ogy, Part D 1 (2006) 300–308www.elsevier.com/locate/cbpd

Comparative Biochemistry and Physiol

Subproteomic analysis of soluble proteins of the microsomalfraction 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, Brazilb 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 2006Available 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 diseasethat affects 12 million people worldwide. With the aim of identifying potential secreted protein targets for further characterization, we have appliedtwo-dimensional gel electrophoresis and mass spectrometry methods to study the soluble protein content of the microsomal fraction from twoLeishmania species, Leishmania L. major and L. L. amazonensis. MALDI-TOF peptide mass fingerprint analysis of 33 protein spots fromL. L. amazonensis and 41 protein spots from L. L. major identified 14 proteins from each sample could be unambiguously assigned. Theseproteins include the nucleotide diphosphate kinase (NDKb), a calpain-like protease, a tryparedoxin peroxidase (TXNPx) and a small GTP-bindingRab1-protein, all of which have a potential functional involvement with secretion pathways and/or environmental responses of the parasite. Theseresults 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

The protozoan parasite Leishmania sp. is the causative agent ofLeishmaniasis which affects approximately 12 million peopleworldwide and places a further 350 million people at risk, themajority of whom live in underdeveloped countries. With 1.5–2 million cases reported each year (Herwaldt, 1999), Leishman-iasis is considered by the World Health Organization to be thesecond most important disease caused by a protozoan parasite.Clinical forms of the disease range from mild cutaneous or de-forming mucocutaneous manifestations to the fatal visceral Leis-hmaniasis, also known as kalazar. Leishmania parasites have adimorphic life-cycle characterized by a free-living promastigote

⁎ Corresponding author. Department of Chemistry Faculdade de Filosofia,Ciências e Letras de Ribeirão Preto, Universidade de São Paulo AvenidaBandeirantes, 3900, CEP 14040-901, Ribeirão Preto-SP, Brazil. Tel.: +55 1636024384; fax: +55 16 36024838.

E-mail address: rjward@ffclrp.usp.br (R.J. Ward).

1744-117X/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.cbd.2006.05.003

form and non-motile amastigote form. After replication in themidgut of sand-fly vector, non-infective promastigote forms dif-ferentiate into infective metacylic promastigotes, which areinoculated into dermis of the mammalian host during the blood-meal of infected sandflies (Sacks, 1989). They are then phago-cytosed by macrophages where they transform into the non-motile, intracellular replicative amastigote form, which is adaptedto survive and replicate within the hostile environment of thephagolysosome. After the amastigote release by macrophagelysis, the amastigote forms can be phagocytosed by adjacentmacrophages (Alexander and Russel, 1992).

The classification of twenty species of the trypanosomatidLeishmania in 2 subgenera, Leishmania (Leishmania) and L.(Viannia), is based on the mode of promastigote developmentwithin the digestive tract of the invertebrate sand fly host (Lainsonand Shaw, 1987). Some Leishmania species are traditionallyassociated with a given clinical form, such as L. L. major whichproduces localized cutaneous lesions (LCL), or L. L. donovanithat is associated with kalazar (VL), L. L. amazonensis with

mailto:rjward@ffclrp.usp.brhttp://dx.doi.org/10.1016/j.cbd.2006.05.003

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diffuse cutaneous Leishmaniasis (DCL) and L. V. braziliensiswith mucocutaneous manifestations (MCL). It should be notedhowever, that the association of some species with atypical formsof the disease has been reported (Barral et al., 1986, 1991;Hernandez et al., 1995; Ponce et al., 1991). The biology of the OldWorld L. L. major and the NewWorld L. L. amazonensis differ atvarious levels, ranging from the distribution of the parasites in thehost parasitophorous vacuoles (Antoine et al., 1998) to the degreeof infectivity in BALB/c mice (Courret et al., 2003).

Comparative genomic studies of Leishmania species with othertrypanosomatids such as Trypanosoma brucei and T. cruzi,together with analyses of gene expression patterns and proteomicstudies in various developmental stages holds much promise forunderstanding the biology, virulence and the variable clinicalmanifestations on infection by these parasites. The transcription ofprotein-encoding genes in trypanosomatids results in policystronicRNAs which are processed by a trans-splicing mechanism tomonocistronic mRNAs that are subsequently translated (LeBowitzet al., 1993). Gene regulation therefore occurs by controlling thestability and translation of specific mRNAs (Graham, 1995; Van-hamme and Pays, 1995). Indeed, recent results from microarrayanalyses of mRNA from different developmental stages of Leish-mania have confirmed that only a relatively modest number ofmRNAs show significant alterations in abundance (Akopyantset al., 2004; Almeida et al., 2004). Furthermore, the proteinsundergo extensive post-translation modification (Mottram et al.,1993; Saas et al., 2000; Soto et al., 2000), which emphasizes theimportant role of post-transcriptional regulation in these parasites.

A proteomic based approach is therefore particularly interest-ing for the investigation of trypanosomatid parasites, and pro-teomic studies in Leishmania have focused on analyzing thedifferences in protein content and post-translation modification inthe different life cycle stages of the parasite. Indeed, changes in theprotein profile as observed in 2D-gels between the promastigotesand axenic amastigote forms of these parasites have been des-cribed (El Fakhry et al., 2002; Bente et al., 2003; Nugent et al.,2004). Proteomic analysis with a methotrexate resistant strain ofL. L. major (Drummelsmith et al., 2003) has directly confirmedthat over-expression of pteridine reductase by an episomal geneamplification mechanism leads to drug tolerance (Bello et al.,1994; Beverley, 1991). Furthermore, characterization of differen-tial protein expression between wild-type and methotrexate re-sistant L. L. major promastigotes identified a series of proteintargets implicated in drug resistance (Drummelsmith et al., 2004).

The majority of proteomic studies in trypanosomatids havebeen performed with general sample preparation protocols thatidentify abundant soluble proteins. The enrichment of other pro-teins such as hydrophobic membrane associated and low copy-number proteins require different sample preparation methods,which may include the pre-fractionation of cells (Gorg et al., 2000;Wu et al., 2003) and the proteomic analysis of cell fractions,commonly called the subproteomic approach, has been employedto investigate secreted virulence factors in pathogenic organisms,such as the Plasmodium falciparum rhoptry-enriched proteome(Sam-Yellowe et al., 2004) and the vesicle enriched-fraction(Gelhaus et al., 2005). Other recent subproteome studies includethe Schistosoma mansoni tegumentary proteome (van Balkom

et al., 2005) and cercarial secretome (Knudsen et al., 2005), theToxoplasma gondii rhoptry proteome (Bradley et al., 2005) and theHeliobacter pylori subproteome analysis of Backert et al. (2005).

Here we describe the subcellular fractionation of the pro-mastigote forms and analysis of the soluble content of totalmicrosomal extracts of L. L. major and L. L. amazonensis. Wedemonstrate that these two Leishmania species present differencesin their microsomal contents, and the identification of solubleproteins presenting functions related to the parasite environmentalresponse indicates that a subproteomic approach is a useful tool foridentifying individual protein targets that may play a role indefining host/parasite interactions.

2. Materials and methods

2.1. Preparation of soluble extract of the microsomes

Promastigote forms of L. L. major (strain LV-39, clone 5, Rho/SU/59/P, (Marchand et al., 1987)) and L. L. amazonensis (MPRO/BR/1972/M1841-LV-79) were grown in M199 culture supplemen-ted with 10% fetal bovine serum. All manipulations of bothLeishmania cell types were conducted within a laboratory confor-ming to the standards defined by international biosecurity level II.The microsomal enriched fraction was separated as previouslydescribed (Ulloa et al., 1995) with somemodifications. Briefly, oneliter of culture containing 1.5×107 parasites per mL was centri-fuged (1000 ×g/10 min/4 °C) and the parasite pellet was washed inphosphate buffered saline pH 7.4 (PBS) and resuspended in 10 mLof lysis buffer (10 mM Tris-Cl pH 7.5, 5 mM phenylmethylsul-phonyl fluoride, 176 μg/mL benzamidine, 200 μg/mL phenanthro-line, 3 mM EDTA and 3 mM EGTA) to give a cell density of1.5×109 parasites/mL. The parasiteswere lysed by five freeze/thawcycles, and after lysis the crude extract was centrifuged (1000 ×g/10 min/4 °C) in order to sediment the nuclear fraction, unlysedparasites and mitochondria. The post-nuclear supernatant (approxi-mately 10 mL, at 1.2 mg of protein/mL) was centrifuged (100,000×g/60 min/4 °C), and the sediment containing the microsomalfractionwas retained fromwhich the soluble proteinswere obtainedby lysis of follows.After resuspension of themicrosomal fraction inlysis buffer, the suspension was treated with 5 ultrasonic pulses(10W/60 s) intercalated by 5-minute intervals on ice. The sonicatedmicrosomal fraction was centrifuged (100,000 × g/60 min/4 °C) inorder to sediment the microsomal membranes, and the supernatant(10 mL) containing soluble fraction of the microsome extract wasretained. The total protein content of the soluble extract of themicrosomes fraction was estimated by the Bradford method(Bradford, 1976) which showed typical yields were 0.45–0.5(L. L. major) and 0.40–0.45 (L. L. amazonensis) mg of protein perliter of culture. The protein soluble extract of microsomes of L. L.amazonensis was analysed by unidimensional eletrophoresis (SDS-PAGE 12%; Laemmli, 1970) and stained with colloidal CoomassieBlue, as previously described (Neuhoff et al., 1988).

2.2. Two-dimensional gel electrophoresis

The protein content of the soluble extracts of the microsomalfraction was precipitated by adding trichloroacetic acid to a final

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concentration of 17%. After 2 h at 4 °C this mixture wascentrifuged (10,000 ×g/40 min/4 °C), and the pellet was washed3 times in chilled acetone. Samples of precipitated proteins (1.5mgof L. L. major and 1.0 mg of L. L. amazonensis) were solubilizedin rehydratation buffer containing 8M urea, 2% (w/v) CHAPS (3-[(3-cholamidoporpyl)-dimethylammonio]-propane-sulphonate),65 mM DTT, 10 mM Tris-Cl, and 0.5%(v/v) ampholyte bufferstock (IPG buffer, Amersham) and 0.001% bromophenol blue(Sigma). The sample was centrifuged (10,000 ×g/5 min/4 °C) andapplied to 13-cm immobilized pH 3–10 gradient strips (Amer-sham Biosciences), and after rehydration (12 h/20 °C), isoelectricfocusing of the proteins was achieved by IPGPhor System (Amer-sham Biosciences; 50 mA per strip, 150 V for 2 h, 500 V for 1 h,8000 V up to about 22,000 V h). The focused strips were equi-librated for 15 min in a buffer containing 50 mM Tris–HCl, 30%(v/v) glycerol, 2%(w/v) SDS, 10 mg/mL DTT and 25 mg/mL ofiodoacetamide, respectively, and a trace of Bromophenol Blue.Subsequently, the strips were transferred to a 12% polyacrylamidegel, and the proteins were separated under denaturating conditionsby electrophoresis (25 mA, 300 V) until the tracking dye ran offthe gel. After electrophoresis, the gels were stained with colloidalCoomassie Blue as previously described (Neuhoff et al., 1988).The gel images were analysed with Image Master 2D software(Amersham Biosciences) using standard molecular weight (SDS-6H, Sigma, USA) and a linear isoeletric point gradient.

2.3. Mass spectrometry

ESI-MS/MS: Protein separated by SDS-PAGE electrophoresiswas submitted to in situ gel band trypsin digestion with 0.5 μg ofmodified trypsin (Promega, USA). The tryptic peptides were de-salted in micro Tip filled with POROS R2 (Perseptive Biosystem,Inc. Foster City, CA) and eluted in 70% methanol, 0.2% Formicacid for MS analysis. The MS analysis of tryptic peptides wascarried out using an electrospray triple-quadrupole mass spectro-meter Quattro II (Micromass, Manchester, UK) by direct infusion(300 nL/min) under the following conditions: capillary voltagemaintained at 2.8 kV, cone voltage 40 V, cone temperature set to100 °C. The parameters for MS1 scanning and daughter-ion scan-ning were optimized with synthetic peptide for the highest signal-to-noise ratio and calibrated with PEG. In the daughter-ion scan-ning mode, the collision energy was set to 25–35 eV, and argonwas used as collision gas at a partial pressure of 3.0×10−3 mTorr.The spectra were collected as an average of 20–50 scans (2 to 5 s/scans) and processed using the MassLynx software v.3.3 (Micro-mass, Manchester, UK). The sequence of tryptic peptides wasdeduced from series of b and y ion fragments produced by collisioninduced dissociation mass spectrometry (CID-MS/MS). Masses oftryptic peptides and fragment ions from CID were used to searchthe Genebank at NCBI using Protein Prospector MS-Fit and MS-Tag (http://prospector.ucsf.edu/). Peptide mass fingerprints werealso obtained from spots separated from 2D-gel electrophoresis byMALDI-TOF-MS (Amersham-Bioscience, Uppsala, Sweden).Samples were desalted with POROS R2 and mixed in 1:1 (v/v)ratio with 10 mg/ml of matrix α-cyano-4-hydroxycinamic acid.Spectra were collected as an average of 10–20 scans, andmonoiso-topic masses were used to search the Genebank protein database.

MALDI-TOF-MS was calibrated with a mixture of syntheticpeptides, Angiotensin II, angiotensin I and a fragment of ACTH(Sigma, Chemical Co. St. Louis, MI).

MALDI-TOF: Spotswere cut from the 2D-gel andwashed threetimes in 100 mM ammonium bicarbonate containing 50% ace-tonitrile, followed by a wash in pure acetonitrile and subsequentlyophilization. Gel plugs were covered with 60 μL of trypsinsolution (4 ng/μL, Promega) and incubated for 18 h at 37 °C, andthe trypsinized gels were incubated for a further 2 hwith 40 μL of a50% acetonitrile/2.5% trichloroacetic acid mixture in order toextract the peptides. After extraction, the supernatant was lyophi-lized and the pellet was resuspended in 10 μL of the same solventmixture. One μL of the sample wasmixed with 1 μL ofα-cyano-4-hydroxycinamic acid, and after air-drying on an inert sample sup-port, the sample was submitted to matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis. Peptide massanalysis was performed in duplicate for each sample on aMALDI-TOF instrument (Ettan MALDI-TOF Pro, Amersham Bioscien-ces). The peptide mass fingerprints obtained from MALDI-TOFwere compared with a protein database generated by automaticannotation of L. L. major Friedlin sequence information availablethrough theWellcome Trust Sanger Institute website (www.sanger.ac.uk/Projects/L_major/), using the program Protein Probe (Bio-Lynx-MassLynx 3.3, Micromass, Manchester, UK), in which thesearch parameters permitted a mass tolerance of ±0.8 Da.

3. Results and discussion

Leishmania and other tryanosomatids are exposed to multipleenvironments since their life cycles have at least one developmentstage in both the insect vector and the vertebrate host. The envi-ronmental changes experienced by trypanosomatids during hostcrossing causes parasite differentiation, an event which is con-comitant with biochemicalmodifications (Zilberstein and Shapira,1994). The Leishmania promastigote form from the insect midguthas the first contact with the vertebrate host bloodstream andsubsequently with the host cell target, which are macrophages inthe case of Leishmania. Adaption of the cell surface compositionof the trypanosomatid is a crucial response to environmentalchanges, and the secretory pathways of these organisms play a keyrole during this process. Biochemical studies, including proteomeanalyses, can provide new information as to the molecular ma-chinery of trypanosomatids, and provide basic insights that maybe of use in the development of novel strategies to combat parasiteinfections.

Subproteomic studies of the microsomal fraction have beenused to identify membrane bound and/or secreted proteins ofeukaryotic cells including human prostate cancer cells (Whright etal., 2003), murine myeloma cells (Alete et al., 2005) and humanlymphoblasts (Filen et al., 2005). Furthermore, microsomal pro-teins from pathogenic organisms such as Theileria parva (Ebelet al., 1997), T. gondii (Chini et al., 2005) and Trypanosoma cruzi(Figueiredo et al., 2005) have been characterized with the aim ofidentify targets for intervention in the parasite life cycle. Here weshow that the protein content of the enriched microsomal fractionsof Leishmania can be used to identify secreted proteins in thepromastigote form of the parasite.

http://prospector.ucsf.edu/http://www.sanger.ac.uk/Projects/L_major/http://www.sanger.ac.uk/Projects/L_major/

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Previous authors have reported the use of sodium carbonatetreatment to prepare both the soluble fraction of vesicle extracts invarious Leishmania species (Noronha et al., 1996) and to rupturemicrosomes from Plasmodium (Florens et al., 2002). Althoughthis procedure is well established in the literature (Fujiki et al.,1982), in the present study the protein yield was not sufficient forvisualization of protein spots with colloidal Coomassie Blue eitherin 1D-SDS-PAGE or 2D-gels of soluble proteins of the micro-somal extracts from L. L. amazonensis and L. L. major (data notshown). In contrast, the extraction of soluble proteins frommicrosomal fractions by ultrasonic lysis yielded 0.49 and 0.45 mgprotein/L of culture from L. L. major and L. L. amazonensis,respectively. In this study, 4–6 L of culturewere typically prepared

Fig. 1. Unidimesional gel of the soluble protein content of themicrosomal fraction fromon a 1D-gel (SDS-PAGE12%) and several of themost abundant protein bands (encoloseMS (see the Materials and methods section for further details). (B) Amino acid sequehighlighted the peptide sequences identified by peptidemass fingerprint of the protein banm/z 679.8[+2H] from the Gp63, showing the amino acid sequence derived from interpre

from which a total of 2 mg (L. L. amazonensis) and 3 mg (L. L.major) of protein could routinely be obtained. However, preci-pitation by trichloroacetic acid and solubilization in the urea bufferfor isoelectric focusing resulted in a loss of approximately 50% ofthe protein.Nevertheless, the final yieldwas approximately 1.0mg(L. L. amazonensis) and 1.5 mg (L. L. major) of protein that wereanalyzed on 2D-gels and visualized by Coomassie Blue staining.The culture volumes necessary to obtain a sufficient quantity ofsolublemicrosomal proteins proved to be themain difficulty in thissubcellular proteomic study, however these large volumes wereessential for the identification of proteins with low abundance.

The viability of the extraction protocol was evaluated by 1D-SDS-PAGE of the microsomal extract fraction of L. amazonensis

L. amazonensis. (A) The soluble proteins of the microsomal fraction were separatedd by the boxes 1–3)were excised from the gel for subsequently analysis byESI-MS/nce of the Gp63 (gi ∣1100213∣ leishmanolysin of L. amazonensis), in which ared frombox #1 in (A). (C)Mass spectrumofCID-MS/MSof the trypsinic peptide oftation of the daughter ion scanning (ions y [M+1H]).

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 thetrichloroacetic acid precipitation, the resuspended soluble proteins of microsomal extracts were separated by isoelectric point (IEF pH 3–10) in the horizontaldimension and by molecular weight in the vertical dimension (SDS-PAGE 12%). The gel was stained using colloidal Coomassie Blue and analysed by Image Master2D Analysis Software (Amersham Biosciences) as described in the Materials and methods section. The spots within the numbered circles were selected for furtheranalysis by MALDI-TOF, and the identified proteins are shown in Table 2A and B.

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Fig. 3. MALDI-TOF analysis of spot #32 from the 2D-gel from the microsomalfraction of L. l. major. (A) Peptide mass fingerprint of the trypsin digested spot #32obtained by MALDI-TOF. (B) Amino acid sequence of the L. major NDKbshowing the localization of the peptides detected by the peptide fragment massfingerprint. The highlighted residues in the protein sequence are from the peptidesindicated in the MALDI-TOF spectrum with masses 1329.74, 926.55, 1612.82,1708.91, 934.45, 939.48 (in order from the N-terminal). See the Materials andmethods section for further details of the mass spectrometry analyses.

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(Fig. 1A). Three major bands were excised from the gel, and aftertrypsin treatment, the digested peptides were submitted to electro-spray mass spectrometry (ESI/MS-MS). The peptide mass finger-print identified these proteins as the membrane GPI-anchoredproteins Gp63 (Leishmanolysin) and Gp46 (PSA surface antigen)and cytosolic nucleoside diphosphate kinase (NDKb). The amino-acid sequence of Gp63 containing the identified peptides is pre-sented in Fig. 1B, in which are highlighted the peptide sequencesgenerated by the CID/MS analysis. A representative spectrumfrom the CID/MS analysis of a peptide (FDLSTVSDAFEK) from

Table 1Identification of proteins by peptide mass fingerprints from MALDI-TOF analysis o

Spotno.

Protein Genomannotat

1 Putative HSP60 chaperonin precursor LmjF32 Putative HSP60.2 chaperonin LmjF33 Putative HSP60.2 chaperonin LmjF37 Hypothetical protein LmjF310 Eukaryotic translation initiation factor 3 (subunit δ) LmjF312 Putative pyruvate dehydrogenase precursor (e1 component,

β subunit)LmjF2

16 25 kDa (1β) Elongation factor (putative) LmjF317 Hypothetical protein conserved (morn repeat protein) LmjF319 20S proteosome α5 subunit LmjF220 20S proteosome α5 subunit LmjF222 Conserved hypothetical protein LmjF325 Small GTP-binding protein Rab1 LmjF228 Putative eukaryotic initiation factor 5a LmjF230 Putative eukaryotic initiation factor 5a LmjF231 MIF-like protein LmjF333 Nucleoside diphosphate kinase b (NDK-b) LmjF3

Gp63 is presented in Fig. 1C. The Gp63 is a 63 kDa-surface-localized metalloprotease involved in the intracellular survivalwithin host macrophages (McGwire and Chang, 1996) and hasbeen described as a 59-kDa secreted form within vesicles that fusewith flagellar pocket membrane (McGwire et al., 2002). Theidentification of the 59-kDa form of the protein in the 1D-gel of theL. amazonensis microsomal fraction (Fig. 1), at higher concentra-tion is consistent with the presence of secreted addressed vesiclesin the extractedmicrosomal fraction. Furthermore, identification ofthe nucleoside diphosphate kinase b (NDKb), which is a secretedprotein implicated in the pathogenicity of Mycobacterium tuber-culosis (Saini et al., 2004) and Vibrio cholera (Punj et al., 2000)confirmed that the microsomal fraction was enriched with vesiclesthat are addressed to the cell membrane and/or for secretion.

Fig. 2A presents the 2D-gel of the soluble fraction of themicrosomal fraction fromL. L. amazonensis, which reveals that themajority of the proteins are detected within a pI range of 4–7 andhave a molecular weight below 66 kD. Approximately 100 proteinspots could be observed on the gel, of which 33 were submitted toMALDI-TOF mass spectrometry. Fig. 2B shows the 2D-gel of thesoluble extract of the microsomal from L. L. majorwhere approxi-mately 130 protein spots could be detected, of which 41 wereanalyzed byMALDI-TOFmass spectrometry. In commonwith theL. L. amazonensis extract, the L. L. major proteins have molecularweights of less than 66 kD, however the distribution of the proteinsin the 2D-gel is significantly different from L. L. amazonensis,demonstrating that the overall protein profile of the microsomalextract differs between the two species. Recent comparisons of theL. L. infantum and L. L. major genomes have revealed a high levelof sequence conservation between protein-coding genes (see http://www.genedb.org/genedb/), therefore the divergent spot patternsobserved in the 2D-gels between L. L. amazonensis and L. L.major may suggest that significant differences arise at the level ofpost-translational modification and/or intracellular compartmenttargeting of the expressed proteins.

Fig. 3 shows the spectrum of spot #32 from the L. L. major 2D-gel (see Fig. 2B), which was identified as the nucleoside

f the soluble protein content of the vesicle fraction from L. l. amazonensis

icion

kDa theor/expt

pI theor/expt

Matched/masses

Protein coverage(%)

6.2020 60.1/65.2 5.7/5.3 8/12 166.2030 59.3/65.1 5.4/5.5 10/22 276.2030 59.3/64.8 5.4/5.7 9/17 210.1520 49.3/54.7 8.1/8.0 3/9 116.3880 45.4/43.3 6.1/5.1 5/6 135.1710c 37.8/39.8 5.8/5.1 4/5 11

4.0820 25.6/26.2 5.2/6.2 3/7 170.3310 40.7/25.8 5.3/5.8 6/10 211.1830 26.7/24.3 5.4/5.3 3/4 161.1830 26.7/24.2 5.4/5.5 4/8 222.1640 23.7/23.2 7.1/6.7 2/4 107.0760 22/21.4 5.6/6.1 2/3 135.0730 17.8/18.9 4.8/4.6 2/5 185.0730 17.8/17.4 4.8/5.4 2/5 183.1750 12.5/15.5 8.2/8.8 2/4 252.2950 16.6/12.3 8.2/7.7 4/8 33

http://www.genedb.org/genedb/http://www.genedb.org/genedb/

Table 2Identification of proteins by peptide mass fingerprints from MALDI-TOF analysis of the soluble protein content of the vesicle fraction from L. l. major

Spotno.

Protein Genomicannotation

kDa theor/expt

pI theor/expt

Matched/masses

Proteincoverage (%)

5 Putative pyruvate dehydrogenase precursor (component e1, subunit α) LmjF18.1380 42.8/42.8 8.2/7.7 4/4 136 Putative pyruvate dehydrogenase precursor (component e1, subunit β) LmjF25.1710 37.8/40.1 5.8/5.3 5/12 178 Proteasome subunit α type I LmjF36.1600 29.6/32.3 5.2/5.2 7/12 2812 20S proteosome α1 subunit LmjF35.4850 27.2/29.7 7.4/7.0 3/5 1513 20S proteosome α5 subunit putative LmjF21.1830 26.7/29.3 5.6/5.6 3/6 1720 Hypothetical protein, conserved, unknown function LmjF27.2200 27.2/24.1 5.6/5.4 3/6 1323 Cell cycle protein Mob1-2, (putative) LmjF06.0960 26/20.2 8.2/8.7 4/8 1824 Hypothetical protein (conserved) LmjF34.2580 22.5/19.9 9.7/7.1 5/9 3125 Hypothetical protein (conserved) LmjF34.2580 22.5/19.8 9.7/8.2 7/12 3526 Hypothetical protein (conserved) LmjF34.2580 22.5/19.7 9.7/8.1 5/10 3027 Hypothetical protein (conserved) LmjF34.2580 22.5/19.7 9.7/7.9 4/8 3229 Cyclophilin a LmjF25.0910 18/17.2 8.1/8.5 3/7 1732 Nucleoside diphosphate kinase b (NDK-b) LmjF32.2950 16/15.9 8.2/8.1 6/11 4533 Hypothetical protein (conserved) LmjF19.1400 25.9/15.8 6.7/7.2 3/6 1134 Nucleoside diphosphate kinase b (NDK-b) LmjF32.2950 16/15.8 8.2/7.5 6/7 4535 Nucleoside diphosphate kinase b (NDK-b) LmjF32.2950 16/15.5 8.2/9.0 4/7 4536 Calpain-like protease LmjF20.1310 14.9/14.5 5.3/5.4 2/5 1637 Hypothetical protein, conserved LmjF13.0450 13.3/13.9 5.2/5.1 5/8 2038 Hypothetical protein LmjF28.1340 20.7/13.8 5.7/4.9 2/3 1241 TRYP (TXNPx), tryparedoxin peroxidase LmjF15.1120 21.1/9.9 6.4/8.8 4/8 22

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diphosphate kinase (NDKb), and which is representative of peptidemass fingerprint data collected in the MALDI-TOF experiments.The MALDI-TOF peptide mass fingerprint data from all theselected spots were compared with the L. L. major sequencedatabases (Tables 1 and 2). Of the total of 33 and 41 protein spotsanalyzed, 12 and 14 proteins could be identified from the L. L.amazonensis and L. L. major extracts, respectively. Several proteinspots were conserved between the extracts from the two species,such as proteins associated with energy metabolism (e.g. piruvatedehydrogenase subunits) and the proteins associated with the 20Ssubunit of the proteasome. However, as suggested by thedifferences in the patterns observed in the 2D-gels, the two extractswere characterized by the presence of different functional classes ofproteins. For example, proteins involved in the cell cycle and cellproliferation were observed, such as translation initiation factor 5a,25 kD elongation factor, translation factor 3 (L. L. amazonensis)and the protein associated with the cell cycle Mob 1–2 (L. L.major). Furthermore, three proteins implicated in the stressresponse, the HSP60 (L. L. amazonensis), the cyclophilin-a andtryparedoxin peroxidase (L. L. major) were also identified. Theidentified protein LmjF20.2310 (L. L. major) presents a high levelof sequence similarity to the C-terminal domain of the META-2protein, a calpain-like paralogue that maps to META1 gene locuswhich is a region that encodes the protein virulence factors found invacuoles located around the flagellar pocket (Ramos et al., 2004;Uliana et al., 1999). Although calpain-like protease paralogues arewidespread in trypanosomatids, their functions are unknown.

Two proteins identified in this study are involved in drug resis-tance. The small GTP-binding Rab1-protein (L. L. amazonensis)and tryparedoxin peroxidase (L. L. major) and were both observedto be differentially expressed in a proteomic studywithLeishmaniamethotrexate resistant strains (Drummelsmith et al., 2004). Thegene encoding the GTP-binding Rab1-protein (L. L. amazonensis)is homologous with YPT, a Rab/GTPase which has been described

in Golgi-vesicles near the plasma membrane (Cappai et al., 1993),and which may be involved in drug resistance in Leishmania(Marchini et al., 2003). The nucleoside diphosphate kinase b(NDKb) was identified in both species and in the case of L. L.major was identified in 3 spots (Fig. 1B), demonstrating a post-translational processing of the protein. A lower molecular mass(12 kDa) was detected for NDKb in the 2D-gel from L. L.amazonensis, which may be related to the previous observation ofboth a16 kDa cytosolic and a 12 kDa truncated membraneassociated form of the protein that have been described from thepathogenic Pseudomonas aeruginosa (Shankar et al., 1996). Thisenzyme is present in different organisms as membranous, cytosolicand secreted forms and plays a pivotal role in the NTP and dNTPregulation (Lascu et al., 2000). Furthermore, the NDKb secretionby pathogenic intracellular organisms such as M. tuberculosis(Saini et al., 2004) has led to the suggestion that in the presence ofATP this protein is a cytotoxic factor against macrophages (Chopraet al., 2003). Thus at least four proteins (NDKb, calpain-likeprotease, tryparedoxin peroxidase and GTP binding Rab-protein)identified in this study show possible functional involvement withsecretion pathways and/or environmental responses of the parasite.In the case of Leishmania, these proteins could be involved in thecommunication with the host cell during the invasion process.

Subcellular fractionation methods coupled with proteomicanalysis provide a powerful approach to investigate the micro-somal proteome. However preparation of isolated organelles istechnically challenging and the presence of cytoplasmatic con-taminants is inevitable, and this may explain the identification ofproteins which are generally believed to be derived from non-vesicular cellular structures. On the other hand, as the number ofsubcellular proteomic studies increases, the identification of pro-teins that are associated with organelles which previously were notconsidered specific to these compartments is becoming a recurrentobservation. It is a point of debate as to whether these proteins are

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contaminants, whether they are associated (albeit temporarily)with the organelles or structures under examination, or whetherthey may be present in hitherto unappreciated subcellular loca-lizations (Warnock et al., 2004). Although the current data do notpermit a detailed description of the subcellular structures fromwhich the proteins identified were derived, the results clearly showthat the two Leishmania species studied present differences in themicrosomal contents. Furthermore, the identification of solubleproteins presenting functions related to the parasite environmentalresponse demonstrates that microsomal subproteomic approachcan be helpful in the discovery of new protein targets for structuraland functional studies, and may be relevant for understanding thebiology of parasite transmission and host/parasite interactions.

Acknowledgments

K.A. Beattie and D. J. Lamont and the FingerPrints ProteomicsFacility, PGMIC,WellcomeTrust Biocentre,University ofDundee,Scotland, UK for their generous assistance with data collection.This work was funded by FAPESP doctorate grant 00/11165-0(AHCO), FAPESP genome SMOLBnet project 01/7537-2 (RJW),FAPESP research grant 99/12403-3 (AKC), CNPq and the Pro-Reitoria de Pesquisa-USP.

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Subproteomic analysis of soluble proteins of the microsomal �fraction from two Leishmania speci.....IntroductionMaterials and methodsPreparation of soluble extract of the microsomesTwo-dimensional gel electrophoresisMass spectrometry

Results and discussionAcknowledgmentsReferences