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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

Cytotoxic effect of crude Daboia r. russelii venom and daboxin P.Microscopic images were photographed at 10X magnification under Inverted microscope (Axio Vert A1., Zeiss) after treatment with venom samples for 24 h (A): HEK-293 cells treated with 0.9% NaCl were considered as negative control (B): HEK-293 cells treated with crude Daboia r. russelii venom (5 μg/ml) (C): HEK-293 cells treated with daboxin P (5 μg/ml) (D): MCF-7 cells treated with 0.9% NaCl were considered as negative control (E): MCF-7 cells treated with crude Daboia r. russelii venom (5 μg/ml) (F): MCF-7 cells treated with daboxin P (5 μg/ml). Percentage cell viability (G): HEK-293; (H): MCF-7 after treatment with crude venom and daboxin P using MTT based colorimetric assay. Percentage cell viability was calculated by considering the cells without venom treatment as 100% viable. The results are mean ± SD of three independent experiments.
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pone.0153770.g006: Cytotoxic effect of crude Daboia r. russelii venom and daboxin P.Microscopic images were photographed at 10X magnification under Inverted microscope (Axio Vert A1., Zeiss) after treatment with venom samples for 24 h (A): HEK-293 cells treated with 0.9% NaCl were considered as negative control (B): HEK-293 cells treated with crude Daboia r. russelii venom (5 μg/ml) (C): HEK-293 cells treated with daboxin P (5 μg/ml) (D): MCF-7 cells treated with 0.9% NaCl were considered as negative control (E): MCF-7 cells treated with crude Daboia r. russelii venom (5 μg/ml) (F): MCF-7 cells treated with daboxin P (5 μg/ml). Percentage cell viability (G): HEK-293; (H): MCF-7 after treatment with crude venom and daboxin P using MTT based colorimetric assay. Percentage cell viability was calculated by considering the cells without venom treatment as 100% viable. The results are mean ± SD of three independent experiments.

Mentions: The crude venom of Daboia r. russelii has shown prominent cytotoxic effect on HEK-293 and MCF-7 cells in a dose dependent manner (Fig 6). At a concentration of 2.8 and 3 μg/ml of crude venom, only 13.09% of HEK-293and 14.97% of MCF-7 cells respectively (Fig 6B, 6E, 6G and 6H) remained viable while the same concentrations of daboxin P did not show any cytotoxic effect, suggesting that the cytotoxic effect of the crude venom is not due to this protein (Fig 6C and 6F–6H).


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)

Cytotoxic effect of crude Daboia r. russelii venom and daboxin P.Microscopic images were photographed at 10X magnification under Inverted microscope (Axio Vert A1., Zeiss) after treatment with venom samples for 24 h (A): HEK-293 cells treated with 0.9% NaCl were considered as negative control (B): HEK-293 cells treated with crude Daboia r. russelii venom (5 μg/ml) (C): HEK-293 cells treated with daboxin P (5 μg/ml) (D): MCF-7 cells treated with 0.9% NaCl were considered as negative control (E): MCF-7 cells treated with crude Daboia r. russelii venom (5 μg/ml) (F): MCF-7 cells treated with daboxin P (5 μg/ml). Percentage cell viability (G): HEK-293; (H): MCF-7 after treatment with crude venom and daboxin P using MTT based colorimetric assay. Percentage cell viability was calculated by considering the cells without venom treatment as 100% viable. The results are mean ± SD of three independent experiments.
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Related In: Results  -  Collection

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

pone.0153770.g006: Cytotoxic effect of crude Daboia r. russelii venom and daboxin P.Microscopic images were photographed at 10X magnification under Inverted microscope (Axio Vert A1., Zeiss) after treatment with venom samples for 24 h (A): HEK-293 cells treated with 0.9% NaCl were considered as negative control (B): HEK-293 cells treated with crude Daboia r. russelii venom (5 μg/ml) (C): HEK-293 cells treated with daboxin P (5 μg/ml) (D): MCF-7 cells treated with 0.9% NaCl were considered as negative control (E): MCF-7 cells treated with crude Daboia r. russelii venom (5 μg/ml) (F): MCF-7 cells treated with daboxin P (5 μg/ml). Percentage cell viability (G): HEK-293; (H): MCF-7 after treatment with crude venom and daboxin P using MTT based colorimetric assay. Percentage cell viability was calculated by considering the cells without venom treatment as 100% viable. The results are mean ± SD of three independent experiments.
Mentions: The crude venom of Daboia r. russelii has shown prominent cytotoxic effect on HEK-293 and MCF-7 cells in a dose dependent manner (Fig 6). At a concentration of 2.8 and 3 μg/ml of crude venom, only 13.09% of HEK-293and 14.97% of MCF-7 cells respectively (Fig 6B, 6E, 6G and 6H) remained viable while the same concentrations of daboxin P did not show any cytotoxic effect, suggesting that the cytotoxic effect of the crude venom is not due to this protein (Fig 6C and 6F–6H).

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