The effect of post-translational modifications on the hemorrhagic activity of snake venom metalloproteinases.
Metalloproteinases (MPs) are Zn(+)-dependent endoproteolytic enzymes, abundant in crotalid and viperid snake venoms. Most snake venom metalloproteinases (svMPs) are active on extracellular matrix components and this effect is thought to result in bleeding as a consequence of the basement membrane disruption in capillaries. Jararhagin and ACLH are hemorrhagic svMPs from Bothrops jararaca and Agkistrodon contortrix laticinctus venom, respectively. Both enzymes demonstrate proteolytic activity on fibrinogen and fibronectin and jararhagin inhibits collagen-induced platelet aggregation in vitro. This work describes the expression, purification and successful refolding of the recombinant ACLH zymogen (rPRO-ACLH) as well as the catalytic domain of jararhagin (rCDJARA). The heterologous proteins were produced in E. coli, an in vivo expression system that does not make post-translational modifications. The recombinant refolded proteins did not show any hemorrhagic activity in mice skin, as well as the native deglycosylated jararhagin and ACLH. However, they preserved their proteolytic activity on fibrinogen and fibronectin. It seems that the hemorrhagic properties of these hemorrhagins are dependent on post-translational modifications, whereas their proteolytic activity is not dependent on such modifications.
García
L T
LT
Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, Rodovia Washington Luis, Km 235, São Carlos, SP, 13565-905, Brazil.
Parreiras e Silva
L T
LT
Ramos
O H P
OH
Carmona
A K
AK
Bersanetti
P A
PA
Selistre-de-Araujo
H S
HS
D017836:Agkistrodon / Q000502:physiology; D000818:Animals; D017837:Bothrops / Q000502:physiology; D003435:Crotalid Venoms / Q000378:metabolism* / Q000633:toxicity*; D006470:Hemorrhage / Q000139:chemically induced*; D008666:Metalloendopeptidases / Q000378:metabolism / Q000633:toxicity; D045726:Metalloproteases / Q000378:metabolism* / Q000633:toxicity*; D051379:Mice; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism / Q000633:toxicity; D012867:Skin / Q000187:drug effects; D000097146:Bothrops jararaca Venom
D003435:Crotalid Venoms; D008666:Metalloendopeptidases; D045726:Metalloproteases; D011994:Recombinant Proteins; D000097146:Bothrops jararaca Venom; C031442:Agkistrodon venoms