Purification, chitooligosaccharide binding properties and thermal stability of CIA24, a new PP2-like phloem exudate lectin from ivy gourd (Coccinia indica)

摘要

PP2-like chitin binding phloem exudate lectins, abundant in the sieve tube of cucurbits, have been implicated to play key roles in wound sealing and antipathogenic responses of the plant. Here we report the affinity purification, macromolecular characterization and carbohydrate binding properties of a new chitooligosaccharide-specific lectin from the phloem exudate of ivy gourds (Coccinia indica). The protein, CIA24, has a subunit mass of 24 kDa. Partial sequence analysis indicated that CIA24 exhibits high homology with CIAI7 and other Cucurbitaceae PP2 proteins whereas CD spectroscopic studies suggested that beta-sheets constitute the predominant secondary structure. Temperature dependent CD spectroscopic and differential scanning calorimetric studies revealed that CIA24 is a highly thermostable protein, which undergoes complete unfolding at similar to 105 degrees C. Isothermal titration calorimetric studies suggested that binding of chitooligosaccharides to CIA24 is a highly exothermic process. The lectin combining site can accommodate upto a tetrasaccharide with the binding stoichiometry (n) close to unity with respect to each protein subunit, whereas for chitohexaose a sharp decrease in the binding stoichiometry (n) to similar to 1:0.5 was observed. This suggests that the protein probably undergoes dimerisation in presence of chitohexaose, wherein two protein molecules bind to the oligosaccharides from the reducing and non-reducing end, respectively. (C) 2018 Published by Elsevier B.V.