Cloning, Characterization, Expression Analysis, and Agglutination Studies of Novel Gene Encoding ß-D-Galactose, N-Acetyl-D-Glucosamine and Lactose-Binding Lectin from Rice Bean (Vigna umbellata).

Lectins are glycoproteins and known for their peculiar carbohydrate-binding activity and their insect-pest-resistant properties. Earlier we have published our research finding on novel gene encoding Bowman-Birk type protease inhibitor with insecticidal properties from rice bean. This paper presents first report on cloning, sequencing, and expression of RbL ORF of 843 bp encoding 280 amino acids long lectin precursor from rice bean (Vigna umbellata) seeds. Blast analysis revealed more than 90% similarity of RbL protein with Vigna aconitifolia and Vigna angularis lectins. Phylogenetic analysis also revealed a close relationship between RbL and other legume lectins. Sequence analysis of genomic DNA revealed intronless nature of RbL gene (GenBank accession No. MT043160). The isolated RbL ORF was expressed in E. coli BL-21(DE3) cells and maximum expression was recorded with 0.5 mM IPTG after 4 h incubation at 37 °C. Western blotting confirmed RbL protein expression in E. coli. Recombinant protein (His6-RbL) of ~ 35 kDa m.wt was purified using Ni-NTA affinity chromatography to the extent of 0.26 mg/ml. In silico analysis characterized RbL protein as acidic, stable, hydrophobic, and secretary protein with one signal peptide cleavage site (A26-A27) and four N-glycosylation sites. Template-based 3D model of RbL was structured using MODELLER tool and validated as good quality model. Structural analysis revealed dominance of ß-pleated sheets and ß-turns in RbL protein structure. ß-D-galactose, N-acetyl-D-glucosamine, and lactose were predicted as putative ligands for RbL protein. Hydrogen bonding and hydrophobic forces were the major interactions between the predicted ligands and RbL protein. Agglutination and agglutination inhibition assays confirmed the binding specificity of RbL protein with the trypsinized rabbit erythrocytes and with the predicted ligands, respectively. Gene ontology analysis functionally annotated RbL protein as a plant defense protein. The novel information generated in the study is not mere pre-experimental findings but could also lay foundation for future research on exploring RbL gene and encoding protein for different biomedical and biotechnological applications.

Research advances and prospects of legume lectins.

Lectins are widely distributed proteins having ability of binding selectively and reversibly with carbohydrates moieties and glycoconjugates. Although lectins have been reported from different biological sources, the legume lectins are the best-characterized family of plant lectins. Legume lectins are a large family of homologous proteins with considerable similarity in amino acid sequence and their tertiary structures. Despite having strong sequence conservation, these lectins show remarkable variability in carbohydrate specificity and quaternary structures. The ability of legume lectins in recognizing glycans and glycoconjugates on cells and other intracellular structures make them a valuable research tool in glycomic research. Due to variability in binding with glycans, glycoconjugates and multiple biological functions, legume lectins are the subject of intense research for their diverse application in different fields such as glycobiology, biomedical research and crop improvement. The present review specially focuses on structural and functional characteristics of legume lectins along with their potential areas of application.