Characterization of plasma membrane bound inorganic pyrophosphatase from Leishmania donovani promastigotes and amastigotes

Abstract

Background

Currently, a major problem in the management of visceral leishmaniasis or kala-azar, especially in the Indian subcontinent, is the growing unresponsiveness to conventional antimonial therapy. Membrane bound pyrophophatase (PPases) do not exist in plasma membrane from mammals. Thus, H⁺-PPases from Leishmania plasma membrane might be potential target in rational chemotherapy of the disease caused by Leishmania parasites.

Objective

To characterize the activities of inorganic pyrophophatase (PPase) in the plasma membrane of Leishmania donovani promastigote and amastigote.

Methods

Culture method of promastigote and amastigote were developed. We assayed PPase activity in isolated plasma membrane of L. donovani.

Results

We characterized K⁺-PPase present in the plasma membrane of Leishmania donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K⁺ ions and sodium orthovanadate, inhibited by pyrophosphate analogs imidodiphosphate and alendronate, KF, DCCD, thiol reagent parachloromercuribenzenesulfonate (PCMBS), N-ethylmaliemide (NEM), phenylarsineoxide, ABC superfamily transport modulator verapamil and was also by F₁F_o-ATPase inhibitor quercetin.

Conclusion

We conclude that there are significant differences within promastigote, amastigote and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as putative targets for rational drug design.

Introduction

Protozoa parasites are responsible for important diseases that threaten the lives of nearly one-quarter of the human population world-wide. Among them, leishmaniasis has become the a leading cause of death, mainly due to the emergence of parasite resistance to conventional drugs¹. Leishmania donovani, the causative agent of visceral leishmaniasis, encounters a wide range of pH values in its life cycle. The gut of the phlebotamine insect vector is extremely alkaline, whereas promastigotes of L. donovani invade host cells via acidic lysosomes². Mechanisms to cope with this varied environmental pH and maintain cytosolic pH homeostasis might involve the use of proton pumps (H⁺-ATPases and H⁺-PP_i ases) on both plasma membrane and internal membranes. These transport proteins specifically and actively mobilize ions, generating chemical gradients across a membrane. This movement of ions is vital for numerous cellular functions ranging from energy production, motility, nutrient uptake, ionic homeostasis, intracellular signaling, and differentiation, to name a few. Membrane-bound proton-translocating inorganic pyrophosphatases (H⁺-PPase; EC 3.6.1.1) belong to a new category of proton pumps, distinct from F-, P-, and V-ATPases, which utilize pyrophosphate hydrolysis as the driving force for H⁺ movement across the biological membranes³. The membrane-bound, proton-pumping inorganic pyrophosphatase was first described⁴ in chromatophores from the photosynthetic bacterium Rhodospirillum rubrum. Under physiological conditions this enzyme can both synthesize and hydrolyze PP_i. It was also demonstrated^5,6 that PP_i could drive a number of energy requiring reactions in chromatophores including ATP synthesis in the dark⁷. H⁺-PP_i synthase is the only known alternative to the well-known ATP synthease in biological electron transport phosphorylation⁸. H⁺-PPase gene have been reported to occur in Leishmania parasites⁹. Eukaryotic cells also possess soluble inorganic pyrophosphatases. Pyrophosphate (PP_i) is formed as a byproduct in several metabolic reactions, for example, DNA and RNA synthesis. It has to be hydrolyzed in order not to stop these reactions. This is a major function of the soluble, cytoplasmic PPases. Membrane bound PPases do not exist in plasma membrane from mammals¹⁰, thus, H⁺-PPases from Leishmania plasma membrane might be potential target in relation to rational chemotherapy of the disease caused by Leishmania parasites¹¹.

In the present work, we demonstrate that L. donovani promastigote and amastigote plasma membrane possesses PPase activity with features in common with the trypanosomatid and plant enzymes⁴. Our results also indicate that PP_i analogues inhibit the PPase of Leishmania parasite.

Methods

Culture methods^{12, 13, 14}

L. donovani promastigote strain MHOM/IN/1978/UR6, a clinical isolate from a confirmed kala-azar patient , was grown at 24°C on blood agar medium, pH 7.5. The cells were washed at 500x g twice in cold Tris-sucrose-salt solution (250 mM sucrose, 50 mM NaCl, 20 mM KCl, 1 mM ethylenediamine tetra acetic acid, 20 mM Tris, pH 7.2) and kept it at 4°C until use. Viability of harvested cells was monitored microscopically by trypan blue exclusion.

Amastigote culture method^{15, 16, 17}

L. donovani amastigote strain MHOM/IN/1978/UR6 was grown and maintained as described by Debrabant A et al. Axenically grown amastigotes of L. donovani are maintained at 37 °C with 5% CO₂ by weekly subpassages in MAA/20 (medium for axenically grown amastigotes) at pH 5.5 in 177 cm² Petridishes. Under these conditions, promastigotes differentiated to amastigotes within 120 hr. Cultures were maintained by 1:3 dilution once a week. The axenic amastigotes remained stable in culture for a long time. Axenic amastigotes were routinely recycled every 10 weeks by differentiation back to promastigotes, and in parallel, initiating a new line of promastigotes. Transformation of amastigotes to promastigotes was performed by centrifugation of amastigotes (1,000×g at room temperature for 10 min), suspension in promastigote medium, and incubation at 25°C. Under these conditions amastigotes differentiate to promastigotes within 72 hr.

Assay of pyrophosphatase activity¹⁸

Pyrophosphatase activity, in terms of P_i release, was assayed according to the method of Katewa and Katyare. The reaction mixture contains buffer B [Sucrose 300mM, KCl 50mM, Tris 50mM, pH 7, EGTA 2mM pH 7], 0.5 mM Magnesium acetate, 0.25 mM pyrophosphate disodium together with 25 µg L. donovani plasma membrane preparation in a final volume of 1 ml. The mixture was incubated at 25°C for 15 min, and the reaction was terminated by adding 0.14 ml of 2.5 M HClO₄. In the blank experiments, the reaction mixture was incubated, and the protein solution was added after reaction termination. The amount of enzyme catalyzing hydrolysis of 1 µmol of pyrophosphate per minute is taken to the unity. Activity was calibrated with a phosphate standard solution. For enzyme inhibition study, inhibitor was added to the reaction mixture containing enzyme but lacking pyrophosphate. The mixture was preincubated for 10 min at 25°C before starting the reaction with pyrophosphate. Activity values shown below represent means ±S.E. of three independent experiment.

Plasma membrane preparation¹⁹

Plasma membrane from L. donovani promastigotes was prepared according to the method as described before. Parasites were harvested at a concentration of 10⁸ cells/ml (5000 x g, 10 min, 4°C) washed twice in PBS, and pooled with ¹²⁵I cells. All subsequent steps were performed on ice or in refrigerated centrifuges. The cell pellet was suspended to 2 x 10⁷ cells/ml in PBS buffer, pH 7.2 that contained 10 mM MgCl₂ and rapidly mixed with equal volume of 1 mg/ml concanavalin A in the same buffer. Cell aggregation was apparent within 1 min. After 5 min, cells were gently spined at 1,000 x g for 1 min to remove excess concanavalin A. The supernate was discarded, and the cell pellet was re-suspended in 12 ml of 10µM Tris-HCl buffer, pH 7.5, that contained 10µg leupeptin/ml and 1mM MgCl₂. After swelling for 10min in that hypotonic buffer, cells were homogenized by 18–20 strokes in tight fitting Dounce-type homogenizer. Cell lyses and formation of membrane sheets were verified by phase-contrast microscopy. The homogenate was layered over a two- step gradient consisting of 8 ml of 0.5 M mannitol over 4 ml 0.58 M sucrose, both in Tris buffer, and spin at 1000 x g for 20 min. For analysis, material remaining at the top of the 0.5 M mannitol was saved. Large crude plasma membrane fragments were separated as a tight pellet at the bottom of the gradient. This pellet was re-suspended in Tris buffer that contained 1M á-methylmannoside and left on ice for 40 min with occasional mixing.

This plasma membrane, free from bulk concanavalin A, were diluted into three volumes of Tris buffer and homogenized by 80 strokes with a glass Dounce.type homogenizer. This second homogenate was layered on a single-step gradient that consisted of 20% sucrose in Tris buffer and spun for 30 min at 500 x g. Scrolls and large plasma membrane sheets above the 20% (w/v) sucrose layer was collected by centrifugation at 40,000 x g for 1 hr. The pellet containing the enriched plasma membranes, was re-suspended in Tris buffer. All samples were either assayed immediately or frozen at −20°C for further use.

Preparation of digitonin permeabilized Leishmania cell

L. donovani promastigote and/or amastigote cells were collected, washed once by buffer A (140mM NaCl, 20mM KCl, 20mM Tris, 1 mM EDTA, pH 7.5) and resuspended in isolation buffer (20 mM MOPS, pH 7.0; 0.3% bovine serum albumin; 350 mM sucrose; 20 mM potassium acetate; 5 mM magnesium acetate and 1 mM EGTA). Cells were permeabilized in separate tube with 50µg digitonin/mg protein and incubated on ice for 10 min. After incubation, the cells were centrifuged at 6,000×g for 7 min. Pellets were re-suspended in assay buffer.

Protein estimation^{20, 21}

LDC protein was determined by the biuret method in the presence of 0.2% deoxycolate. 1 mg of promastigote protein corresponds to 1.75 × 10⁸ cells and 1 mg of amastigote protein corresponds to 1.14 × 10⁸ cells. Cytosolic, mitochondrial and the plasma membrane protein were determined by modified method of Lowry et al.

Materials

All the biochemicals unless otherwise mentioned were from Sigma-Aldrich (St. Louis, MO, USA). Panmede was purchased from Paines and Byrne (Greenford, Middlesex, UK).

Statistical analysis

All experiments were performed in triplicate, with similar results obtained in at least three experiments. Statistical significance was determined by student.s t test. Significance was considered as P < 0.05.

Results

Isolation of plasma membrane

Development of a method based on cell disruption by osmotic swelling in presence of a plasma membrane specific lectin concanvalin A and differential centrifugation has proved to be ideal for obtaining a plasma membrane fraction from L. donovani promastigote¹⁹. This membrane fraction consists of open membrane sheets with microtubules attached to the membranes²². The utility of the method was assessed by assaying marker enzymes. The activity of tartarate-resistant acid phosphatase, a plasma membrane marker of L. donovani²³ was enriched in this fraction. An enrichment of 28 fold was obtained when cell surface plasma membrane was labeled with ¹²⁵.

PPase activity in plasma membrane

Activity of PPase was optimum at pH 6.8 (data not shown). KCl greatly stimulated PPase activity. Replacing 50 mM KCl with 50 mM NaCl or 50 mM choline chloride in the buffer resulted in substantial loss of PPase activity as shown in figure.1.

Figure1.

Effect of salt concentration on plasma membrane PPase activity. The values represent the means ± S. D. of three independent experiments

Table 1 shows that activity of PPase in plasma membrane is located at endoplasmic face of plasma membrane. Permeabilized L. donovani cell showed substantial PPase activity, whereas nonpermeabilized cell did not. However, very littie ecto-PPase activity have been observed at exoplasmic face of the plasma membrane.

Table 1.

Substrate specificity of pyrophosphatase in L. donovani promastigote membrane^a

Addition (Substrate)	Enzyme activity in DPCb	Enzyme activity in LDCb	Enzyme activity in PMb
PPi (0.25 mM)	47 ± 6	10 ± 0.8	117 ± 18

^aAssay of pyrophosphatase activity have been described in Materials and methods. Values represent mean ± S.E.M. of three independent experiments.

^bEnzyme activity (nmol phosphate/min/mg protein)

Figure 2 shows PPase activity and the amount of plasma membrane proteins added was linear. Linearity of PPase activity was also observed for digitonin permeabilized cell. It is also evident from Figure 3 that in digitonin permeabilized amastigote cell, PPase activity was lesser than promastigote.

Figure 2.

Effect of plasma membrane protein concentration on the rate of PP_i hydrolysis by PPase. Each point represents the average value of three experiments.

Figure 3.

Effect of digitonin permeabilized Leishmania cell protein concentration on the rate of PP_i (●), hydrolysis by promastigote PPase, and amastigote PPase (▲). Each point represents the average value of three experiments.

Specific inhibitors and stimulators of PPase activity

PP_i hydrolysis in the plasma membrane and digitonin permeabilized L. donovani promastigote cell was inhibited, in a dose-dependent manner by KF as shown in figure 4. PP_i hydrolysis was inhibited by the PP_i analogues, IDP and alendronate as shown in figure 5B. The sulphydryl group inhibitors p-chloromercuribenzensulphonate (PCMBS) and phenylarsineoxide (PAO) showed potent inhibition on PPase activity. Some compounds used to inhibit H⁺-ATPase activity, such as N-ethylmalimide (NEM) and DCCD²⁶, are also able to inhibit the plasma membrane PPase. F₁-ATPase and F₁F₀ ATPase inhibitor sodium azide²⁷ did not inhibited the PPase activity as shown in figure 5B. The bioflavonoid quercetin which can inhibit F₀F₁-ATPase strongly at low concentration and F₁-ATPase moderately at high concentration²⁸ had inhibited plasma membrane bound PPase moderately at high concentration. Sodium orthovanadate, a P-type H⁺-ATPase inhibitor²² and P-type ABC superfamily of transporters, such as P-glycoprotein involved in the multidrug resistance²⁹, stimulated PPase activity. Effects of stimulators and inhibitors on PPase in digitonin permeabilized amastigotes were very similar to the effect on PPase of promostigote plasma membrane (data not shown). Verapamil, a P-gp ATPase stimulator³⁰, inhibited PPase activity.

Figure 5.

Concentration-response curve describing the effect of effectors on promastigote plasma membrane PPase.

Each point represents the average value of three experiments. The effectors were added to plasma membrane 10 min before the addition of substrates.

Discussion

In the present study, we have identified and characterized PPase activity in the plasma membrane of L. donovani promastigote. This is the first report demonstrating biochemically the presence of a PPase in the plasma membrane of L. donovani promastigote. Our results suggest that K⁺ was necessary for PPase activity, but substantial amount of K⁺ and Na⁺ independent PPase was also present in the plasma membrane. Since IDP is a bisphosphonate, we tested other bisphosphonate (alendronate) used clinically in the treatment of bone resorbtion disorders²⁴ for its inhibitory effect on PP_i hydrolysis. The chemical modification with the trivalent arseniqal reagent PAO indicates the involvement in PPase activity of a viscenal sulfhydryl group²⁵.

The significance of the presence of a PPase in the amastigote plasma membrane may be proton pumping to maintain neutral cytosolic pH ³¹ in an environment of acidic pH of phagolysosome³². Data provided evidence that "pH formation in promastigote and amastigote was partially inhibited by H⁺-ATPase inhibitor DCCD. This fact suggested that mechanisms in addition to that inhibited by DCCD may also be involved in controlling homeostasis of pH_i ³¹ It appears from our observations that PPase may also play an important role in the extrusion of proton from the cytosol.

Conclusion

In conclusion, this is the first report of a PPase in plasma membrane of an organism different from plants. Since membrane bound PPases are apparently absent from vertebrates, analysis of the role of PPase in pH regulatory mechanisms of L. donovani amastigote might serve as potential target for putative therapeutic intervention.

Abbreviations used

PPase

Pyrophosphatase

NEM

N-ethylmaliemide

PAO

Phenylarsineoxide

KF

Potassium fluoride

DCCD

N,N?-dicclohexylcarbodiimide

PCMBS

Parachloromer curibenzenesulfonate

SA

Sodium azide

SOV

Sodium orthovanadate

IDP

Imidodiphosphate

QTN

Quercetin

LDC

Leishmania donovani cell

DPC

Digitonin permeabilized cell

PM

Plasma membrane

PP_i

Pyrophosphate

gp

Glycoprotein

Pro

Promastigote

Am

Amastigote