Structural and functional characterization of a P-III metalloproteinase, leucurolysin-B, from Bothrops leucurus venom.
Leucurolysin-B (leuc-B) is an hemorrhagic metalloproteinase found in the venom of Bothrops leucurus (white-tailed-jararaca) snake. By means of liquid chromatography consisting of gel filtration on Sephracryl S-200, S-300 and ion-exchange on DEAE Sepharose, leuc-B was purified to homogeneity. The proteinase has an apparent molecular mass of 55kDa as revealed by the reduced SDS-PAGE, and represents approximately 1.2% of the total protein in B. leucurus venom. The partial amino acid sequence of leuc-B was determined by automated Edman sequencing of peptides derived from digests of the S-reduced and alkylated protein with trypsin. Leuc-B exhibits the characteristic motif of metalloproteinases, HEXXHXXGXXH and a methionine-containing turn of similar conformation ("Met-turn"), which forms a hydrophobic basis for the zinc ions and the three histidine residues involved as ligands. Leuc-B has been characterized as a P-III metalloproteinase and possesses a multidomain structure including a metalloproteinase, a disintegrin-like (ECD sequence instead of the typical RGD motif) and a cysteine-rich C-terminal domain. Leuc-B contains three potential sites of N-glycosylation. The enzyme only cleaves the Ala14-Leu15 peptide bond of the oxidized insulin B-chain and preferentially hydrolyzes the Aalpha-chain of fibrinogen and the alpha-chain of fibrin. Its proteolytic activity was completely inhibited by metal chelating agents but not by other typical proteinase inhibitors. In addition, its enzymatic activity was stimulated by the divalent cations Ca2+ and Mg2+ but inhibited by Zn2+ and Cu2+. The catalytic activity of leuc-B on extracellular matrix proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites (MHD=30ng in rabbit), with alterations in platelet function. In summary, here we report the isolation and the structure-function relationship of a P-III snake venom metalloproteinase.
Sanchez
Eladio F
EF
Research and Development Center, Ezequiel Dias Foundation, 30510-010 Belo Horizonte, Brazil.
Gabriel
Lucilene M
LM
Gontijo
Sileia
S
Gremski
Luiza H
LH
Veiga
Silvio S
SS
Evangelista
Karla S
KS
Eble
Johannes A
JA
Richardson
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops / Q000378:metabolism*; D003435:Crotalid Venoms / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D045726:Metalloproteases / Q000737:chemistry* / Q000145:classification / Q000648:ultrastructure*; D008969:Molecular Sequence Data
D003435:Crotalid Venoms; D045726:Metalloproteases