Subproteomic Analysis of Soluble Proteins of the Microsomal Fraction from Two Leishmania Species

Comparative Biochemistry and Physiology, Part D 1 (2006) 300–308 www.elsevier.com/locate/cbpd

Subproteomic analysis of soluble proteins of the microsomal fraction from two Leishmania species

Arthur H.C. de Oliveira a, Jerônimo C. Ruiz b, Angela K. Cruz b, ⁎ Lewis J. Greene b,c, José César Rosa b,c, Richard J. Ward a,

a Departamento de Química, FFCLRP-USP, Universidade de São Paulo, Ribeirão Preto-SP, Brazil b Departamento de Biologia Celular e Molecular e Celular e Bioagentes Patogênicos, FMRP-USP, Ribeirão Preto-SP, Brazil c Centro de Química de Proteínas, FMRP-USP, Ribeirão Preto-SP, Brazil Received 17 November 2005; received in revised form 26 May 2006; accepted 27 May 2006 Available online 3 June 2006

Abstract

Parasites of the genus Leishmania are the causative agents of a range of clinical manifestations collectively known as Leishmaniasis, a disease that affects 12 million people worldwide. With the aim of identifying potential secreted protein targets for further characterization, we have applied two-dimensional gel electrophoresis and mass spectrometry methods to study the soluble protein content of the microsomal fraction from two Leishmania species, Leishmania L. major and L. L. amazonensis. MALDI-TOF peptide mass fingerprint analysis of 33 protein spots from L. L. amazonensis and 41 protein spots from L. L. major identified 14 proteins from each sample could be unambiguously assigned. These proteins include the nucleotide diphosphate kinase (NDKb), a calpain-like protease, a tryparedoxin peroxidase (TXNPx) and a small GTP-binding Rab1-protein, all of which have a potential functional involvement with secretion pathways and/or environmental responses of the parasite. These results complement ongoing genomic studies in Leishmania, and are relevant to further understanding of host/parasite interactions. © 2006 Elsevier Inc. All rights reserved.

Keywords: 2D-PAGE; Leishmania; Microsome; MALDI-TOF; Subproteome; Trypanosomatidae

1. Introduction form and non-motile amastigote form. After replication in the midgut of sand-fly vector, non-infective promastigote forms dif- The protozoan parasite Leishmania sp. is the causative agent of ferentiate into infective metacylic promastigotes, which are Leishmaniasis which affects approximately 12 million people inoculated into dermis of the mammalian host during the blood- worldwide and places a further 350 million people at risk, the meal of infected sandflies (Sacks, 1989). They are then phago- majority of whom live in underdeveloped countries. With 1.5– cytosed by macrophages where they transform into the non- 2 million cases reported each year (Herwaldt, 1999), Leishman- motile, intracellular replicative amastigote form, which is adapted iasis is considered by the World Health Organization to be the to survive and replicate within the hostile environment of the second most important disease caused by a protozoan parasite. phagolysosome. After the amastigote release by macrophage Clinical forms of the disease range from mild cutaneous or de- lysis, the amastigote forms can be phagocytosed by adjacent forming mucocutaneous manifestations to the fatal visceral Leis- macrophages (Alexander and Russel, 1992). hmaniasis, also known as kalazar. Leishmania parasites have a The classification of twenty species of the trypanosomatid dimorphic life-cycle characterized by a free-living promastigote Leishmania in 2 subgenera, Leishmania (Leishmania)andL. (Viannia), is based on the mode of promastigote development within the digestive tract of the invertebrate sand fly host (Lainson ⁎ Corresponding author. Department of Chemistry Faculdade de Filosofia, and Shaw, 1987). Some Leishmania species are traditionally Ciências e Letras de Ribeirão Preto, Universidade de São Paulo Avenida Bandeirantes, 3900, CEP 14040-901, Ribeirão Preto-SP, Brazil. Tel.: +55 16 associated with a given clinical form, such as L. L. major which 36024384; fax: +55 16 36024838. produces localized cutaneous lesions (LCL), or L. L. donovani E-mail address: [email protected] (R.J. Ward). that is associated with kalazar (VL), L. L. amazonensis with

1744-117X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2006.05.003 A.H.C. de Oliveira et al. / Comparative Biochemistry and Physiology, Part D 1 (2006) 300–308 301 diffuse cutaneous Leishmaniasis (DCL) and L. V. braziliensis et al., 2005) and cercarial secretome (Knudsen et al., 2005), the with mucocutaneous manifestations (MCL). It should be noted Toxoplasma gondii rhoptry proteome (Bradley et al., 2005)andthe however, that the association of some species with atypical forms Heliobacter pylori subproteome analysis of Backert et al. (2005). of the disease has been reported (Barral et al., 1986, 1991; Here we describe the subcellular fractionation of the pro- Hernandez et al., 1995; Ponce et al., 1991). The biology of the Old mastigote forms and analysis of the soluble content of total World L. L. major and the New World L. L. amazonensis differ at microsomal extracts of L. L. major and L. L. amazonensis.We various levels, ranging from the distribution of the parasites in the demonstrate that these two Leishmania species present differences host parasitophorous vacuoles (Antoine et al., 1998) to the degree in their microsomal contents, and the identification of soluble of infectivity in BALB/c mice (Courret et al., 2003). proteins presenting functions related to the parasite environmental Comparative genomic studies of Leishmania species with other response indicates that a subproteomic approach is a useful tool for trypanosomatids such as Trypanosoma brucei and T. cruzi, identifying individual protein targets that may play a role in together with analyses of gene expression patterns and proteomic defining host/parasite interactions. studies in various developmental stages holds much promise for understanding the biology, virulence and the variable clinical 2. Materials and methods manifestations on infection by these parasites. The transcription of protein-encoding genes in trypanosomatids results in policystronic 2.1. Preparation of soluble extract of the microsomes RNAs which are processed by a trans-splicing mechanism to monocistronic mRNAs that are subsequently translated (LeBowitz Promastigote forms of L. L. major (strain LV-39, clone 5, Rho/ et al., 1993). Gene regulation therefore occurs by controlling the SU/59/P, (Marchand et al., 1987)) and L. L. amazonensis (MPRO/ stability and translation of specific mRNAs (Graham, 1995; Van- BR/1972/M1841-LV-79) were grown in M199 culture supplemen- hamme and Pays, 1995). Indeed, recent results from microarray ted with 10% fetal bovine serum. All manipulations of both analyses of mRNA from different developmental stages of Leish- Leishmania cell types were conducted within a laboratory confor- mania have confirmed that only a relatively modest number of ming to the standards defined by international biosecurity level II. mRNAs show significant alterations in abundance (Akopyants The microsomal enriched fractionwasseparatedaspreviously et al., 2004; Almeida et al., 2004). Furthermore, the proteins described (Ulloa et al., 1995) with some modifications. Briefly, one undergo extensive post-translation modification (Mottram et al., liter of culture containing 1.5×107 parasites per mL was centri- 1993; Saas et al., 2000; Soto et al., 2000), which emphasizes the fuged (1000 ×g/10 min/4 °C) and the parasite pellet was washed in important role of post-transcriptional regulation in these parasites. phosphate buffered saline pH 7.4 (PBS)andresuspendedin10mL A proteomic based approach is therefore particularly interest- of lysis buffer (10 mM Tris-Cl pH 7.5, 5 mM phenylmethylsul- ing for the investigation of trypanosomatid parasites, and pro- phonyl fluoride, 176 μg/mL benzamidine, 200 μg/mL phenanthro- teomic studies in Leishmania have focused on analyzing the line, 3 mM EDTA and 3 mM EGTA) to give a cell density of differences in protein content and post-translation modification in 1.5×109 parasites/mL. The parasites were lysed by five freeze/thaw the different life cycle stages of the parasite. Indeed, changes in the cycles, and after lysis the crude extract was centrifuged (1000 ×g/ protein profile as observed in 2D-gels between the promastigotes 10 min/4 °C) in order to sediment the nuclear fraction, unlysed and axenic amastigote forms of these parasites have been des- parasites and mitochondria. The post-nuclear supernatant (approxi- cribed (El Fakhry et al., 2002; Bente et al., 2003; Nugent et al., mately 10 mL, at 1.2 mg of protein/mL) was centrifuged (100,000 2004). Proteomic analysis with a methotrexate resistant strain of ×g/60 min/4 °C), and the sediment containing the microsomal L. L. major (Drummelsmith et al., 2003) has directly confirmed fraction was retained from which the soluble proteins were obtained that over-expression of pteridine reductase by an episomal gene by lysis of follows. After resuspension of the microsomal fraction in amplification mechanism leads to drug tolerance (Bello et al., lysis buffer, the suspension was treated with 5 ultrasonic pulses 1994; Beverley, 1991). Furthermore, characterization of differen- (10 W/60 s) intercalated by 5-minute intervals on ice. The sonicated tial protein expression between wild-type and methotrexate re- microsomal fraction was centrifuged (100,000 × g/60 min/4 °C) in sistant L. L. major promastigotes identified a series of protein order to sediment the microsomal membranes, and the supernatant targets implicated in drug resistance (Drummelsmith et al., 2004). (10 mL) containing soluble fraction of the microsome extract was The majority of proteomic studies in trypanosomatids have retained. The total protein content of the soluble extract of the been performed with general sample preparation protocols that microsomes fraction was estimated by the Bradford method identify abundant soluble proteins. The enrichment of other pro- (Bradford, 1976) which showed typical yields were 0.45–0.5 teins such as hydrophobic membrane associated and low copy- (L. L. major) and 0.40–0.45 (L. L. amazonensis)mgofproteinper number proteins require different sample preparation methods, liter of culture. The protein soluble extract of microsomes of L. L. which may include the pre-fractionation of cells (Gorg et al., 2000; amazonensis was analysed by unidimensional eletrophoresis (SDS- Wu et al., 2003) and the proteomic analysis of cell fractions, PAGE 12%; Laemmli, 1970) and stained with colloidal Coomassie commonly called the subproteomic approach, has been employed Blue, as previously described (Neuhoff et al., 1988). to investigate secreted virulence factors in pathogenic organisms, such as the Plasmodium falciparum rhoptry-enriched proteome 2.2. Two-dimensional gel electrophoresis (Sam-Yellowe et al., 2004) and the vesicle enriched-fraction (Gelhaus et al., 2005). Other recent subproteome studies include The protein content of the soluble extracts of the microsomal the Schistosoma mansoni tegumentary proteome (van Balkom fraction was precipitated by adding trichloroacetic acid to a final 302 A.H.C. de Oliveira et al. / Comparative Biochemistry and Physiology, Part D 1 (2006) 300–308 concentration of 17%. After 2 h at 4 °C this mixture was MALDI-TOF-MS was calibrated with a mixture of synthetic centrifuged (10,000 ×g/40 min/4 °C), and the pellet was washed peptides, Angiotensin II, angiotensin I and a fragment of ACTH 3 times in chilled acetone. Samples of precipitated proteins (1.5 mg (Sigma, Chemical Co. St. Louis, MI). of L. L. major and 1.0 mg of L. L. amazonensis) were solubilized MALDI-TOF: Spots were cut from the 2D-gel and washed three in rehydratation buffer containing 8 M urea, 2% (w/v) CHAPS (3- times in 100 mM ammonium bicarbonate containing 50% ace- [(3-cholamidoporpyl)-dimethylammonio]-propane-sulphonate), tonitrile, followed by a wash in pure acetonitrile and subsequent 65 mM DTT, 10 mM Tris-Cl, and 0.5%(v/v) ampholyte buffer lyophilization. Gel plugs were covered with 60 μLoftrypsin stock (IPG buffer, Amersham) and 0.001% bromophenol blue solution (4 ng/μL,Promega)andincubatedfor18hat37°C,and (Sigma). The sample was centrifuged (10,000 ×g/5 min/4 °C) and the trypsinized gels were incubated for a further 2 h with 40 μLofa applied to 13-cm immobilized pH 3–10 gradient strips (Amer- 50% acetonitrile/2.5% trichloroacetic acid mixture in order to sham Biosciences), and after rehydration (12 h/20 °C), isoelectric extract the peptides. After extraction, the supernatant was lyophi- focusing of the proteins was achieved by IPGPhor System (Amer- lized and the pellet was resuspended in 10 μLofthesamesolvent sham Biosciences; 50 mA per strip, 150 V for 2 h, 500 V for 1 h, mixture. One μL of the sample was mixed with 1 μLofα-cyano-4- 8000 V up to about 22,000 V h). The focused strips were equi- hydroxycinamic acid, and after air-drying on an inert sample sup- librated for 15 min in a buffer containing 50 mM Tris–HCl, 30% port, the sample was submitted to matrix-assisted laser desorption/ (v/v) glycerol, 2%(w/v) SDS, 10 mg/mL DTT and 25 mg/mL of ionization-time-of-flight (MALDI-TOF) analysis. Peptide mass iodoacetamide, respectively, and a trace of Bromophenol Blue. analysis was performed in duplicate for each sample on a MALDI- Subsequently, the strips were transferred to a 12% polyacrylamide TOF instrument (Ettan MALDI-TOF Pro, Amersham Bioscien- gel, and the proteins were separated under denaturating conditions ces). The peptide mass fingerprints obtained from MALDI-TOF by electrophoresis (25 mA, 300 V) until the tracking dye ran off were compared with a protein database generated by automatic the gel. After electrophoresis, the gels were stained with colloidal annotation of L. L. major Friedlin sequence information available Coomassie Blue as previously described (Neuhoff et al., 1988). through the Wellcome Trust Sanger Institute website (www.sanger. The gel images were analysed with Image Master 2D software ac.uk/Projects/L_major/), using the program Protein Probe (Bio- (Amersham Biosciences) using standard molecular weight (SDS- Lynx-MassLynx 3.3, Micromass, Manchester, UK), in which the 6H, Sigma, USA) and a linear isoeletric point gradient. search parameters permitted a mass tolerance of ±0.8 Da.

2.3. Mass spectrometry 3. Results and discussion

ESI-MS/MS: Protein separated by SDS-PAGE electrophoresis Leishmania and other tryanosomatids are exposed to multiple was submitted to in situ gel band trypsin digestion with 0.5 μgof environments since their life cycles have at least one development modified trypsin (Promega, USA). The tryptic peptides were de- stage in both the insect vector and the vertebrate host. The envi- salted in micro Tip filled with POROS R2 (Perseptive Biosystem, ronmental changes experienced by trypanosomatids during host Inc. Foster City, CA) and eluted in 70% methanol, 0.2% Formic crossing causes parasite differentiation, an event which is con- acid for MS analysis. The MS analysis of tryptic peptides was comitant with biochemical modifications (Zilberstein and Shapira, carried out using an electrospray triple-quadrupole mass spectro- 1994). The Leishmania promastigote form from the insect midgut meter Quattro II (Micromass, Manchester, UK) by direct infusion has the first contact with the vertebrate host bloodstream and (300 nL/min) under the following conditions: capillary voltage subsequently with the host cell target, which are macrophages in maintained at 2.8 kV, cone voltage 40 V, cone temperature set to the case of Leishmania. Adaption of the cell surface composition 100 °C. The parameters for MS1 scanning and daughter-ion scan- of the trypanosomatid is a crucial response to environmental ning were optimized with synthetic peptide for the highest signal- changes, and the secretory pathways of these organisms play a key to-noise ratio and calibrated with PEG. In the daughter-ion scan- role during this process. Biochemical studies, including proteome ning mode, the collision energy was set to 25–35 eV, and argon analyses, can provide new information as to the molecular ma- was used as collision gas at a partial pressure of 3.0×10−3 mTorr. chinery of trypanosomatids, and provide basic insights that may The spectra were collected as an average of 20–50scans(2to5s/ be of use in the development of novel strategies to combat parasite scans) and processed using the MassLynx software v.3.3 (Micro- infections. mass, Manchester, UK). The sequence of tryptic peptides was Subproteomic studies of the microsomal fraction have been deduced from series of b and y ion fragments produced by collision used to identify membrane bound and/or secreted proteins of induced dissociation mass spectrometry (CID-MS/MS). Masses of eukaryotic cells including human prostate cancer cells (Whright et tryptic peptides and fragment ions from CID were used to search al., 2003), murine myeloma cells (Aleteetal.,2005)andhuman the Genebank at NCBI using Protein Prospector MS-Fit and MS- lymphoblasts (Filen et al., 2005). Furthermore, microsomal pro- Tag (http://prospector.ucsf.edu/). Peptide mass fingerprints were teins from pathogenic organisms such as Theileria parva (Ebel also obtained from spots separated from 2D-gel electrophoresis by et al., 1997), T. gondii (Chini et al., 2005)andTrypanosoma cruzi MALDI-TOF-MS (Amersham-Bioscience, Uppsala, Sweden). (Figueiredo et al., 2005) have been characterized with the aim of SamplesweredesaltedwithPOROSR2andmixedin1:1(v/v) identify targets for intervention in the parasite life cycle. Here we ratio with 10 mg/ml of matrix α-cyano-4-hydroxycinamic acid. show that the protein content of the enriched microsomal fractions Spectra were collected as an average of 10–20 scans, and monoiso- of Leishmania can be used to identify secreted proteins in the topic masses were used to search the Genebank protein database. promastigote form of the parasite. A.H.C. de Oliveira et al. / Comparative Biochemistry and Physiology, Part D 1 (2006) 300–308 303

Previous authors have reported the use of sodium carbonate from which a total of 2 mg (L. L. amazonensis)and3mg(L. L. treatment to prepare both the soluble fraction of vesicle extracts in major) of protein could routinely be obtained. However, preci- various Leishmania species (Noronha et al., 1996) and to rupture pitation by trichloroacetic acid and solubilization in the urea buffer microsomes from Plasmodium (Florens et al., 2002). Although for isoelectric focusing resulted in a loss of approximately 50% of this procedure is well established in the literature (Fujiki et al., the protein. Nevertheless, the final yield was approximately 1.0 mg 1982), in the present study the protein yield was not sufficient for (L. L. amazonensis)and1.5mg(L. L. major)ofproteinthatwere visualization of protein spots with colloidal Coomassie Blue either analyzed on 2D-gels and visualized by Coomassie Blue staining. in 1D-SDS-PAGE or 2D-gels of soluble proteins of the micro- The culture volumes necessary to obtain a sufficient quantity of somal extracts from L. L. amazonensis and L. L. major (data not soluble microsomal proteins proved to be the main difficulty in this shown). In contrast, the extraction of soluble proteins from subcellular proteomic study, however these large volumes were microsomal fractions by ultrasonic lysis yielded 0.49 and 0.45 mg essential for the identification of proteins with low abundance. protein/L of culture from L. L. major and L. L. amazonensis, The viability of the extraction protocol was evaluated by 1D- respectively. In this study, 4–6 L of culture were typically prepared SDS-PAGE of the microsomal extract fraction of L. amazonensis

Fig. 1. Unidimesional gel of the soluble protein content of the microsomal fraction from L. amazonensis. (A) The soluble proteins of the microsomal fraction were separated on a 1D-gel (SDS-PAGE 12%) and several of the most abundant protein bands (encolosed by the boxes 1–3) were excised from the gel for subsequently analysis by ESI-MS/ MS (see the Materials and methods section for further details). (B) Amino acid sequence of the Gp63 (gi ∣1100213∣ leishmanolysin of L. amazonensis), in which are highlighted the peptide sequences identified by peptide mass fingerprint of the protein band from box #1 in (A). (C) Mass spectrum of CID-MS/MS of the trypsinic peptide of m/z 679.8[+2H] from the Gp63, showing the amino acid sequence derived from interpretation of the daughter ion scanning (ions y [M+1H]). 304 A.H.C. de Oliveira et al. / Comparative Biochemistry and Physiology, Part D 1 (2006) 300–308

Fig. 2. Bidimensional polyacrylamide gel of the soluble protein content of the microsomal fraction from L. L. amazonensis (A) and L. L. major (B). After the trichloroacetic acid precipitation, the resuspended soluble proteins of microsomal extracts were separated by isoelectric point (IEF pH 3–10) in the horizontal dimension and by molecular weight in the vertical dimension (SDS-PAGE 12%). The gel was stained using colloidal Coomassie Blue and analysed by Image Master 2D Analysis Software (Amersham Biosciences) as described in the Materials and methods section. The spots within the numbered circles were selected for further analysis by MALDI-TOF, and the identified proteins are shown in Table 2A and B. A.H.C. de Oliveira et al. / Comparative Biochemistry and Physiology, Part D 1 (2006) 300–308 305

Gp63 is presented in Fig. 1C. The Gp63 is a 63 kDa-surface- localized metalloprotease involved in the intracellular survival within host macrophages (McGwire and Chang, 1996) and has been described as a 59-kDa secreted form within vesicles that fuse with flagellar pocket membrane (McGwire et al., 2002). The identification of the 59-kDa form of the protein in the 1D-gel of the L. amazonensis microsomal fraction (Fig. 1), at higher concentration is consistent with the presence of secreted addressed vesicles in the extracted microsomal fraction. Furthermore, identification of the nucleoside diphosphate kinase b (NDKb), which is a secreted protein implicated in the pathogenicity of Mycobacterium tuberculosis (Sainietal.,2004)andVibrio cholera (Punj et al., 2000) confirmed that the microsomal fraction was enriched with vesicles that are addressed to the cell membrane and/or for secretion. Fig. 2A presents the 2D-gel of the soluble fraction of the microsomal fraction from L. L. amazonensis, which reveals that the majority of the proteins are detected within a pI range of 4–7and have a molecular weight below 66 kD. Approximately 100 protein spots could be observed on the gel, of which 33 were submitted to Fig. 3. MALDI-TOF analysis of spot #32 from the 2D-gel from the microsomal MALDI-TOF mass spectrometry. Fig. 2B shows the 2D-gel of the fraction of L. l. major. (A) Peptide mass fingerprint of the trypsin digested spot #32 soluble extract of the microsomal from L. L. major where approxi- obtained by MALDI-TOF. (B) Amino acid sequence of the L. major NDKb showing the localization of the peptides detected by the peptide fragment mass mately 130 protein spots could be detected, of which 41 were fingerprint. The highlighted residues in the protein sequence are from the peptides analyzed by MALDI-TOF mass spectrometry. In common with the indicated in the MALDI-TOF spectrum with masses 1329.74, 926.55, 1612.82, L. L. amazonensis extract, the L. L. major proteins have molecular 1708.91, 934.45, 939.48 (in order from the N-terminal). See the Materials and weights of less than 66 kD, however the distribution of the proteins methods section for further details of the mass spectrometry analyses. in the 2D-gel is significantly different from L. L. amazonensis, demonstrating that the overall protein profile of the microsomal (Fig. 1A). Three major bands were excised from the gel, and after extract differs between the two species. Recent comparisons of the trypsin treatment, the digested peptides were submitted to electro- L. L. infantum and L. L. major genomes have revealed a high level spray mass spectrometry (ESI/MS-MS). The peptide mass finger- of sequence conservation between protein-coding genes (see http:// print identified these proteins as the membrane GPI-anchored www.genedb.org/genedb/), therefore the divergent spot patterns proteins Gp63 (Leishmanolysin) and Gp46 (PSA surface antigen) observed in the 2D-gels between L. L. amazonensis and L. L. and cytosolic nucleoside diphosphate kinase (NDKb). The amino- major may suggest that significant differences arise at the level of acid sequence of Gp63 containing the identified peptides is pre- post-translational modification and/or intracellular compartment sented in Fig. 1B, in which are highlighted the peptide sequences targeting of the expressed proteins. generated by the CID/MS analysis. A representative spectrum Fig. 3 shows the spectrum of spot #32 from the L. L. major 2D- from the CID/MS analysis of a peptide (FDLSTVSDAFEK) from gel (see Fig. 2B), which was identified as the nucleoside

Table 1 Identification of proteins by peptide mass fingerprints from MALDI-TOF analysis of the soluble protein content of the vesicle fraction from L. l. amazonensis Spot Protein Genomic kDa theor/ pI theor/ Matched/ Protein coverage no. annotation expt expt masses (%) 1 Putative HSP60 chaperonin precursor LmjF36.2020 60.1/65.2 5.7/5.3 8/12 16 2 Putative HSP60.2 chaperonin LmjF36.2030 59.3/65.1 5.4/5.5 10/22 27 3 Putative HSP60.2 chaperonin LmjF36.2030 59.3/64.8 5.4/5.7 9/17 21 7 Hypothetical protein LmjF30.1520 49.3/54.7 8.1/8.0 3/9 11 10 Eukaryotic translation initiation factor 3 (subunit δ) LmjF36.3880 45.4/43.3 6.1/5.1 5/6 13 12 Putative pyruvate dehydrogenase precursor (e1 component, LmjF25.1710c 37.8/39.8 5.8/5.1 4/5 11 β subunit) 16 25 kDa (1β) Elongation factor (putative) LmjF34.0820 25.6/26.2 5.2/6.2 3/7 17 17 Hypothetical protein conserved (morn repeat protein) LmjF30.3310 40.7/25.8 5.3/5.8 6/10 21 19 20S proteosome α5 subunit LmjF21.1830 26.7/24.3 5.4/5.3 3/4 16 20 20S proteosome α5 subunit LmjF21.1830 26.7/24.2 5.4/5.5 4/8 22 22 Conserved hypothetical protein LmjF32.1640 23.7/23.2 7.1/6.7 2/4 10 25 Small GTP-binding protein Rab1 LmjF27.0760 22/21.4 5.6/6.1 2/3 13 28 Putative eukaryotic initiation factor 5a LmjF25.0730 17.8/18.9 4.8/4.6 2/5 18 30 Putative eukaryotic initiation factor 5a LmjF25.0730 17.8/17.4 4.8/5.4 2/5 18 31 MIF-like protein LmjF33.1750 12.5/15.5 8.2/8.8 2/4 25 33 Nucleoside diphosphate kinase b (NDK-b) LmjF32.2950 16.6/12.3 8.2/7.7 4/8 33 306 A.H.C. de Oliveira et al. / Comparative Biochemistry and Physiology, Part D 1 (2006) 300–308

Table 2 Identification of proteins by peptide mass fingerprints from MALDI-TOF analysis of the soluble protein content of the vesicle fraction from L. l. major Spot Protein Genomic kDa theor/ pI theor/ Matched/ Protein no. annotation expt expt masses coverage (%) 5 Putative pyruvate dehydrogenase precursor (component e1, subunit α) LmjF18.1380 42.8/42.8 8.2/7.7 4/4 13 6 Putative pyruvate dehydrogenase precursor (component e1, subunit β) LmjF25.1710 37.8/40.1 5.8/5.3 5/12 17 8 Proteasome subunit α type I LmjF36.1600 29.6/32.3 5.2/5.2 7/12 28 12 20S proteosome α1 subunit LmjF35.4850 27.2/29.7 7.4/7.0 3/5 15 13 20S proteosome α5 subunit putative LmjF21.1830 26.7/29.3 5.6/5.6 3/6 17 20 Hypothetical protein, conserved, unknown function LmjF27.2200 27.2/24.1 5.6/5.4 3/6 13 23 Cell cycle protein Mob1-2, (putative) LmjF06.0960 26/20.2 8.2/8.7 4/8 18 24 Hypothetical protein (conserved) LmjF34.2580 22.5/19.9 9.7/7.1 5/9 31 25 Hypothetical protein (conserved) LmjF34.2580 22.5/19.8 9.7/8.2 7/12 35 26 Hypothetical protein (conserved) LmjF34.2580 22.5/19.7 9.7/8.1 5/10 30 27 Hypothetical protein (conserved) LmjF34.2580 22.5/19.7 9.7/7.9 4/8 32 29 Cyclophilin a LmjF25.0910 18/17.2 8.1/8.5 3/7 17 32 Nucleoside diphosphate kinase b (NDK-b) LmjF32.2950 16/15.9 8.2/8.1 6/11 45 33 Hypothetical protein (conserved) LmjF19.1400 25.9/15.8 6.7/7.2 3/6 11 34 Nucleoside diphosphate kinase b (NDK-b) LmjF32.2950 16/15.8 8.2/7.5 6/7 45 35 Nucleoside diphosphate kinase b (NDK-b) LmjF32.2950 16/15.5 8.2/9.0 4/7 45 36 Calpain-like protease LmjF20.1310 14.9/14.5 5.3/5.4 2/5 16 37 Hypothetical protein, conserved LmjF13.0450 13.3/13.9 5.2/5.1 5/8 20 38 Hypothetical protein LmjF28.1340 20.7/13.8 5.7/4.9 2/3 12 41 TRYP (TXNPx), tryparedoxin peroxidase LmjF15.1120 21.1/9.9 6.4/8.8 4/8 22 diphosphate kinase (NDKb), and which is representative of peptide in Golgi-vesicles near the plasma membrane (Cappai et al., 1993), mass fingerprint data collected in the MALDI-TOF experiments. and which may be involved in drug resistance in Leishmania The MALDI-TOF peptide mass fingerprint data from all the (Marchini et al., 2003).Thenucleosidediphosphatekinaseb selected spots were compared with the L. L. major sequence (NDKb) was identified in both species and in the case of L. L. databases (Tables 1 and 2). Of the total of 33 and 41 protein spots major was identified in 3 spots (Fig. 1B), demonstrating a post- analyzed, 12 and 14 proteins could be identified from the L. L. translational processing of the protein. A lower molecular mass amazonensis and L. L. major extracts, respectively. Several protein (12 kDa) was detected for NDKb in the 2D-gel from L. L. spots were conserved between the extracts from the two species, amazonensis, which may be related to the previous observation of such as proteins associated with energy metabolism (e.g. piruvate both a16 kDa cytosolic and a 12 kDa truncated membrane dehydrogenase subunits) and the proteins associated with the 20S associated form of the protein that have been described from the subunit of the proteasome. However, as suggested by the pathogenic Pseudomonas aeruginosa (Shankar et al., 1996). This differences in the patterns observed in the 2D-gels, the two extracts enzyme is present in different organisms as membranous, cytosolic were characterized by the presence of different functional classes of and secreted forms and plays a pivotal role in the NTP and dNTP proteins. For example, proteins involved in the cell cycle and cell regulation (Lascu et al., 2000). Furthermore, the NDKb secretion proliferation were observed, such as translation initiation factor 5a, by pathogenic intracellular organisms such as M. tuberculosis 25 kD elongation factor, translation factor 3 (L. L. amazonensis) (Sainietal.,2004) has led to the suggestion that in the presence of and the protein associated with the cell cycle Mob 1–2(L. L. ATP this protein is a cytotoxic factor against macrophages (Chopra major). Furthermore, three proteins implicated in the stress et al., 2003). Thus at least four proteins (NDKb, calpain-like response, the HSP60 (L. L. amazonensis), the cyclophilin-a and protease, tryparedoxin peroxidase and GTP binding Rab-protein) tryparedoxin peroxidase (L. L. major) were also identified. The identified in this study show possible functional involvement with identified protein LmjF20.2310 (L. L. major) presents a high level secretion pathways and/or environmental responses of the parasite. of sequence similarity to the C-terminal domain of the META-2 InthecaseofLeishmania, these proteins could be involved in the protein, a calpain-like paralogue that maps to META1 gene locus communication with the host cell during the invasion process. which is a region that encodes the protein virulence factors found in Subcellular fractionation methods coupled with proteomic vacuoles located around the flagellar pocket (Ramos et al., 2004; analysis provide a powerful approach to investigate the micro- Uliana et al., 1999). Although calpain-like protease paralogues are somal proteome. However preparation of isolated organelles is widespread in trypanosomatids, their functions are unknown. technically challenging and the presence of cytoplasmatic con- Two proteins identified in this study are involved in drug resis- taminants is inevitable, and this may explain the identification of tance. The small GTP-binding Rab1-protein (L. L. amazonensis) proteins which are generally believed to be derived from non- and tryparedoxin peroxidase (L. L. major) and were both observed vesicular cellular structures. On the other hand, as the number of to be differentially expressed in a proteomic study with Leishmania subcellular proteomic studies increases, the identification of pro- methotrexate resistant strains (Drummelsmith et al., 2004). The teins that are associated with organelles which previously were not gene encoding the GTP-binding Rab1-protein (L. L. amazonensis) considered specific to these compartments is becoming a recurrent is homologous with YPT, a Rab/GTPase which has been described observation. It is a point of debate as to whether these proteins are A.H.C. de Oliveira et al. / Comparative Biochemistry and Physiology, Part D 1 (2006) 300–308 307 contaminants, whether they are associated (albeit temporarily) Bente, M., Harder, S., Wiesgigl, M., Heukeshoven, J., Gelhaus, C., Krause, E., with the organelles or structures under examination, or whether Clos, J., Bruchhaus, I., 2003. Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani. Proteomics 3, 1811–1829. they may be present in hitherto unappreciated subcellular loca- Beverley, S.M., 1991. Gene amplification in Leishmania. Annu. Rev. Microbiol. lizations (Warnock et al., 2004). Although the current data do not 45, 417–444. permit a detailed description of the subcellular structures from Bradford, M.M., 1976. 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