Autolysis of patagonfibrase, a metalloproteinase isolated from Philodryas patagoniensis venom (Serpentes: Colubridae)

MARÍA ELISA PEICHOTO; OFELIA ACOSTA; MARCELO LARAMI SANTORO

San Pablo

Encuentro; XI Annual Scientific Meeting of Butantan Institute; 2009

Instituto Butantan

Introduction: Patagonfibrase is a 57.5-kDa hemorrhagic metalloproteinase isolated from the venom of the South American colubrid snake Philodryas patagoniensis. Metalloproteinases isolated from Bothrops venoms with molecular mass similar to patagonfibrase are known to undergo autolysis under different conditions, giving rise mainly to one fragment lacking the metalloproteinase domain, that is, containing only disintegrin-like/cysteine-rich domains. Objectives: Taking into consideration that patagonfibrase is the only metalloproteinase isolated to date in native form from a colubrid snake venom that inhibits platelet aggregation, and that this enzyme exhibits various biological activities similar to those exhibited by Bothrops venom metalloproteinases with similar molecular mass, in this study we aimed to evaluate the autoproteolytic activity of patagonfibrase. Methods: Patagonfibrase was incubated at 37ºC at a concentration of 0.5 mg/mL in 50 mM Tris-HCl buffer, pH 7.4, for different intervals of time (0, 5, 15, 30, 60 and 120 min; 18 h), in the presence and  absence of 1 mM CaCl2. Immediately after incubation, autolysis was interrupted by addition of SDS-PAGE sample buffer containing β-mercaptoethanol. Autolysis was visualized by SDS-PAGE (12% running gel) and silver staining. Residual proteolytic activity of patagonfibrase (30 µg/mL), was also evaluated using azocasein as substrate. Results: Patagonfibrase was able to undergo autolysis at 37ºC. The analysis of electrophoretic migration pattern showed a progressive fainting of the proteinase band and an increase in the staining density of bands below 45 kDa over incubation up to 18 h. At this period,of incubation, the enzyme was almost totally autolysed, and thus it failed to hydrolyze azocasein. However, in the presence of 1 mM CaCl2, patagonfibrase was only partially autolysed, even after 18 h of incubation, giving rise mainly to one fragment of 52.2 kDa. In addition, preincubation of patagonfibrase at 37ºC for 18 h in the presence of 1 mM CaCl2 caused an increase in about 50% of its azocaseinolytic activity. Discussion: Taken together, these results imply that the calcium ions are important for structural stabilization of patagonfibrase, as demonstrated for other snake venom metalloproteinases found in different ophidian taxa. Moreover, this work demonstrates for the first time the autoproteolytic processing of a colubrid snake venom metalloproteinase, which will contribute to a better understanding of the structural and mechanistic bases of this type of proteins that are widely distributed among snake venoms.