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

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• 作者：Kunal R. Shah^a ; Dhaval K. Patel^a ; Anju Pappachan^a ; C. Ratna Prabha^b ; Desh Deepak Singh^a ; ^c ; ^{ddsingh@iiar.res.in" class="auth_mail" title="E-mail the corresponding author} ;
• 关键词：Serine protease inhibitor ; Protein-carbohydrate interaction ; Lectin ; β-Trefoil fold ; Fluorescence spectroscopy ; Circular dichroism
• 刊名：International Journal of Biological Macromolecules
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：83
• 期：Complete
• 页码：259-269
• 全文大小：2129 K

文摘

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 (K_d) 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.