Characterization of an extracellular serine protease ofLeishmania (Leishmania) amazonensis

Abstract

A serine protease was purified 942-fold from culture supernatant of L. amazonensis promastigotes using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose and continuous elution electrophoresis by Prep Cell, yielding a total recovery of 61%. The molecular mass of the active enzyme estimated by SDS-PAGE under conditions of reduction was 56 kDa and 115 kDa under conditions of non-reduction, suggesting that the protease is a dimeric protein. Additionally, it was found to be a non-glycosylated enzyme, with a pI of 5.0. The optimal pH and temperature of the enzyme were 7.5 and 28 degrees C respectively, using alpha-N-rho-tosyl-L-arginine-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 61% of the enzyme activity was preserved after 1 h of pre-treatment at 42 degrees C. Haemoglobin, bovine serum albumin (BSA), ovalbumin, fibrinogen, collagen, gelatin and peptide substrates containing arginine in an ester bond and amide substrates containing hydrophobic residues at the P1 site were hydrolysed by this extracellular protease. The insulin beta-chain was also hydrolysed by the enzyme and many peptidic bonds were susceptible to the protease action, and 4 of them (L11-V12, E3-A14, L15-Y16 and Y16-L17) were identified. Inhibition studies suggested that the enzyme belongs to the serine protease class inhibited by calcium and manganese and activated by zinc. These findings show that this enzyme of L. amazonensis is a novel serine protease, which differs from all known flagellate proteases characterized.

Epoxy-α-Lapachone Has In Vitro and In Vivo Anti-Leishmania (Leishmania) amazonensis Effects and Inhibits Serine Proteinase Activity in This Parasite

“…Other serine proteinases of various molecular sizes (i.e., 115 kDa [40], 68 kDa [41,42], and 56 kDa [43]) were previously identified in L. (L.) amazonensis and may also be affected by the compound. Additionally, a serine proteinase named OPB has been described for other Leishmania species and has been found to play roles in many essential events for the parasites in their mammalian hosts (19,44).…”

Effects of serine protease inhibitors on viability and morphology of Leishmania (Leishmania) amazonensis promastigotes

“…Enzymes employed in this study (LSPI, LSPII, and LSPIII) were purified in accordance with the protocols previously described (Silva-Lopez and Giovanni De Simone 2004a, b;Silva-Lopez et al 2005). The effect of ShPI-I (10 −5 , 10 −6 , and 10 −7 M) on the LSPI, LSPII, and LSPIII enzymatic activities was determined using L-TAME (N-ρ-tosyl-Larginine methyl ester) as substrate.…”

“…Inhibitor choice was based on capacity to impede Leishmania serine proteases (Silva-Lopez and Giovanni De Simone 2004a, b;Silva-Lopez et al 2005). Therefore, we employed three classical low-molecularweight serine protease inhibitor, which reacts with the active site of protease: benzamidine (Bza), which reacts with Asp residue of the serine protease's active sites and is able to competitively inhibit, with high specificity, trypsinlike and chymotrypsin-like serine proteases (Sturzebecher et al 1992), N-tosyl-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK).…”

“…TPCK and TLCK are irreversible inhibitors that have Phe and Lys residues, respectively, in chloromethyl ketone linkage and react with His of the active site of the active enzyme (Powers et al 2002). We have previously reported that Bza completely inhibited the LSPI activity (SilvaLopez and Giovanni De Simone 2004a) and 80% LSPIII activity (Silva-Lopez et al 2005) but did not affect the LSPII (Silva-Lopez and Giovanni De Simone 2004b), while TPCK inhibited all enzymes in different degrees, TLCK had a moderate influence on the LSP II and LSP III activity but did not affect the detergent-soluble Leishmania serine protease (Silva-Lopez and Giovanni De Simone 2004a, b;Silva-Lopez et al 2005). Furthermore, we also analyzed the effect of a high molecular weight inhibitor from sea anemone Stichodactyla helianthus (ShPI-I), which is a Kunitz-type inhibitor of 6,110.6 Da that exhibits a broad specificity for serine, cysteine, and aspartic proteases (Delfin et al 1996).…”

Proteases de Leishmania: novos alvos para o desenvolvimento racional de fármacos

“…[64][65][66] As enzimas foram purificadas de três extratos distintos: um intracelular hidrossolúvel, um intracelular solúvel em detergente e um extrato extracelular, e todas apresentam características cinéti-cas e bioquímicas distintas entre si, sugerindo que por conta destas diferenças, estas proteases possam desempenhar funções distintas na Leishmania. Estas serino-proteases são expressas tanto em promastigotas, quanto em amastigotas e são encontradas na bolsa flagelar, em vesículas da via endocítica/exocítica, nos megassomas, na superfície celular e na membrana de organelas.…”