding mechanism (Brewer and Brown, 1979; Mandal et al., 1994), which was later confirmed by x-ray crystallography (Moothoo et al., 1999). The unique recognition mode observed in all our crystal structures refutes this explanation for PAL, as does the observed 1:1 stoichiometry in ITC experiments with GlcNAc(1–2)Man(1–3)[GlcNAc(1–2)Man(1–6)]Man (Buts et al., 2006), Man-5, Man-9, and SBA (when concentration is expressed in SBA monomers). This 1:1 stoichiometry is also at odds with the potential multivalence of Man-9 (three terminal Mans that each could interact with a different molecule of PAL).

View larger version (9K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. A, Titration of 6.2 mM PAL into 0.14 mM SBA. The solid line represents the power required to maintain the calorimeter cell at constant temperature. Each dip in the line corresponds to the injection of a constant amount of PAL, resulting in heat production by the binding reaction and a temporary reduction in the power requirement. Integration of the surface area for each peak yields the total heat production corresponding to the formation of a known amount of complex. The positive final peaks correspond to the small heat of dilution of PAL into the saturated solution. B, Integrated and dilution-corrected heats of binding for the titrations of PAL into SBA (white circles, solid line) and Man-9 (black triangles, dotted line). The lines represent the fits for a 1:1 binding model.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS AND DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Sugar and Protein

All carbohydrates were obtained from DEXTRA laboratories. The nomenclature used to identify the Man residues in high Man oligosaccharides is taken according to Koles et al. (2004). PAL was purified from mature seeds by affinity chromatography. Extracts from uncoated ground and defatted Pterocarpus angolensis seeds were fractionated with ammonium sulfate. The 30% to 60% ammonium sulfate fraction was suspended in 100 mM NaCl + 10 mM phosphate buffer pH 7.4 and then dialyzed extensively against the same buffer. After removal of all remaining insoluble material by centrifugation (30 min at 24000g), the resulting solution was applied to a fetuin-sepharose column. After extensive washing to remove all unbound protein, the lectin was eluted using 0.3 M Man in 100 mM NaCl + 10 mM phosphate buffer pH 7.4. The protein was subsequently extensively dialyzed against 50 mM phosphate pH 7.2 + 150 mM NaCl. Protein concentrations were determined from UV absorption spectroscopy at 280 nm using the specific absorption coefficient of 37,410 M^–1 cm^–1 as calculated according to Gill and von Hippel (1989).

Crystallization and Data Collection

Cocrystallization of PAL with the different oligomannoses is not possible due to the amounts of sugar needed. For each sugar, only 20 µg was available (except for Man-9 [1 mg] and Man-5 [5 mg]), which is sufficient for just one soaking experiment in a 2 µL drop. Therefore, the complexes were prepared starting from crystals of PAL in complex with Man(1–3)Man, which were obtained as described previously (Loris et al., 2005).

To produce complexes with high Man oligosaccharides, these crystals were first desoaked by transferring them to artificial mother liquor devoid of sugar (100 mM Na-cacodylate pH 6.5, 200 mM Ca-acetate, 20% [w/v] PEG-8000) for at least 1 week and refreshing the soaking solution twice. This treatment leads to crystals in which the binding site of subunit-B becomes empty (Loris et al., 2005; Buts et al., 2006). Desoaked crystals were then transferred to 1 µL drops containing 20 µg of the desired sugar. After an overnight soak, the crystals were then mounted in thin-walled glass capillaries for data collection at room temperature.

X-ray data were collected on EMBL beamlines X11 and X13 (DESY) as well as on ESRF beamline ID14-1 (ESRF). The data were processed with DENZO and SCALEPACK (Otwinowski and Minor, 1997). Data were integrated with DENZO, merged with SCALEPACK, and converted to structure factor amplitudes using the CCP4 program TRUNCATE (CCP4, 1994). The statistics of the data collections are given in Table I.

Structure Determination

The crystal structure of the PAL:Man(1–3)Man complex, stripped of water molecules, metal ions, and carbohydrate ligands, was used as the starting model in refinement with CNS 1.0 (Brünger et al., 1998). After an initial rigid body refinement, a slow cool stage was used to uncouple R and R_free. From then on restrained positional and B-factor refinements were alternated with manual fitting in electron density maps using TURBO (Roussel and Cambillau, 1989). The refinement statistics are given in Table I. Superpositions of crystal structures were done using TURBO. Contact surfaces between carbohydrate and protein were calculated using NACCESS (S.J. Hubbard and J. Thornthon, University College, London, 1993). All figures were produced using MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Bacon, 1997).

ITC

The heat accompanying the binding of PAL to oligosaccharides or SBA was measured using an Omega isothermal titration calorimeter (MicroCal) at a temperature of 25°C. Binding of Man-5 was measured by a direct titration with a PAL solution (0.97 mM in 50 mM phosphate pH 7.5, 150 mM NaCl) in the calorimeter cell and an oligosaccharide solution of 14.8 mM in the same buffer being injected from a 250 µL syringe (32 injections of 10 µL at 240 s intervals).

Because of limitations in material availability, binding of Man-9 and SBA was assessed via reverse titrations by placing the oligosaccharide (0.12 mM) or glycoprotein (0.14 mM) ligand in the calorimeter cell and titrating with PAL (3.1 mM or 6.3 mM). After the titration, the mixed solution was retrieved, stored at 4 degrees for 24 h, and centrifuged at 20,000g for 15 min. The PAL:Man-5 and PAL:Man-9 mixtures remained completely free of precipitation, whereas a large pellet was observed for the PAL:SBA cross-linked complex.

The cell and syringe concentrations [C]₀ and [S]₀ were chosen to ensure that the product c = n x K_a x [C]₀ was between 1 and 10 and the ratio [S]₀/n x [C]₀ was 12.5 for the anticipated number of binding sites n. Each injection generated a heat burst, with the area under the curve being proportional to the heat of interaction. The heat effects accompanying ligand dilution were measured by titration into pure buffer and subtracted. After comparing different fitting models, all data were finally fitted with a Wiseman isotherm for a 1:1 binding model using a nonlinear regression procedure (Wiseman et al., 1989).

Modeling Calculations

Energy maps are calculated using the TRIPOS force field (Clark et al., 1989) together with the PIM parameterization (Imberty et al., 1999) developed for carbohydrates as a function of the and dihedral angles defined as: = O5 – C1 – O1 – Cx' and = C1 – O1 – Cx' – C(x + 1)' for the (1–2). For (1–6) linkages, three dihedral angles = O5 – C1 – O1 – C6', = C1 – O1 – C6' – C5', and = O1 – C6' – C5' – O5' were used.

Atomic coordinates and structure factor data were submitted to the Protein Data Bank and are available as entries 2PHX, 2PHF, 2PHR, 2PHT, 2PHU, and 2PHW.

	ACKNOWLEDGMENTS

 
The authors acknowledge the use of beamtime at the EMBL beamlines at the DESY (Hamburg, Germany) and ESRF (Grenoble, France) synchrotrons.

Received April 13, 2007; accepted June 3, 2007; published June 7, 2007.

	FOOTNOTES

 
¹ This work was supported by the Vlaams Interuniversitair Instituut voor Biotechnologie, the Onderzoeksraad of the Vrije Universiteit Brussel, and the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (post-doc fellowship to L.B.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Remy Loris (reloris{at}vub.ac.be).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.100867

^* Corresponding author; e-mail reloris{at}vub.ac.be; fax 32–2–6291963.

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