Understanding structural and functional aspects of PII snake venom metalloproteinases: characterization of BlatH1, a hemorrhagic dimeric enzyme from the venom of Bothriechis lateralis.
A new homodimeric PII metalloproteinase, named BlatH1, was purified from the venom of the Central American arboreal viperid snake Bothriechis lateralis by a combination of anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. BlatH1 is a glycoprotein of 84 kDa. The mature protein contains a metalloproteinase domain, with the characteristic zinc-binding motif (HEXXHXXGXXH) followed by the sequence CIM at the Met-turn. In the disintegrin domain, the tripeptide sequence TDN substitutes the characteristic RGD motif found in many disintegrins. BlatH1 hydrolyzed azocasein, gelatin and fibrinogen, and exerts a potent local and systemic hemorrhagic activity in mice. The hemorrhagic activity of BlatH1 is not inhibited by the plasma proteinase inhibitor α2-macroglobulin, although the SVMP is able to cleave this plasma inhibitor, generating a 90 kDa product. BlatH1 inhibits ADP- and collagen-induced human platelet aggregation (IC50 = 0.3 μM and 0.7 μM for ADP and collagen, respectively). This activity is abrogated when the enzyme is preincubated with the metalloproteinase inhibitor Batimastat, implying that it depends on proteolysis. In agreement, a synthetic peptide containing the sequence TDN of the disintegrin domain is unable to inhibit platelet aggregation. BlatH1 is a valuable tool to understand the structural determinants of toxicity in PII SVMPs.
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
Erika
E
Camacho
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
Eva
E
Villalobos
Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
Libia
L
Sanz
Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
Alicia
A
Pérez
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
Teresa
T
Escalante
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
Bruno
B
Lomonte
Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain; Departamento de Biotecnología, Universidad Politécnica de Valencia, Spain.
Juan J
JJ
Calvete
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
José María
JM
Gutiérrez
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address: alexandra.rucavado@ucr.ac.cr.
Alexandra
A
Rucavado
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000201:enzymology* / Q000633:toxicity; D006470:Hemorrhage / Q000139:chemically induced; D006801:Humans; D007928:Lethal Dose 50; D008168:Lung / Q000187:drug effects / Q000473:pathology; D045726:Metalloproteases / Q000737:chemistry* / Q000502:physiology / Q000633:toxicity; D051379:Mice; D008969:Molecular Sequence Data; D010974:Platelet Aggregation / Q000187:drug effects; D020836:Protein Structure, Quaternary; D059748:Proteolysis; D030162:Reptilian Proteins / Q000737:chemistry* / Q000502:physiology / Q000633:toxicity; D020539:Sequence Analysis, Protein; D017819:Viperidae; D000511:alpha-Macroglobulins / Q000737:chemistry
D003435:Crotalid Venoms; D030162:Reptilian Proteins; D000511:alpha-Macroglobulins; D045726:Metalloproteases
Bothriechis lateralis; Hemorrhagic activity; PII SVMP; Platelet aggregation; Snake venom metalloproteinase; α2-Macroglobulin