Molecular Cloning and Expression of a Functional Snake Venom Serine Proteinase, with Platelet Aggregating Activity, from the Cerastes cerastes Viper

Autor: Cassian Bon, Anne Wisner, Hafedh Dekhil, Habib Karoui, Naziha Marrakchi, Mohamed El Ayeb
Přispěvatelé: Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Institut Pasteur de Tunis, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Venins, Institut Pasteur [Paris], This work was supported by Secretariat d’Etat a la Recherche Scientifique et Technique PNM Grant P95/SP27GH and by Comite Mixte Franco-Tunisien pour la Cooperation Universitaire Grant 96/F09023., We gratefully acknowledge Professor Koussay Dellagi for his constant interest in this work and for his helpful encouragement. We thank Dr. Ben Lasfar Zakaria, from the Serpentarium of Institut Pasteur de Tunis, for providing C. cerastes snakes and venoms., Institut Pasteur [Paris] (IP)
Rok vydání: 2003
Předmět:
Models
Molecular

Platelet Aggregation
MESH: Sequence Homology
Amino Acid

[SDV]Life Sciences [q-bio]
MESH: Viperidae
MESH: Rabbits
Venom
MESH: Amino Acid Sequence
MESH: Base Sequence
Biochemistry
MESH: Fibrinolysis
MESH: Recombinant Proteins
MESH: Thrombin
Serine
Viperidae
MESH: Animals
MESH: Serine Endopeptidases
Cloning
Molecular

Peptide sequence
MESH: Platelet Aggregation
0303 health sciences
MESH: Kinetics
biology
Fibrinolysis
Serine Endopeptidases
030302 biochemistry & molecular biology
Thrombin
Recombinant Proteins
Snake venom
Rabbits
MESH: Viper Venoms
MESH: Models
Molecular

medicine.drug
DNA
Complementary

Molecular Sequence Data
Cerastocytin
Viper Venoms
03 medical and health sciences
biology.animal
medicine
Animals
MESH: Cloning
Molecular

Amino Acid Sequence
030304 developmental biology
MESH: Molecular Sequence Data
Base Sequence
Sequence Homology
Amino Acid

Fibrinogen
MESH: DNA
Complementary

Cerastes cerastes
biology.organism_classification
Molecular biology
Kinetics
MESH: Fibrinogen
Zdroj: Biochemistry
Biochemistry, American Chemical Society, 2003, 42 (36), pp.10609-10618. ⟨10.1021/bi034790b⟩
Biochemistry, 2003, 42 (36), pp.10609-10618. ⟨10.1021/bi034790b⟩
ISSN: 1520-4995
0006-2960
DOI: 10.1021/bi034790b
Popis: International audience; The venoms of Viperidae snakes contain numerous serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen and platelets. Among them, a platelet proaggregant protein, cerastocytin, has been isolated from the venom of the Tunisian viper Cerastes cerastes. Using the RACE-PCR technique, we isolated and identified the complete nucleotide sequence of a cDNA serine proteinase precursor. The recombinant protein was designated rCC-PPP (for C. cerastes platelet proaggregant protein), since its deduced amino acid sequence is more than 96% identical to the partial polypeptide sequences that have been determined for natural cerastocytin. The structure of the rCC-PPP cDNA is similar to that of snake venom serine proteinases. The expression of rCC-PPP in Escherichia coli system allowed, for the first time, the preparation and purification of an active protein from snake venom with platelet proaggregant and fibrinogenolytic activities. Purified rCC-PPP efficiently activates blood platelets at nanomolar (8 nM) concentrations, as do natural cerastocytin (5 nM) and thrombin (1 nM). It is able to clot purified fibrinogen and to hydrolyze alpha-chains. Thus, rCC-PPP could be therefore considered a cerastocytin isoform. By comparison with other snake venom serine proteinases, a Gly replaces the conserved Cys(42). This implies that rCC-PPP lacks the conserved Cys(42)-Cys(58) disulfide bridge. A structural analysis performed by molecular modeling indicated that the segment of residues Tyr(67)-Arg(80) of rCC-PPP corresponds to anion-binding exosite 1 of thrombin that is involved in its capacity to induce platelet aggregation. Furthermore, the surface of the rCC-PPP molecule is characterized by a hydrophobic pocket, comprising the 90 loop (Phe(90)-Val(99)), Tyr(172), and Trp(215) residues, which might be involved in the fibrinogen clotting activity of rCC-PPP.
Databáze: OpenAIRE