Journal Article

Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

Mattaparthi, Venkata Satish Kumar, Doley, Robin, Iyer, Janaki Krishnamurthy, Mukhopadhyay, Rupak, Sharma, Maitreyee, Shih, Norrapat, Majumder, Munmi

2016

Overview

In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Affinity chromatography / Alkylation / Amino Acid Sequence / Amino acids / Animals / Anticoagulants / Anticoagulants - pharmacology / Biology and Life Sciences / Biotechnology / Blood coagulation / Blood Coagulation - drug effects / Blood coagulation factors / Blood Coagulation Tests / Blood platelets / Catalysis / Catalytic activity / Chromatography / Coagulation factors / Cytotoxicity / Daboia / Daboia - physiology / Daboia russelii russelii / Edman degradation / Emission spectroscopy / Enzymes / Factor X - antagonists & inhibitors / Factor X - metabolism / Factor Xa - chemistry / Factor Xa - metabolism / Fluorescence / Gel filtration / Genetic aspects / Health aspects / High performance liquid chromatography / Hydrogen ions / Liquid chromatography / Mass spectrometry / Mass spectroscopy / Medicine and Health Sciences / Molecular biology / Molecular docking / Molecular Docking Simulation / Molecular Sequence Data / Naja nigricollis / pH effects / Phospholipase / Phospholipase A2 / Phospholipases / Phospholipases A2 - chemistry / Phospholipases A2 - isolation & purification / Phospholipases A2 - pharmacology / Phospholipids / Properties / Protein structure / Protein Structure, Secondary / Proteins / Prothrombinase / Research and Analysis Methods / Researchers / Russell's viper / Secondary structure / Sequence Homology, Amino Acid / Serine / Snake venoms / Snakes / Spectroscopy / Structure-Activity Relationship / Venom / Viper Venoms - enzymology / Vipera / Vipera ammodytes ammodytes