Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom.
A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.
Bernardes
Carolina P
CP
Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, 38400-902 Uberlândia-MG, Brazil.
Santos-Filho
Norival A
NA
Costa
Tássia R
TR
Gomes
Mário S R
MS
Torres
Fernanda S
FS
Costa
Júnia
J
Borges
Márcia H
MH
Richardson
Michael
M
dos Santos
Daniel M
DM
de Castro Pimenta
Adriano M
AM
Homsi-Brandeburgo
Maria I
MI
Soares
Andreimar M
AM
de Oliveira
Fábio
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017837:Bothrops / Q000378:metabolism*; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000201:enzymology*; D005340:Fibrinogen / Q000037:antagonists & inhibitors*; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data
D003435:Crotalid Venoms; D005340:Fibrinogen; D045726:Metalloproteases