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

Sharma M, Iyer JK, Shih N, Majumder M, Mattaparthi VS, Mukhopadhyay R, Doley R - PLoS ONE (2016)

Bottom Line: It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur-784028, Assam, India.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

3D ribbon model of the docked complex of FXa and daboxin P.The interface surface residues involved in the interaction were predicted by PDBsum. The Ca+2 binding loop (Trp30, Gly31, Gly32); helix C (Asp48, Tyr51, Gly52); anticoagulant region (Asn58) and C-terminal region (Phe113) of daboxin P interact with the heavy chain of FXa (Thr132, Arg165, Lys169, Asn166, Leu170, Tyr225 and Arg125) are represented in scaled ball and stick. Inset: Diagram illustrating the interaction of the seven residues of chain B (heavy chain of FXa) with eight residues of chain C (daboxin P) as predicted by PDBsum server. Orange line denotes non-bonded contacts and blue line denote hydrogen bond.
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pone.0153770.g012: 3D ribbon model of the docked complex of FXa and daboxin P.The interface surface residues involved in the interaction were predicted by PDBsum. The Ca+2 binding loop (Trp30, Gly31, Gly32); helix C (Asp48, Tyr51, Gly52); anticoagulant region (Asn58) and C-terminal region (Phe113) of daboxin P interact with the heavy chain of FXa (Thr132, Arg165, Lys169, Asn166, Leu170, Tyr225 and Arg125) are represented in scaled ball and stick. Inset: Diagram illustrating the interaction of the seven residues of chain B (heavy chain of FXa) with eight residues of chain C (daboxin P) as predicted by PDBsum server. Orange line denotes non-bonded contacts and blue line denote hydrogen bond.

Mentions: The best protein-protein docked model of daboxin P and FXa chosen in terms of minimum free energy and maximum surface contact area, was obtained from PatchDock web server (Fig 12). Daboxin P interacts with the heavy chain (B-Chain) of FXa with a geometric shape complementarity score of 12234. Geometric scoring refers to good molecular shape complementarity between the docked chains due to optimal fit with wide interface areas and lesser steric clashes [49]. The approximate interface area of the complex was found to be 1837 Å2 with atomic contact energy (ACE) of 472.62 kcal/mol.


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

Sharma M, Iyer JK, Shih N, Majumder M, Mattaparthi VS, Mukhopadhyay R, Doley R - PLoS ONE (2016)

3D ribbon model of the docked complex of FXa and daboxin P.The interface surface residues involved in the interaction were predicted by PDBsum. The Ca+2 binding loop (Trp30, Gly31, Gly32); helix C (Asp48, Tyr51, Gly52); anticoagulant region (Asn58) and C-terminal region (Phe113) of daboxin P interact with the heavy chain of FXa (Thr132, Arg165, Lys169, Asn166, Leu170, Tyr225 and Arg125) are represented in scaled ball and stick. Inset: Diagram illustrating the interaction of the seven residues of chain B (heavy chain of FXa) with eight residues of chain C (daboxin P) as predicted by PDBsum server. Orange line denotes non-bonded contacts and blue line denote hydrogen bond.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835082&req=5

pone.0153770.g012: 3D ribbon model of the docked complex of FXa and daboxin P.The interface surface residues involved in the interaction were predicted by PDBsum. The Ca+2 binding loop (Trp30, Gly31, Gly32); helix C (Asp48, Tyr51, Gly52); anticoagulant region (Asn58) and C-terminal region (Phe113) of daboxin P interact with the heavy chain of FXa (Thr132, Arg165, Lys169, Asn166, Leu170, Tyr225 and Arg125) are represented in scaled ball and stick. Inset: Diagram illustrating the interaction of the seven residues of chain B (heavy chain of FXa) with eight residues of chain C (daboxin P) as predicted by PDBsum server. Orange line denotes non-bonded contacts and blue line denote hydrogen bond.
Mentions: The best protein-protein docked model of daboxin P and FXa chosen in terms of minimum free energy and maximum surface contact area, was obtained from PatchDock web server (Fig 12). Daboxin P interacts with the heavy chain (B-Chain) of FXa with a geometric shape complementarity score of 12234. Geometric scoring refers to good molecular shape complementarity between the docked chains due to optimal fit with wide interface areas and lesser steric clashes [49]. The approximate interface area of the complex was found to be 1837 Å2 with atomic contact energy (ACE) of 472.62 kcal/mol.

Bottom Line: It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur-784028, Assam, India.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus