Journal
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 83, Issue -, Pages 259-269Publisher
ELSEVIER
DOI: 10.1016/j.ijbiomac.2015.11.068
Keywords
Serine protease inhibitor; Protein-carbohydrate interaction; Lectin; beta-Trefoil fold; Fluorescence spectroscopy; Circular dichroism
Funding
- DST
- DBT-DISC, Govt. Of India
- Puri Foundation for Education in India
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Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20 kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5 kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640 H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80 degrees C. Mannose and galactose interacted with the PotHg with a dissociation constant (K-d) of 1.5 x 10(-3) M and 2.8 x 10(-3) M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly beta sheets (similar to 45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation. (C) 2015 Elsevier B.V. All rights reserved.
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