Structure of Dioclea virgata lectin: Relations between carbohydrate binding site and nitric oxide production

The lectin of Dioclea virgata (DvirL), both native and complexed with X-man, was submitted to X-ray diffraction analysis and the crystal structure was compared to that of other Diocleinae lectins in order to better understand differences in biological properties, especially with regard to the ability of lectins to induce nitric oxide (NO) production. An association was observed between the volume of the carbohydrate recognition domain (CRD), the ability to induce NO production and the relative positions of Tyr12, Arg228 and Leu99. Thus, differences in biological activity induced by Diocleinae lectins are related to the configuration of amino acid residues in the carbohydrate binding site and to the structural conformation of subsequent regions capable of influencing site-ligand interactions. In conclusion, the ability of Diocleinae lectins to induce NO production depends on CRD configuration.     

Crystal structure of Dioclea rostrata lectin: insights into understanding the pH-dependent dimer-tetramer equilibrium and the structural basis for carbohydrate recognition in Diocleinae lectins

The legume lectins from the subtribe Diocleinae, often referred to as concanavalin A-like lectins, are a typical example of highly similar proteins that show distinct biological activities. The pH-dependent oligomerization that some of these lectins undergo and the relative position of amino acids within the carbohydrate-binding site are factors that have been reported to contribute to these differences in the activities of Diocleinae lectins. In the present work, we determined the amino acid sequence and the crystal structure of the lectin of Dioclea rostrata seeds (DRL), with the aim of investigating the structural bases of the different behavior displayed by this lectin in comparison to other Diocleinae lectins and determining the reason for the distinct pH-dependent dimer-tetramer equilibrium. In addition, we discovered a novel multimeric arrangement for this lectin.     

Demonstration of a conserved histidine and two water ligands at the Mn2+ site in Diocleinae lectins by pulsed EPR spectroscopy

Lectins from the Diocleinae subtribe, including Canavalia brasiliensis, Canavalia bonariensis, Canavalia grandiflora, Cratylia floribunda, Dioclea grandiflora, Dioclea guianensis, Dioclea rostrata, Dioclea violacea, and Dioclea virgata, have been recently isolated and characterized in terms of their carbohydrate binding specificities. Although all of the lectins are Man/Glc specific, they possess different biological activities. In the present study, electron paramagnetic resonance (EPR) spectroscopy demonstrates that all nine Diocleinae lectins contain Mn2+. The spectra of C. floribunda and D. rostrata suggest Mn2+ site symmetry different from that of the other seven lectins. However, electron spin-echo envelope modulation (ESEEM) spectroscopy indicates that all nine lectins are coordinated to a histidyl imidazole, with similar electron-nuclear coupling to the Mn2+-bound imidazole nitrogen. ESEEM also demonstrates ligation of two water molecules to Mn2+ in all nine Diocleinae lectins. Thus, the EPR and ESEEM data indicate the presence of a Mn2+ binding site in the above Diocleinae lectins with a conserved histidine residue and two water ligands.     

Molecular characterization and crystallization of Diocleinae lectins

Molecular characterization of seven Diocleinae lectins was assessed by sequence analysis, determination of molecular masses by mass spectrometry, and analytical ultracentrifugation equilibrium sedimentation. The lectins show distinct pH-dependent dimer-tetramer equilibria, which we hypothesize are due to small primary structure differences at key positions. Lectins from Dioclea guianensis, Dioclea virgata, and Cratylia floribunda seeds have been crystallized and preliminary X-ray diffraction analyses are reported.     

Thermodynamic binding studies of lectins from the diocleinae subtribe to deoxy analogs of the core trimannoside of asparagine-linked oligosaccharides

Lectins from seven different species of the Diocleinae subtribe have been recently isolated and characterized in terms of their carbohydrate binding specificities (Dam, T. K., Cavada, B. S., Grangeiro, T. B., Santos, C. F., de Sousa, F. A. M., Oscarson, S., and Brewer, C. F. (1998) J. Biol. Chem. 273, 12082-12088). The lectins included those from Canavalia brasiliensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. All of the lectins exhibited specificity for Man and Glc residues, but much higher affinities for the branched chain trimannoside, 3,6-di-O-(alpha-d-mannopyranosyl)-d-mannose, which is found in the core region of all asparagine-linked carbohydrates. In the present study, isothermal titration microcalorimetry is used to determine the binding thermodynamics of the above lectins, including a new lectin from Canavalia grandiflora, to a complete series of monodeoxy analogs of the core trimannoside. From losses in the affinity constants and enthalpies of binding of certain deoxy analogs, assignments are made of the hydroxyl epitopes on the trimannoside that are involved in binding to the lectins. The pattern of binding of the deoxy analogs is similar for all seven lectins, and similar to that of concanavalin A which is also a member of the Diocleinae subtribe. However, differences in the magnitude of the thermodynamic binding parameters of the lectins are observed, even though the lectins possess conserved contact residues in many cases, and highly conserved primary sequences. The results indicate that non-contact residues in the lectins, even those distant from the binding sites, modulate their thermodynamic binding parameters.     

Insights into the structural basis of the pH-dependent dimer-tetramer equilibrium through crystallographic analysis of recombinant Diocleinae lectins

The structural ground underlying the pH-dependency of the dimer-tetramer transition of Diocleinae lectins was investigated by equilibrium sedimentation and X-ray crystal structure determination of wild-type and site-directed mutants of recombinant lectins. Synthetic genes coding for the full-length alpha-chains of the seed lectins of Dioclea guianensis (termed r-alphaDguia) and Dioclea grandiflora (termed r-alphaDGL) were designed and expressed in Escherichia coli. This pioneering approach, which will be described in detail in the present paper, yielded recombinant lectins displaying carbohydrate-binding activity, dimer-tetramer equilibria and crystal structures indistinguishable from their natural homologues. Conversion of the pH-stable tetrameric r-alphaDGL into a structure exhibiting pH-dependent dimer-tetramer transition was accomplished through mutations that abolished the interdimeric interactions at the central cavity of the tetrameric lectins. Both the central and the peripheral interacting regions bear structural information for formation of the canonical legume lectin tetramer. We hypothesize that the strength of the ionic contacts at these sites may be modulated by the pH, leading to dissociation of those lectin structures that are not locked into a pH-stable tetramer through interdimeric contacts networking the central cavity loops.     

In vivo lymphocyte activation and apoptosis by lectins of the Diocleinae subtribe

This paper reports the overall effects of three lectins, extracted from Canavalia brasiliensis, Dioclea violacea, and D. grandiflora, on BALB/c mice popliteal draining lymph nodes. These lectins have presented high stimulatory capacity on lymph node T cells. Additionally, they were able to induce apoptosis and inflammation (frequently associated with high endothelial venule necrosis). The data presented here suggest that the Diocleinae lectins studied can stimulate in vivo T cell activation and apoptosis, as well as present important side effects.     

Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer-tetramer association.

Diocleinae legume lectins are a group of oligomeric proteins whose subunits display a high degree of primary structure and tertiary fold conservation but exhibit considerable diversity in their oligomerisation modes. To elucidate the structural determinants underlaying Diocleinae lectin oligomerisation, we have determined the crystal structures of native and cadmium-substituted Dioclea guianensis (Dguia) seed lectin. These structures have been solved by molecular replacement using concanavalin (ConA) coordinates as the starting model, and refined against data to 2.0 A resolution. In the native (Mn/Ca-Dguia) crystal form (P4(3)2(1)2), the asymmetric unit contains two monomers arranged into a canonical legume lectin dimer, and the tetramer is formed with a symmetry-related dimer. In the Cd/Cd-substituted form (I4(1)22), the asymmetric unit is occupied by a monomer. In both crystal forms, the tetrameric association is achieved by the corresponding symmetry operators. Like other legume lectins, native D. guianensis lectin contains manganese and calcium ions bound in the vicinity of the saccharide-combining site. The architecture of these metal-binding sites (S1 and S2) changed only slightly in the cadmium/cadmium-substituted form. A highly ordered calcium (native lectin) or cadmium (Cd/Cd-substituted lectin) ion is coordinated at the interface between dimers that are not tetrameric partners in a similar manner as the previously identified Cd(2+) in site S3 of a Cd/Ca-ConA. An additional Mn(2+) coordination site (called S5), whose presence has not been reported in crystal structures of any other homologous lectin, is present in both, the Mn/Ca and the Cd/Cd-substituted D. guianensis lectin forms. On the other hand, comparison of the primary and quaternary crystal structures of seed lectins from D. guianensis and Dioclea grandiflora (1DGL) indicates that the loop comprising residues 117-123 is ordered to make interdimer contacts in the D. grandiflora lectin structure, while this loop is disordered in the D. guianensis lectin structure. A single amino acid difference at position 131 (histidine in D. grandiflora and asparagine in D. guianensis) drastically reduces interdimer contacts, accounting for the disordered loop. Further, this amino acid change yields a conformation that may explain why a pH-dependent dimer-tetramer equilibrium exists for the D. guianensis lectin but not for the D. grandiflora lectin.     

Structural analysis of ConBr reveals molecular correlation between the carbohydrate recognition domain and endothelial NO synthase activation

Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.     

Differential recognition of natural and remodeled glycotopes by three Diocleae lectins

The carbohydrate specificities of Dioclea grandiflora lectins DGL-I¹ and DGL-II, and Galactia lindenii lectin II (GLL-II) were explored by use of remodeled glycoproteins as well as by the lectin hemagglutinating activity against erythrocytes from various species with different glycomic profiles. The three lectins exhibited differences in glycan binding specificity but also showed overlapping recognition of some glycotopes (i.e. Tα glycotope for the three lectins; IIβ glycotope for DGL-II and GLL-II lectins); in many cases the interaction with distinct glycotopes was influenced by the structural context, i.e., by the neighbouring sugar residues. Our data complement and expand the existing knowledge about the binding specificity of these three Diocleae lectins, and taken together with results of previous studies, allow us to suggest a functional map of the carbohydrate recognition which illustrate the impact of modification of basic glycotopes enhancing, permiting, or inhibiting their recognition by each lectin.     

Thermodynamic, kinetic, and electron microscopy studies of concanavalin A and Dioclea grandiflora lectin cross-linked with synthetic divalent carbohydrates

The jack bean lectin concanavalin A (ConA) and the Dioclea grandiflora lectin (DGL) are highly homologous Man/Glc-specific members of the Diocleinae subtribe. Both lectins bind, cross-link, and precipitate with carbohydrates possessing multiple terminal nonreducing Man residues. The present study investigates the binding and cross-linking interactions of ConA and DGL with a series of synthetic divalent carbohydrates that possess spacer groups with increasing flexibility and length between terminal alpha-mannopyranoside residues. Isothermal titration microcalorimetry was used to determine the thermodynamics of binding of the two lectins to the divalent analogs, and kinetic light scattering and electron microscopy studies were used to characterize the cross-linking interactions of the lectins with the carbohydrates. The results demonstrated that divalent analogs with flexible spacer groups between the two terminal Man residues possess higher affinities for the two lectins as compared with those with inflexible spacer groups. Furthermore, despite their high degree of homology, ConA and DGL exhibit differences in their kinetics of cross-linking and precipitation with the divalent analogs. Electron microscopy shows the loss of organized cross-linked lattices of the two lectins with analogs possessing increased distance between the terminal Man residues. The loss of lattice patterns with the analogs is distinct for each lectin. These results have important implications for the interactions of lectins with multivalent carbohydrate receptors in biological systems.     

Binding and cross-linking properties of galectins

The galectins are a family of animal lectins that possess similar carbohydrate binding specificities and conserved consensus sequences. The biological properties of mammalian galectins include the regulation of inflammation, cell adhesion, cell proliferation and cell death. Evidence suggests that the biological activities of the galectins are related to their multivalent binding properties since most galectins possess two carbohydrate recognition domains and are therefore bivalent. For example, galectin-1, which is dimeric, binds and cross-links specific glycoprotein counter-receptors on the surface of human T-cells leading to apoptosis [J. Immunol. 163 (1999) 3801]. Different galectin-1 counter-receptors associated with specific phosphatase or kinase activities formed separate clusters on the surface of the cells as a result of the lectin binding to the carbohydrate chains of the respective glycoproteins. Importantly, monovalent galectin-1 is inactive in this system. This indicates that the separation and organization of signaling molecules that result from galectin-1 binding is involved in the apoptotic signal. The separation of specific glycoprotein receptors induced by galectin-1 binding was modeled on the basis of molecular and structural studies of the binding of lectins to multivalent carbohydrates resulting in the formation of specific two- and three-dimensional cross-linked lattices [Biochemistry 36 (1997) 15073]. In this article, the binding and cross-linking properties of galectin-1 and other lectins are reviewed as a model for the biological signal transduction properties of the galectin family of animal lectins.     

Relationships in subtribe Diocleinae (Leguminosae; Papilionoideae) inferred from internal transcribed spacer sequences from nuclear ribosomal DNA

The complete sequences of nuclear ribosomal DNA (nrDNA) internal transcribed spacer regions (ITS/5.8S) were determined for species belonging to six genera from the subtribe Diocleinae as well as for the anomalous genera Calopogonium and Pachyrhizus. Phylogenetic trees constructed by distance matrix, maximum parsimony and maximum likelihood methods showed that Calopogonium and Pachyrhizus were outside the clade Diocleinae (Canavalia, Camptosema, Cratylia, Dioclea, Cymbosema, and Galactia). This finding supports previous morphological, phytochemical, and molecular evidence that Calopogonium and Pachyrhizus do not belong to the subtribe Diocleinae. Within the true Diocleinae clade, the clustering of genera and species were congruent with morphology-based classifications, suggesting that ITS/5.8S sequences can provide enough informative sites to allow resolution below the genus level. This is the first evidence of the phylogeny of subtribe Diocleinae based on nuclear DNA sequences.     

Purification and partial characterization of a lectin from Canavalia grandiflora benth. seeds

A D-glucose/D-mannose specific lectin from seeds of Canavalia grandiflora (ConGF) was purified by affinity chromatography on Sephadex G-50. By SDS-PAGE ConGF yielded three protein bands with apparent molecular masses of 29-30 kDa (alpha chain), 16-18 kDa (beta fragment) and 12-13 kDa (gamma fragment), like other related lectins from the genus Canavalia (Leguminosae). ConGF strongly agglutinates rabbit erythrocytes, has a high content of ASP and SER, and its N-terminal sequence (30 residues) is highly similar to the sequences of other related lectins from subtribe Diocleinae.     

A database analysis of jacalin-like lectins: sequence-structure-function relationships

Lectins are known to be important for many biological processes, due to their ability to recognize cell surface carbohydrates with high specificity. Plant lectins have been model systems to study protein-carbohydrate recognition, because individually they exhibit high sensitivity and as a group large diversity in recognizing carbohydrate structures. Although extensive studies have been carried out for legume lectins that have led to interesting insights into the sequence determinants of sugar recognition in them, frameworks with such specific correlations are not available for other plant lectin families. This study reports a large-scale data acquisition and extensive analysis of sequences and structures of beta-prism-I or jacalin-related lectins (JRLs) and shows that hypervariability in the binding site loops generates carbohydrate recognition diversity, a strategy analogous to that in legume lectins. Analyses of the size, conformation, and sequence variability in key regions reveal the existence of a common theme, encoded as a set of structural features over a common scaffold, in defining specificity. This study also points to the remarkable range of domain architectures, often arising out of gene duplication events in lectins of this family. The data analyzed here also indicate a spectacular variety of quaternary associations possible in this family of lectins that have implications for glycan recognition. These results thus provide sequence-structure-function correlations, an understanding of the molecular basis of carbohydrate recognition by beta-prism-I lectins, and also a rationale for engineering specific recognition capabilities in relevant molecules.     

Crystal structures of Cratylia floribunda seed lectin at acidic and basic pHs. Insights into the structural basis of the pH-dependent dimer-tetramer transition

Structural determinants underlaying the pH-dependent dimer-tetramer transition of Diocleinae lectins were investigated from the structures of Cratylia floribunda seed lectin crystallized in conditions where it exist as a dimer (pH 4.6) or as a tetramer (pH 8.5). The acidic (aCFL) and the basic (bCFL) tetramers superimpose with overall r.m.s.d. of 0.53 A, though interdimer contacts are drastically reduced in aCFL, and the r.m.s.d. for the superposition of the 117-120 loops of aCFL vs. the bCFL tetramer is 1.29 A. Our data support the view that His51 plays a role in determining the conformation of the central cavity loops and that interdimer contacts involving ordered loop residues stabilize the canonical, pH-dependent tetramer. In the bCFL tetramer, hydrogen bonds between Asn118 and Thr120 of monomers A and D and residues Ser66, Ser108, Ser110, and Thr49 of the opposite monomer stabilize the canonical, pH-dependent tetrameric lectin structure. In CFL, Asn131 makes intradimer contacts with Asn122 and Ala123. In comparison, His131 in Dioclea grandiflora lectin establishes a network of interdimer interactions bridging the four central loops of the pH-independent tetramer. Our data provide new insights into the participation of specific amino acid residues in the mechanism of the quaternary association of Diocleinae lectins.     

Isolation and characterization of a lectin from the seeds of Dioclea lehmanni.

Affinity chromatography of the globulin fraction from the seeds of Dioclea lehmanni on Sephacryl S-200 yielded two lectins, one slightly retarded and another strongly bound. The latter, which was a glucose/mannose specific lectin, was purified and the following properties were determined: pI, Mr of subunits, carbohydrate content, A, aminoacid composition, hemagglutination and inhibition patterns, N-terminal sequence and mitogenic activity. These properties of the lectin were very similar to those of the Con A and Dioclea grandiflora lectins.     

The first crystal structure of a Mimosoideae lectin reveals a novel quaternary arrangement of a widespread domain

The crystal structures of the apo and mannose-bound Parkia platycephala seed lectin represent the first structure of a Mimosoideae lectin and a novel circular arrangement of beta-prism domains, and highlight the adaptability of the beta-prism fold as a building block in the evolution of plant lectins. The P.platycephala lectin is a dimer both in solution and in the crystals. Mannose binding to each of the three homologous carbohydrate-recognition domains of the lectin occurs through different modes, and restrains the flexibility of surface-exposed loops and residues involved in carbohydrate recognition. The planar array of carbohydrate-binding sites on the rim of the toroid-shaped structure of the P.platycephala lectin dimer immediately suggests a mechanism to promote multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host strategy against phytopredators and pathogens. The cyclic structure of the P.platycephala lectin points to the convergent evolution of a structural principle for the construction of lectins involved in host defense or in attacking other organisms.     

Structural basis for both pro- and anti-inflammatory response induced by mannose-specific legume lectin from Cymbosema roseum

Legume lectins, despite high sequence homology, express diverse biological activities that vary in potency and efficacy. In studies reported here, the mannose-specific lectin from Cymbosema roseum (CRLI), which binds N-glycoproteins, shows both pro-inflammatory effects when administered by local injection and anti-inflammatory effects when by systemic injection. Protein sequencing was obtained by Tandem Mass Spectrometry and the crystal structure was solved by X-ray crystallography using a Synchrotron radiation source. Molecular replacement and refinement were performed using CCP4 and the carbohydrate binding properties were described by affinity assays and computational docking. Biological assays were performed in order to evaluate the lectin edematogenic activity. The crystal structure of CRLI was established to a 1.8 Å resolution in order to determine a structural basis for these differing activities. The structure of CRLI is closely homologous to those of other legume lectins at the monomer level and assembles into tetramers as do many of its homologues. The CRLI carbohydrate binding site was predicted by docking with a specific inhibitory trisaccharide. CRLI possesses a hydrophobic pocket for the binding of α-aminobutyric acid and that pocket is occupied in this structure as are the binding sites for calcium and manganese cations characteristic of legume lectins. CRLI route-dependent effects for acute inflammation are related to its carbohydrate binding domain (due to inhibition caused by the presence of α-methyl-mannoside), and are based on comparative analysis with ConA crystal structure. This may be due to carbohydrate binding site design, which differs at Tyr12 and Glu205 position.     

Human RegIV Protein Adopts a Typical C-Type Lectin Fold but Binds Mannan with Two Calcium-Independent Sites

Human RegIV protein, which contains a sequence motif homologous to calcium-dependent (C-type) lectin-like domain, is highly expressed in mucosa cells of the gastrointestinal tract during pathogen infection and carcinogenesis and may be applied in both diagnosis and treatment of gastric and colon cancers. Here, we provide evidence that, unlike other C-type lectins, human RegIV binds to polysaccharides, mannan, and heparin in the absence of calcium. To elucidate the structural basis for carbohydrate recognition by NMR, we generated the mutant with Pro91 replaced by Ser (hRegIV-P91S) and showed that the structural property and carbohydrate binding ability of hRegIV-P91S are almost identical with those of wild-type protein. The solution structure of hRegIV-P91S was determined, showing that it adopts a typical fold of C-type lectin. Based on the chemical shift perturbations of amide resonances, two calcium-independent mannan-binding sites were proposed. One site is similar to the calcium-independent sugar-binding site on human RegIII and Langerin. Interestingly, the other site is adjacent to the conserved calcium-dependent site at position Ca-2 of typical C-type lectins. Moreover, model-free analysis of ¹⁵N relaxation parameters and simplified Carr-Purcell-Meiboom-Gill relaxation dispersion experiments showed that a slow microsecond-to-millisecond time-scale backbone motion is involved in mannan binding by this site, suggesting a potential role for specific carbohydrate recognition. Our findings shed light on the sugar-binding mode of Reg family proteins, and we postulate that Reg family proteins evolved to bind sugar without calcium to keep the carbohydrate recognition activity under low-pH environments in the gastrointestinal tract.