title: Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity.
abstract: 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°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10(-3) M and 2.8×10(-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 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.
authors.affiliation: Department of Bioinformatics and Structural Biology, Indian Institute of Advanced Research, Koba, Gandhinagar 382007, Gujarat, India.
authors.forename: Kunal R
authors.initials: KR
authors.lastname: Shah
authors.affiliation: Department of Bioinformatics and Structural Biology, Indian Institute of Advanced Research, Koba, Gandhinagar 382007, Gujarat, India.
authors.forename: Dhaval K
authors.initials: DK
authors.lastname: Patel
authors.affiliation: Department of Bioinformatics and Structural Biology, Indian Institute of Advanced Research, Koba, Gandhinagar 382007, Gujarat, India.
authors.forename: Anju
authors.initials: A
authors.lastname: Pappachan
authors.affiliation: Department of Biochemistry, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, India.
authors.forename: C Ratna
authors.initials: CR
authors.lastname: Prabha
authors.affiliation: Department of Bioinformatics and Structural Biology, Indian Institute of Advanced Research, Koba, Gandhinagar 382007, Gujarat, India; Onlien Centre for Genetic Disease and Molecular Medicine, Central University of Punjab, Bathinda 151001, Punjab, India. Electronic address: ddsingh@iiar.res.in.
authors.forename: Desh Deepak
authors.initials: DD
authors.lastname: Singh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001974:Bromosuccinimide / Q000494:pharmacology; D050260:Carbohydrate Metabolism; D019295:Computational Biology; D006384:Hemagglutination / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D037121:Plant Lectins / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D011817:Rabbits; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D011198:Solanum tuberosum / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D037121:Plant Lectins; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D001974:Bromosuccinimide
keywords: Circular dichroism; Fluorescence spectroscopy; Lectin; Protein-carbohydrate interaction; Serine protease inhibitor; β-Trefoil fold