A Serine Protease from a Detergent-Soluble
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A Serine Protease from a Detergent-soluble Extract of Leishmania (Leishmania) amazonensis Raquel Elisa da Silva Lopeza,* and Salvatore Giovanni De Simonea,b a Laborato´ rio de Bioquı´mica de Proteı´nas e Peptı´deos, Departamento de Bioquı´mica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21045-900, Rio de Janeiro, RJ, Brasil. Fax: +55215903495. E-mail: [email protected] b Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Nitero´ i, RJ, Brasil * Author for correspondence and reprint requests Z. Naturforsch. 59c, 590Ð598 (2004); January 22/March 18, 2004 Proteases mediate important crucial functions in parasitic diseases, and their characteriza- tion contributes to the understanding of host-parasite interaction. A serine protease was purified about 43-fold with a total recovery of 60% from a detergent-soluble extract of pro- mastigotes of Leishmania amazonensis. The purification procedures included aprotinin-agar- ose affinity chromatography and gel filtration high performance liquid chromatography. The molecular mass of active enzyme was 110 kDa by native gel filtration HPLC and by SDS- PAGE gelatin under non-reducing conditions. Under conditions of reduction using SDS- PAGE gelatin analyses the activity of enzyme was observed in two proteins of 60 and 45 kDa, suggesting that the enzyme may be considered as a dimer. The Leishmania protease was not glycosylated, and its isoelectric point (pI) was around 4.8. The maximal protease activity was α ρ at pH 7.0 and 28 ∞C, using -N- -tosyl-L-arginyl-methyl ester (l-TAME) as substrate. Assays of thermal stability indicated that this enzyme was totally denatured after pre-treatment at 42 ∞C for 12 min and preserved only 20% of its activity after pre-treatment at 37 ∞C for 24 h, in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin and gela- tin were hydrolyzed by Leishmania protease. Inhibition studies indicated that the enzyme belonged to a serine protease class because of a significant impediment by serine protease inhibitors such as benzamidine, aprotinin, and antipain. The activity of the present serine protease is negatively modulated by calcium and zinc and positively modulated by manganese ions. This is the first study that reports the purification of a protease from a detergent-soluble extract of Leishmania species. Key words: Leishmania (Leishmania) amazonensis, Serine Protease, Characterization Introduction ics and subtropics, and the increment of Protozoan parasites of the genus Leishmania Leishmania transmission and dissemination has are associated with a broad spectrum of diseases been mainly attributed to blood transfusion and ranging from self-healing cutaneous lesions to le- human immunodeficiency virus (HIV) co-infec- thal visceral consequences (Alexander and Rus- tion (Desjeux, 1999). Leishmania are dimorphic sell, 1992). Leishmaniasis are endemic in the trop- obligate intracellular parasites: Flagellated pro- mastigotes replicate in the gut of the sandfly vec- tor, and their transmission to humans or other ver- α Abbreviations: ACN, acetonitrile; BAME, N- -benzoyl- tebrate hosts occurs when the vector feeds on L-arginyl-methyl ester; BHI, brain heart infusion; Bz, benzoyl; CHAPS, 3-[(3-cholamidopropyl) dimethylam- blood. These promastigotes are internalized by monio]-1-propanesulfonate; DTT, dithiothreitol; EDTA, mononuclear phagocytic system cells and undergo ethylenediaminetetraacetic acid; E-64, L-trans-epoxysuc- transformation into the nonmotile amastigote cinyleucylamido-(4-guanidino) butane; HPLC, high per- form that maintain the infection, leading to the formance liquid chromatography; PBS, phosphate buffer saline; pI, isoelectric point; PMSF, phenylmethylsulfonyl destruction of host tissues and invasion of new fluoride; ρ-NA, paranitroanilide; SBTI, soybean trypsin cells (Colmenares et al., 2001). Consequently the inhibitor; SDS-PAGE, polyacrylamide gel electrophore- expression of many molecules is responsible for α ρ sis containing sodium dodecyl sulfate; l-TAME, -N- - mechanisms triggered by parasites in order to tosyl-L-arginyl-methyl ester; TLCK, N-tosyl-lysine chlo- romethyl ketone; TFA, trifluoroacetic acid; TPCK, N- guarantee survival within the host. Among these tosyl-L-phenylalanine chloromethyl ketone. molecules proteases have received paramount at- 0939Ð5075/2004/0700Ð0590 $ 06.00 ” 2004 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D R. E. da Silva Lopez and S. Giovanni De Simone · A Serine Protease from Leishmania 591 tention due to their crucial roles in the parasite life has great activity against human connective tissue cycle and disease pathogenesis (Rosenthal, 1999; proteins, enabling parasite penetration of the host Sadij and McKerrow, 2002). They are involved in tissues (Ghendler et al., 1996). Furthermore, a chy- the host-parasite interaction, including digestion motrypsin-like protease of S. mansoni cercariae is of exogenous proteins for nutritive purposes (Ro- essential for host skin penetration and can be senthal, 1999), invasion of host cells and tissues found both in cercariae secretions and membranes (Roggwiller et al., 1996) and modification of host (Salter et al., 2000). Herein we describe the purifi- proteins (Caler et al., 1998) which are very impor- cation procedure used and give some characteris- tant for parasite survival. Leishmania proteases tics of serine protease from a detergent-soluble ex- have been studied for almost three decades, and tract of Leishmania amazonensis promastigotes, some functions have been proposed for these en- an enzyme which definitely differs from the other zymes (Coombs, 1982). The importance of Leish- known serine peptidases of this parasite (Ribeiro mania proteases has been confirmed by the find- de Andrade et al., 1998). ings that specific protease inhibitors killed Leishmania parasites and reduced the evolution of Materials and Methods leishmaniatic lesions (Sadij and McKerrow, 2002). Parasites Serine proteases (EC 3.4.21) are among the most extensively studied enzymes and participate Promastigote forms of L. amazonensis (IOC in numerous physiological phenomena such as 575; IFLA/BR/67/PH8) were maintained at 28 ∞C blood clotting and complement activation (Raw- in brain heart infusion (BHI) medium (Difco, ling and Barret, 1994). Serine proteases of patho- Detroit, USA) supplemented with 10% (v/v) genic parasites play crucial roles in host-parasite heat-inactivated fetal-calf serum. For large-scale interaction, and their characterization is crucial to cultivation (2 l), the cultures were maintained at elucidate their functions in parasite physiology room temperature (25 ∞C) in Roller bottles using and parasite-host interaction. The majority of par- a Cel-Gro Rotator (Lab-Line Model, Thomas Sci- asite serine proteases has been isolated from entific, New Jersey, USA). Cell growth was esti- water-soluble extracts and is involved in mecha- mated by counting the parasites in a Neubauer nisms of host invasion (Roggwiller et al., 1996; chamber. Caler et al., 1998). The most notorious protozoan serine proteases involved in host cell invasion are Purification of Leishmania serine protease associated with Plasmodium falciparum and Try- The parasites (4.8 ¥ 1010) were harvested by cen- panossoma cruzi. Malarial proteases digest cyto- trifugation (3,000 ¥ g for 15 min at 4 ∞C) at the log plasmatic membrane proteins of red blood cells phase (4th day of cultivation) and washed three (glycophorin and band 3 proteins) thereby afford- times in cold phosphate buffer saline (PBS) ing invasion and infection by the parasite, whereas (pH 7.2) by centrifugation (3,000 ¥ g for 15 min at specific proteases inhibitors prevent such (Rogg- 4 ∞C). Parasite lysates were prepared in PBS by willer et al., 1996). Mammalian cell invasion by T. seven cycles of freezing and thawing (Ð 80 ∞C/ cruzi is mediated by a serine oligopeptidase B 37 ∞C). Finally, the cell lysate was centrifuged which is responsible for the generation of a pep- (100,000 ¥ g for 60 min at 4 ∞C), the supernatant tide hormone-like factor that triggers Ca2+ signal- was discarded and the pellet was ressuspended ing, facilitating parasite infection (Caler et al., using 10 ml of extraction buffer containing 0.5% 1998). Deletion of the gene encoding oligopepti- CHAPS, 0.01 m Tris-HCl, pH 7.5, and centrifuged dase B results in a marked deficiency for host cell (100,000 ¥ g for 60 min at 4 ∞C). The clear super- invasion, as well as reducing the establishment of natant was dialyzed overnight at 4 ∞C against infections in mice (Caler et al., 1998). 0.01 m Tris-HCl, pH 7.5, containing 0.1% CHAPS. Proteases of membrane or membrane-associ- Insoluble material was removed by centrifugation ated proteases require detergents for their extrac- (100,000 ¥ g, 60 min) and the clear supernatant tion, isolation and purification, because of the low loaded on to an aprotinin-agarose affinity column solubility in aqueous systems. Schistosoma man- (2.5 ml gel) previously equilibrated with 0.01 m soni has a membrane-associated serine protease of Tris-HCl, pH 7.5, 0.1% CHAPS containing 5 mm 28 kDa. This enzyme is anchored to the tegu- CaCl2. After exhaustive washing (20 bed volumes) mental membrane of schistosomula by lipids and the active material was eluted with 0.01 m Tris- 592 R. E. da Silva Lopez and S. Giovanni De Simone · A Serine Protease from Leishmania HCl, pH 7.5, containing 1.5 m NaCl. Fractions of Determination of optimal pH, temperature and 1 ml were collected on ice, the absorption at heat stability 280 nm of effluents was monitored to detect the The assays for pH dependency were carried out protein peak and the enzymatic activity of the incubating the enzyme (2 µg) for 30 min at room fractions was assayed using α-N-ρ-tosyl- -arginyl- L temperature with 0.25 mml-TAME in solutions methyl ester (l-TAME) as substrate. The enzyme with different pH values. The buffers used were active fractions were pooled and concentrated in as follows: 100 mm sodium citrate (pH 5.0/6.0) and Microcon (Amicon) concentrators (3-kDa cut-off Tris-HCl (pH 7.0/9.0), all buffers containing 0.1% membrane) at 4 ∞C.