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CCSV-MPase, a Novel Procoagulant Metalloproteinase from Cerastes cerastes Venom: Purification, Biochemical Characterization and Protein Identification

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Abstract

A procoagulant metalloproteinase called CCSV-MPase was purified from C. cerastes venom by successive chromatographic methods starting with gel-filtration through Sephadex G-75; ion-exchange DEAE-Cellulose A-50; affinity chromatography on Benzamidine Sepharose 6B and RP-HPLC on a C8 column. CCSV-MPase has been isolated to an extent of about tenfolds and its molecular mass was evaluated at 70 kDa by SDS–PAGE. CCSV-MPase hydrolyzes casein and fibrinogene as natural substrates. Its proteolytic activity was inhibited by EDTA and 1.10-phenanthroline, a chelators of bivalent cation metals and Zn2+ respectively. CCSV-MPase is therefore a Zn2+-metalloproteinase with fibrinogenolytic but not hemorrhagic activity. It greatly decreased levels of plasmatic fibrinogen when administered to rats for 24 h. This fibrinogenase hydrolyzes the Bβ chain of human fibrinogen in vitro releasing fibrinopeptide B only. LC MS/MS analysis of tryptic fragments of CCSV-MPase demonstrated that it showed some sequence similarities with four other venom metalloproteinases. CCSV-MPase could be considered as a potential therapeutic agent as it is a non-hemorrhagic enzyme and could be useful in thrombotic diseases because of its defibrinogenation of blood.

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Abbreviations

ACN:

Acetonitrile

BAEE:

Benzoyl-l-arginine ethyl ester hydrochloride

CCSV-MPase:

Cerastes cerastes snake venom metalloproteinase

CID:

Collision ion dissociation

DEAE:

Di ethyl amino ethylene

EDTA:

Ethylene diamine-tetra-acetic acid

HPLC:

High performance liquid chromatography

PMSF:

Phenyl methyl sulfonyl fluoride

IAM:

2-Iodoacetamide

MHD:

Minimal hemorrhage diameter

TCEP:

Tris (2-carboxyethyl)phosphine hydrochloride

TFA:

Tri fluorophosphate acid

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Authors and Affiliations

  1. Laboratoire de Biologie Cellulaire et Moléculaire, Faculté des Sciences Biologiques, Université des Sciences et de la Technologie Houari Boumediene, BP 32, El-Alia Bab Ezzouar, 16111, Algiers, Algeria

    Fatah Chérifi & Fatima Laraba-Djebari

  2. Laboratoire de Recherche et de Développement sur les Venins, Institut Pasteur d’Algérie, Algiers, Algeria

    Fatah Chérifi & Fatima Laraba-Djebari

  3. Plate-Forme de Protéomique, CNRS URA 2185, Institut Pasteur, 75724, Paris Cedex15, France

    Jean-Claude Rousselle & Abdelkader Namane

Authors
  1. Fatah Chérifi
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  2. Jean-Claude Rousselle
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  3. Abdelkader Namane
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  4. Fatima Laraba-Djebari
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Correspondence to Fatima Laraba-Djebari.

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Chérifi, F., Rousselle, JC., Namane, A. et al. CCSV-MPase, a Novel Procoagulant Metalloproteinase from Cerastes cerastes Venom: Purification, Biochemical Characterization and Protein Identification. Protein J 29, 466–474 (2010). https://doi.org/10.1007/s10930-010-9273-1

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