Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity

Abstract

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80 degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 microg/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.

Antibacterial and Hemolytic Activity of a new Lectin Purified from the Seeds of Sterculia Foetida L.

“…Lectin isolated from Sophora alopecuroides has a molecular weight of 39.0 kDa [11]. R. vivipara lectin has a molecular mass of 49.5 kDa [27]. The data on the specific activity of all purification steps are shown in Table 1.…”

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

“…2B). The stability of the tarin quaternary structure, evaluated by the ability to easily maintain or recover itself after exposure to extreme temperature and pH conditions, has also been reported for other monocot mannose-binding lectins from species of Araceae [31,35]. Tarin's stability over a wide pH range confers resistance to degradation within the insect digestive tract, increasing its potential against insects [18,36].…”

Lectins: production and practical applications

“…The lectin contents in some parts of plants are higher, e.g., 390 and 75 mg of the purified lectin was recovered from 100 g Remusatia vivipara tubers (Bhat et al 2010) and Astragalus mongholicus roots (Yan et al 2005), respectively. Lectins are also found in seeds.…”