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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

Percentage residual amidolytic activity of various serine proteases and complexes pre-incubated with daboxin P.(A): Residual activity of FXIIa, FXIa, FXa, FIXa and FVIIa; (B): Activity of extrinsic tenase complex (ETC) (C): intrinsic tenase complex (ITC), in the presence or absence of phospholipid and alkylated daboxin P (indicated by *). (D): Residual activity of prothrombinase complex. Daboxin P was either pre-incubated with FXa followed by addition of FVa (pre-complex) or after reconstitution of FXa-FVa complex (post-complex). The rate of hydrolysis of respective chromogenic substrates for all the assays was measured at 405 nm using Multiskan Go spectrophotometer. Activity of the serine protease/complex without daboxin P was considered as 100%. The results are mean ± SD of three independent experiments.
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pone.0153770.g009: Percentage residual amidolytic activity of various serine proteases and complexes pre-incubated with daboxin P.(A): Residual activity of FXIIa, FXIa, FXa, FIXa and FVIIa; (B): Activity of extrinsic tenase complex (ETC) (C): intrinsic tenase complex (ITC), in the presence or absence of phospholipid and alkylated daboxin P (indicated by *). (D): Residual activity of prothrombinase complex. Daboxin P was either pre-incubated with FXa followed by addition of FVa (pre-complex) or after reconstitution of FXa-FVa complex (post-complex). The rate of hydrolysis of respective chromogenic substrates for all the assays was measured at 405 nm using Multiskan Go spectrophotometer. Activity of the serine protease/complex without daboxin P was considered as 100%. The results are mean ± SD of three independent experiments.

Mentions: Screening for inhibitory effect of daboxin P on the amidolytic activity of various serine proteases involved in the extrinsic (FVIIa), intrinsic (FXIIa, FXIa, FIXa) and common (FXa) pathway revealed that it did not inhibit any of these serine proteases when assayed using respective synthetic chromogenic substrates (Fig 9A). However, it exerted inhibitory effect on both the extrinsic (ETC) (IC50 = 0.43 nM) and intrinsic (ITC) (IC50 = 39.20 nM) tenase complexes causing hindrance in the activation of FX to FXa (Fig 10). The residual activity of ETC and ITC was observed to be 9.05% ± 1.9 and 2.8% ± 1.6 respectively upon treatment with 3 μM of daboxin P (Fig 9B & 9C). Further, when the tenase complexes were reconstituted without phospholipid, the residual activity of ETC and ITC was found to be 25% ± 2.7 and 20.05% ± 2.3 respectively. Moreover, when the tenase complexes were treated with alkylated daboxin P (3 μM), only 23% ± 1.8 (ETC) and 19.03% ± 1.8 (ITC) of residual activity remained which is comparable to the residual activity of both the complexes reconstituted without phospholipids (Fig 9B & 9C). Interestingly, the thrombin formation by the prothrombinase complex was unaffected when daboxin P was added after the formation of the FXa-FVa complex. However, only 11.28% of residual activity remained when it was pre-incubated with FXa followed by addition of FVa for the formation of prothrombinase complex (Fig 9D).


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)

Percentage residual amidolytic activity of various serine proteases and complexes pre-incubated with daboxin P.(A): Residual activity of FXIIa, FXIa, FXa, FIXa and FVIIa; (B): Activity of extrinsic tenase complex (ETC) (C): intrinsic tenase complex (ITC), in the presence or absence of phospholipid and alkylated daboxin P (indicated by *). (D): Residual activity of prothrombinase complex. Daboxin P was either pre-incubated with FXa followed by addition of FVa (pre-complex) or after reconstitution of FXa-FVa complex (post-complex). The rate of hydrolysis of respective chromogenic substrates for all the assays was measured at 405 nm using Multiskan Go spectrophotometer. Activity of the serine protease/complex without daboxin P was considered as 100%. The results are mean ± SD of three independent experiments.
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Related In: Results  -  Collection

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pone.0153770.g009: Percentage residual amidolytic activity of various serine proteases and complexes pre-incubated with daboxin P.(A): Residual activity of FXIIa, FXIa, FXa, FIXa and FVIIa; (B): Activity of extrinsic tenase complex (ETC) (C): intrinsic tenase complex (ITC), in the presence or absence of phospholipid and alkylated daboxin P (indicated by *). (D): Residual activity of prothrombinase complex. Daboxin P was either pre-incubated with FXa followed by addition of FVa (pre-complex) or after reconstitution of FXa-FVa complex (post-complex). The rate of hydrolysis of respective chromogenic substrates for all the assays was measured at 405 nm using Multiskan Go spectrophotometer. Activity of the serine protease/complex without daboxin P was considered as 100%. The results are mean ± SD of three independent experiments.
Mentions: Screening for inhibitory effect of daboxin P on the amidolytic activity of various serine proteases involved in the extrinsic (FVIIa), intrinsic (FXIIa, FXIa, FIXa) and common (FXa) pathway revealed that it did not inhibit any of these serine proteases when assayed using respective synthetic chromogenic substrates (Fig 9A). However, it exerted inhibitory effect on both the extrinsic (ETC) (IC50 = 0.43 nM) and intrinsic (ITC) (IC50 = 39.20 nM) tenase complexes causing hindrance in the activation of FX to FXa (Fig 10). The residual activity of ETC and ITC was observed to be 9.05% ± 1.9 and 2.8% ± 1.6 respectively upon treatment with 3 μM of daboxin P (Fig 9B & 9C). Further, when the tenase complexes were reconstituted without phospholipid, the residual activity of ETC and ITC was found to be 25% ± 2.7 and 20.05% ± 2.3 respectively. Moreover, when the tenase complexes were treated with alkylated daboxin P (3 μM), only 23% ± 1.8 (ETC) and 19.03% ± 1.8 (ITC) of residual activity remained which is comparable to the residual activity of both the complexes reconstituted without phospholipids (Fig 9B & 9C). Interestingly, the thrombin formation by the prothrombinase complex was unaffected when daboxin P was added after the formation of the FXa-FVa complex. However, only 11.28% of residual activity remained when it was pre-incubated with FXa followed by addition of FVa for the formation of prothrombinase complex (Fig 9D).

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