Experimental Parasitology 116 (2007) 182–189

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Research brief

Degradation of pteridine reductase 1 (PTR1) enzyme during growth phase in the protozoan parasite Leishmania donovani

Pranav Kumar a, Shyam Sundar b, Neeloo Singh a,¤

a Drug Target Discovery and Development Division, Central Drug Research Institute, Lucknow, Indiab Institute of Medical Sciences, BHU, Varanasi, India

Received 4 July 2006; received in revised form 8 December 2006; accepted 12 December 2006Available online 30 December 2006

Abstract

Pteridine reductase 1 (PTR1) is an essential enzyme of pterin and folate metabolism in the protozoan parasite Leishmania. The presentwork is focused on the degradation of PTR1 during growth phase in Leishmania donovani. Western blot analysis with PTR1-GFP trans-fected promastigotes revealed that PTR1 protein was degraded in the stationary phase of growth at the time when the parasites wereundergoing metacyclogenesis. Fluorescence microscopy revealed cytoplasmic localization of GFP tagged protein extending to the Xagel-lum in these stationary phase promastigotes, implying that degradation of the protein was not by the usual multivesicular tubule lyso-some (MVT) pathway. A probable destruction box of nine amino acids Q63ADLSNVAK71 and possible lysine residue K156 wasidentiWed in L. donovani PTR1 to be the site for ubiquitin conjugation. This suggests that PTR1 degradation during the stationary phaseof growth is mediated by the proteasome. This leads to lower levels of H4-biopterin, which favors metacyclogenesis, and subsequentlyresults in a highly infective stage of the parasite. Therefore, this Wnding has importance to identify new target molecule like the protea-some for therapeutic intervention.© 2007 Elsevier Inc. All rights reserved.

Index Descriptors and Abbreviations: Leishmania donovani; Clinical isolate; Pteridine reductase 1; Protein degradation; Proteasome; Metacyclogenesis;PTR1, pteridine reductase 1; GFP, green Xuorescent protein; MVT, multivesicular tubule lysosome; PBS, phosphate-buVered saline; HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); HFBS, heat-inactivated fetal bovine serum; ECL, electrochemiluminescence; FACS, Xuorescence acti-vated cell sorting; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis

1. Introduction

During its infectious cycle pathogenic Leishmania spp.alternates between Xagellated promastigote forms, whichgrow within the alimentary tract of the sand Xy vector andaXagellate amastigotes, which replicates within the acidiWedphagolysosomes of the vertebrate host macrophages. Meta-cyclogenesis is a critical step, which involves the diVerentia-tion of the noninfective procyclic promastigotes to highlyinfective metacyclic forms within the sand Xy (Sacks andPerkins, 1984). Metacyclogenesis also occurs during growthin in vitro cultures, wherein non-infective logarithmic phase

* Corresponding author. Fax: +91 522 2223405.E-mail address: neeloo888@yahoo.com (N. Singh).

0014-4894/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.exppara.2006.12.008

procyclics diVerentiate into infective metacyclics, derived atthe stationary phase of the culture. Both the metacyclic andprocyclic forms generated in vitro are morphologically sim-ilar to their insect counterparts (Sacks and Perkins, 1984;Pimenta et al., 1994). This diVerentiation process (metacy-clogenesis) is accompanied by changes in parasite morphol-ogy, gene expression and structural modiWcations (Saraivaet al., 1995). These modiWcations are likely to reXect thebiochemical adaptations of each stage to its immediateenvironment and also involve selective degradation of cyto-plasmic proteins.

In eukaryotic cells, the turnover of intracellular proteinsis mediated mainly by the ubiquitin-proteasome system(Goldberg et al., 1997). Following ubiquitination, proteinsare unfolded and proteolytically degraded by 26S protea-some, a large multisubunit and multicatalytic complex

P. Kumar et al. / Experimental Parasitology 116 (2007) 182–189 183

located in the cytosolic and nuclear compartments. InLeishmania, proteasome has been reported in Leishmaniamexicana (Robertson, 1999) Leishmania major (Bouzatet al., 2000) Leishmania chagasi (Silva-Jardim et al., 2004)and Leishmania donovani (Christensen et al., 2000). InLeishmania genome data bank (gene DB, www.genedb.org.)the presence of homologues to eukaryotic proteasome andubiquitin genes have been reported.

Pteridine reductase 1 (PTR1) is a novel broad-spec-trum enzyme of pterin and folate metabolism in the pro-tozoan parasite Leishmania. Biochemical studies indicatethat this enzyme is an NADPH dependent pterin reduc-tase and active as a tetramer (Nare et al., 1997; Wanget al., 1997; Kumar et al., 2004). PTR1 reduces biopterinto H2-biopterin and H4-biopterin; it is also capable ofreducing folate to 7,8-dihydrofolate and tetrahydrofo-late. PTR1 is regulated with the growth stage of the L.donovani promastigote with high activity in the logarith-mic stage of the parasite and lower activity (approxi-mately 70% of the log phase values) in the stationaryphase (Cunningham and Beverley, 2001). Unlike promas-tigotes, amastigote PTR1 level does not decline in sta-tionary phase in L. mexicana (Cunningham and Beverley,2001). PTR1 contributes about 10% of the reduction offolates in wild-type cells while the remaining 90% is dueto the activity of dihydrofolate reductase-thymidylatesynthase (Nare et al., 1997). Since PTR1 is signiWcantlyless inhibited by methotrexate than dihydrofolate reduc-tase-thymidylate synthase (Nare et al., 1997), this activityallows it to act as a metabolic by-pass of dihydrofolatereductase-thymidylate synthase enzyme. The novelty andpossible uniqueness of the pathway in which PTRI isinvolved opens the possibility of developing speciWcinhibitors, which in combination with DHFR (dihydro-folate reductase) inhibitors could be highly eVectiveagainst Leishmania (Bello et al., 1994). Deletion of thePTR1 gene is lethal to the insect stage promastigotes butcan be oVset by provision of reduced pterins but notfolates, indicating an essential role for H4-biopterin(Nare et al., 1997; Wang et al., 1997; Bello et al., 1994).While H4-biopterin is an essential cofactor in many reac-tions including ether lipid cleavage, aromatic amino acidhydroxylations, molybdopterin synthesis and nitric oxidesynthesis in higher eukaryotes (Blakley and Benkovic,1985; Tietz et al., 1964; Tayeh and Marletta, 1989; Kwonet al., 1989), the role(s) of H4-biopterin in Leishmania hasnot been clearly established, recently its role in the regu-lation of parasite diVerentiation has been conWrmed.Mutants lacking PTR1 had a low level of H4-biopterin,leading to diVerentiation of parasites within the sand Xyvector to the highly infective metacyclic promastigotestage (Cunningham et al., 2001). This suggests that regu-lation of PTR1 is stage-speciWc, at least in part of thegrowth stage-dependent degradation of the PTR1 pro-tein. Here, we present degradation of pteridine reductase1 (PTR1) in the growth phase by making PTR1-GFP chi-mera in which PTR1 is fused to GFP at its N-terminus.

2. Materials and methods

2.1. Leishmania growth

Leishmania donovani promastigote form of drug sensi-tive (2001) isolate (Singh, 2002) were cultured in M-199medium (Sigma, USA) containing 25 mM HEPES, 40 mg/lgentamicin and supplemented with 10% HFBS at 25 °C.For growth phase analysis 10£ 106 parasites wereinoculated initially in 5 ml of medium, and samples weretaken after each desired growth period. Cell concentra-tions were determined by daily counts with a hemocy-tometer at 20£ magniWcation after adequate dilution inphosphate-buVered saline (PBS) containing 2% parafor-maldehyde.

2.2. DNA construct

The Leishmania expression vector pXG-GFP and pXG-GFP+2 were obtained from Dr. Stephen Beverley [Wash-ington University St. Louis, MO (Ha et al., 1996)]. pXG-GFP vector was transfected in Leishmania promastigotes asa control to see the expression of GFP protein throughoutthe growth phase of Leishmania parasites. To determine thecellular localization of PTR1 and whether its degradation isgrowth phase dependent, we generated a PTR1 proteinfused to GFP at its N-terminal end to the pXG-GFP+2vector, which is a GFP variant similar to pXG-GFP. Cod-ing sequence for PTR1 was ampliWed from genomic DNAisolated from a drug sensitive (2001) isolate of L. donovanipromastigotes. The primer sequences used were, forwardprimer: 5�-CGCGGATCCACCATGACTGCTCCGACC-3� and reverse Primer: 5�-GGCGGATCCTCAGGCCCGGGTAAGGCTG-3�. The nucleotides in bold in the for-ward and reverse primers signify a BamHI site to facilitatecloning in pXG-GFP+2 vector and ‘Kozak’ sequence(underlined) for expression in Leishmania. PCR was per-formed as reported previously (Kumar et al., 2004). ThePCR product was puriWed using Qiagen kit. Ligation wasdone by inserting the puriWed PCR product (PTR1 gene) inthe appropriate reading frame in the poly-linker located 3�-ward of the GFP+2 gene. Right orientation of PTR1-GFPconstruct was conWrmed by PCR using GFP gene speciWcforward primer: 5�-CATGGTCCTGCTGGAGTTCGTGA-3� and PTR1 gene speciWc reverse primer: 5�-GGCGGATCCTCAGGCCCGGGTAAGGCTG-3�. Out of eightclones selected on the basis of BamH1 restriction digestion,three clones were found to be in the right orientation byPCR. A clone was selected and sequenced by automaticsequencer ABI Prism (Version 3.0. Model 3100) fromUDSC Department of Biochemistry, University of Delhi,South campus, New Delhi and the result conWrmed thepresence of PTR1 gene in-frame with N-terminal GFP inpXG-GFP+2 multiple cloning site. The PTR1 gene ampli-Wed from genomic DNA of L. donovani clinical isolatesdescribed in this report have been submitted to Gene bankand assigned the Accession no: AY547305.

184 P. Kumar et al. / Experimental Parasitology 116 (2007) 182–189

2.3. Transfection of the parasites

The promastigote form of L. donovani clinical isolate2001 was transfected with GFP and GFP-PTR1 chimera.BrieXy, late log phase promastigotes were harvested by cen-trifugation at (3000g, 1 min) washed once with transfectionbuVer (HEPES 1.25 g, CaCl2 0.0004 g, MgCl2 0.250 g, KCl2.22 g, NaH2PO4 0.02 g, glucose 0.27 g in 250 ml of sterilewater) and Wnally resuspended in the same buVer to 5£107

cells/ml. About 300 �l of cell suspension was chilled on icefor 10 min before adding 10–20�g of plasmid DNA. Cellswere immediately electroporated in Genepulser II appara-tus (Bio-Rad Laboratories) with capacitance extender at0.45 kV and 500 �F in 2 mm electroporation cuvette. Cellswere placed on ice for 2 min before being transferred to liq-uid medium. Geneticin disulWde (G418; Sigma) was addedto the liquid medium for the selection of transfected pro-mastigote stage of the parasite. Parasites were examined forgrowth and survival in increasing concentration of G418(up to 150�g/ml) for about 14–20 days. The parasites wereroutinely grown in 150�g/ml of G418 because furtherincreasing the drug concentration did not result in anincrease in arbitrary Xuorescence units. GFP Xuorescencein the cultures was regularly monitored by FACS CaliburXow cytometer (Becton Dickinson, NJ, USA) and epiXuo-rescence microscopy.

2.4. Fluorescence microscopy

Parasites expressing GFP and GFP-PTR1 were visual-ized with a Leica upright microscope (Model Leica DM5000B) equipped with a Leica DFC 320 digital camera.Promastigotes were pelleted by centrifugation (3000g,1 min) and washed with chilled phosphate-buVered saline(PBS) and than resuspended in PBS. Live promastigoteswere immobilized for epiXuorescence microscopy in poly-L-lysine coated coverslips. Coverslips were immersed in 4%paraformaldehyde (30 min, RT) to Wx the L. donovani pro-mastigotes. After washing in PBS (three times), coverslipswere mounted with 90% glycerol containing n-propyl gal-late (Sigma) as antifade agent for epiXouresence micros-copy. Visualization of the Xuorophore was achieved byGFP excitation/emission Wlter. Samples were scanned forgreen Xuorescence using a 63£ oil emersion objective.Images of 512£ 512 pixels were obtained using Leica FW4000 software and processed using Adobe Photoshop soft-ware.

2.5. Western blotting

Leishmania promastigotes of diVerent growth phasewere harvested, washed in PBS and lysed directly in the 5£sample buVer (0.313 M Tris–HCl pH 6.8, 10% SDS, 0.05%bromophenol blue, 50% glycerol and 0.5 M DTT). Proteinswere separated by SDS–PAGE and transferred onto nitro-cellulose Hybond membrane using Hoefer SemiPhor(Amersham Pharmacia Biotech). The cells (4£ 106) were

used for each lane. Western blotting was performed withpolyclonal anti-GFP antibody (Biovision) and monoclonalanti-�-tubulin antibody (Sigma) and detected by horserad-ish peroxidase-conjugated secondary antibodies and ECLchemiluminescent reagent (Amersham Pharmacia Biotech).

3. Results

To determine the degradation of PTR1 protein ingrowth phase, we generated a PTR1 protein fused to GFPat its N-terminal end to the pXG-GFP+2 vector (Fig. 1a)and the resulting chimera was found to be active. The pXGvector substitutes generic Xanking regions and allowexpression of the protein at relatively constant levelsthroughout the growth phase. The promastigote form of L.donovani clinical isolate 2001 was transfected with this chi-mera. After transfection we proceeded to study the growthkinetics of these promastigotes. Based on growth curveanalysis (Fig. 1b) 2£ 106 promastigotes/ml were used forinitial seeding. At this seeding concentration it was seenthat mid log phase reached at 48 h and stationary phasereached at 96 h of growth. So for further study we took agrown culture at 24 h as early log phase, 48 h as mid logphase, 72 h as late log phase and 96 h as a stationary phase.

We performed Western blot to determine the growthphase dependent degradation of PTR1 protein. We probedwhole-cell protein extracts of parasites isolated at diVerentphase of growth with anti-GFP antibody. The theoreticalsize of the PTR1-GFP chimera is 57 kDa and Western blotanalysis of proteins isolated from early (24 h), mid (48 h),and late (72 h) logarithmic phase cells highlighted only57 kDa band (Fig. 1c, Lanes 1–3), while stationary phase(96 h) cells highlighted two major proteins bands of 57 and27 kDa (Fig. 1c, Lane 4). The 57-kDa band corresponded toGFP-PTR1 chimera, while smaller reacting band of 27 kDacorresponds to GFP. Indeed this band had the same size asthe GFP protein detected in GFP transfected Leishmaniapromastigotes cells in both logarithmic and stationaryphase (Fig. 1d, Lanes 5 and 6). Intensity of the GFP proteinband was found to be less in the GFP-PTR1 transfectedpromastigotes as compared to GFP transfected promasti-gote. To check further whether the eVect increases as theparasite progresses into the stationary phase we probedGFP-PTR1 transfected promastigote with anti GFP anti-body at the 48 h of stationary phase (Fig. 1e, Lane 7). Thelevel of �-tubulin was analyzed to assure equal loading(Fig. 1f).

To further verify the posttranslational regulation ofPTR1 protein and its cellular localization, we carried outepiXuorescence microscopic analysis. EpiXuoresence micro-scopic analysis of the GFP-PTR1 chimera transfected cellsat early (24 h), mid (48 h), late (72 h) logarithmic phase ofgrowth revealed cytoplasmic localization of the protein(Fig. 2, Panels 1a, 2a and 3a). Interestingly no Xuorescencewas found in the Xagellum in the early (24 h), mid (48 h) andlate (72 h) logarithmic phase of growth. As the parasitesreached stationary (96 h), phase of growth, the protein could

P. Kumar et al. / Experimental Parasitology 116 (2007) 182–189 185

still be found in the cytoplasm; however, the Xuorescencehad a distinct localization in the Xagellum as well (Fig. 2,Panel 4a). This observation generates a question as to whyXuorescence had a distinct localization in the Xagellum inthe 96 h of growth? Is this due to the speciWc degradation ofPTR1 protein in the cytoplasm in the stationary phase (96 h)and migration of protease resistant GFP towards the Xagel-lum? For this purpose, we compared the Xuorescence imagesof GFP transfected promastigotes (control) with the PTR1-GFP transfected promastigotes. EpiXuorescence imagesshowed that GFP alone in the control cell was uniformlydistributed including Xagellum and showed no diVerence inlocalization of GFP protein between early logarithmic (24 h)and stationary phase (96 h) of the parasite growth (Fig. 2,Panels 5a and 6a). In the PTR1-GFP transfected promasti-

gotes GFP was found to be localized in the cytoplasm inearly (24 h), mid (48 h) and late (72 h) logarithmic phase ofgrowth (Fig. 2, Panels 1a, 2a, and 3a) while uniform distri-bution of GFP including the Xagellum was found to be inthe stationary phase (96 h) of growth (Fig. 2, Panel 4a). Thisexplains that the PTRI protein degrades in the cytoplasm inthe stationary phase of growth and after its degradationchimera becomes similar to GFP transfected cell becausepXG-GFP+2 vector (PTR1 protein fused to GFP at itsN-terminal end) is a similar GFP variant of pXG-GFPvector. Fluorescence microscopy and Western blot analysiswith polyclonal anti GFP antibody of cells taken at diVerentphases of growth verify that degradation of PTR1 proteinwas growth phase dependent and PTR1 protein degrades inthe stationary phase of growth.

Fig. 1. Localization and degradation of PTR1 in the growth phase of the parasite. (a) Construct of GFP-PTR1 chimera. PTR1 protein fused to GFP at itsN-terminal end to the pXG-GFP+2 vector. (b) Growth curve analysis of GFP-PTR1 transfected promastigotes. (c) Levels of the PTR1-GFP proteinalong the growth phase of the parasites were monitored by Western blot using an anti-GFP antibody. Total protein from PTR1-GFP transfectants isshown at 24 h (lane 1), 48 h (lane 2), 72 h (lane 3) and 96 h of growth (lane 4). (d) Total protein from GFP transfectant is shown at 24 h (lane 5) and 96 h ofgrowth (lane 6). (e) Total protein from GFP-PTR1 transfectant is shown at 48 h of stationary phase (lane 7). For Western blots, total protein from 4£ 106

parasites were loaded in each lane. (f) The levels of �-tubulin were analyzed to assure equal loading. Molecular masses were determined using the ECLprotein molecular weight markers (Amersham Biosciences, Cat # RPN 2107).

186 P. Kumar et al. / Experimental Parasitology 116 (2007) 182–189

4. Discussion

Pteridine reductase 1 (PTR1) is part of a novel metabolicpathway in Leishmania associated with folate metabolism.Its main function is to salvage pterins but a second one is to

reduce folates. Due to the central role of pterins in Leish-mania biology, intuitively, one would expect that PTR1 nullmutant could be attenuated and would give rise to reducedinfection in animal models, but surprisingly, a L. majorPTR1 null mutant was more infective in the mouse model

Fig. 2. Fluorescence microscopy of GFP-PTR1 chimera transfectants (Xuorescence images Panels 1a–4a, merge images Panels 1c–4c). Fluorescencemicroscopy of GFP transfectants (Xuorescence images Panels 5a and 6a, merge images Panels 5c and 6c). PTR1-GFP transfectant are shown at 24 h (Panel1a), 48 h (Panel 2a), 72 h (Panel 3a) and 96 h of growth (Panel 4a). GFP transfectants are shown at 24 h (Panel 5a) and 96 h of growth (Panel 6a).

P. Kumar et al. / Experimental Parasitology 116 (2007) 182–189 187

(Cunningham et al., 2001). This increased virulence appearsto be due to regulation of metacyclogenesis by H4-biop-terin, as both BT1 and PTR1 null mutants of L. major dis-played lower H4-biopterin levels and a speciWc increase inthe proportion of metacyclic parasites (Cunningham et al.,2001). The mechanism by which decreased levels ofH4-biopterin increase metacyclogenesis is unknown. Onepossibility is that it is a cofactor for an enzyme involved incontrolling diVerentiation; alternatively, it could beinvolved in a novel-signaling pathway. We propose thatthis is a manifestation of a cellular process occurring nor-mally during development, and that decreased pteridinelevels accountable for metacyclogenesis is due to the downregulation of PTR1 in the growth phase.

We studied this growth phase dependent regulation ofPTR1 in the promastigotes by forming a chimera of thisprotein with GFP at the N-terminal. PTR1 is a cytosolicenzyme and cytosolic proteins do not possess any signalsequence (Nelson and Cox, 2005), which was also con-Wrmed by our bioinformatic analysis-using program LOC-tree (Nair and Rost, 2005). Therefore PTR1 fusion either atthe C-terminal or N-terminal of GFP does not alter thelocalization of this cytosolic protein in any way.

Our Western blot analysis with GFP-PTR1 transfectedpromastigotes using anti GFP antibody proved that PTR1protein was degraded in the stationary phase of growth(96 h) although the eVect was less as compared to GFPtransfected promastigotes. To check further whether eVectincreases or not beyond the stationary phase when the par-asite is undergoing metacyclogenesis (>96 h of growthphase), we probed whole cell lysate with anti GFP antibodyand found that intensity of 27kDa band corresponding toGFP increases in the chimera.

To prove this further we analyzed the epiXuorescenceimages taken at diVerent phase of the growth. The questionarises why Xuorescence in the chimera was observed only inthe cytoplasm in early (24 h), mid (48 h) and late (72 h) logphase of the growth whereas in the stationary phase (96 h)of growth Xuorescence was observed in the Xagellum aswell. This could be due to the beginning of speciWc degrada-tion of PTR1 protein in the cytoplasm in the stationaryphase (96 h), leading to the generation of only GFP protein,which migrates towards the Xagellum. Cytosolic proteinsynthesis takes place by the free ribosome in the cytoplasm.The activity of PTR1 was found to be maximum in the logphase (Cunningham and Beverley, 2001) i.e. 48 h of growth

and active PTR1 is homotetramer of 30 kDa subunit(Kumar et al., 2004). This homotetramer of PTR1 resiststhe chimera from migrating towards the Xagellum. Wespeculate that destruction box and Lys156 residue for poly-ubiquitylation required for proteasome-mediated degrada-tion is present at the N-terminal of PTR1 enzyme invarious Leishmania species (Fig. 3). This large proteasomecomplex remains in the cytoplasm thereby also restrictingthe enzyme within the cytoplasm. That is why GFP-PTR1chimera Xuorescence has distinct localization in the cyto-plasm upto the late log phase (72 h) of the growth, afterwhich due to speciWc degradation of PTR1 by the proposedproteasome-ubiqutin conjugating system in the stationary(96 h) phase of growth, free GFP from the chimera which isresistant to proteases (Richard et al., 2004; Dodge et al.,2004; Mullin et al., 2001) migrates to the Xagellum.

The multivesicular tubule lysosome (MVT) has beensuggested to be a site where considerable protein degrada-tion takes place (Ghedin et al., 2001). At least three otherLeishmania proteins, the endoplasmic reticulum glycosyl-transferase dolichol-phosphate- mannose synthase, multi-drug resistance protein 1 and folate transporter 5 appear tobe growth stage regulated with retargeting to the multive-sicular tubule-lysosome followed by speciWc degradation(Richard et al., 2004; Dodge et al., 2004; Mullin et al., 2001).However, our result showed that PTR1 was not targeted toMVT and it remains in the cytoplasm suggesting diVerentmode of posttranslational regulation other than describedabove.

In eukaryotic cells, most proteins in the cytoplasm andnucleus are degraded not in lysosomes, but within protea-somes, after they are marked for destruction by covalentattachment of ubiquitin molecules (Orlowski, 1990; Couxet al., 1996; Rechsterner et al., 1993; Hilt and Wolf, 1996).Regulatory proteins ranging from committed step enzymeswithin metabolic pathways to components of signal trans-duction cascades generally possess short half-lives that aredetermined by structural features encoded within theiramino acid sequences, but can be further modulated byligand binding and posttranslational modiWcation. One ofthe Sea urchin cyclin B has a 9-amino acid region,RAALGNISN (single-letter amino acid code), termed the‘destruction box’. Lysine residue terminal to destructionbox are the sites for ubiquitin conjugation (Glotzer et al.,1991) Sacchromyces cerevisie cyclins, CLN (Hadwiger et al.,1989) contains the sequence RMGLVINAK, has week

Fig. 3. The amino acid sequence of PTR1 of various Leishmania species was aligned with the destruction box of Sea urchin Cyclin B using CLUSTAL W(1.83) multiple sequence alignment software. (¤), indicates identical sequences; (·), indicates conserved substitution; (:), indicates semi conserved substitu-tion. The regions Q63 to K71 are probable destruction box of PTR1 presented in various Leishmania species. K156 represented by box is possible site forpolyubiquitylation.

188 P. Kumar et al. / Experimental Parasitology 116 (2007) 182–189

homology to the destruction box. Similarly, the yeast tran-scriptional regulation has the motif RDILVFLSR in adomain involved in destabilizing the protein (Tatchell et al.,1981). Amino acid sequence analysis of L. donovani PTR1protein revealed probable destruction box of nine aminoacids QADLSNVAK and found to be conserved in thePTR1 of other Leishmania species also (Fig. 3). This nineamino acids Q63ADLSNVAK71 sequence of PTR1 hashomology with Sea urchin cyclin B destruction box (Glot-zer et al., 1991). Gonda and coworkers showed that ubiqui-tin mediated destruction of proteins whose N-terminalamino acid is recognized by ubiquitin-conjugating enzymesrequire nearby lysine residue to act as acceptor site forubiquitin conjugation (Gonda et al., 1989; Bachmair andVarshavsky, 1989; Chau et al., 1989). We speculate that thelysine residue K156 terminal to the destruction box (Fig. 3)is the site for ubiquitin conjugation, which leads to protea-some mediated degradation of the PTR1 protein.

Proteolysis plays an important role in Leishmania tomaintain the protein turnover and also in parasite diVeren-tiation. The work presented here clearly demonstrates thatdegradation of PTR1 protein in the clinical isolate of L.donovani is growth phase dependent. EpiXuorescensemicroscopy revealed cytoplasmic localization of PTR1 pro-tein. Western blot analysis with anti GFP antibody showedthat PTR1 degrades in the stationary phase of growth.Putative destruction box was identiWed in the amino acidsequence of PTR1 when aligned with known destructionbox of Sea urchin cyclin B. This suggests that PTR1degrades during the stationary phase when the parasiteswere undergoing metacyclogenesis and is mediated by theproteasome. This proteolytic pathway controls a broadarray of cellular functions including cell cycle progression,protein turnover, and speciWc degradation of protein dur-ing metacyclogenesis in order to increase the virulence ofthe parasites. This Wnding has importance to identify newtarget molecule like proteasome for the development ofattenuated vaccine for Leishmania.

Acknowledgments

This work was supported by grant from CSIR Indiafunded network project SMM003 ‘Molecular biology ofselected pathogens for developing drug targets’. Weacknowledge Mr. Ali Kausar of photography section ofCentral Drug Research Institute, Lucknow, for editing thephotographs and Wgures. P.K. acknowledges Council ofScientiWc and Industrial Research, India for fellowship.

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