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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

Primary structure of daboxin P.(A): Amino acid sequence was deciphered by Edman degradation sequencing and ESI-LC MS/MS. The peptide sequences obtained from N-terminal sequencing and chemical cleavage by BNPS-skatole and hydroxylamine hydrochloride are shown with two headed solid arrows whereas peptide sequences obtained after ESI-LC MS/MS of the tryptic digested fragments are indicated with two headed doted arrows. (B): Multiple sequence alignment of daboxin P with the PLA2 enzymes from different subspecies of Daboia russelii (24638087:D. r. russelii, 408407675:D. r. siamensis, 31615955:D. r. pulchella, 49259309:D. r. russelii, 31615954:D r.pulchella, 109157490:D. r. pulchella, 48425253:D. r. pulchella, 298351762:D. r. russelii, 81174981:D. r. russelii). The conserved cys residues are highlighted in grey and the amino acid substitutions in daboxin P are underlined. * indicates the His residue at the active site. The predicted anticoagulant region is highlighted with a solid black line.
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pone.0153770.g003: Primary structure of daboxin P.(A): Amino acid sequence was deciphered by Edman degradation sequencing and ESI-LC MS/MS. The peptide sequences obtained from N-terminal sequencing and chemical cleavage by BNPS-skatole and hydroxylamine hydrochloride are shown with two headed solid arrows whereas peptide sequences obtained after ESI-LC MS/MS of the tryptic digested fragments are indicated with two headed doted arrows. (B): Multiple sequence alignment of daboxin P with the PLA2 enzymes from different subspecies of Daboia russelii (24638087:D. r. russelii, 408407675:D. r. siamensis, 31615955:D. r. pulchella, 49259309:D. r. russelii, 31615954:D r.pulchella, 109157490:D. r. pulchella, 48425253:D. r. pulchella, 298351762:D. r. russelii, 81174981:D. r. russelii). The conserved cys residues are highlighted in grey and the amino acid substitutions in daboxin P are underlined. * indicates the His residue at the active site. The predicted anticoagulant region is highlighted with a solid black line.

Mentions: The first 30 residues were determined by N-terminal sequencing (Fig 3A). The cleavage of pyridylethylated daboxin P with BNPS-skatole yielded two peptide fragments of molecular mass 11.7 kDa and 3.6 kDa (data not shown) while cleavage with hydroxylamine hydrochloride resulted into two peptide fragments of molecular mass 6.6 kDa and 8.6 kDa (data not shown). Sequencing of these peptides by Automated Edman degradation revealed the rest of the amino acid residues (Fig 3A). On the other hand from tandem mass spectrometry, eleven peptide fragments of daboxin P were obtained (Table A in S1 File). These peptide fragments obtained by chemical and enzymatic cleavage were assembled and overlapped to decipher the complete sequence of daboxin P (Fig 3A). The homology of the protein sequence was analyzed by multiple sequence alignment with PLA2 enzymes using online blastp algorithm as represented in Fig 3B.


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)

Primary structure of daboxin P.(A): Amino acid sequence was deciphered by Edman degradation sequencing and ESI-LC MS/MS. The peptide sequences obtained from N-terminal sequencing and chemical cleavage by BNPS-skatole and hydroxylamine hydrochloride are shown with two headed solid arrows whereas peptide sequences obtained after ESI-LC MS/MS of the tryptic digested fragments are indicated with two headed doted arrows. (B): Multiple sequence alignment of daboxin P with the PLA2 enzymes from different subspecies of Daboia russelii (24638087:D. r. russelii, 408407675:D. r. siamensis, 31615955:D. r. pulchella, 49259309:D. r. russelii, 31615954:D r.pulchella, 109157490:D. r. pulchella, 48425253:D. r. pulchella, 298351762:D. r. russelii, 81174981:D. r. russelii). The conserved cys residues are highlighted in grey and the amino acid substitutions in daboxin P are underlined. * indicates the His residue at the active site. The predicted anticoagulant region is highlighted with a solid black line.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153770.g003: Primary structure of daboxin P.(A): Amino acid sequence was deciphered by Edman degradation sequencing and ESI-LC MS/MS. The peptide sequences obtained from N-terminal sequencing and chemical cleavage by BNPS-skatole and hydroxylamine hydrochloride are shown with two headed solid arrows whereas peptide sequences obtained after ESI-LC MS/MS of the tryptic digested fragments are indicated with two headed doted arrows. (B): Multiple sequence alignment of daboxin P with the PLA2 enzymes from different subspecies of Daboia russelii (24638087:D. r. russelii, 408407675:D. r. siamensis, 31615955:D. r. pulchella, 49259309:D. r. russelii, 31615954:D r.pulchella, 109157490:D. r. pulchella, 48425253:D. r. pulchella, 298351762:D. r. russelii, 81174981:D. r. russelii). The conserved cys residues are highlighted in grey and the amino acid substitutions in daboxin P are underlined. * indicates the His residue at the active site. The predicted anticoagulant region is highlighted with a solid black line.
Mentions: The first 30 residues were determined by N-terminal sequencing (Fig 3A). The cleavage of pyridylethylated daboxin P with BNPS-skatole yielded two peptide fragments of molecular mass 11.7 kDa and 3.6 kDa (data not shown) while cleavage with hydroxylamine hydrochloride resulted into two peptide fragments of molecular mass 6.6 kDa and 8.6 kDa (data not shown). Sequencing of these peptides by Automated Edman degradation revealed the rest of the amino acid residues (Fig 3A). On the other hand from tandem mass spectrometry, eleven peptide fragments of daboxin P were obtained (Table A in S1 File). These peptide fragments obtained by chemical and enzymatic cleavage were assembled and overlapped to decipher the complete sequence of daboxin P (Fig 3A). The homology of the protein sequence was analyzed by multiple sequence alignment with PLA2 enzymes using online blastp algorithm as represented in Fig 3B.

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