Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

• Published in: Toxicon (Oxford) 2012-07, Vol.60 (1), p.70-82
• Main Authors: Cintra, A.C.O., De Toni, L.G.B., Sartim, M.A., Franco, J.J., Caetano, R.C., Murakami, M.T., Sampaio, S.V.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animal poisons toxicology. Antivenoms; Animals; anion exchange chromatography; binding sites; Biological and medical sciences; Bothrops; Bothrops atrox; Bothrops atrox venom; Bothrops moojeni; collagen; Crotalid Venoms - enzymology; crystal structure; cystine; disulfide bonds; EDTA (chelating agent); ethylene glycol tetraacetic acid; extracellular matrix; fibrin; fibrinogen; Fibrinolysis; Fibrinolytic and thrombolytic activity; fibronectins; gel chromatography; Isoelectric Point; Mass Spectrometry; Medical sciences; Metalloproteases - chemistry; Metalloproteases - metabolism; metalloproteinases; Models, Molecular; Molecular Sequence Data; molecular weight; plasminogen; sequence analysis; Sequence Homology, Amino Acid; Snake venom metalloproteinase; snakes; Substrate specificity; Toxicology; venoms; zinc
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2012.03.018

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Pará) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C164I165M166 motif that is associated with a “Met-turn” structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMPα-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10μg, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both α and β-chains of the fibrinogen molecule, and it can be inhibited by EDTA, EGTA and β-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity. ► Batroxase is a weakly hemorrhagic metalloproteinase. ► Represent 1.2% (w/w) of the crude snake venom, pI 7.5 and molecular mass 22.9kDa. ► Batroxase presented thrombolytic and fibrin(ogen)olytic activity. ► Batroxase was characterized as a PIb class SVMP. ► Clinical use as fibrinolytic agent for the treatment of coagulation disorders.