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Leishmania (L.) Amazonensis Peptidase Activities Inside the Living Cells and in Their Lysates

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Leishmania (L.) Amazonensis Peptidase Activities Inside the Living Cells and in Their Lysates Molecular & Biochemical Parasitology 184 (2012) 82–89 Contents lists available at SciVerse ScienceDirect Molecular & Biochemical Parasitology Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates a a b c a Elide E. Caroselli , Diego M. Assis , Clara L. Barbiéri , Wagner A.S. Júdice , Maria A. Juliano , d a,∗ Marcos L. Gazarini , Luiz Juliano a Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil b Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil c Centro Interdisciplinar de Investigac¸ ão Bioquímica, Universidade de Mogi das Cruzes, Av. Dr. Cândido Xavier de Almeida Souza 200, 08780-911 Mogi das Cruzes, Brazil d Department of Biosciences, Universidade Federal de São Paulo, Santos, Brazil a r t i c l e i n f o a b s t r a c t Article history: In this study we investigated the peptidase activity in Leishmania (L.) amazonensis live amastigote by con- Received 24 November 2011 focal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fluorophore Received in revised form 13 March 2012 inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and ser- Accepted 27 April 2012 ine peptidases. It was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA, Available online 6 May 2012 Z-RR-MCA) or inhibitors (E64, TLCK) containing positively charged groups. The peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA Keywords: substrates and inhibitors in the pH range 4.5–9.0. The effects of temperature and different salts were also Cysteine protease Peptidase included in this study. The hydrolytic activities of supernatants on Z-FR-MCA clearly indicate the pres- ence of different cysteine peptidases that adapted to work in different environment conditions. Intact Oligopeptidase B Fluorescent peptides Leishmania cells incorporated Z-RR-MCA, the hydrolysis of which was inhibited only by TLCK indicating the presence of at least one serine peptidase. The pH profile of Z-RR-MCA hydrolysis by amastigotes and promastigotes lysate supernatants, and the hydrolysis time course of the FRET peptide Abz-AGRRRAQ- EDDnp at R A bond, followed by removal of the two C-termini R to yield Abz-AGR-OH that is a unique characteristic of oligopeptidase B, indicate its presence in the parasite. © 2012 Elsevier B.V. Open access under the Elsevier OA license. 1. Introduction sequence [13] http://merops.sanger.ac.uk/. The best characterized peptidases in the genus Leishmania are the cysteine peptidases Peptidases are critical for the survival and pathogenicity of par- (CPs) designated as CPA, CPB and CPC [14] that belong to clan asites, playing roles in the recycling and metabolism of proteins CA. The cysteine peptidases present stage-regulated levels, are [1–6], invasion of host cells and tissues [7,8], parasite nutri- important virulence factors, modulate mammalian host immune tion, modification of host proteins [8,9], parasite immune evasion cells and facilitate tissue host invasion and constitute an attrac- and differentiation [10–12]. The set of peptidases described in tive potential target for chemotherapy [12,14]. Leishmania parasites Leishmania species includes aspartic, cysteine, metallo, serine and have two forms in their life cycle: the promastigote, a flagellate threonine peptidases, and represent around 1.8% of the genome extracellular organism living in the gut of phlebotominae insect and an obligate intracellular oval amastigote. Amastigotes are aci- dophiles and adapted to live inside the parasitophorous vacuole of vertebrate host macrophages [7,15]. The activities of cysteine pep- Abbreviations: DTT, 1,4-dithiothreitol; MCA, [7-amino-4-methyl] coumarin; tidases are considerably higher in amastigote than promastigote E64, 1-[l-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane; Abz, ortho- form [12], particularly CPB-like cysteine peptidase activities that aminobenzoic acid; EDDnp[, N-(2,4-dinitrophenyl) ethylenediamine]; TLCK, increase during the differentiation of internalized promastigote tosyl-lysine chloromethyl ketone; FRET, fluorescence resonance energy transfer; into amastigote within macrophages [7]. Other identified cysteine PMSF, phenylmethylsulfonyl fluoride; CPB, cysteine peptidase B; pFF-MCA, here p d ␧ means proline as -stereoisomer, -NH2-caproyl-C(SBzl)-C(SBzl)-MCA, here SBzl peptidases that differ fundamentally from the clan CA enzymes means benzyl bound to sulfydryl group; CPB 2.8 CTE, cloned cysteine peptidase B have been assigned to the clan CD that includes metacaspase, sep- with deleted C-terminal extension. arase and GPI6 [16]. The best studied metallo peptidase in several ∗ Corresponding author at: Department of Biophysics, Universidade Federal de Leishmania species is leishmanolysin (gp63, MSP), a surface zinc São Paulo, 100 Rua Tres de Maio, SP, Brazil. Tel.: +55 11 5576 4450; endopeptidase, that maximizes promastigote invasion and intra- fax: +55 11 5575 9617. E-mail address: [email protected] (L. Juliano). macrophage survival of amastigote form [17,18]. Leishmania sp. has 0166-6851 © 2012 Elsevier B.V. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.molbiopara.2012.04.012 E.E. Caroselli et al. / Molecular & Biochemical Parasitology 184 (2012) 82–89 83 at least two aspartic peptidases, a soluble cathepsin D-simile and streptomycin, and 10% heat inactivated fetal bovine serum (Gibco- one membrane associated presenelin-simile (PS1) [13]. Another BRL). The promastigotes were isolated from stationary growth peptidase called signal peptidase (SPP) has been identified [19,20]. phase (5 to 6-day-old) cultures [33]. Genes that encode serine peptidases in Leishmania include pro- Promastigotes and amastigotes lysates were prepared by cen- lyloligopeptidase (POP), and oligopeptidase B, which are related trifugation of the parasites at 2000 × g for 10 min, separated into 9 ◦ to parasite virulence [21,22]. Oligopeptidase B pertains to prolyl small flasks containing 1 × 10 cells/ml and frozen at −20 C. The oligopeptidase family (clan SC, family S9) but is highly specific for pellets were suspended in 100 ␮l of NaCl (0.9%) containing 0.1% basic amino acids [23,24]. Triton X 100 and kept on ice, sonicated (Ultrasonic Sonicator XL The aim of this study was to investigate the peptidase activ- 2020, Misonic Inc., NY, USA) for 3 cycles during 20 s with 5 s inter- ity in live Leishmania (L.) amazonensis amastigotes by confocal vals between the cycles. The solution was diluted to 200 ␮l and microscopy. Z-FR-MCA and Z-RR-MCA were used as substrates, centrifuged at 6000 × g for 10 min. The supernatant was separated whose hydrolysis inside the cells releases the MCA fluorophore, from the pellet and used in the peptidase activity assays. resulting in intense fluorescence that enabled quantization of the peptidase activities. Z-FR-MCA is a peptide substrate that is effi- 2.2. Detection of intracellular proteolysis activity with confocal ciently hydrolyzed by serine and cysteine peptidases [25] while microscopy Z-RR-MCA is mainly hydrolyzed by serine peptidases that prefer 9 or even require a pair of basic residues as reported for oligopepti- L. (L.) amazonensis amastigotas (1 × 10 cells/ml) were sus- dase B [24,26], dengue and yellow fever virus NS2B-NS3 proteases pended in buffer containing 75 mM TRIS, 25 mM glycine, 25 mM [27,28]. The peptidase activities in the supernatants of amastig- MES and 25 mM acetic acid, with 10 mM CaCl2, 116 mM NaCl, otes and promastigotes lysates were also evaluated with the same 0.8 mM MgSO4, 0.8 mM KCl and 55 mM glucose, pH 7.0, at room peptidyl-MCA substrates in the presence and the absence of the temperature and placed in a glass bottom dish for microscopy (Mat- inhibitors in the pH range 4.5–9.0. The effects of temperature and Tek Corp., USA) pre-treated for 1 h with poly-lysine solution for cell different salts on the peptidase activities of amastigotes and pro- adhesion. Dynamic imaging was performed with a LSM 510 laser- mastigotes lysates were also investigated in order to explore if the scanning microscope (Carl Zeiss) using LSM 510 software, version × enzymes are differentially adapted to environmental conditions. 2.5. The Axiovert 100 M microscope is equipped with a 63 water The significant hydrolysis of Z-RR-MCA inside the parasites immersion objective. Parasites were plated onto microscopy cover l and in the supernatant of amastigote and promastigote lysates slips (MatTek Corp.) pretreated for 1 h with -polylysine solution mainly at alkaline pH suggested the expression of an oligopep- and excited at 351 nm (Abz or MCA). Emitted light was collected tidase B, which was previously cloned [29]. These results led us through a band pass filter at 387–470 nm (Abz or MCA). Trans- to assay as substrate the fluorescence resonance energy transfer mitted light observations were performed during the experiments (FRET) peptide Abz-AGRRRAQ-EDDnp that characteristically is in order to follow the integrity of the cells. Fluorescence arbitrary hydrolyzed by Trypanosoma cruzi and Trypanosoma brucei oligopep- units were acquired from an average of selected whole parasite tidase B at the R A bond (Abz-AGRRR↓AQ-EDDnp) followed areas. The results are representative of at least three experiments by removal of the two C-terminus R from the Abz-AGRRR- adding 10 ␮M of peptide
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