Ascorbate Peroxidase Acts As a Novel Determiner of Redox Homeostasis in Leishmania

FORUM REVIEW ARTICLE

Ascorbate Peroxidase Acts As a Novel Determinerof Redox Homeostasis in Leishmania

Subrata Adak and Swati Pal

Abstract

Significance: Reactive oxygen species (ROS) are produced as natural byproducts of metabolism and respiration.While physiological levels of ROS are required for vital cellular functions (e.g., development and proliferation), aliving organism is faced with constant challenges due to accumulation or overproduction of ROS throughout itslife. The life cycle of Leishmania parasite has led it to confront the highly oxidizing environment in the macro-phage phagosomes, necessitating ROS homeostasis and signaling as key strategies for successful survival andpathogenicity. Recent Advances: Ascorbate peroxidase from Leishmania major (LmAPX) is the only heme per-oxidase identified so far in Leishmania. Structural analysis and functional characterization of LmAPX haveyielded interesting and novel insight on this enzyme. The protein has been found to be a hybrid of cytochrome cperoxidase and ascorbate peroxidase. This enzyme is colocalized with cytochrome c in the inner mitochondrialmembrane facing the intermembrane space and shows higher activity toward cytochrome c oxidation. CriticalIssues: Overexpression of LmAPX in L. major cells confers tolerance to oxidative stress-mediated cardiolipinoxidation and consequently protects cells from extensive protein damage. LmAPX - / - mutants show higherintracellular hydrogen peroxide (H2O2), which might signal for cellular transformation from noninfective pro-cyclic to infective metacyclic form and ultimately apoptosis. Future Directions: Manipulation of LmAPX ex-pression has significantly added to the present understanding of the parasite’s defense network against oxidativedamage caused by H2O2. The future investigations will address more exactly the signaling pathways involved inredox homeostasis. Antioxid. Redox Signal. 19, 746–754.

Introduction

Leishmaniasis is a disease caused by the trypanosomatidparasites belonging to the genus Leishmania spp. and is

transmitted by the bite of certain species of sandfly (6). Theburden of leishmaniasis is 2 million new cases annually and 14million infected people worldwide (www.who.int/gb/ebwha/pdf_files/EB118/B118_4-en.pdf. Accessed on 19th December2008). Generally, Leishmania species exist as flagellated ex-tracellular promastigotes in the alimentary tract of the sandflyvector (39) and as aflagellar intracellular amastigotes withinthe phagolysosomes of the macrophages of their vertebratehosts (11). In the gut of the female Phlebotomine sandflies, thepromastigotes go through various morphological stages.During infection, the complement pathway triggers pro-grammed cell death in non-infective procyclics, while the in-fective metacyclics escape the assault, get themselves attachedto the surface of the macrophages, and are phagocytosed (62),whereupon they transform into aflagellated amastigotes. Theprocyclic to metacyclic differentiation is associated with in-creased infectivity to the mammalian host, where the parasiteinvades and multiplies in specialized phagocytic cells like

macrophages and thus has to resist the microbiostatic andmicrobicidal mechanisms, for example, oxidative burst, forsuccessful colonization. However, unlike most eukaryotes,Leishmania lack catalase and selenium-containing glutathioneperoxidase (GPX), enzymes capable of rapidly metabolizinghydrogen peroxide (H2O2). Nevertheless, Leishmania areequipped with a robust trypanothione-dependent H2O2-detoxifying system (10). But, interestingly, detoxification ofH2O2 by amastigotes is markedly attenuated by aminotriazoleand sodium azide, which are well-known inhibitors ofheme-containing enzymes like catalase or peroxidase (12).Throughout this review, the ascorbate peroxidase fromLeishmania major (LmAPX) will be presented from a structure–function point of view, emphasizing on its contribution to theredox homeostasis of the parasite.

Redox Homeostasis in Leishmania

Reactive oxygen species (ROS) in Leishmania are generatedby means of cellular metabolism, uncoupled electron transferin mitochondria, drug metabolism, exogenous agents, etc.Right after phagocytosis, as the first line of defense, the

Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, Kolkata, India.

ANTIOXIDANTS & REDOX SIGNALINGVolume 19, Number 7, 2013ª Mary Ann Liebert, Inc.DOI: 10.1089/ars.2012.4745

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macrophage produces ROS, like superoxide anion (O2� - ) and

H2O2, with the help of NADPH oxidase, a phenomenonknown as oxidative burst. Once the infection is established,the proinflammatory cytokines like tumor necrosis factor-a(TNF-a) and interferon-c activate the macrophage to producereactive nitrogen species (RNS) by upregulating induciblenitric oxide synthase expression (25). Also, the production ofhypohalide by myeloperoxidase or eosinophil peroxidase andperoxynitrite (RNS) as secondary oxidants is observed as aresult of oxidative burst (21). Small (physiological) amounts ofROS are a cellular requirement, because they are involved insignaling pathways, inducing and regulating a variety ofcellular activities for example, growth, differentiation, andgene expression (32). However, excessive ROS have the po-tential to induce significant biological damage and, hence,cells possess many antioxidant systems for maintaining theROS threshold at physiological concentrations (Fig. 1). Oxi-dative stress can occur when ROS production is increased orwhen the mechanisms involved in maintaining the normalreductive cellular milieu are impaired. Redox active thiolgroups in proteins, superoxide dismutase (SOD), catalase,and low-molecular mass compounds, such as ascorbic acid,tocopherol, thiols including glutathione, and uric acid playkey roles as redox buffers that balance any disturbance of theintracellular redox state (43, 60). Depending on the level andextension of the oxidative stress, the cells may establish short-or long-term adaptive responses (40). The redox homeostasisin Leishmania parasites appears to be efficiently regulatedsince they can successfully withstand the oxidative burstduring host infection and perfectly adapt to the differentmetabolic and environmental conditions imposed by theirdigenetic life cycle (40). The Leishmania are equipped withSOD, tryparedoxin (TXN), TXN-dependent peroxidases andascorbate peroxidase (APX) to maintain cellular redox equi-librium (3, 8, 9, 24, 26, 27, 52, 63). In addition, Leishmaniaproduces significant amounts of low-molecular mass thiolssuch as trypanothione, glutathione, and ovothiol A (2, 16, 24).Of these, only glutathione is present in cells of the host (30, 49).

All the three thiols are directly or indirectly maintained in areduced state by trypanothione reductase (TR) (64). Most ofthe glutathione content in Leishmania is found in the formof trypanothione (N1,N8-bis(glutathionyl) spermidine), aunique thiol consisting of two glutathione molecules joinedby a spermidine linker that is produced by trypanothionesynthetase (51). As in other organisms, in Leishmania also, theenzymes c-glutamylcysteine synthetase and glutathione syn-thetase successively act for the synthesis of glutathione (49).The second order rate constants for reactions with H2O2 haveproved ovothiol A to be functionally less efficient than try-panothione; however, in promastigote stages of L. major, L.Mexicana, and L. aethiopica it can be considered as the principalthiol, particularly in the late logarithmic and stationary phasesof growth (2). Ovothiol A and trypanothione can reduce H2O2

nonenzymatically. The anti-oxidative armamentaria of Leish-mania also include TXN and tryparedoxin peroxidases. TXNsbelong to the thioredoxin-fold family but trypanosomatidslack thioredoxin reductase (58). Unlike typical thioredoxins,which are directly reduced by NADPH-dependent flavor-eductases, Leishmania TXNs are oxidoreductases requiringtrypanothione as the mediator to take up electrons from theflavoprotein TR. Two classes of peroxidases occur in trypa-nosomatids: the non-selenium peroxiredoxin and the cysteineGPX members, which reduce H2O2 using electrons fromTXN (9). Leishmania can synthesize ascorbic acid, which is apowerful antioxidant (4, 68). Very recently, in L. major anascorbate-dependent peroxidase has been detected as a part ofthe organism’s antioxidant defense system. As biochemicaland functional characterization of LmAPX is the subjectmatter of this review, a brief introduction about peroxidases isgiven below.

Peroxidases: An Overview

Peroxidases hold a venerable position in enzymology. Thisis one of the most extensively studied group of enzymes andliterature has been enriched with the reviews and a large

FIG. 1. The key players involved in redoxhomeostasis of Leishmania life cycle.

LEISHMANIA ASCORBATE PEROXIDASE 747

number of basic research articles dating back to the early partof the 19th century. Peroxidases are mainly classified intoheme- and non-heme-containing peroxidases. The nativeheme peroxidases contain a heme prosthetic group, usuallyferriprotoporphyrin IX, with four pyrrole nitrogens bound tothe Fe (III). The fifth coordination position on the proximalside of the heme is usually the imidazole side chain of a his-tidine residue. The sixth coordination position is vacant in thenative enzyme on the distal side of the heme. The distal cavityis the region in which the peroxidase reactions occur. Theperoxidative reaction cycle follows the initial formation ofcompound I followed by compound II formation and regen-eration of native enzyme. Compound I and compound II arethe two and one electron-oxidizing equivalents higher thannative ferric state of peroxidase respectively (21). Hugo The-orell was awarded Nobel Prize in 1955 for the discovery ofhorseradish peroxidase (HRP) compound I. A major break-through was the determination of the X-ray crystal structureof yeast cytochrome c peroxidase (CCP) in the year 1980.These discoveries established both HRP and CCP as modelperoxidases, based on which structure-function studies of otherperoxidases are carried out. Heme peroxidases are distributedthroughout plant and animal kingdom. All currently knownheme-containing peroxidases can be divided in two main su-perfamilies (peroxidase–cyclooxygenase and peroxidase–catalase superfamily) and three families (di-heme peroxidase,dyp-type heme peroxidase, and haloperoxidase) (73). Theperoxidase–catalase superfamily is further divided into threedistinct classes. Class I peroxidases include intracellular per-oxidases of prokaryotic origin-like plant chloroplast and cyto-solic APX, yeast CCP, and gene duplicated catalase-peroxidase.Analyses of the amino acid sequences of catalase-peroxidaseindicate that the double length of the bacterial peroxidases canbe ascribed to gene duplication (66). Class II peroxidases com-prise fungal secretory peroxidases like lignin and manganeseperoxidases from Phanerochaete chrysosporium and the ink capmushroom peroxidase from Coprinus cinereus (21). Examples ofClass III peroxidases are endoplasmic reticulum mediatedclassical secretory plant peroxidases like HRP isoenzyme C.During 1940–1960s a large number of animal peroxidases werecharacterized and their physiological functions revealed. Plantascorbate dependent peroxidase was discovered in 1979 (29).APX from pea is well characterized biochemically and thecrystal structure has been solved in great detail in the year 1995(55). In plants, different isoforms of APX are shown to localizein chloroplast, cytosol, and microbody where they eliminatephotosynthetically generated H2O2 by glutathione/ascorbatecycle and control H2O2-mediated redox signaling (47).

GPXs and vanadium haloperoxidases lack heme in theiractive sites. GPXs share a common catalytic core formed byselenocysteine (Sec), tryptophan, and a glutamine residue.However, in some GPXs, a Cys replaces the Sec residue. Suchsubstitution, characteristic of the so-called nonselenium GPX-like enzymes, confers decreased peroxidase activity to theseproteins (33). Vanadium haloperoxidase contains a vanadateprosthetic group and utilize H2O2 to oxidize a halide ion into areactive electrophilic intermediate (69).

Ascorbate and APX in Leishmania

l-Ascorbic acid or vitamin C acts as a pivotal antioxidantmolecule. It is also used as a cofactor in various enzymatic

reactions. Humans lack the ability to synthesize ascorbate,whereas a vast majority of plants and animals can synthesizeascorbic acid de novo. Although, presence of ascorbate haslong been detected in trypanosomatids (15), only recently thebiosynthetic pathway has been demonstrated in Trypanosomabrucei, T. cruzi, and L. donovani (4, 68). Biosynthesis of ascor-bate in both the kinetoplastids takes place within glycosomalcompartment. Detection of ascorbate-dependent peroxidaseactivity in T. cruzi epimastigote extracts dates back to 1980 (5)and in 2002, an endoplasmic reticulum-located unusual plant-like APX enzyme has been discovered in the organism (67).But, until recently, apart from the Leishmania genome data-base, which predicted the presence of the putative enzyme inthe organism, nothing was known about the enzyme inLeishmania. Our laboratory has reported, for the first time, thepresence of an ascorbate-dependent peroxidase from L. majorin 2005. LmAPX is more related to T. cruzi APX than to plantAPX. In contrast to plant APX, LmAPX lacks arginine 172residue at the ascorbate-binding site.

Physical and Spectral Characteristics of LmAPX

LmAPX primary sequence has the high level of sequenceidentity with both yeast CCP (*35%) and the pea APX(*36%) (70). In silico analysis of LmAPX sequence (TargetPV1.0) predicts (22) that the N-terminal 12 amino acids con-stitute a signal sequence, which is followed by a stretch of 22amino acids containing a hydrophobic region resembling atransmembrane domain (Fig. 2A). All the key residues onboth distal and proximal sites of the heme are conservedamong LmAPX, yeast CCP, and plant APX. However, it lacksboth the cytochrome c-binding residues (Asp34/Glu35,Tyr39, and Glu290 in yeast CCP) and critical ascorbate-binding residue (Arg 172 in pea APX) (7, 56, 59). The proximalcation (K + ) binding loop of the LmAPX is more similar to peaAPX, whereas the c-terminal insertion and proximal Trpradical stabilizing Met residues of LmAPX are more similar to

FIG. 2. Physical characteristics of LmAPX. (A) Shows thedifference between premature and mature protein. (B) Rep-resents the UV-visible spectra of ascorbate-free LmAPX be-fore (solid line) and after addition of equimolar H2O2 (dottedline), and after addition of 5 lM ascorbate (dashed line).H2O2, hydrogen peroxide; LmAPX, ascorbate peroxidasefrom Leishmania major.

748 ADAK AND PAL

CCP (35, 70). The gel filtration results indicate that D34LmAPX (34 amino acids deleted from the N-terminus se-quence of LmAPX) is a monomeric enzyme with a molecularweight of *33 kDa (1). The UV-visible spectra of ascorbate-free D34 LmAPX shows the Soret peak at 408 nm with chargetransfer peaks at around 500 and 640 nm (Fig. 2B). The cal-culated purity number, Rz (A408/A280), for D34 LmAPX is 0.98(Fig. 2B). Addition of an equimolar H2O2 to resting state ofD34 LmAPX produces compound I* within 0.75 ms absorbingat 420 nm at Soret region with visible peaks at 532 and 560 nm.Extensive spectroscopic studies have demonstrated that theSoret band of compound I* is similar to an oxyferryl trypto-phan cation radical of yeast CCP compound I species (23).

Catalytic Mechanism of LmAPX

Based on previous yeast CCP, plant APX, and currentLmAPX results (35, 54, 70, 72), a plausible mechanism for theredox reaction catalyzed by LmAPX may be suggested asshown in Figure 3. In the presence of low-molecular mass(micromolecule) electron donors like ascorbate or guaiacol (2-methoxyphenol), the reaction of H2O2 with the resting form ofLmAPX (FeIII,P,Trp) leads to the formation of CMPI (FeIV =

O,P� + ,Trp), which contains an oxyferryl heme FeIV = O and aporphyrin p-cation radical. Low-molecular mass electrondonor initially reduces p-cation radical of CMPI (FeIV =O,P� + ,Trp) to produce compound II (FeIV = O,P,Trp), whichcontains an oxyferryl heme FeIV = O. A second molecule of theelectron donor then reduces the oxyferryl heme in CMPII(FeIV = O,P,Trp) to form the resting enzyme (FeIII,P,Trp).However, in the presence of cytochrome c, the situation isaltered. Indeed, one of the curious features of LmAPX is that,in contrast to plant APX, its CMPI (FeIV = O,P� + ,Trp) is mostprobably comparatively unstable and, it leads to the genera-tion of another species CMPI* (FeIV = O,P,Trp� + ), which con-tains an oxyferryl heme FeIV = O and a Trp� + radical cationlocated on the indole ring of Trp-208. Ferrocytochrome c ini-tially reduces the Trp-208 radical cation in CMPI* (FeIV =O,P,Trp� + ) to produce compound II (FeIV = O,P,Trp). By in-ternal electron transfer in CMPII (FeIV = O,P,Trp) the Trpradical cation in CMPII* (FeIII,P,Trp� + ) is regenerated, whichis then reduced by cytochrome c to form the native en-zyme (48).

Pseudocatalase Activity of LmAPX

In addition to peroxidase activity of lactoperoxidase andthyroid peroxidase, several reports have shown that in case ofiodide as electron donor, the intermediate enzyme withbound hypoiodous acid reacts with a second molecule ofH2O2 in a catalase-like reaction, with liberation of O2 (34, 44,45). The pseudohalide, SCN - , cannot replace I - as a promoterof pseudocatalase activity, even though SCN - is readily oxi-dized by thyroid peroxidase or lactoperoxidase in presence ofH2O2 (34, 44, 45). In this case, H2O2 is utilized solely for theoxidation of SCN - , in contrast to the catalytic cleavage ofH2O2, which occurs in the presence of low I - concentration(34, 44, 45). Like well known peroxidases, LmAPX catalyzesthe degradation of H2O2 with concomitant formation of O2

under low ascorbate concentration at physiological pH (18).The reaction is initiated by its peroxidase activity with thegeneration of ascorbate-free radicals via one-electron-transfermechanism and then regeneration of ascorbate by reductionof ascorbate radical with H2O2. The requirement of enzyme,H2O2 and ascorbate indicates that monodehydroascorbateplays a vital role in free radical mediated H2O2 consump-tion, rather than the disproportionation reaction of the freeradicals.

Structural Investigation of the CatalyticDomain of LmAPX

The structure of the distal pocket, proximal pocket, and thecation-binding loop in LmAPX, yeast CCP, and plant pea APXis shown in Figure 4. The 1.76 A resolution of LmAPX struc-ture demonstrates 10 alpha helical bundle folds, characteristicof peroxidases (35). The distal Arg-Trp-His and proximal His-Asp-Trp arrangements in LmAPX are typical of Class I per-oxidases, which include CCP and APX. The electron densityof LmAPX shows two cations, a proximal K + that is visible upto 20 r in the Fo-Fc difference map and a distal Ca2 + visible upto 26 r in the same map (35). As expected from sequencealignments, LmAPX can be considered as a hybrid peroxidasethat shares structural features with various classes of hemeperoxidases. Like class III peroxidases such as HRP, LmAPXcontains two cation-binding sites. The following structural

FIG. 3. Universal model for the macro- and micro-molecule oxidation by heme peroxidase. A common he-meferryl protoporphyrin p-cation radical ( + �P FeIV = O Trp)intermediate can react with a micromolecule (RH). If theelectron is transferred from the proximal tryptophan to theferryl protoporphyrin p-cation radical, a tryptophan radical(P FeIV = O Trp + �) is generated, that can react with themacromolecule ferrocytochrome c (Cyt CII). This ensures theroute of electron transfer from the ferrocytochrome c to hemeiron via proximal tryptophan residue. RH, FeIV = O, P, Trpindicate low-molecular mass electron donors, ferryl hemeiron, porphyrin, and proximal tryptophan residue respec-tively.


features are similar between yeast CCP and LmAPX (35): (i)presence of the triple beta strand, (ii) the proximal Met resi-dues (Met248 and Met249) that are responsible for formationof the stable proximal Trp208 radical (35, 72), and (iii) a stretchof acidic residues (D225, E226, and D227) before ac-terminal sequence insertion (228–240 aa), one of them (E226)is responsible for proper maintenance of active site confor-mation (71). Again, some plant APX-like structural elements,present in LmAPX are: (i) the insert region between A and Bhelices, which provides contacts with cytochrome c in theCCP–cytochrome c complex (56), is much shorter in LmAPXand forms part of the ascorbate binding pocket and (ii) themonovalent nature of the proximal cation (35). Analysis of theproximal K + cation-binding loop in LmAPX indicates thatthree main-chain ligands, three side-chain ligands, and onewater ligand are bonded to K + (35). Those seven coordina-tions inside the loop are formed by residues 193 (164 in peaAPX and 176 in yeast CCP) to 218 (189 in pea APX and 201 inyeast CCP) in LmAPX. In LmAPX, T193 (side chain OH, backbone CO), T209 (side chain OH), D211 (back bone CO), G214(back bone CO), and S218 (side chain OH) amino acid resi-dues, and H2O ligand contribute to form coordinate bondswith K + . LmAPX and pea APX differ in coordination bondformation: (i) H2O molecule and D211 residue in LmAPXform one coordinate bond each with K + , whereas the corre-sponding residue N182 in pea APX forms two coordinate

bonds. (ii) One coordinate bond is formed by S218 in LmAPXbut the corresponding residue S189 in pea APX does notform coordinate bond. Instead, D187 residue in pea APXtakes part in coordinate bonding. However, LmAPX hasPhe201 residue in the ascorbate-binding site instead of Argresidue (Arg172 in plant pea APX) (56, 59), which mayaccount for the lower APX activity of LmAPX, 32 min - 1

compared with *250 s - 1 for plant APX (42). The mainstructural dissimilarity between LmAPX and CCP involvesthe presence of Cys197 in LmAPX, whereas the analogousresidue in CCP is Thr180. Mutational and electron para-magnetic resonance studies suggest that this major structuraldifference plays a key role in LmAPX for stabilization ofproximal Trp radical in the presence of K + ion (35). Otherreports reveal that in plant APX and LmAPX, the K + -bindingresidues function in maintaining the protein structure in theheme vicinity to favor the enzymatic activity by controllingthe specific structural conformation of the proximal and distalhistidines (14, 53). Recently, a model of the L. major APX–cytochrome c complex, using the yeast system as a guide,suggests that its complex is very close to the yeast CCP–cytochrome c crystal structure (36). KM and the efficiency(kcat/KM) of LmAPX for L. major cytochrome c are 6 lM and1.6 · 108 M - 1 s - 1, respectively, similar to those exhibited byCCP in the yeast system (36, 38). It has been suggested that thebiological function of LmAPX is to act as a CCP (36).

FIG. 4. X-ray crystal structure of LmAPX, pea APX, and yeast CCP. The PDB coordinates were taken from publishedLmAPX (PDB entry code: 3RIV), pea APX (PDB entry code: 1APX), and yeast CCP (PDB entry code: 2CCP). (A–C) Showedcartoon structures of LmAPX, yeast CCP, and pea APX respectively, where the heme group is superimposed. (D–F) Showsthe active site residues of LmAPX, yeast CCP, and pea APX respectively. Structures were illustrated by using PyMOL (17)software. The alternative designation of LmAPX is LmP (peroxidase from L. major). APX, ascorbate peroxidase; CCP,cytochrome c peroxidase; PDB, protein data bank.

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Localization and Regulation of LmAPX

Localization and regulation of a protein provides vital in-formation for a group of many co-operating proteins, whichare responsible for an integrated physiological function. Thus,exact information regarding the localization of LmAPX will besupportive for predicting the possible physiological functionof this protein. TargetP 1.1 Server predicts that the N-terminalsignal sequence of LmAPX is a mitochondrial targeting signal.Subcellular fractionation, Western-blot analysis, and confocalmicroscopy suggest that the enzyme is localized in the mito-chondrion of Leishmania (19). Mitoplast (mitochondria with-out outer membrane) preparation by treatment of digitonin,sub-mitochondrial fractionation, and activity measurement ofkynurenine hydroxylase (outer membrane marker), succinatedehydrogenase (inner membrane marker), and malate dehy-drogenase (matrix marker) enzymes have revealed thatLmAPX is localized in the inner membrane of the mitochon-dria with its catalytic domain in the intermembrane space(19), which is similar to the location of CCP in yeast (13, 28,37). Therefore, LmAPX is more likely to be accessible toscavenge endogenous ROS produced in the mitochondrialinter membrane space. Although, chloroplasts and peroxi-somes in plants are the main organelles in terms of both ROSformation and breakdown (50), yet intermembrane space ofmitochondria is clearly the major site of ROS generation inyeast and animal systems. The expression of yeast CCP geneand plant APX gene was also increased on treatment withvarious oxidative stressors (41, 61). Similarly, upregulation ofLmAPX gene expression by H2O2 suggests that the para-site sequentially uses this enzyme to overcome oxidativestress (19).

Physiological Function of LmAPX

In a wide variety of species, ranging from bacteria to highermammals, the mechanisms of H2O2-detoxification are grosslydistributed into variations of two mechanisms: mediated ei-ther by the heme proteins and/or by non-heme proteinslike selenium-containing GPX. Detailed cell biological ap-proaches have successfully established the biochemistry andphysiological significance of LmAPX enzyme in Leishmaniapromastigotes (see working model on Fig. 5) (19, 20, 52).Overexpression of LmAPX in L. major protects promastigotecells against oxidative stress-induced apoptosis (19, 20).Clearly, knocking the gene out of the cell has thrown theparasite under a constant level of oxidative stress as evi-denced by the higher intracellular H2O2 content (52). Thisbasal level of oxidative stress acts as the driving force for theearly onset of metacyclogenesis producing infective meta-cyclic promastigotes from non-infective procyclic ones. Un-less favorable condition (high temperature and low pH,

characteristic of the condition prevalent in the mammalianhost) is reached, the metacyclics proceed toward apoptosis; inthis respect, metacyclogenesis is comparable to terminal dif-ferentiation. The infectious parasite population in the midgutof infected sandfly or in vitro stationary phase cultures containboth metacyclic and apoptotic parasites and a combination ofthese two types of cells are needed for infectivity of the par-asite within host macrophages (65). In accordance with this,deprived of LmAPX, the stationary phase culture contains ahigher number of metacyclic and apoptotic parasites com-pared with both wild-type and LmAPXverexpressing cells(52). The apoptotic cells suppress macrophage activation bydownregulating the inflammatory cytokines like TNF-a andupregulating the anti-inflammatory cytokines like trans-forming growth factor-b, while the virulent metacyclics pro-ceed to invade macrophages and cause disease. These dataestablish vital contributions of LmAPX: (i) the enzyme has adirect role in effective detoxification of ROS, which is signif-icant enough considering the absence of any other potent ROSscavenging heme enzyme in Leishmania. (ii) The indirect effectof the enzyme was revealed on creation of the knock outmutant, where its absence enhances cellular differentiationand virulence mediated by oxidative stress. Generally, at-tention is given to the genes whose loss leads to decreasedvirulence, pathogenicity, or infectivity. LmAPX represents anew class of genes whose deletion helps in the infectivity ofthe parasite. Thus, the enzyme possesses great evolutionarysignificance, being effectively utilized by the parasite at dif-ferent conditions, which is not surprising, considering thehigh degree of adaptability of the phylum, protozoa. Hence,our research presents an ideal model system to study parasitevirulence.

Potential of the Defense System InvolvingLmAPX in Oxidative Stress

Like yeast CCP, one of the electron donors of LmAPX wasfound to be ferrocytochrome c (18), which is co-localized atintermembrane space side of the inner membrane. The factthat cytochrome c is a much better substrate than ascorbate,as observed from the kcat values for the reaction of thesemolecules with LmAPX, suggests that the biological func-tion of the enzyme is similar to yeast CCP (35, 36, 72). SinceLmAPX can oxidize ferrocytochrome c in presence of en-dogenous H2O2, it can be considered as an excellent candi-date for playing key functions in mitochondria-related redoxcontrol, such as elimination of H2O2 and O2

- �, and resis-tance to mitochondrial ROS. Although the relative contri-bution of mitochondria to ROS production in green plants isvery low (57) yet mitochondria is the major source of ROS innon-photosynthetic organisms (31). The plausible antioxi-dant role of mitochondrial LmAPX in Leishmania (catalaseabsent) is illustrated in Figure 6. The processes possibly in-volved in the LmAPX-linked defense mechanism are indi-cated in this model. LmAPX could detoxify H2O2 in thepresence of reduced cytochrome c. The reduction of cyto-chrome c could occur either by NADH-cytochrome b5 re-ductase-cytochrome b5 pathway or O2

- �, which wasgenerated by complexes I and III of the respiratory chain.Thus, the cytochrome c-LmAPX system appears to play animportant antioxidant role in L. major cells by scavengingboth H2O2 and O2

- �.

FIG. 5. Physiological functions of LmAPX in Leishmaniapromastigotes. J represents mitochondrial membranepotential.


Conclusion

Living cells require ROS for their normal growth and pro-liferation and at the same time must possess appropriatescavenging system to get rid of the excess ROS, which, oth-erwise, might be detrimental. So, there is a constant strugglefor maintaining the balance, leading to a variety of adapta-tions, honed through evolution, which is evident from theredundancy of antioxidant systems as well as intelligent useof a single protein in diverse physiological processes. Single-celled trypanosomatids are adroit adaptors, which is not un-expected considering their digenetic life cycle, exposing themto very different environments. Structural and functionalanalysis of LmAPX has pointed to its novel evolutionaryconsequence as a hybrid of APX and CCP (35, 70, 72). LmAPXis located in the intermembrane space side of inner mito-chondrial membrane, where cytochrome c is present (19). Thehigh in vitro oxidation rate for cytochrome c (35, 36, 72) sug-gests that this molecule likely is the probable physiologicalsubstrate of the enzyme. Also, the enzyme has been shown tohave pseudocatalase activity (18) and its role in protection ofthe cell against oxidative stress mediated cardiolipin oxida-tion, extensive protein damage, and apoptosis (19, 20, 52) hasalso been established. It is also possible that the loss ofLmAPX activity has secondary effects on leishmanial geneexpression caused by an alteration in the redox balance in theprotozoa. As a consequence, modulation of surface lipopho-sphoglycan (LPG) of L. major (an approximate doubling inlength of LPG and capping of majority of the galactose sugarswith arabinose residues) and simultaneous differentiation ofthe promastigotes into infective metacyclic form may occur(46, 52). Thus, in L. major APX acts to protect the cells fromROS-mediated apoptosis and may have a major role in cel-

lular differentiation. Future works will more precisely deter-mine the signaling pathways involved in the processes.

Acknowledgments

This work was supported by the Council of Scientific andIndustrial Research (CSIR) Network Project NWP 0038. Dr.Swati Pal is supported by an individual fellowship from theCSIR, Government of India.

References

1. Adak S and Datta AK. Leishmania major encodes an unusualperoxidase that is a close homologue of plant ascorbateperoxidase: a novel role of the transmembrane domain.Biochem J 390: 465–474, 2005.

2. Ariyanayagam MR and Fairlamb AH. Ovothiol and trypa-nothione as antioxidants in trypanosomatids. Mol BiochemParasitol 115: 189–198, 2001.

3. Barr SD and Gedamu L. Cloning and characterization ofthree differentially expressed peroxidoxin genes fromLeishmania chagasi. Evidence for an enzymatic detoxificationof hydroxyl radicals. J Biol Chem 276: 34279–34287, 2001.

4. Biyani N and Madhubala R. Leishmania donovani encodes afunctional enzyme involved in vitamin C biosynthesis:arabino-1,4-lactone oxidase. Mol Biochem Parasitol 180: 76–85,2011.

5. Boveris A, Sies H, Martino EE, Docampo R, Turrens JF, andStoppani AO. Deficient metabolic utilization of hydro-gen peroxide in Trypanosoma cruzi. Biochem J 188: 643–648,1980.

6. Bray RS. Leishmaniasis. Trop Dis Bull 66: 501–507, 1969.7. Bursey EH and Poulos TL. Two substrate binding sites in

ascorbate peroxidase: the role of arginine 172. Biochemistry39: 7374–7379, 2000.

8. Castro H, Romao S, Carvalho S, Teixeira F, Sousa C, andTomas AM. Mitochondrial redox metabolism in trypanoso-matids is independent of tryparedoxin activity. PLoS One 5:e12607, 2010.

9. Castro H, Sousa C, Novais M, Santos M, Budde H, Cordeiro-da-Silva A, Flohe L, and Tomas AM. Two linked genes ofLeishmania infantum encode tryparedoxins localised to cyto-sol and mitochondrion. Mol Biochem Parasitol 136: 137–147,2004.

10. Castro H and Tomas AM. Peroxidases of trypanosomatids.Antioxid Redox Signal 10: 1593–1606, 2008.

11. Chang KP. Leishmania donovani: promastigote—macrophagesurface interactions in vitro. Exp Parasitol 48: 175–189, 1979.

12. Channon JY and Blackwell JM. A study of the sensitivity ofLeishmania donovani promastigotes and amastigotes to hy-drogen peroxide. II. Possible mechanisms involved in pro-tective H2O2 scavenging. Parasitology 91 (Pt 2): 207–217,1985.

13. Charizanis C, Juhnke H, Krems B, and Entian KD. The mi-tochondrial cytochrome c peroxidase Ccp1 of Saccharomycescerevisiae is involved in conveying an oxidative stress signalto the transcription factor Pos9 (Skn7). Mol Gen Genet 262:437–447, 1999.

14. Cheek J, Mandelman D, Poulos TL, and Dawson JH. A studyof the K( + )-site mutant of ascorbate peroxidase: mutationsof protein residues on the proximal side of the heme causechanges in iron ligation on the distal side. J Biol Inorg Chem 4:64–72, 1999.

15. Clark D, Albrecht M, and Arevalo J. Ascorbate variationsand dehydroascorbate reductase activity in Trypanosoma

FIG. 6. The predicted schematic presentation for theH2O2 and O2

�2 scavenging role of mitochondrial LmAPXin Leishmania promastigotes. Electrons move from NADHto O2 via a series of redox reactions in the mitochondrialelectron transport chain. Three proton pumps (I, III, and IV)create a proton gradient across the mitochondrial innermembrane for synthesizing ATP via ATP synthase. A smallpercentage of electrons directly leak to O2 without comple-tion of the whole series, resulting in the generation of O2

2�.Fb5, b5, I, III, IV, Cytc3 + , and Cytc2 + denoted cytochrome b5reductase, cytochrome b5, NADH-CoQ reductase, CoQH2-cytochrome c reductase, cytochrome c oxidase, oxidized cy-tochrome c, and reduced cytochrome c, respectively.

752 ADAK AND PAL

cruzi epimastigotes and trypomastigotes. Mol Biochem Para-sitol 66: 143–145, 1994.

16. Colotti G and Ilari A. Polyamine metabolism in Leishmania:from arginine to trypanothione. Amino Acids 40: 269–285,2011.

17. DeLano WL. (2002) The PyMOL molecular graphics system.www.pymol.org/ (last accessed date 30 Nov, 2007).

18. Dolai S, Yadav RK, Datta AK, and Adak S. Effect of thio-cyanate on the peroxidase and pseudocatalase activities ofLeishmania major ascorbate peroxidase. Biochim Biophys Acta1770: 247–256, 2007.

19. Dolai S, Yadav RK, Pal S, and Adak S. Leishmania majorascorbate peroxidase overexpression protects cells againstreactive oxygen species-mediated cardiolipin oxidation. FreeRadic Biol Med 45: 1520–1529, 2008.

20. Dolai S, Yadav RK, Pal S, and Adak S. Overexpression ofmitochondrial Leishmania major ascorbate peroxidase en-hances tolerance to oxidative stress-induced programmedcell death and protein damage. Eukaryot Cell 8: 1721–1731,2009.

21. Dunford HB. Heme Peroxidases, New York: John Wiley &Sons, Inc. 1999.

22. Emanuelsson O, Nielsen H, Brunak S, and von Heijne G.Predicting subcellular localization of proteins based on theirN-terminal amino acid sequence. J Mol Biol 300: 1005–1016,2000.

23. Erman JE, Vitello LB, Mauro JM, and Kraut J. Detection of anoxyferryl porphyrin pi-cation-radical intermediate in thereaction between hydrogen peroxide and a mutant yeastcytochrome c peroxidase. Evidence for tryptophan-191 in-volvement in the radical site of compound I. Biochemistry 28:7992–7995, 1989.

24. Fairlamb AH, Blackburn P, Ulrich P, Chait BT, and CeramiA. Trypanothione: a novel bis(glutathionyl)spermidine co-factor for glutathione reductase in trypanosomatids. Science227: 1485–1487, 1985.

25. Gantt KR, Goldman TL, McCormick ML, Miller MA, Jer-onimo SM, Nascimento ET, Britigan BE, and Wilson ME.Oxidative responses of human and murine macrophagesduring phagocytosis of Leishmania chagasi. J Immunol 167:893–901, 2001.

26. Getachew F and Gedamu L. Leishmania donovani iron su-peroxide dismutase A is targeted to the mitochondria by itsN-terminal positively charged amino acids. Mol BiochemParasitol 154: 62–69, 2007.

27. Ghosh S, Goswami S, and Adhya S. Role of superoxidedismutase in survival of Leishmania within the macrophage.Biochem J 369: 447–452, 2003.

28. Goltz S, Kaput J, and Blobel G. Isolation of the yeast nucleargene encoding the mitochondrial protein, cytochrome cperoxidase. J Biol Chem 257: 11186–11190, 1982.

29. Groden D and Beck E. H2O2 destruction by ascorbate-dependent systems from chloroplasts. Biochim Biophys Acta546: 426–435, 1979.

30. Grondin K, Haimeur A, Mukhopadhyay R, Rosen BP, andOuellette M. Co-amplification of the gamma-glutamylcys-teine synthetase gene gsh1 and of the ABC transporter genepgpA in arsenite-resistant Leishmania tarentolae. EMBO J 16:3057–3065, 1997.

31. Halliwell B and Gutteridge JMC. Free Radicals in Biology andMedicine, 2nd edn. Clarendon: Oxford, 1989.

32. Hensley K, Robinson KA, Gabbita SP, Salsman S, and FloydRA. Reactive oxygen species, cell signaling, and cell injury.Free Radic Biol Med 28: 1456–1462, 2000.

33. Herbette S, Roeckel-Drevet P, and Drevet JR. Seleno-independent glutathione peroxidases. More than simpleantioxidant scavengers. FEBS J 274: 2163–2180, 2007.

34. Huwiler M and Kohler H. Pseudo-catalytic degradation ofhydrogen peroxide in the lactoperoxidase/H2O2/iodidesystem. Eur J Biochem 141: 69–74, 1984.

35. Jasion VS, Polanco JA, Meharenna YT, Li H, and Poulos TL.Crystal structure of Leishmania major peroxidase and char-acterization of the compound I Tryptophan radical. J BiolChem 286: 24608–24615, 2011.

36. Jasion VS and Poulos TL. Leishmania major peroxidase is acytochrome c peroxidase. Biochemistry 51: 2453–2460, 2012.

37. Jiang H and English AM. Phenotypic analysis of theccp1Delta and ccp1Delta-ccp1W191F mutant strains of Sac-charomyces cerevisiae indicates that cytochrome c peroxidasefunctions in oxidative-stress signaling. J Inorg Biochem 100:1996–2008, 2006.

38. Kang CH, Ferguson-Miller S, and Margoliash E. Steady statekinetics and binding of eukaryotic cytochromes c with yeastcytochrome c peroxidase. J Biol Chem 252: 919–926, 1977.

39. Killick-Kendrick R, Molyneux DH, Hommel M, Leaney AJ,and Robertson ES. Leishmania in phlebotomid sandflies. V.The nature and significance of infections of the pylorus andileum of the sandfly by leishmaniae of the braziliensiscomplex. Proc R Soc Lond B Biol Sci 198: 191–199, 1977.

40. Krauth-Siegel RL and Comini MA. Redox control in trypa-nosomatids, parasitic protozoa with trypanothione-basedthiol metabolism. Biochim Biophys Acta 1780: 1236–1248, 2008.

41. Kwon M, Chong S, Han S, and Kim K. Oxidative stresseselevate the expression of cytochrome c peroxidase in Sac-charomyces cerevisiae. Biochim Biophys Acta 1623: 1–5, 2003.

42. Lad L, Mewies M, and Raven EL. Substrate binding andcatalytic mechanism in ascorbate peroxidase: evidence fortwo ascorbate binding sites. Biochemistry 41: 13774–13781, 2002.

43. Lillig CH and Holmgren A. Thioredoxin and related mole-cules—from biology to health and disease. Antioxid RedoxSignal 9: 25–47, 2007.

44. Magnusson RP and Taurog A. Iodide-dependent catalaticactivity of thyroid peroxidase and lactoperoxidase. BiochemBiophys Res Commun 112: 475–481, 1983.

45. Magnusson RP, Taurog A, and Dorris ML. Mechanism ofiodide-dependent catalatic activity of thyroid peroxidaseand lactoperoxidase. J Biol Chem 259: 197–205, 1984.

46. McConville MJ, Turco SJ, Ferguson MA, and Sacks DL.Developmental modification of lipophosphoglycan duringthe differentiation of Leishmania major promastigotes to aninfectious stage. EMBO J 11: 3593–3600, 1992.

47. Miller G, Suzuki N, Rizhsky L, Hegie A, Koussevitzky S, andMittler R. Double mutants deficient in cytosolic and thyla-koid ascorbate peroxidase reveal a complex mode of inter-action between reactive oxygen species, plant development,and response to abiotic stresses. Plant Physiol 144: 1777–1785,2007.

48. Miller MA, Vitello L, and Erman JE. Regulation of inter-protein electron transfer by Trp 191 of cytochrome c perox-idase. Biochemistry 34: 12048–12058, 1995.

49. Mukherjee A, Roy G, Guimond C, and Ouellette M. Thegamma-glutamylcysteine synthetase gene of Leishmania isessential and involved in response to oxidants. Mol Microbiol74: 914–927, 2009.

50. Noctor G, Veljovic-Jovanovic S, and Foyer CH. Peroxideprocessing in photosynthesis: antioxidant coupling and re-dox signalling. Philos Trans R Soc Lond B Biol Sci 355: 1465–1475, 2000.


51. Oza SL, Shaw MP, Wyllie S, and Fairlamb AH. Trypa-nothione biosynthesis in Leishmania major. Mol BiochemParasitol 139: 107–116, 2005.

52. Pal S, Dolai S, Yadav RK, and Adak S. Ascorbate peroxidasefrom Leishmania major controls the virulence of infectivestage of promastigotes by regulating oxidative stress. PLoSOne 5: e11271, 2010.

53. Pal S, Yadav RK, and Adak S. Role of K + binding residuesin stabilization of heme spin state of Leishmania major per-oxidase. Biochim Biophys Acta 1824: 1002–1007, 2012.

54. Pappa H, Patterson WR, and Poulos TL. The homologoustryptophan critical for cytochrome c peroxidase function isnot essential for ascorbate peroxidase activity. J Biol InorgChem 1: 61–66, 1996.

55. Patterson WR and Poulos TL. Crystal structure of recombi-nant pea cytosolic ascorbate peroxidase. Biochemistry 34:4331–4341, 1995.

56. Pelletier H and Kraut J. Crystal structure of a complex be-tween electron transfer partners, cytochrome c peroxidaseand cytochrome c. Science 258: 1748–1755, 1992.

57. Purvis A. Role of the alternative oxidase in limiting super-oxide production by plant mitochondria. Physiol Plant 100:165–170, 1997.

58. Romao S, Castro H, Sousa C, Carvalho S, and Tomas AM.The cytosolic tryparedoxin of Leishmania infantum is essentialfor parasite survival. Int J Parasitol 39: 703–711, 2009.

59. Sharp KH, Mewies M, Moody PC, and Raven EL. Crystalstructure of the ascorbate peroxidase-ascorbate complex. NatStruct Biol 10: 303–307, 2003.

60. Shelton MD, Chock PB, and Mieyal JJ. Glutaredoxin: role inreversible protein s-glutathionylation and regulation of re-dox signal transduction and protein translocation. AntioxidRedox Signal 7: 348–366, 2005.

61. Shigeoka S, Ishikawa T, Tamoi M, Miyagawa Y, Takeda T,Yabuta Y, and Yoshimura K. Regulation and function ofascorbate peroxidase isoenzymes. J Exp Bot 53: 1305–1319,2002.

62. Spath GF, Garraway LA, Turco SJ, and Beverley SM. Therole(s) of lipophosphoglycan (LPG) in the establishment ofLeishmania major infections in mammalian hosts. Proc NatlAcad Sci U S A 100: 9536–9541, 2003.

63. Spies HS and Steenkamp DJ. Thiols of intracellular patho-gens. Identification of ovothiol A in Leishmania donovani andstructural analysis of a novel thiol from Mycobacterium bovis.Eur J Biochem 224: 203–213, 1994.

64. Taylor MC, Kelly JM, Fairlamb AH, and Miles MA. Thetrypanothione reductase gene of Leishmania donovani. Bio-chem Soc Trans 18: 869–870, 1990.

65. van Zandbergen G, Bollinger A, Wenzel A, Kamhawi S, VollR, Klinger M, Muller A, Holscher C, Herrmann M, Sacks D,Solbach W, and Laskay T. Leishmania disease developmentdepends on the presence of apoptotic promastigotes in thevirulent inoculum. Proc Natl Acad Sci U S A 103: 13837–13842, 2006.

66. Welinder KG. Bacterial catalase-peroxidases are geneduplicated members of the plant peroxidase superfamily.Biochim Biophys Acta 1080: 215–220, 1991.

67. Wilkinson SR, Obado SO, Mauricio IL, and Kelly JM. Try-panosoma cruzi expresses a plant-like ascorbate-dependent

hemoperoxidase localized to the endoplasmic reticulum.Proc Natl Acad Sci U S A 99: 13453–13458, 2002.

68. Wilkinson SR, Prathalingam SR, Taylor MC, Horn D, andKelly JM. Vitamin C biosynthesis in trypanosomes: a role forthe glycosome. Proc Natl Acad Sci U S A 102: 11645–11650,2005.

69. Winter JM and Moore BS. Exploring the chemistry and bi-ology of vanadium-dependent haloperoxidases. J Biol Chem284: 18577–18581, 2009.

70. Yadav RK, Dolai S, Pal S, and Adak S. Role of tryptophan-208 residue in cytochrome c oxidation by ascorbate peroxi-dase from Leishmania major-kinetic studies on Trp208Phemutant and wild type enzyme. Biochim Biophys Acta 1784:863–871, 2008.

71. Yadav RK, Dolai S, Pal S, and Adak S. Role of C-terminalacidic cluster in stabilization of heme spin state of ascorbateperoxidase from Leishmania major. Arch Biochem Biophys 495:129–135, 2010.

72. Yadav RK, Pal S, Dolai S, and Adak S. Role of proximalmethionine residues in Leishmania major peroxidase. ArchBiochem Biophys 515: 21–27, 2011.

73. Za’mocky M and Obinger C. Molecular phylogeny of hemeperoxidases. In: Biocatalysis Based on Heme Peroxidases, editedby Torres E, Ayala M. Heidelberg: Springer-Verlag Berlin,2010, pp. 7–35, 2010.

Address correspondence to:Dr. Subrata Adak

Division of Structural Biology and Bio-informaticsCSIR-Indian Institute of Chemical Biology

4, Raja S.C. Mullick RoadKolkata 700032

India

E-mail: [email protected]

Date of first submission to ARS Central, June 8, 2012; date ofacceptance, June 15, 2012.

Abbreviations Used

APX¼ ascorbate peroxidaseCCP¼ cytochrome c peroxidase

H2O2¼hydrogen peroxideHRP¼horseradish peroxidase

LmAPX¼ ascorbate peroxidase from L. majorLPG¼ lipophosphoglycanO2�-¼ superoxide anion

PDB¼protein data bankRNS¼ reactive nitrogen speciesROS¼ reactive oxygen species

Sec¼ selenocysteineSOD¼ superoxide dismutase

TNF-a¼ tumor necrosis factor-aTR¼ trypanothione reductase

TXN¼ tryparedoxin

754 ADAK AND PAL

Documents

Ascorbate Peroxidase Acts As a Novel Determiner of Redox Homeostasis in Leishmania