Purification, biochemical/biophysical characterization and chitooligosaccharide binding to BGL24, a new PP2-type phloem exudate lectin from bottle gourd (Lagenaria siceraria)

Abstract

Phloem Protein 2 (PP2), highly abundant in the sieve elements of plants, plays a significant role in wound sealing and anti-pathogenic responses. In this study, we report the purification and characterization of a new PP2-type lectin, BGL24 from the phloem exudate of bottle gourd (Lagenaria siceraria). BGL24 is a homodimer with a subunit mass of ~24 kDa and exhibits high specificity for chitooligosaccharides. The isoelectric point of BGL24 was estimated from zeta potential measurements as 5.95. Partial amino acid sequence obtained by mass spectrometric studies indicated that BGL24 exhibits extensive homology with other PP2-type phloem exudate lectins. CD spectroscopic measurements revealed that the lectin contains predominantly β-sheets, with low α-helical content. CD spectroscopic and DSC studies showed that BGL24 exhibits high thermal stability with an unfolding temperature of ~82 °C, and that its secondary structure is essentially unaltered between pH 3.0 and 8.0. Fluorescence titrations employing 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside as an indicator ligand revealed that the association constants for BGL24-chitooligosaccharide interaction increase considerably when the ligand size is increased from chitotriose to chitotetraose, whereas only marginal increase was observed for chitopentaose and chitohexaose. BGL24 exhibited moderate cytotoxicity against MDA-MB-231 breast cancer cells, whereas its effect on normal splenocytes was marginal.