GNA-related Lectins Research Articles

Purification of GNA-Related Lectins from Natural Sources.

The Galanthus nivalis lectin, abbreviated as GNA, is the model protein for a large group of mannose-binding lectins. Here, we describe the purification of GNA starting from dry bulbs. Using a combination of ion exchange chromatography and affinity chromatography on mannose-Sepharose, a highly pure preparation of GNA can be obtained.

Sauromatum guttatum lectin: Spectral studies, lectin-carbohydrate interaction, molecular cloning and in silico analysis

Non-immune carbohydrate binding proteins are broadly defined as lectins. Having been reported from all kingdoms of life, phytolectins are the most widely studied group of lectins. Sauromatum guttatum agglutinin (SGA) was isolated from the plant tubers and characterized for structural variations due to solvent perturbation using polarimetry, fluorescence and light scattering. For the β-sheet rich SGA, a pH and temperature induced molten globule like intermediate was identified. In isothermal titration microcalorimetry, SGA demonstrated cooperative binding to a complex glycoprotein in enthalpically driven mechanism. Fine sugar specificity exploration identified core pentasaccharide as the most common and highest binding motif with complex N-glycans and fucosylated core N-glycans as additional motifs. Molecular cloning of SGA which has previously been demonstrated to have anti-cancer and anti-insect activities is being reported for the first time. Full length cDNA sequence was obtained with RACE-PCR based upon the conserved carbohydrate recognition site [QXDXNXVXY] present in all GNA-related lectins. Quaternary structure was proposed by homology modeling and an attempt was made to explain the structure-function relationship by in silico analysis.

High-resolution crystal structures of Colocasia esculenta tarin lectin.

Tarin, the Colocasia esculenta lectin from the superfamily of α-d-mannose-specific plant bulb lectins, is a tetramer of 47 kDa composed of two heterodimers. Each heterodimer possesses homologous monomers of ~11.9 (A chain) and ~12.7 (B chain) kDa. The structures of apo and carbohydrate-bound tarin were solved to 1.7 Å and 1.91 Å, respectively. Each tarin monomer forms a canonical β-prism II fold, common to all members of Galanthus nivalis agglutinin (GNA) family, which is partially stabilized by a disulfide bond and a conserved hydrophobic core. The heterodimer is formed through domain swapping involving the C-terminal β-strand and the β-sheet on face I of the prism. The tetramer is assembled through the dimerization of the B chains from heterodimers involving face II of each prism. The 1.91 Å crystal structure of tarin bound to Manα(1,3)Manα(1,6)Man reveals an expanded carbohydrate-binding sequence (QxDxNxVxYx4/6WX) on face III of the β-prism. Both monomers possess a similar fold, except for the length of the loop, which begins after the conserved tyrosine and creates the binding pocket for the α(1,6)-terminal mannose. This loop differs in size and amino-acid composition from 10 other β-prism II domain proteins, and may confer carbohydrate-binding specificity among members of the GNA-related lectin family.

The pepper GNA-related lectin and PAN domain protein gene, CaGLP1, is required for plant cell death and defense signaling during bacterial infection

Carbohydrate-binding proteins, commonly referred to as lectins or agglutinins, function in defense responses to microbial pathogens. Pepper (Capsicum annuum) GNA-related lectin and PAN-domain protein gene CaGLP1 was isolated and functionally characterized from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). CaGLP1 contained an amine-terminus prokaryotic membrane lipoprotein lipid attachment site, a Galanthus nivalis agglutinin (GNA)-related lectin domain responsible for the recognition of high-mannose N-glycans, and a carboxyl-terminus PAN/apple domain. RNA gel blot and immunoblot analyses determined that CaGLP1 was strongly induced in pepper by compatible and incompatible Xcv infection. CaGLP1 protein localized primarily to the plasma membrane and exhibited mannose-binding specificity. CaGLP1-silenced pepper plants were more susceptible to compatible or incompatible Xcv infection compared with that of non-silenced control plants. CaGLP1 silencing in pepper leaves did not accumulate H2O2 and induce cell death during incompatible Xcv infection. Defense-related CaDEF1 (defensin) gene expression was significantly reduced in CaGLP1-silenced pepper plants. CaGLP1-overexpression in Arabidopsis thaliana enhanced resistance to Pseudomonas syringae pv. tomato. Defense-related AtPDF1.2 expression was elevated in CaGLP1-overexpression lines. Together, these results suggest that CaGLP1 is required for plant cell death and defense responses through the reactive oxygen species burst and downstream defense-related gene expression in response to bacterial pathogen challenge.

Structural analysis and binding properties of isoforms of tarin, the GNA-related lectin from Colocasia esculenta

The lectins, a class of proteins that occur widely in animals, plants, fungi, lichens and microorganisms, are known for their ability to specifically bind to carbohydrates. Plant lectins can be classified into 12 families including the Galanthus nivalis agglutinin (GNA)-related lectin superfamily, which is widespread among monocotyledonous plants and binds specifically to mannose, a behavior that confers remarkable anti-tumor, anti-viral and insecticidal properties on these proteins. The present study characterized a mitogenic lectin from this family, called tarin, which was purified from the crude extract from taro (Colocasia esculenta). The results showed that tarin is a glycoprotein with 2–3% carbohydrate content, composed of least 10 isoforms with pIs ranging from 5.5 to 9.5. The intact protein is a heterotetramer of 47kDa composed of two non-identical and non-covalently associated polypeptides, with small subunits of 11.9kDa and large subunits of 12.6kDa. The tarin structure is stable and recovers or maintains its functional structure following treatments at different temperatures and pH. Tarin showed a complex carbohydrate specificity, binding with high affinity to high-mannose and complex N-glycans. Many of these ligands can be found in viruses, tumor cells and insects, as well as in hematopoietic progenitor cells. Chemical modifications confirmed that both conserved and non-conserved amino acids participate in this interaction. This study determined the structural and ligand binding characteristics of a GNA-related lectin that can be exploited for several different purposes, particularly as a proliferative therapeutic molecule that is able to enhance the immunological response.

Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK pathway in human MCF-7 breast cancer cells

Polygonatum odoratum lectin (POL), a mannose-binding GNA-related lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which POL induces cancer cell death are still elusive. In the current study, we found that POL could induce both apoptosis and autophagy in human MCF-7 breast cancer cells. Subsequently, we found that POL induced MCF-7 cell apoptosis via the mitochondrial pathway. Additionally, we also found that POL induces MCF-7 cell apoptosis via EGFR-mediated Ras-Raf-MEK-ERK pathway, suggesting that POL may be a potential EGFR inhibitor. Finally, we used proteomics analyses for exploring more possible POL-induced pathways with EGFR, Ras, Raf, MEK and ERK, some of which were consistent with our in silico network prediction. Taken together, these results demonstrate that POL induces MCF-7 cell apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK signaling pathway, which would provide a new clue for exploiting POL as a potential anti-neoplastic drug for future cancer therapy.

Molecular switch role of Akt in Polygonatum odoratum lectin-induced apoptosis and autophagy in human non-small cell lung cancer A549 cells.

Polygonatum odoratum lectin (POL), isolated from traditional Chinese medicine herb (Mill.) Druce, has drawn rising attention due to its wide biological activities. In the present study, anti-tumor effects, including apoptosis- and autophagy-inducing properties of POL, were determined by a series of cell biology methods such as MTT, cellular morphology observation, flow cytometry, immunoblotting. Herein, we found that POL could simultaneously induce apoptosis and autophagy in human non-small cell lung cancer A549 cells. POL initiated apoptosis through inhibiting Akt-NF-κB pathway, while POL triggered autophagy via suppressing Akt-mTOR pathway, suggesting the molecular switch role of Akt in regulating between POL-induced apoptosis and autophagy. Moreover, ROS was involved in POL-induced inhibition of Akt expression, and might therefore mediate both apoptosis and autophagy in A549 cells. In addition, POL displayed no significant cytotoxicity toward normal human embryonic lung fibroblast HELF cells. Due to the anti-tumor activities, POL might become a potent anti-cancer drug in future therapy, which might pave the way for exploring GNA-related lectins into effective drugs in cancer treatment.

Anti-tumor and anti-viral activities of Galanthus nivalis agglutinin (GNA)-related lectins

Galanthus nivalis agglutinin (GNA)-related lectin family, a superfamily of strictly mannose-binding specific lectins widespread among monocotyledonous plants, is well-known to possess a broad range of biological functions such as anti-tumor, anti-viral and anti-fungal activities. Herein, we mainly focused on exploring the precise molecular mechanisms by which GNA-related lectins induce cancer cell apoptotic and autophagic death targeting mitochondria-mediated ROS-p38-p53 apoptotic or autophagic pathway, Ras-Raf and PI3K-Akt anti-apoptotic or anti-autophagic pathways. In addition, we further discussed the molecular mechanisms of GNA-related lectins exerting anti-viral activities by blocking the entry of the virus into its target cells, preventing transmission of the virus as well as forcing virus to delete glycan in its envelope protein and triggering neutralizing antibody. In conclusion, these findings may provide a new perspective of GNA-related lectins as potential drugs for cancer and virus therapeutics in the future.

Molecular modeling, docking and dynamics simulations of GNA-related lectins for potential prevention of influenza virus (H1N1)

The Galanthus nivalis agglutinin (GNA)-related lectin family exhibit significant anti-HIV and anti-HSV properties that are closely related to their carbohydrate-binding activities. However, there is still no conclusive evidence that GNA-related lectins possess anti-influenza properties. The hemagglutinin (HA) of influenza virus is a surface protein that is involved in binding host cell sialic acid during the early stages of infection. Herein, we studied the 3D-QSARs (three-dimensional quantitative structure-activity relationships) of lectin- and HA-sialic acid by molecular modeling. The affinities and stabilities of lectin- and HA-sialic acid complexes were also assessed by molecular docking and molecular dynamics simulations. Finally, anti-influenza GNA-related lectins that possess stable conformations and higher binding affinities for sialic acid than HAs of human influenza virus were screened, and a possible mechanism was proposed. Accordingly, our results indicate that some GNA-related lectins, such as Yucca filamentosa lectin and Polygonatum cyrtonema lectin, could act as drugs that prevent influenza virus infection via competitive binding. In conclusion, the GNA-related lectin family may be helpful in the design of novel candidate agents for preventing influenza A infection through the use of competitive combination against sialic acid specific viral infection.

Polygonatum cyrtonema lectin induces murine fibrosarcoma L929 cell apoptosis via a caspase-dependent pathway as compared to Ophiopogon japonicus lectin

Galanthus nivalis agglutinin (GNA)-related lectin family, a superfamily of strictly mannose-binding specific lectins, has been well-known to possess several biological functions including apoptosis-inducing activities. However, the precise mechanisms of GNA-related lectins to induce apoptosis remains to be clarified. In this study, we showed that Polygonatum cyrtonema lectin (PCL) and Ophiopogon japonicus lectin (OJL), the two mannose-binding GNA-related lectins, could induce murine fibrosarcoma L929 cell apoptosis. In addition, we found that there was a close link between their sugar-binding and apoptosis-inducing activities. Interestingly, we further confirmed that the mechanism of lectin-induced apoptosis was a caspase-dependent pathway. Moreover, we found that the two lectins could amplify tumor necrosis factor α (TNFα)-induced apoptosis. Taken together, these findings would open a new perspective for GNA-related lectins as potential anti-tumor agents.

Crystal structures of a novel anti-HIV mannose-binding lectin from Polygonatum cyrtonema Hua with unique ligand-binding property and super-structure

Polygonatum cyrtonema lectin (PCL) is a novel anti-HIV mannose-binding lectin from Galanthus nivalis agglutinin (GNA)-related lectin family. Crystal structures of ligand-free PCL and its complexes with monomannoside and alpha1-3 dimannoside have been determined. The ligand-free PCL is dimeric, with both subunits adopt the beta-prism II fold. PCL subunit binds mannose using a potential bivalent mode instead of the usual trivalent mode, in which carbohydrate-binding site (CBS) I and CBS III adopt the conserved mannose-binding motif of QXDXNXVXY (X is one of any amino acid residues) as observed in other structurally characterized GNA-related lectins, while CBS II adopts a modified motif with residues Gln58 and Asp60, which are critical for mannose-binding, substituted by His58 and Asn60, respectively. As a result, CBS II is unfit for mannose-binding. In the mannoside complexes, ligand-bindings only occur at CBS I which provides the specificity for alpha1-3 dimannoside. CBS II and CBS III are cooperatively occupied by a well-ordered sulfate ion, through which the individual dimers are cross-linked to form a unique super-structure of 3(2) helical lattice. Surveying the sequences of GNA-related lectins revealed that the modified binding motif of CBS II is widely distributed in the Liliaceae family as an intrinsic structural element. There is evidence that other GNA-related lectins will also adopt the similar super-structure as PCL. Thus PCL structure, unique in ligand-binding mode, may represent a novel type of structure of GNA-related lectins. Comparative analyses indicated that the dimer-based super-structure may play a primary role in the anti-HIV property of PCL.

Expression and characterization of two domains of Pinellia ternata agglutinin (PTA), a plant agglutinin from Pinellia ternata with antifungal activity

Pinellia ternata agglutinin (PTA) from P. ternata f. angustata is a two-domain GNA-related lectin. The current study indicates that the PTA gene encodes a precursor consisting of two tandemly arrayed domains, N-terminal domain (PTA-DOM1) and C-terminal domain (PTA-DOM2). Both domains and the precursor without signal peptide (PTA-P) present different number of activity mannose-binding sites which play key roles for the lectin function. Analyses of the three fusion proteins, PTA-DOM1, PTA-DOM2 and PTA-P, expressed in Escherichia coli revealed that one mannose-binding site the agglutination activity while the additional sites do not possess such activity. However, the number of carbohydrate-binding sites suggests some significant properties on the antifungal effectiveness. In addition, each of the PTA domains has the same function when compared with the natural PTA (N-PTA). The information on PTA gene obtained in this study will served as baseline information in developing this protein as a form of transgenic plant protection.

Galanthus nivalis Agglutinin (GNA)-Related Lectins: Traditional Proteins, Burgeoning Drugs?

Galanthus nivalis agglutinin (GNA)-related lectin family, a superfamily of strictly mannose-binding specific lectins widespread among monotyledonous plants, is well-known to possess a broad range of biological functions such as anti-tumor, anti-fungal and anti-viral activities. Growing insights into the investigations of them provide us a principal force to start this review with a variety of evidences to landmark their biological activities, molecular mechanisms and potential therapeutic applications. Herein, we mainly focused on a comprehensive study on a great number of reported GNA-related lectins with the anti-tumor activity. On the trail of such clues, we studied some GNA-related lectins with anti-tumor effects and explored the apoptosis- and autophagy-induced activities and related molecular mechanisms. These inspiring results further provide us more convincing evidence to understand the anti-cancer molecular mechanisms implicated in apoptosis and autophagy. In addition, GNA-related lectins also possess marked anti-HIV activities and the specific molecular mechanisms are also explored herein. To sum up, GNA-related lectin family would play a key role as a potential drug in future therapeutics. Keywords: GNA-related lectins, anti-tumor activity, apoptosis, autophagy, drug

Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells

Background The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNA-related lectins induce cell death are still only rudimentarily understood. Methods In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)-induced L929 cell apoptosis. Results Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis. General significance These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations.

Related lectins from snowdrop and maize differ in their carbohydrate-binding specificity

Searches in an EST database from maize revealed the expression of a protein related to the Galanthus nivalis (GNA) agglutinin, referred to as GNA maize. Heterologous expression of GNA maize in Pichia pastoris allowed characterization of the first nucleocytoplasmic GNA homolog from plants. GNA maize is a tetrameric protein which shares 64% sequence similarity with GNA. Glycan microarray analyses revealed important differences in the specificity. Unlike GNA, which binds strongly to high-mannose N-glycans, the lectin from maize reacts almost exclusively with more complex glycans. Interestingly, GNA maize prefers complex glycans containing β1-2 GlcNAc residues. The obvious difference in carbohydrate-binding properties is accompanied by a 100-fold reduced anti-HIV activity. Although the sequences of GNA and GNA maize are clearly related they show only 28% sequence identity. Our results indicate that gene divergence within the family of GNA-related lectins leads to changes in carbohydrate-binding specificity, as shown on N-glycan arrays.

Localization and Topogenesis Studies of Cytoplasmic and Vacuolar Homologs of the Galanthus nivalis Agglutinin

The Galanthus nivalis agglutinin (GNA) is synthesized as a preproprotein. To corroborate the role of the different targeting peptides in the topogenesis of GNA and related proteins, different constructs were made whereby both the complete original GNA gene and different truncated sequences were coupled to the enhanced green fluorescent protein (EGFP). In addition, a GNA ortholog from rice that lacks the signal peptide and C-terminal propeptide sequence was fused to EGFP. These fusion constructs were expressed in tobacco BY-2 cells and their localization analyzed by confocal fluorescence microscopy. We observed that the processed preproprotein of GNA was directed towards the vacuolar compartment, whereas both the truncated forms of GNA corresponding to the mature lectin polypeptide and the rice ortholog of GNA were located in the nucleus and the cytoplasm. It can be concluded, therefore, that removal of the C-terminal propeptide and the signal peptide is sufficient to change the subcellular targeting of a normally vacuolar protein to the nuclear/cytoplasmic compartment of the BY-2 cells. These findings support the proposed hypothesis that cytoplasmic/nuclear GNA-like proteins and their vacuolar homologs are evolutionarily related and that the classical GNA-related lectins might have evolved from cytoplasmic orthologs through an evolutionary event involving the insertion of a signal peptide and a C-terminal propeptide.

Phylogenetic and specificity studies of two-domain GNA-related lectins: generation of multispecificity through domain duplication and divergent evolution

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.