Mannose-binding Jacalin-related Lectin Research Articles

ZG16p, an Animal Homologue of Plant β-Prism Fold Lectins: Purification Methods of Natural and Recombinant ZG16p and Inhibition Assay of Cancer Cell Growth Using ZG16p.

ZG16p is a soluble 16-kDa protein abundantly expressed in the pancreas and gut, and has a β-prism fold structure similar to that of mannose-binding Jacalin-related lectins (mJRLs) such as BanLec, Heltuba, and Artocarpin. ZG16p binds to mannose via the well-conserved GXXXD loop among mJRLs and sulfated glycosaminoglycans (e.g., heparin and heparan sulfate) via the basic patch of molecular surface. In addition to the above binding activities, ZG16p has inhibitory activity against proliferation of colon cancer cells. This manuscript describes purification of rat pancreatic ZG16p and recombinant ZG16p expressed in Escherichia coli expression system, and cell growth inhibition assay using ZG16p as an inhibitor.

Overexpression of rice jacalin-related mannose-binding lectin (OsJAC1) enhances resistance to ionizing radiation in Arabidopsis

BackgroundJacalin-related lectins in plants are important in defense signaling and regulate growth, development, and response to abiotic stress. We characterized the function of a rice mannose-binding jacalin-related lectin (OsJAC1) in the response to DNA damage from gamma radiation.ResultsTime- and dose-dependent changes of OsJAC1 expression in rice were detected in response to gamma radiation. To identify OsJAC1 function, OsJAC1-overexpressing transgenic Arabidopsis plants were generated. Interestingly, OsJAC1 overexpression conferred hyper-resistance to gamma radiation in these plants. Using comparative transcriptome analysis, genes related to pathogen defense were identified among 22 differentially expressed genes in OsJAC1-overexpressing Arabidopsis lines following gamma irradiation. Furthermore, expression profiles of genes associated with the plant response to DNA damage were determined in these transgenic lines, revealing expression changes of important DNA damage checkpoint and perception regulatory components, namely MCMs, RPA, ATM, and MRE11.ConclusionsOsJAC1 overexpression may confer hyper-resistance to gamma radiation via activation of DNA damage perception and DNA damage checkpoints in Arabidopsis, implicating OsJAC1 as a key player in DNA damage response in plants. This study is the first report of a role for mannose-binding jacalin-related lectin in DNA damage.

Cloning and characterization of mannose binding jacalin related lectin encoding gene from Indian Cycas (Cycas annaikalensis Singh and Radha)

Mannose specific jacalin-related lectins or agglutinins (mJRLs) constitute an important superfamily of proteins known to play vital roles in various biological processes. In the present study, a cDNA having 876 bp open reading frame (ORF) coding for mJRL of 291 amino acids residues was cloned from pinna of Cycas annaikalensis which is endemic to Western Ghats, India and designated as C. annaikalensis pinna lectin (CAPL). Expression of the coding sequence under the control of a T7 promoter in E. coli produced 31 kDa protein. The purified recombinant protein had shown agglutination with erythrocytes of rabbit blood. The deduced amino acid sequence of CAPL showed two sugar binding sites (also determined to be jacalin-like lectin domains) and 95% similarity with C. revoluta leaf lectin (CRLL) protein. Further, a monomeric protein of CAPL consisting of mannose binding residues and jacalin motifs that are having 35–90% similarities with mJRLs which have already been reported. A phylogenetic tree exhibited the grouping of CAPL into a subclade different from that of the CRLL. Also, a model of cycas leaf lectin was built by homology modeling using 1ZGRA (Parkia platycephala seed lectin) as a template for the construction of three-dimensional structures. Structural modeling and docking studies were completed using Discovery studio version 2.1. This study, first of its kind, reports mJRLs from the Indian gymnosperm.

Two carbohydrate recognizing domains from Cycas revoluta leaf lectin show the distinct sugar-binding specificity-A unique mannooligosaccharide recognition by N-terminal domain.

Cycas revoluta leaf lectin (CRLL) of mannose-recognizing jacalin-related lectin (mJRL) has two tandem repeated carbohydrate recognition domains, and shows the characteristic sugar-binding specificity toward high mannose-glycans, compared with other mJRLs. We expressed the N-terminal domain and C-terminal domain (CRLL-N and CRLL-C) separately, to determine the fine sugar-binding specificity of each domain, using frontal affinity chromatography, glycan array and equilibrium dialysis. The specificity of CRLL toward high mannose was basically derived from CRLL-N, whereas CRLL-C had affinity for α1-6 extended mono-antennary complex-type glycans. Notably, the affinity of CRLL-N was most potent to one of three Man 8 glycans and Man 9 glycan, whereas the affinity of CRLL-C decreased with the increase in the number of extended α1-2 linked mannose residue. The recognition of the Man 8 glycans by CRLL-N has not been found for other mannose recognizing lectins. Glycan array reflected these specificities of the two domains. Furthermore, it was revealed by equilibrium dialysis method that the each domain had two sugar-binding sites, similar with Banlec, banana mannose-binding Jacalin-related lectin.

Early diagnosis of blast fungus, Magnaporthe oryzae, in rice plant by using an ultra-sensitive electrically magnetic-controllable electrochemical biosensor

As one of the most destructive and widespread disease of rice, Magnaporthe oryzae (also called Magnaporthe grisea) has a significant negative impact on rice production. Therefore, it is still in high demand to develop extremely sensitive and accurate methods for the early diagnosis of Magnaporthe oryzae (M. oryzae). In this study, we developed a novel magnetic-controllable electrochemical biosensor for the ultra sensitive and specific detection of M. oryzae in rice plant by using M. oryzae’s chitinases (Mgchi) as biochemical marker and a rice (Oryza sativa) cDNA encoding mannose-binding jacalin-related lectin (Osmbl) as recognition probe. The proposed biosensor combined with the merits of chronoamperometry, electrically magnetic-controllable gold electrode and magnetic beads (MBs)-based palladium nano-particles (PdNPs) catalysis amplification, has an ultra-high sensitivity and specificity for the detection of trace M. oryzae in rice plant. It could be used to detect M. oryzae in rice plant in the initial infection stage (before any symptomatic lesions were observed) to help farmers timely manage the disease. In comparison with previous methods, the proposed method has notable advantages such as higher sensitivity, excellent specificity, short analysis time, robust resistibility to complex matrix and low cost etc. The success in this study provides a reliable approach for the early diagnosis and fast screening of M. oryzae in rice plant.

A Sunflower Lectin with Antifungal Properties and Putative Medical Mycology Applications

Lectins are carbohydrate-binding proteins with a high specificity for a variety of glycoconjugate sugar motifs. The jacalin-related lectins (JRL) are considered to be a small sub-family composed of galactose- and mannose-specific members. Using a proteomics approach, we have detected a 16 kDa protein (Helja) in sunflower seedlings that were further purified by mannose-agarose affinity chromatography. The aim of this work was to characterize the biological activity of Helja and to explore potential applications for the antifungal activity of this plant lectin against medically important yeasts. To initially assess the agglutination properties of the lectin, Saccharomyces cerevisiae cells were incubated with increasing concentrations of the purified lectin. At a concentration of 120 μg/ml, Helja clearly agglutinated these cells. The ability of different sugars to inhibit S. cerevisiae cell agglutination determined its carbohydrate-specificity. Among the monosaccharides tested, D-mannose had the greatest inhibitory effect, with a minimal concentration of 1.5 mM required to prevent cell agglutination. The antifungal activity was evaluated using pathogenic fungi belonging to the Candida and Pichia genera. We demonstrate that 200 μg/ml of Helja inhibited the growth of all yeasts, and it induced morphological changes, particularly through pseudohyphae formation on Candida tropicalis. Helja alters the membrane permeability of the tested fungi and is also able to induce the production of reactive oxygen species in C. tropicalis cells. We concluded that Helja is a mannose-binding JRL with cell agglutination capabilities and antifungal activity against yeasts. The biological properties of Helja may have practical applications in the control of human pathogens.

The early diagnosis and fast detection of blast fungus, Magnaporthe grisea, in rice plant by using its chitinase as biochemical marker and a rice cDNA encoding mannose-binding lectin as recognition probe

As one of the most destructive and widespread disease of rice, Magnaporthe grisea (M. grisea) has a significant negative impact on rice production. Therefore, it is significant to develop a method for sensitive and high-throughput detection of M. grisea in order to help farmers to find and control blast in the early stage. We herein first cloned and expressed the M. grisea's chitinases (Mgchi) and a rice cDNA encoding mannose-binding jacalin-related lectin (Osmbl) with Mgchi-related gene. We demonstrated that Mgchi could be used as a biochemical marker for the detection of M. grisea and there was a specific interaction between Osmbl and Mgchi. Using Mgchi as biochemical marker and Osmbl as recognition probe, we developed a visual method for the specific and sensitive detection of M. grisea based on the PdNPs-catalyzed TMB/H2O2 system. The proposed method could be used to detect Mgchi as low as 7.5×10−9M by naked eye observation and 2.5×10–11M Mgchi by the micro-plate reader. With the help of the method, we had successfully detected M. grisea in the real M. grisea-infected rice plant with recoveries of 88–109% and RSD<7%. The proposed method was sensitive, specific, potentially high-throughput and cost-effective. The success in this study provides a promising substitution for the early diagnosis and fast screening of M. grisea in rice plant.

Cloning and expression of a novel cDNA encoding a mannose-specific jacalin-related lectin from Oryza sativa

Lectin plays an important role in defense signaling in plants. A few genes of this family have been cloned. Here we report on a mannose-specific jacalin-related lectin in rice. Using sequence information of wheat gene VER2, which we had previously cloned, we were able to amplify a cDNA of OsJAC1 from Oryza sativa by RT-PCR. The cDNA of OsJAC1 was 1172 bp and contained a 921-bp open reading frame (ORF) encoding dirigent (amino acids 26–139) and jacalin (amino acids 175–305) domains of 306 amino acids. Comparison of the OsJAC1 sequence with those of other lectins (jacalin) from rice, wheat and other species revealed that OsJAC1 had the 12 amino acid positions conserved in all mannose-binding lectins. Semi-quantitative RT-PCR revealed that OsJAC1 expression was present in stems, leaves and young spikes but not young roots; the expression was high in leaves and low in stems and spikes. And methyl jasmonate could induce the expression of OsJAC1. To test the activity of OsJAC1, the jacalin domain at the C-terminal was expressed in E. coli. BL21 (DE3). The purified recombinant protein could agglutinate red blood cells of rabbit, and the agglutination activity was strongly inhibited by mannose compared with other carbohydrates. These results indicate that lectin with dirigent and jacalin domains exists in rice as well as wheat. This is the first report of cDNA cloning of mannose-binding jacalin-related lectin with a dirigent domain in N-terminal region from O. sativa.

Overall carbohydrate-binding properties of Castanea crenata agglutinin (CCA)

The carbohydrate-binding properties of Castanea crenata agglutinin (CCA) were investigated by an enzyme-linked lectin absorbent assay. The binding ability of each carbohydrate was compared using IC 50 values. CCA exhibited mannose/glucose specificity, as observed with many mannose-binding jacalin-related lectins. For oligosaccharides containing glucose, it has been shown that the degree of polymerization and the linkage mode of glucose residues have no effect on CCA–carbohydrate interaction; thus, only the non-reducing end glucose unit in glucooligosaccharides may be involved in the interaction with CCA. Among mannooligosaccharides, CCA strongly recognized α-(1→3)- d-Man-[α- d-Man-(1→6)]- d-Man, which is a core in N-linked carbohydrate chains. By considering the results with glycoproteins, it is likely that CCA binds preferentially to mono- or non-sialylated biantennary carbohydrate chains. We also obtained K d values by analysis of the dependency of the IC 50 on CCA concentration, based on the hypothesis that CCA has a single binding site or two equivalent binding sites. The estimated K d values for mannose, glucose and α-(1→3)- d-Man-[α- d-Man-(1→6)]- d-Man were 2.39, 7.19 and 0.483 mM, respectively. The relative binding abilities showed good agreement with the relative inhibition intensities. Isothermal calorimetric titration was carried out to directly estimate the dissociation constants of CCA for mannose and for α- d-Man-(1→3)- d-Man. The values were 2.34 mM for mannose and 0.507 mM α- d-Man-(1→3)- d-Man. These results suggest that the relative inhibition intensity represents the ratio of K d values and that CCA has a single or two equivalent binding sites.

The Crystal Structure of the Calystegia sepium Agglutinin Reveals a Novel Quaternary Arrangement of Lectin Subunits with a β-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.

The galactose-binding and mannose-binding jacalin-related lectins are located in different sub-cellular compartments

A galactose-specific and a mannose-specific lectin of the family of the jacalin-related lectins have been localized by immunofluorescence microscopy. The present localization studies provide for the first time unambiguous evidence for the cytoplasmic location of the mannose-specific jacalin-related lectin from rhizomes of Calystegia sepium, which definitely differs from the vacuolar location of the galactose-specific jacalin from Artocarpus integrifolia. These observations support the hypothesis that the galactose-specific jacalin-related lectins evolved from their mannose-specific homologues through the acquisition of vacuolar targeting sequences.

Isolation and characterization of a jacalin-related mannose-binding lectin from salt-stressed rice (Oryza sativa) plants.

A novel plant lectin was isolated from salt-stressed rice (Oryza sativa L.) plants and partially characterized. The lectin occurs as a natural mixture of two closely related isoforms consisting of two identical non-covalently linked subunits of 15 kDa. Both isoforms are best inhibited by mannose and exhibit potent mitogenic activity towards T-lymphocytes. Biochemical analyses and sequence comparisons further revealed that the rice lectins belong to the subgroup of mannose-binding jacalin-related lectins. In addition, it could be demonstrated that the lectins described here correspond to the protein products of previously described salt-stress-induced genes. Our results not only identify the rice lectin as a stress protein but also highlight the possible importance of protein-carbohydrate interactions in stress responses in plants.