Leaves of the Lamiaceae species Glechoma hederacea (ground ivy) contain a lectin that is structurally and evolutionary related to the legume lectins

A novel lectin has been isolated and cloned from leaves of Glechoma hederacea (ground ivy), a typical representative of the plant family Lamiaceae. Biochemical analyses indicated that the G. hederacea agglutinin (Gleheda) is a tetrameric protein consisting of four subunits pairwise linked through an interchain disulphide bridge and exhibits a preferential specificity towards N-acetylgalactosamine. Cloning of the corresponding gene and molecular modeling of the deduced sequence demonstrated that Gleheda shares high sequence similarity with the legume lectins and exhibits the same overall fold and three-dimensional structure as the classical legume lectins. The identification of a soluble and active legume lectin ortholog in G. hederacea not only indicates that the yet unclassified Lamiaceae lectins belong to the same lectin family as the legume lectins, but also sheds a new light on the specificity, physiological role and evolution of the classical legume lectins.     

The crystal structure of the Calystegia sepium agglutinin reveals a novel quaternary arrangement of lectin subunits with a beta-prism fold

The high number of quaternary structures observed for lectins highlights the important role of these oligomeric assemblies during carbohydrate recognition events. Although a large diversity in the mode of association of lectin subunits is frequently observed, the oligomeric assemblies of plant lectins display small variations within a single family. The crystal structure of the mannose-binding jacalin-related lectin from Calystegia sepium (Calsepa) has been determined at 1.37-A resolution. Calsepa exhibits the same beta-prism fold as identified previously for other members of the family, but the shape and the hydrophobic character of its carbohydrate-binding site is unlike that of other members, consistent with surface plasmon resonance analysis showing a preference for methylated sugars. Calsepa reveals a novel dimeric assembly markedly dissimilar to those described earlier for Heltuba and jacalin but mimics the canonical 12-stranded beta-sandwich dimer found in legume lectins. The present structure exemplifies the adaptability of the beta-prism building block in the evolution of plant lectins and highlights the biological role of these quaternary structures for carbohydrate recognition.     

Carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus

The seed lectin (DBL) from the leguminous plant Dolichos biflorus has a unique specificity among the members of the legume lectin family because of its high preference for GalNAc over Gal. In addition, precipitation of blood group A+H substance by DBL is slightly better inhibited by a blood group A trisaccharide (GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal) containing pentasaccharide, and about 40 times better by the Forssman disaccharide (GalNAc(alpha1-3)GalNAc) than by GalNAc. We report the crystal structures of the DBL-blood group A trisaccharide complex and the DBL-Forssman disaccharide complex.A comparison with the binding sites of Gal-binding legume lectins indicates that the low affinity of DBL for Gal is due to the substitution of a conserved aromatic residue by an aliphatic residue (Leu127). Binding studies with a Leu127Phe mutant corroborate these conclusions. DBL has a higher affinity for GalNAc because the N-acetyl group compensates for the loss of aromatic stacking in DBL by making a hydrogen bond with the backbone amide group of Gly103 and a hydrophobic contact with the side-chains of Trp132 and Tyr104.Some legume lectins possess a hydrophobic binding site that binds adenine and adenine-derived plant hormones, i.e. cytokinins. The exact function of this binding site is unknown, but adenine/cytokinin-binding legume lectins might be involved in storage of plant hormones or plant growth regulation. The structures of DBL in complex with adenine and of the dimeric stem and leaf lectin (DB58) from the same plant provide the first structural data on these binding sites. Both oligomers possess an unusual architecture, featuring an alpha-helix sandwiched between two monomers. In both oligomers, this alpha-helix is directly involved in the formation of the hydrophobic binding site. DB58 adopts a novel quaternary structure, related to the quaternary structure of the DBL heterotetramer, and brings the number of know legume lectin dimer types to four.     

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.     

豆科凝集素研究进展

豆科凝集素是植物凝集素中最丰富,也是研究最多的一类凝集素。在生理条件下豆科凝集素大多是以二聚体或四聚体的形式存在,这种低聚物的形式给予豆科凝集素较强的糖专一性和大分子结构的稳定性。豆科凝集素除作为植物储存物质的作用外,还具有识别糖蛋白、糖肽及生物膜中碳水化合物和作为植物与微生物的共生介质等生理功能。现对豆科凝集素的结构、功能及其在生物学、农业和医学方面的应用进行了综述。     

植物凝集素研究进展

植物凝集素广泛分布于植物界,它可以根据不同性质进行分类,按进化及结构相关性可以分为七个家族;豆科凝集素,单子叶植物甘露糖结构凝集素,含橡胶素结构域的几丁质结合凝集素,2型核糖体失活蛋白,葫芦科韧皮部凝集素,木菠萝素相关凝集素和苋科凝集素,在长期的进化过程中,它们形成几种不同的结合模体来识别一些外源多糖,在植物中未发现合适的内源性多糖受体。植物凝集素在生物学研究,农业和医学上有广泛的应用。     

Weak protein-protein interactions in lectins: the crystal structure of a vegetative lectin from the legume Dolichos biflorus

The legume lectins are widely used as a model system for studying protein-carbohydrate and protein-protein interactions. They exhibit a fascinating quaternary structure variation, which becomes important when they interact with multivalent glycoconjugates, for instance those on cell surfaces. Recently, it has become clear that certain lectins form weakly associated oligomers. This phenomenon may play a role in the regulation of receptor crosslinking and subsequent signal transduction. The crystal structure of DB58, a dimeric lectin from the legume Dolichos biflorus reveals a separate dimer of a previously unobserved type, in addition to a tetramer consisting of two such dimers. This tetramer resembles that formed by DBL, the seed lectin from the same plant. A single amino acid substitution in DB58 affects the conformation and flexibility of a loop in the canonical dimer interface. This disrupts the formation of a stable DBL-like tetramer in solution, but does not prohibit its formation in suitable conditions, which greatly increases the possibilities for the cross-linking of multivalent ligands. The non-canonical DB58 dimer has a buried symmetrical alpha helix, which can be present in the crystal in either of two antiparallel orientations. Two existing structures and datasets for lectins with similar quaternary structures were reconsidered. A central alpha helix could be observed in the soybean lectin, but not in the leucoagglutinating lectin from Phaseolus vulgaris. The relative position and orientation of the carbohydrate-binding sites in the DB58 dimer may affect its ability to crosslink mulitivalent ligands, compared to the other legume lectin dimers.     

Plant lectins: targeting programmed cell death pathways as antitumor agents

Lectins, a group of highly diverse, carbohydrate-binding proteins of non-immune origin that are ubiquitously distributed in plants, animals and fungi, are well-characterized to have numerous links a wide range of pathological processes, most notably cancer. In this review, we present a brief outline of the representative plant lectins including Ricin-B family, proteins with legume lectin domains and GNA family that can induce cancer cell death via targeting programmed cell death pathways. Amongst these above-mentioned lectins, we demonstrate that mistletoe lectins (MLs), Ricin, Concanavalin A (ConA) and Polygonatum cyrtonema lectin (PCL) can lead to cancer cell programmed death via targeting apoptotic pathways. In addition, we show that ConA and PCL can also result in cancer cell programmed death by targeting autophagic pathways. Moreover, we summarize the possible anti-cancer therapeutic implications of plant lectins such as ConA, Phaseolus vulgaris lectin (PHA) and MLs that have been utilized at different stages of preclinical and clinical trials. Together, these findings can provide a comprehensive perspective for further elucidating the roles of plant lectins that may target programmed cell death pathways in cancer pathogenesis and therapeutics. And, this research may, in turn, ultimately help cancer biologists and clinicians to exploit lectins as potential novel antitumor drugs in the future.     

Use of codons in plant lectins

Codon usage in the coding region of mature lectins has been examined for 11 plant species (8 leguminoseae, 1 euphorbiaceae, 2 gramineae). The different legume lectins exhibit nearly the same codon usage pattern whereas the choice for the silent position of codons is non-random.     

Accessibility of the high-mannose glycans of glycoprotein gp120 from human immunodeficiency virus type 1 probed by in vitro interaction with mannose-binding lectins

The direct interaction of mannose-specific plant lectins with gp120 of HIV-1 was studied by surface plasmon resonance. Inhibition experiments indicated that exposed high mannose type glycans play a key role in the interaction. Most of the lectins specifically accommodate outer alpha1,2-, alpha1,3-, or alpha1,6-linked di- or trimannosides, and especially legume lectins, also interact with the trimannoside core of the complex type glycans. The unexpected affinity of some lectins towards gp120 presumably results from conformational differences in their binding sites. These results demonstrate that mannose-specific plant lectins are powerful tools to study the accessibility and elucidate the function of the gp120 glycans in the recognition and infection of the host cells by HIV-1.     

Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process

Mannose-specific lectins are widely distributed in higher plants and are believed to play a role in recognition of high-mannose type glycans of foreign micro-organisms or plant predators. Structural studies have demonstrated that the mannose-binding specificity of lectins is mediated by distinct structural scaffolds. The mannose/glucose-specific legume (e.g., Con A, pea lectin) exhibit the canonical twelve-stranded beta-sandwich structure. In contrast to legume lectins that interact with both mannose and glucose, the monocot mannose-binding lectins (e.g., the Galanthus nivalis agglutinin or GNA from bulbs) react exclusively with mannose and mannose-containing N-glycans. These lectins possess a beta-prism structure. More recently, an increasing number of mannose-specific lectins structurally related to jacalin (e.g., the lectins from the Jerusalem artichoke, banana or rice), which also exhibit a beta-prism organization, were characterized. Jacalin itself was re-defined as a polyspecific lectin which, in addition to galactose, also interacts with mannose and mannose-containing glycans. Finally the B-chain of the type II RIP of iris, which has the same beta-prism structure as all other members of the ricin-B family, interacts specifically with mannose and galactose. This structural diversity associated with the specific recognition of high-mannose type glycans highlights the importance of mannose-specific lectins as recognition molecules in higher plants.     

Mutational analysis of the sugar-binding site of pea lectin

Comparison of x-ray crystal structures of several legume lectins, co-crystallized with sugar molecules, showed a strong conservation of amino acid residues directly involved in ligand binding. For pea (Pisum sativum) lectin (PSL), these conserved amino acids can be classified into three groups: (I) D81 and N125, present in all legume lectins studied so far; (II) G99 and G216, conserved in almost all legume lectins; and (III) A217 and E218, which are only found in Vicieae lectins and are possibly determinants of sugar-binding specificity. Each of these amino acids in PSL was changed by site-directed mutagenesis, resulting in PSL molecules with single substitutions: for group I D81A, D81N, N125A; for group II G99R, G216L; and for group III A217L, E218Q, respectively. PSL double mutant Y124R; A126S was included as a control. The modified PSL molecules appeared not to be affected in their ability to form dimeric proteins, whereas the sugar-binding activity of each of the PSL mutants, with the exception of the control mutant (as shown by haemagglutination assays), was completely eliminated. These results confirm the model of the sugar-binding site of Vicieae lectins as deduced from X-ray analysis.     

Plant lectins: the ties that bind in root symbiosis and plant defense

Lectins are a diverse group of carbohydrate-binding proteins that are found within and associated with organisms from all kingdoms of life. Several different classes of plant lectins serve a diverse array of functions. The most prominent of these include participation in plant defense against predators and pathogens and involvement in symbiotic interactions between host plants and symbiotic microbes, including mycorrhizal fungi and nitrogen-fixing rhizobia. Extensive biological, biochemical, and molecular studies have shed light on the functions of plant lectins, and a plethora of uncharacterized lectin genes are being revealed at the genomic scale, suggesting unexplored and novel diversity in plant lectin structure and function. Integration of the results from these different types of research is beginning to yield a more detailed understanding of the function of lectins in symbiosis, defense, and plant biology in general.     

Legume lectins--a large family of homologous proteins

More than 70 lectins from leguminous plants belonging to different suborders and tribes have been isolated, mostly from seeds, and characterized to varying degrees. Although they differ in their carbohydrate specificities, they resemble each other in their physicochemical properties. They usually consist of two or four subunits (25-30 kDa), each with one carbohydrate binding site. Interaction with carbohydrates requires tightly bound Ca2+ and Mn2+ (or another transition metal). The primary sequences of more than 15 legume lectins have been established by chemical or molecular genetic techniques. They exhibit remarkable homologies, with a significant number of invariant amino acid residues, among them most of those involved in metal binding. The 3-dimensional structures of the legume lectins are similar, too, and are characterized by a high content of beta-sheets and a lack of alpha-helix. The location of the metal and carbohydrate binding sites, established unequivocally in concanavalin A by high resolution X-ray crystallography, appears to be the same in the other legume lectins. Several of the lectin genes have been cloned and expressed in heterologous systems. This opens the way for the application of molecular genetics to the investigation of the atomic structure of the carbohydrate binding sites of the lectins, and of the relationship between their structure and biological activity. The new approaches may also provide information on the mechanisms that control gene expression in plants and on the role of lectins in nature.     

A novel family of lectins evolutionarily related to class V chitinases: an example of neofunctionalization in legumes

A lectin has been identified in black locust (Robinia pseudoacacia) bark that shares approximately 50% sequence identity with plant class V chitinases but is essentially devoid of chitinase activity. Specificity studies indicated that the black locust chitinase-related agglutinin (RobpsCRA) preferentially binds to high-mannose N-glycans comprising the proximal pentasaccharide core structure. Closely related orthologs of RobpsCRA could be identified in the legumes Glycine max, Medicago truncatula, and Lotus japonicus but in no other plant species, suggesting that this novel lectin family most probably evolved in an ancient legume species or possibly an earlier ancestor. This identification of RobpsCRA not only illustrates neofunctionalization in plants, but also provides firm evidence that plants are capable of developing a sugar-binding domain from an existing structural scaffold with a different activity and accordingly sheds new light on the molecular evolution of plant lectins.     

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.     

Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins.The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework.