Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity

• Published in: International journal of biological macromolecules 2016-02, Vol.83, p.259-269
• Main Authors: Shah, Kunal R., Patel, Dhaval K., Pappachan, Anju, Prabha, C. Ratna, Singh, Desh Deepak
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Bromosuccinimide - pharmacology; Carbohydrate Metabolism; Circular dichroism; Computational Biology; Fluorescence spectroscopy; Hemagglutination - drug effects; Hydrogen-Ion Concentration; Lectin; Molecular Sequence Data; Molecular Weight; Peptides - chemistry; Peptides - isolation & purification; Peptides - metabolism; Peptides - pharmacology; Plant Lectins - metabolism; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Plant Proteins - pharmacology; Protein-carbohydrate interaction; Rabbits; Serine protease inhibitor; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - isolation & purification; Serine Proteinase Inhibitors - metabolism; Serine Proteinase Inhibitors - pharmacology; Solanum tuberosum - chemistry; Substrate Specificity; β-Trefoil fold
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2015.11.068

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 20kDa, 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 (15kDa and 5kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4–9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10−3M and 2.8×10−3M, 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 β sheets (∼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.