Life Sciences 144 (2016) 218–225
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Identification and characterization of a Leishmania donovani serine protease inhibitor: Possible role in regulation of host serine proteases Md Nur Alam, Partha Das, Tripti De, Tapati Chakraborti ⁎ Department of Biochemistry and Biophysics, University of Kalyani, Nadia 741235, West Bengal, India
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Article history: Received 25 February 2015 Received in revised form 30 September 2015 Accepted 1 December 2015 Available online 2 December 2015 Keywords: Leishmania donovani Serine protease inhibitor Ecotin Serine protease
a b s t r a c t Aims: This study aims to identify, purify, and characterize an endogenous serine protease inhibitor from an Indian strain of Leishmania donovani, which causes the fatal visceral leishmaniasis. Main methods: (i) Reverse zymography was used to identify the serine protease inhibitor by inhibiting the gelatinolytic activity of serine protease. (ii) Purification was performed by combining heat treatment, ultracentrifugation, and affinity and gel permeation chromatography. (iii) Spectrophotometric assays were conducted to quantify and compare the inhibitory activity of the L. donovani serine protease inhibitor (LdISP). (iv) Further, the protein was identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS). Key findings: An endogenous inhibitor with an apparent molecular weight of 21.8 kDa, which is acidic in nature, having a pI of 5.9 was identified. The Ki value of the inhibitor for trypsin was determined to be in the nanomolar range. The protein has the following features: (i) ecotin-like nature, (ii) cross-organism functionality, that is, an inhibitory effect on the serine proteases of higher organisms other than its own, and (iii) homology with other such proteins from a different species of Leishmania on conducting protein mass fingerprinting after MALDI ToF MS. Significance: The inhibitor shows varying and entirely contrasting efficacies toward serine proteases of its own as well as of higher organisms. This indicates that it accelerates disease progression and drives parasite survival as it inhibits the activities of the host serine proteases. © 2015 Published by Elsevier Inc.
1. Introduction Digenic flagellated eukaryotic parasites belonging to the genus Leishmania cause a spectrum of human diseases called leishmaniases. These range from fatal visceral infections to severely disfiguring cutaneous ulcers, which affect around 350 million people in 88 countries, with about two million new cases reported every year [1]. Members of the Leishmania genus live as promastigotes in the digestive tract of sand flies and as amastigotes in the phagolysosomes of mammalian macrophages. Many studies have stated that leishmanial proteases are most likely to be involved in parasite metabolism and host–parasite interactions, such as host tissue invasion, parasite nutrition, and evasion of host immune responses. Conversely, macrophage-derived proteases damage the parasite if not interfered by proper inhibitors. Many Abbreviations: BApNA, N-α-benzoyl–DL-arginyl-p-nitroanilide; BSA, bovine serum albumin; CBB, Coomassie brilliant blue; ICP, cysteine protease inhibitor; ISPs, serine protease inhibitors; LdISP, Leishmania donovani serine protease inhibitor; MS, mass spectrometry; PBS, phosphate-buffered saline; PMF, protein mass fingerprinting; SP-Ld, Leishmania donovani serine protease. ⁎ Corresponding author at: Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India. E-mail address: [email protected] (T. Chakraborti).
http://dx.doi.org/10.1016/j.lfs.2015.12.004 0024-3205/© 2015 Published by Elsevier Inc.
metabolic and/or pathophysiological conditions are affected when the balance between proteases and inhibitors is disturbed, which in turn could affect the progression of infection. Therefore, regulation is imperative to minimize unnecessary damages to both the parasite and the host [2,3]. Protease inhibitors are found in tissues of all life-forms, ranging from viruses to humans. These inhibitors have evolved to regulate the respective proteases for management of a wide variety of physiological processes [4]. The regulation and function of the proteases are achieved through the narrow spectrum of substrate specificity and by a diverse class of protease inhibitors in both the host and the parasite [3]. In addition to regulating their own proteases, parasite-derived protease inhibitors are also known to protect the invading organism from degradation by exogenous host proteases in the hostile environment of the host [3]. Parasites encode several protease inhibitors including inhibitors of serine protease (ISPs), which inhibit serine proteases (SPs) of the immune response system and manipulate the host physiological processes to ensure their survival [5]. These inhibitors regulate such processes as coagulation, inflammation, and immunity [6]. Like several other parasites, eukaryotic protozoans transcribe serpins (serine protease inhibitor or ISPs), which are involved in protecting the parasite from the hostile enzymatic activities of the
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Fig. 1. Evolutionary tree of Leishmania, ☆ showing adjacency of Leishmania infantum and Leishmania donovani, which cause VL (adapted by permission from Nicolas Fasel 2012. Front Cell Infect Microbiol.)
host [7,8]. Serpins form a very large class of protease inhibitors, which change in shape uniquely when inhibiting the target proteases. The modes of action of different inhibitors also vary [9]. The classical Kunitz and Kazal type is the most abundant class of serpins [10,11]. Kazal-type inhibitors are also called canonical inhibitors. They are extremely variable at their reactive sites. Further, they bind to their cognate enzymes similar to an efficient substrate, forming a canonical protease– inhibitor loop. However, the Kunitz family possesses Kunitz domains, which are relatively small active domains of proteins that bind strongly to the protease, blocking its active site and instantly forming an irreversible compound (http://smart.embl-heidelberg.de/). Similar to other parasites, Leishmania species also encode protease inhibitors in their genome, for example, Leishmania major cysteine protease inhibitor (ICP), which is predicted to participate in the host– parasite interaction [12]. The L. major genome has been shown to consist of three genes encoding ecotin-like ISPs [13,14]. Different species of Leishmania cause varying disease symptoms; however, it is interesting to note that both Leishmania infantum and Leishmania donovani cause visceral leishmaniasis (VL), suggesting their evolutionary adjacency (Fig. 1). Our previous reports showed that SPs were one of the virulence factors in L. donovani [15,16]. Consequently; we sought to detect the presence of endogenous inhibitor(s), if any, in L. donovani promastigotes and their role in parasite biology. As part of our present study to elucidate the role of L. donovani-derived serine protease inhibitor (LdISP), we chose to purify the inhibitor to determine its biochemical characteristics, as various Leishmania species differ substantially in their genomewide gene expression [17].
Fig. 2. Reverse zymography for detection of serine protease inhibitors (LdISPs). Lane 1: heat-treated Leishmania donovani cell lysate, Lane 2: molecular weight markers.
Evidently, a comprehensive understanding of parasite biology involving ISPs is imperative to define their role in the pathogenesis of VL. More importantly, they can also act as drug targets for further novel therapies. Hereinafter, the endogenous ISP from the Indian strain of L. donovani is designated as L. donovani serine protease inhibitor (LdISP). 2. Materials and methods All the chemicals used in the present study were purchased from Sigma-Aldrich, unless stated otherwise. 2.1. Culture of L. donovani promastigotes L. donovani strains (MHOM/IN/1983/AG83) were isolated from Indian patients with VL. Promastigotes were cultured at 22 °C in Medium 199 with Hank's salt containing 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), L-glutamine, 12% fetal calf serum, 50 U/ml of penicillin, and 50 μg/ml of streptomycin. The cell viability was assessed by the trypan blue exclusion method [15], and the cell growth was estimated by counting the parasites in the Neubauer chamber. The cell count was further confirmed by a simple cell count in a flow cytometer (BD FACSCalibur).
Fig. 3. Affinity chromatogram of LdISP on trypsin–agarose column (1 × 10 cm) equilibrated with 0.1 mM HCl and eluted with different gradients of HCl (10–100 mM) with a constant flow rate of 0.25 ml/min. Absorbance at 280 nm was monitored for active fractions.
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2.3. Purification of the intracellular ISP from an avirulent strain of L. donovani
Fig. 4. Comparative inhibitory activity assessment of LdISP against the 52.2-kDa fraction. Trypsin (25 μg/ml) was preincubated with equimolar inhibitors at 37 °C for 30 min. The hydrolysis of BApNA is recorded and scanned for 260 s at A410nm.
2.2. Reverse zymography To detect ISPs in L. donovani, reverse zymography was performed as described by Naohiko Koshikawa et al. [18] The samples were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on polyacrylamide gels containing 0.1% (w/v) SDS and 1 mg/ ml of gelatin under nonreducing conditions. Then they were renatured in 50 mM Tris–HCl (pH 7.5) containing 2.5% (v/v) Triton X-100 and 0.1 M NaCl. The renatured gels were incubated at 37 °C for 18 h in 12 ml of the reaction mixture consisting of 50 mM Tris–HCl (pH 7.5) and 10 ng/ml of bovine trypsin as the indicator enzyme. The resultant gels were stained with Coomassie brilliant blue (CBB). The trypsin inhibitors separated on the gels were identified as CBB-stained bands by blocking the gelatinolytic activity of trypsin.
Long-passage cells of L. donovani promastigotes were harvested by centrifugation at 8000 g for 15 min at 4 °C. The pellets were washed twice with 50 mM phosphate-buffered saline (PBS) (pH 7.4) at 5000 g for 3 min. The cell pellets were resuspended with 50 mM PBS (pH 7.4) and subjected to sonication at an amplitude of 0.6 (60%) and 0.25 cycles for four pulses of 10-s interval alternately using Hielscher UP50H (Hielscher Ultrasonics, GmbH) under ice. The lysed suspension was centrifuged at 15,000 g for 15 min to collect the cytosolic protein-rich supernatant separating from the cell debris. The supernatant was heated at 80 °C using a water bath to unbind the protein–protein interactions and to denature several other heat-sensitive cytosolic proteins as well. The heat-treated fractions were again ultracentrifuged to separate the denatured proteins at 105,000 g for 30 min. The supernatant was then passed through a 0.22-μm membrane filter (Millipore polyethersulfone membrane) and loaded onto a trypsin–agarose affinity column (1 × 10 cm) equilibrated with 0.1 mM HCl. The column was eluted with different gradients of HCl (10–100 mM). The fractions were pooled, concentrated by ultrafiltration (Amicon, YM-10 membrane), and dialyzed overnight against 10 mM Tris–HCl (pH 7.5). The fractions were comparatively assayed for inhibitory activity as described in section 2.7. The fraction with higher activity was loaded onto the 75-ml G-75 column, which was preequilibrated with 50 mM NaPO 4 buffer (pH 7.4) containing 150 mM NaCl and 0.02% NaN 3 . The fraction was eluted (at a flow rate of 0.5 ml/min) with the same buffer. In each tube, a 1-ml fraction was collected, and the protein peak was detected simultaneously by monitoring the absorbance of each fraction at 280 nm (GE AKTA prime FPLC assembly). Fractions with inhibitory activity were pooled, concentrated, and used for further studies.
2.4. Protein measurement The protein content was estimated using the Pierce Micro BCA Protein Assay Kit with bovine serum albumin as the standard.
Fig. 5. Elution profile of LdISP in G-75 gel filtration chromatography. The column was equilibrated and eluted with 50 mM NaPO4 buffer (pH 7.4) containing 150 mM NaCl and 0.02% NaN3. The fractions under the peak were pooled and concentrated.
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Fig. 6. Chromatogram of LdISP in reverse-phase HPLC. A C18 reverse-phase column (SRT SEC-300, 4.6 × 300 mm) was used with 50 mM phosphate buffer, containing 150 mM NaCl as the mobile phase, at a pH of 7.0 and a flow rate of 0.35 ml/min.
2.5. Qualitative assessment of the protein
2.7. Enzymatic assays
The protein fraction purified after gel filtration chromatography was concentrated using a 10-kDa cutoff membrane (YM10 membrane, Amicon) and run in the reverse-phase C18 column (SRT SEC-300, 4.6 × 300 mm) with 50 mM phosphate buffer, containing 150 mM NaCl as the mobile-phase buffer, at a pH of 7.0 and a flow rate of 0.35 ml/min maintained with a high-performance liquid chromatography (HPLC) system (JASCO UV-2075/2080).
Enzymatic assays were carried out to quantify the activity and to study the nature of the inhibition. BApNA (N-α-benzoyl–DL-arginyl-pnitroanilide), TAME (tosyl arginine methyl ester), or BAEE (benzoyl arginine ethyl ester) can be used as a substrate. Here, BApNA (0.02 M) was used as a substrate and bovine trypsin (10 and 20 μg/ml in 0.001 N HCl) as SP. The assay was carried out in 0.046 M Tris–HCl basic buffer (pH 8.1) containing 0.115 M CaCl2. The inhibitors were pre-incubated with proteases for 30 min at 37 °C in the reaction mixture for quantifying the residual activity. The inhibition assay of the endogenous inhibitor was also carried out using intracellular SP-Ld and with neutrophil elastase. The inhibitory activity of the fractions was determined by quantifying the amount of p-nitroaniline release by reading the absorbance at 410 nm (Hitachi U-1900).
2.6. Identification and purification of L. donovani serine protease The L. donovani aprotinin-sensitive serine protease (SP-Ld) of molecular mass 58 kDa obtained from the promastigotes of an Indian strain was purified as described by Chowdhury et al. [15] Briefly, the L. donovani promastigotes (second-passaged) were grown to the late log phase, and the cells (~5 × 10 [9]) were harvested by centrifugation at 3000 g for 15 min at 4 °C. Then, the clear soluble supernatant obtained after cell lysis was precipitated with ammonium sulfate at 40% saturation and subjected to aprotinin–agarose affinity chromatography, followed by continuous elution electrophoresis. The purified SP was further characterized [15,16].
2.8. Protein mass fingerprinting The proteins were identified and validated by mass spectrometry (MS). The sample was introduced into the mass spectrometer through matrix-assisted laser desorption/ionization (MALDI), and the time of flight (TOF) of different fragmented peptides with different m/Z was recorded (Bruker Daltonics FLEX). The proteins were validated by
Table 1 Purification profile of serine protease inhibitor from Leishmania donovani (LdISP). Steps
Total protein (mg)
Total activity (TIU) × 106
Specific activity (TIU/mg) × 103
Yield (%)
Crude extract (cell lysate) post precipitation Heat treatment + ultracentrifugation Trypsin–agarose (affinity chromatography) Gel filtration (FPLC) G-75
1884.5 208.6 1.87 0.24
33.28 22.38 17.07 12.66
6.71 8.71 9.13 18.21
100 67.24 51.29 38.04
For the last three steps, the mean values of triplicate readings were taken.
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Fig. 7. Two-dimensional electrophoresis was carried out to determine the pI value of the protein, which was found to be 5.9. The 3.0–10.0 ready (Bio-Rad) IPG strips were used for electrofocusing.
targeted MS and quantitative approaches accordingly. The data from MS/MS were then searched using Mascot-based database search engines.
Fig. 9. Comparative inhibitory capacity of LdISP: the purified LdISP was incubated with trypsin, elastase, and SP-Ld. The inhibitor LdISP substantially inhibits trypsin and elastase but not the SP-Ld. The mean values of triplicate readings for each coordinate were employed and plotted.
3.2. Purification of the L. donovani endogenous ISP 3. Results 3.1. Identification of LdISP The serine protease inhibitors (LdISPs) of L. donovani promastigotes were identified by subjecting the heat-treated cell lysate to highly sensitive reverse zymography. The reverse zymogram of the heat treated cell lysate showed two bands with molecular weights of approximately 52.2 and 21.8 kDa (Fig. 2). The band with low molecular weight (21.8 kDa) was of much higher intensity, designated as LdISP and chosen for further studies.
To understand the biological functions of LdISP, the inhibitor was purified by extracting the L. donovani cell pellets by sonication, heat treatment, ultracentrifugation, affinity binding, and gel filtration. The eluted fractions from the trypsin–agarose column showed two peaks at A280 (Fig. 3). Each major fraction (i.e., 21.8 and 52.2 kDa) was subjected to an enzyme assay. The smaller peptide LdISP (21.8 kDa) was chosen for further studies due to its inhibitory effect on S1A class enzymes (Fig. 4). The same fraction was pooled, concentrated, and purified further by gel filtration. The G-75 column elution profile showed a single peak (Fig. 5). The gel-purified (G-75) protein was further analyzed in
Fig. 8. Trypsin inhibition assay plot by LdISP: Trypsin activity was assayed subsequent to incubation with varying concentrations of LdISP. For 30 min, 4 nM of trypsin was incubated with ), 0.5 nM LdISP ( ), 2.5 nM ( ), and equimolar 4.0 nM LdISP ( ) prior to assaying. A410 of the N-benzoyl-arginine para-nitroaniline hydrolysis reaction mixture no LdISP ( was measured at various time intervals over the 200-s time course. The reaction rate is related to change in A410, and it is indicative of trypsin activity. Values are mean ± SD of triplicate measurements.
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Fig. 10. Protein mass fingerprinting (PMF): identification of protein using mass spectrometry data or with protein mass fingerprinting using Mascot software (Matrix Science) after being subjected MALDI/ToF mass spectrometry using a Bruker Daltonics FLEX. The PMF result of LdISP shows some resemblance with the ecotin-like protein from another Leishmania species, and it also presents the putative sequences of the peptide fragments.
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reverse-phase HPLC to confirm its purity. The HPLC analysis revealed a single intense peak at a retention time of ~16 min (Fig. 6). This method resulted in a 38.04% yield (Table 1) of the purified LdISP. 3.3. Two-dimensional electrophoresis On subjecting the purified LdISP to two-dimensional (2D) electrophoresis, the pI of the inhibitor was found to be 5.9 (Fig. 7) using PD Quest software, which indicates the acidic nature of the inhibitor. 3.4. Inhibition assay The inhibitory activity of LdISP against trypsin was determined by measuring A410 of the hydrolysis of BApNA in a reaction mixture with varying concentrations of LdISP (0.5–4 nM) (Fig. 8). The comparative inhibitory activity of LdISP against trypsin, elastase, and purified SP-Ld was determined by measuring the hydrolytic activity toward BApNA. LdISP showed a strong inhibitory effect against bovine trypsin and elastase but not against SP-Ld (Fig. 9). 3.5. Partial sequence elucidation MALDI/ToF MS analysis reveals the match scores of the fragmented peptide sequences of LdISP with the other peptide sequences in the data bank. In silico protein mass fingerprinting (PMF) using the Matrix Science software was conducted to determine the matching scores, if any, with other peptide sequences in protein/peptide sequence data banks. The PMF results of LdISP are highly similar to those of an ecotin-like protein from another Leishmania species (Fig. 10). 4. Discussion ISPs are ubiquitous proteins found in plants, mammals, bacteria, and parasites, with several functions such as strictly regulating proteases. Serpins were first investigated when the human plasma proteins antithrombin and anti-trypsin were studied in depth for their key roles in controlling blood coagulation and inflammation. Gradually, several new characteristics of ISPs were discovered, such as their cross-class functionality, wherein serpins could inhibit classes of protease other than SPs. Inhibitors were widely studied for their role in protease regulation and for their therapeutic implications until Chung C.H19 purified ecotin with cross-organism functionality, that is, inhibitor(s) of different origins acting on protease(s) of different species. Most parasites, if not all, have been hypothesized to possess these inhibitors of host proteases to ensure their survival. Thus, we primarily attempted to detect, identify, and characterize LdISP from promastigotes. It is highly interesting that these inhibitors (LdISP) inhibit SPs of higher organisms instead of their own ISPs. On assessment, LdISP was found to inhibit neutrophil elastase and bovine trypsin, whereas it did not have a marked effect on endogenous SP-Ld, which has been purified earlier [16]. Several similar studies lead us to the hypothesis that the protein is ecotin-like [19] and may be involved in the regulation of host SPs rather than inhibiting its own endogenous SPs. Recent studies on the Leishmania genome reveal that S1A class SPs are not transcribed in Leishmania, whereas they are transcribed by the mammalian host cell [20]. Thus, the presence of an ecotin-like protein in an Indian strain of L. donovani might provide insight into its immunomodular mechanisms and resulting implications. Although the genes transcribing the ecotin-like protein have long been identified in other Leishmania species [21] and in other related parasites, to the best of our knowledge, an endogenous ISP from an Indian strain of L. donovani (MHOM/IN/1983/AG83) promastigotes has not been characterized yet. Herein, we report the presence of an ecotinlike protein LdISP in the unicellular protozoan parasite L. donovani promastigote. In addition, we determined the pI value, heat stability, and inhibitory activity of LdISP to elucidate its role in infectivity.
The presence and function of LdISP-like proteins are uncharacteristic, as they are found in eukaryotic parasites, trypanosomatids, and many bacteria, with an apparent action on host proteases. This varied distribution provides an insight into evolutionary pathways and confirms the assumed horizontal gene transfer from prokaryotes to eukaryotic protozoans [20]. The inhibitory activity of LdISP against several host SPs (when studied in vitro) is functionally similar to other such inhibitory proteins from different prokaryotes and eukaryotic protozoans (www.uniprot.org). Given that LdISPs are now known to exist in an Indian strain of Leishmania, the action and regulation of the protein in infection progression within this parasite or in other unicellular parasites can be explored, because the ecotin-like protein does inhibit SPs of higher organisms. On the contrary, the endogenous inhibitors of many higher multicellular organisms regulate their own proteases. Genomic studies have proven that L. major has 13 SPs belonging to six families but not the S1A family, which is present in higher multicellular organisms [21]. At present, the possible location of the protein and its activity at different stages of the Indian strain of L. donovani is being investigated for its significant role in understanding the regulation of SPs and the progression of infection. In a new direction, Ramalho-Ortigão JM [22] stated that these parasitic ISPs not only inhibit the host SPs but also play an important role in inhibiting the tryptic enzymes of vectors while maturing in their gut. This notion further proves the evolution of intricate survival strategies of unicellular eukaryotic parasites. Eggers et al. [23] showed that several host SPs that can be inhibited by periplasmic ecotin include those expressed by the innate immune system such as the complement system and the enzymes involved in coagulation cascades. This warrants further investigation into the various other roles of LdISP. Thus, these strong hypotheses and the conclusive results further our understanding of Leishmania pathophysiology, survivability, and etiology, with new implications for LdISP. In addition, these studies can also be exploited to design target-specific drugs, leading to novel therapeutic trends to combat VL. 5. Conclusion The present study demonstrated the identification, purification, and biochemical characterization of an endogenous ISP in L. donovani with an apparent molecular weight of ~21.8 kDa. This acidic inhibitor inhibits SP of higher organisms but not its own SPs. Apparently; enzymatic studies indicate that the inhibitor acts on host SPs to inhibit their proteolytic activity. Thus, the inhibitor proves the evolution of parasite survival strategies with a significant role in disease progression. Conflict of interest None of the authors have any potential financial conflict of interest related to this manuscript. Acknowledgment The study was supported by the University Grants Commission (UGC) (F no.-11-91/2008/BSR) /RFSMS and Council of Scientific and Industrial Research (CSIR), Government of India (37(1537)/12-EMR-II). The authors are grateful to the Indian Institute of Chemical Biology (IICB), Kolkata, and Sandor Proteomics, Hyderabad, for providing the cell lines and mass spectrometry facilities, respectively. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.lfs.2015.12.004.
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Identification and characterization of a Leishmania donovani serine protease inhibitor: Possible role in regulation of host serine proteases Md Nur Alam, Partha Das, Tripti De, Tapati Chakraborti ⁎ Department of Biochemistry and Biophysics, University of Kalyani, Nadia 741235, West Bengal, India
a r t i c l e
i n f o
Article history: Received 25 February 2015 Received in revised form 30 September 2015 Accepted 1 December 2015 Available online 2 December 2015 Keywords: Leishmania donovani Serine protease inhibitor Ecotin Serine protease
a b s t r a c t Aims: This study aims to identify, purify, and characterize an endogenous serine protease inhibitor from an Indian strain of Leishmania donovani, which causes the fatal visceral leishmaniasis. Main methods: (i) Reverse zymography was used to identify the serine protease inhibitor by inhibiting the gelatinolytic activity of serine protease. (ii) Purification was performed by combining heat treatment, ultracentrifugation, and affinity and gel permeation chromatography. (iii) Spectrophotometric assays were conducted to quantify and compare the inhibitory activity of the L. donovani serine protease inhibitor (LdISP). (iv) Further, the protein was identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS). Key findings: An endogenous inhibitor with an apparent molecular weight of 21.8 kDa, which is acidic in nature, having a pI of 5.9 was identified. The Ki value of the inhibitor for trypsin was determined to be in the nanomolar range. The protein has the following features: (i) ecotin-like nature, (ii) cross-organism functionality, that is, an inhibitory effect on the serine proteases of higher organisms other than its own, and (iii) homology with other such proteins from a different species of Leishmania on conducting protein mass fingerprinting after MALDI ToF MS. Significance: The inhibitor shows varying and entirely contrasting efficacies toward serine proteases of its own as well as of higher organisms. This indicates that it accelerates disease progression and drives parasite survival as it inhibits the activities of the host serine proteases. © 2015 Published by Elsevier Inc.
1. Introduction Digenic flagellated eukaryotic parasites belonging to the genus Leishmania cause a spectrum of human diseases called leishmaniases. These range from fatal visceral infections to severely disfiguring cutaneous ulcers, which affect around 350 million people in 88 countries, with about two million new cases reported every year [1]. Members of the Leishmania genus live as promastigotes in the digestive tract of sand flies and as amastigotes in the phagolysosomes of mammalian macrophages. Many studies have stated that leishmanial proteases are most likely to be involved in parasite metabolism and host–parasite interactions, such as host tissue invasion, parasite nutrition, and evasion of host immune responses. Conversely, macrophage-derived proteases damage the parasite if not interfered by proper inhibitors. Many Abbreviations: BApNA, N-α-benzoyl–DL-arginyl-p-nitroanilide; BSA, bovine serum albumin; CBB, Coomassie brilliant blue; ICP, cysteine protease inhibitor; ISPs, serine protease inhibitors; LdISP, Leishmania donovani serine protease inhibitor; MS, mass spectrometry; PBS, phosphate-buffered saline; PMF, protein mass fingerprinting; SP-Ld, Leishmania donovani serine protease. ⁎ Corresponding author at: Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India. E-mail address: [email protected] (T. Chakraborti).
http://dx.doi.org/10.1016/j.lfs.2015.12.004 0024-3205/© 2015 Published by Elsevier Inc.
metabolic and/or pathophysiological conditions are affected when the balance between proteases and inhibitors is disturbed, which in turn could affect the progression of infection. Therefore, regulation is imperative to minimize unnecessary damages to both the parasite and the host [2,3]. Protease inhibitors are found in tissues of all life-forms, ranging from viruses to humans. These inhibitors have evolved to regulate the respective proteases for management of a wide variety of physiological processes [4]. The regulation and function of the proteases are achieved through the narrow spectrum of substrate specificity and by a diverse class of protease inhibitors in both the host and the parasite [3]. In addition to regulating their own proteases, parasite-derived protease inhibitors are also known to protect the invading organism from degradation by exogenous host proteases in the hostile environment of the host [3]. Parasites encode several protease inhibitors including inhibitors of serine protease (ISPs), which inhibit serine proteases (SPs) of the immune response system and manipulate the host physiological processes to ensure their survival [5]. These inhibitors regulate such processes as coagulation, inflammation, and immunity [6]. Like several other parasites, eukaryotic protozoans transcribe serpins (serine protease inhibitor or ISPs), which are involved in protecting the parasite from the hostile enzymatic activities of the
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Fig. 1. Evolutionary tree of Leishmania, ☆ showing adjacency of Leishmania infantum and Leishmania donovani, which cause VL (adapted by permission from Nicolas Fasel 2012. Front Cell Infect Microbiol.)
host [7,8]. Serpins form a very large class of protease inhibitors, which change in shape uniquely when inhibiting the target proteases. The modes of action of different inhibitors also vary [9]. The classical Kunitz and Kazal type is the most abundant class of serpins [10,11]. Kazal-type inhibitors are also called canonical inhibitors. They are extremely variable at their reactive sites. Further, they bind to their cognate enzymes similar to an efficient substrate, forming a canonical protease– inhibitor loop. However, the Kunitz family possesses Kunitz domains, which are relatively small active domains of proteins that bind strongly to the protease, blocking its active site and instantly forming an irreversible compound (http://smart.embl-heidelberg.de/). Similar to other parasites, Leishmania species also encode protease inhibitors in their genome, for example, Leishmania major cysteine protease inhibitor (ICP), which is predicted to participate in the host– parasite interaction [12]. The L. major genome has been shown to consist of three genes encoding ecotin-like ISPs [13,14]. Different species of Leishmania cause varying disease symptoms; however, it is interesting to note that both Leishmania infantum and Leishmania donovani cause visceral leishmaniasis (VL), suggesting their evolutionary adjacency (Fig. 1). Our previous reports showed that SPs were one of the virulence factors in L. donovani [15,16]. Consequently; we sought to detect the presence of endogenous inhibitor(s), if any, in L. donovani promastigotes and their role in parasite biology. As part of our present study to elucidate the role of L. donovani-derived serine protease inhibitor (LdISP), we chose to purify the inhibitor to determine its biochemical characteristics, as various Leishmania species differ substantially in their genomewide gene expression [17].
Fig. 2. Reverse zymography for detection of serine protease inhibitors (LdISPs). Lane 1: heat-treated Leishmania donovani cell lysate, Lane 2: molecular weight markers.
Evidently, a comprehensive understanding of parasite biology involving ISPs is imperative to define their role in the pathogenesis of VL. More importantly, they can also act as drug targets for further novel therapies. Hereinafter, the endogenous ISP from the Indian strain of L. donovani is designated as L. donovani serine protease inhibitor (LdISP). 2. Materials and methods All the chemicals used in the present study were purchased from Sigma-Aldrich, unless stated otherwise. 2.1. Culture of L. donovani promastigotes L. donovani strains (MHOM/IN/1983/AG83) were isolated from Indian patients with VL. Promastigotes were cultured at 22 °C in Medium 199 with Hank's salt containing 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), L-glutamine, 12% fetal calf serum, 50 U/ml of penicillin, and 50 μg/ml of streptomycin. The cell viability was assessed by the trypan blue exclusion method [15], and the cell growth was estimated by counting the parasites in the Neubauer chamber. The cell count was further confirmed by a simple cell count in a flow cytometer (BD FACSCalibur).
Fig. 3. Affinity chromatogram of LdISP on trypsin–agarose column (1 × 10 cm) equilibrated with 0.1 mM HCl and eluted with different gradients of HCl (10–100 mM) with a constant flow rate of 0.25 ml/min. Absorbance at 280 nm was monitored for active fractions.
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2.3. Purification of the intracellular ISP from an avirulent strain of L. donovani
Fig. 4. Comparative inhibitory activity assessment of LdISP against the 52.2-kDa fraction. Trypsin (25 μg/ml) was preincubated with equimolar inhibitors at 37 °C for 30 min. The hydrolysis of BApNA is recorded and scanned for 260 s at A410nm.
2.2. Reverse zymography To detect ISPs in L. donovani, reverse zymography was performed as described by Naohiko Koshikawa et al. [18] The samples were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on polyacrylamide gels containing 0.1% (w/v) SDS and 1 mg/ ml of gelatin under nonreducing conditions. Then they were renatured in 50 mM Tris–HCl (pH 7.5) containing 2.5% (v/v) Triton X-100 and 0.1 M NaCl. The renatured gels were incubated at 37 °C for 18 h in 12 ml of the reaction mixture consisting of 50 mM Tris–HCl (pH 7.5) and 10 ng/ml of bovine trypsin as the indicator enzyme. The resultant gels were stained with Coomassie brilliant blue (CBB). The trypsin inhibitors separated on the gels were identified as CBB-stained bands by blocking the gelatinolytic activity of trypsin.
Long-passage cells of L. donovani promastigotes were harvested by centrifugation at 8000 g for 15 min at 4 °C. The pellets were washed twice with 50 mM phosphate-buffered saline (PBS) (pH 7.4) at 5000 g for 3 min. The cell pellets were resuspended with 50 mM PBS (pH 7.4) and subjected to sonication at an amplitude of 0.6 (60%) and 0.25 cycles for four pulses of 10-s interval alternately using Hielscher UP50H (Hielscher Ultrasonics, GmbH) under ice. The lysed suspension was centrifuged at 15,000 g for 15 min to collect the cytosolic protein-rich supernatant separating from the cell debris. The supernatant was heated at 80 °C using a water bath to unbind the protein–protein interactions and to denature several other heat-sensitive cytosolic proteins as well. The heat-treated fractions were again ultracentrifuged to separate the denatured proteins at 105,000 g for 30 min. The supernatant was then passed through a 0.22-μm membrane filter (Millipore polyethersulfone membrane) and loaded onto a trypsin–agarose affinity column (1 × 10 cm) equilibrated with 0.1 mM HCl. The column was eluted with different gradients of HCl (10–100 mM). The fractions were pooled, concentrated by ultrafiltration (Amicon, YM-10 membrane), and dialyzed overnight against 10 mM Tris–HCl (pH 7.5). The fractions were comparatively assayed for inhibitory activity as described in section 2.7. The fraction with higher activity was loaded onto the 75-ml G-75 column, which was preequilibrated with 50 mM NaPO 4 buffer (pH 7.4) containing 150 mM NaCl and 0.02% NaN 3 . The fraction was eluted (at a flow rate of 0.5 ml/min) with the same buffer. In each tube, a 1-ml fraction was collected, and the protein peak was detected simultaneously by monitoring the absorbance of each fraction at 280 nm (GE AKTA prime FPLC assembly). Fractions with inhibitory activity were pooled, concentrated, and used for further studies.
2.4. Protein measurement The protein content was estimated using the Pierce Micro BCA Protein Assay Kit with bovine serum albumin as the standard.
Fig. 5. Elution profile of LdISP in G-75 gel filtration chromatography. The column was equilibrated and eluted with 50 mM NaPO4 buffer (pH 7.4) containing 150 mM NaCl and 0.02% NaN3. The fractions under the peak were pooled and concentrated.
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Fig. 6. Chromatogram of LdISP in reverse-phase HPLC. A C18 reverse-phase column (SRT SEC-300, 4.6 × 300 mm) was used with 50 mM phosphate buffer, containing 150 mM NaCl as the mobile phase, at a pH of 7.0 and a flow rate of 0.35 ml/min.
2.5. Qualitative assessment of the protein
2.7. Enzymatic assays
The protein fraction purified after gel filtration chromatography was concentrated using a 10-kDa cutoff membrane (YM10 membrane, Amicon) and run in the reverse-phase C18 column (SRT SEC-300, 4.6 × 300 mm) with 50 mM phosphate buffer, containing 150 mM NaCl as the mobile-phase buffer, at a pH of 7.0 and a flow rate of 0.35 ml/min maintained with a high-performance liquid chromatography (HPLC) system (JASCO UV-2075/2080).
Enzymatic assays were carried out to quantify the activity and to study the nature of the inhibition. BApNA (N-α-benzoyl–DL-arginyl-pnitroanilide), TAME (tosyl arginine methyl ester), or BAEE (benzoyl arginine ethyl ester) can be used as a substrate. Here, BApNA (0.02 M) was used as a substrate and bovine trypsin (10 and 20 μg/ml in 0.001 N HCl) as SP. The assay was carried out in 0.046 M Tris–HCl basic buffer (pH 8.1) containing 0.115 M CaCl2. The inhibitors were pre-incubated with proteases for 30 min at 37 °C in the reaction mixture for quantifying the residual activity. The inhibition assay of the endogenous inhibitor was also carried out using intracellular SP-Ld and with neutrophil elastase. The inhibitory activity of the fractions was determined by quantifying the amount of p-nitroaniline release by reading the absorbance at 410 nm (Hitachi U-1900).
2.6. Identification and purification of L. donovani serine protease The L. donovani aprotinin-sensitive serine protease (SP-Ld) of molecular mass 58 kDa obtained from the promastigotes of an Indian strain was purified as described by Chowdhury et al. [15] Briefly, the L. donovani promastigotes (second-passaged) were grown to the late log phase, and the cells (~5 × 10 [9]) were harvested by centrifugation at 3000 g for 15 min at 4 °C. Then, the clear soluble supernatant obtained after cell lysis was precipitated with ammonium sulfate at 40% saturation and subjected to aprotinin–agarose affinity chromatography, followed by continuous elution electrophoresis. The purified SP was further characterized [15,16].
2.8. Protein mass fingerprinting The proteins were identified and validated by mass spectrometry (MS). The sample was introduced into the mass spectrometer through matrix-assisted laser desorption/ionization (MALDI), and the time of flight (TOF) of different fragmented peptides with different m/Z was recorded (Bruker Daltonics FLEX). The proteins were validated by
Table 1 Purification profile of serine protease inhibitor from Leishmania donovani (LdISP). Steps
Total protein (mg)
Total activity (TIU) × 106
Specific activity (TIU/mg) × 103
Yield (%)
Crude extract (cell lysate) post precipitation Heat treatment + ultracentrifugation Trypsin–agarose (affinity chromatography) Gel filtration (FPLC) G-75
1884.5 208.6 1.87 0.24
33.28 22.38 17.07 12.66
6.71 8.71 9.13 18.21
100 67.24 51.29 38.04
For the last three steps, the mean values of triplicate readings were taken.
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Fig. 7. Two-dimensional electrophoresis was carried out to determine the pI value of the protein, which was found to be 5.9. The 3.0–10.0 ready (Bio-Rad) IPG strips were used for electrofocusing.
targeted MS and quantitative approaches accordingly. The data from MS/MS were then searched using Mascot-based database search engines.
Fig. 9. Comparative inhibitory capacity of LdISP: the purified LdISP was incubated with trypsin, elastase, and SP-Ld. The inhibitor LdISP substantially inhibits trypsin and elastase but not the SP-Ld. The mean values of triplicate readings for each coordinate were employed and plotted.
3.2. Purification of the L. donovani endogenous ISP 3. Results 3.1. Identification of LdISP The serine protease inhibitors (LdISPs) of L. donovani promastigotes were identified by subjecting the heat-treated cell lysate to highly sensitive reverse zymography. The reverse zymogram of the heat treated cell lysate showed two bands with molecular weights of approximately 52.2 and 21.8 kDa (Fig. 2). The band with low molecular weight (21.8 kDa) was of much higher intensity, designated as LdISP and chosen for further studies.
To understand the biological functions of LdISP, the inhibitor was purified by extracting the L. donovani cell pellets by sonication, heat treatment, ultracentrifugation, affinity binding, and gel filtration. The eluted fractions from the trypsin–agarose column showed two peaks at A280 (Fig. 3). Each major fraction (i.e., 21.8 and 52.2 kDa) was subjected to an enzyme assay. The smaller peptide LdISP (21.8 kDa) was chosen for further studies due to its inhibitory effect on S1A class enzymes (Fig. 4). The same fraction was pooled, concentrated, and purified further by gel filtration. The G-75 column elution profile showed a single peak (Fig. 5). The gel-purified (G-75) protein was further analyzed in
Fig. 8. Trypsin inhibition assay plot by LdISP: Trypsin activity was assayed subsequent to incubation with varying concentrations of LdISP. For 30 min, 4 nM of trypsin was incubated with ), 0.5 nM LdISP ( ), 2.5 nM ( ), and equimolar 4.0 nM LdISP ( ) prior to assaying. A410 of the N-benzoyl-arginine para-nitroaniline hydrolysis reaction mixture no LdISP ( was measured at various time intervals over the 200-s time course. The reaction rate is related to change in A410, and it is indicative of trypsin activity. Values are mean ± SD of triplicate measurements.
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Fig. 10. Protein mass fingerprinting (PMF): identification of protein using mass spectrometry data or with protein mass fingerprinting using Mascot software (Matrix Science) after being subjected MALDI/ToF mass spectrometry using a Bruker Daltonics FLEX. The PMF result of LdISP shows some resemblance with the ecotin-like protein from another Leishmania species, and it also presents the putative sequences of the peptide fragments.
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reverse-phase HPLC to confirm its purity. The HPLC analysis revealed a single intense peak at a retention time of ~16 min (Fig. 6). This method resulted in a 38.04% yield (Table 1) of the purified LdISP. 3.3. Two-dimensional electrophoresis On subjecting the purified LdISP to two-dimensional (2D) electrophoresis, the pI of the inhibitor was found to be 5.9 (Fig. 7) using PD Quest software, which indicates the acidic nature of the inhibitor. 3.4. Inhibition assay The inhibitory activity of LdISP against trypsin was determined by measuring A410 of the hydrolysis of BApNA in a reaction mixture with varying concentrations of LdISP (0.5–4 nM) (Fig. 8). The comparative inhibitory activity of LdISP against trypsin, elastase, and purified SP-Ld was determined by measuring the hydrolytic activity toward BApNA. LdISP showed a strong inhibitory effect against bovine trypsin and elastase but not against SP-Ld (Fig. 9). 3.5. Partial sequence elucidation MALDI/ToF MS analysis reveals the match scores of the fragmented peptide sequences of LdISP with the other peptide sequences in the data bank. In silico protein mass fingerprinting (PMF) using the Matrix Science software was conducted to determine the matching scores, if any, with other peptide sequences in protein/peptide sequence data banks. The PMF results of LdISP are highly similar to those of an ecotin-like protein from another Leishmania species (Fig. 10). 4. Discussion ISPs are ubiquitous proteins found in plants, mammals, bacteria, and parasites, with several functions such as strictly regulating proteases. Serpins were first investigated when the human plasma proteins antithrombin and anti-trypsin were studied in depth for their key roles in controlling blood coagulation and inflammation. Gradually, several new characteristics of ISPs were discovered, such as their cross-class functionality, wherein serpins could inhibit classes of protease other than SPs. Inhibitors were widely studied for their role in protease regulation and for their therapeutic implications until Chung C.H19 purified ecotin with cross-organism functionality, that is, inhibitor(s) of different origins acting on protease(s) of different species. Most parasites, if not all, have been hypothesized to possess these inhibitors of host proteases to ensure their survival. Thus, we primarily attempted to detect, identify, and characterize LdISP from promastigotes. It is highly interesting that these inhibitors (LdISP) inhibit SPs of higher organisms instead of their own ISPs. On assessment, LdISP was found to inhibit neutrophil elastase and bovine trypsin, whereas it did not have a marked effect on endogenous SP-Ld, which has been purified earlier [16]. Several similar studies lead us to the hypothesis that the protein is ecotin-like [19] and may be involved in the regulation of host SPs rather than inhibiting its own endogenous SPs. Recent studies on the Leishmania genome reveal that S1A class SPs are not transcribed in Leishmania, whereas they are transcribed by the mammalian host cell [20]. Thus, the presence of an ecotin-like protein in an Indian strain of L. donovani might provide insight into its immunomodular mechanisms and resulting implications. Although the genes transcribing the ecotin-like protein have long been identified in other Leishmania species [21] and in other related parasites, to the best of our knowledge, an endogenous ISP from an Indian strain of L. donovani (MHOM/IN/1983/AG83) promastigotes has not been characterized yet. Herein, we report the presence of an ecotinlike protein LdISP in the unicellular protozoan parasite L. donovani promastigote. In addition, we determined the pI value, heat stability, and inhibitory activity of LdISP to elucidate its role in infectivity.
The presence and function of LdISP-like proteins are uncharacteristic, as they are found in eukaryotic parasites, trypanosomatids, and many bacteria, with an apparent action on host proteases. This varied distribution provides an insight into evolutionary pathways and confirms the assumed horizontal gene transfer from prokaryotes to eukaryotic protozoans [20]. The inhibitory activity of LdISP against several host SPs (when studied in vitro) is functionally similar to other such inhibitory proteins from different prokaryotes and eukaryotic protozoans (www.uniprot.org). Given that LdISPs are now known to exist in an Indian strain of Leishmania, the action and regulation of the protein in infection progression within this parasite or in other unicellular parasites can be explored, because the ecotin-like protein does inhibit SPs of higher organisms. On the contrary, the endogenous inhibitors of many higher multicellular organisms regulate their own proteases. Genomic studies have proven that L. major has 13 SPs belonging to six families but not the S1A family, which is present in higher multicellular organisms [21]. At present, the possible location of the protein and its activity at different stages of the Indian strain of L. donovani is being investigated for its significant role in understanding the regulation of SPs and the progression of infection. In a new direction, Ramalho-Ortigão JM [22] stated that these parasitic ISPs not only inhibit the host SPs but also play an important role in inhibiting the tryptic enzymes of vectors while maturing in their gut. This notion further proves the evolution of intricate survival strategies of unicellular eukaryotic parasites. Eggers et al. [23] showed that several host SPs that can be inhibited by periplasmic ecotin include those expressed by the innate immune system such as the complement system and the enzymes involved in coagulation cascades. This warrants further investigation into the various other roles of LdISP. Thus, these strong hypotheses and the conclusive results further our understanding of Leishmania pathophysiology, survivability, and etiology, with new implications for LdISP. In addition, these studies can also be exploited to design target-specific drugs, leading to novel therapeutic trends to combat VL. 5. Conclusion The present study demonstrated the identification, purification, and biochemical characterization of an endogenous ISP in L. donovani with an apparent molecular weight of ~21.8 kDa. This acidic inhibitor inhibits SP of higher organisms but not its own SPs. Apparently; enzymatic studies indicate that the inhibitor acts on host SPs to inhibit their proteolytic activity. Thus, the inhibitor proves the evolution of parasite survival strategies with a significant role in disease progression. Conflict of interest None of the authors have any potential financial conflict of interest related to this manuscript. Acknowledgment The study was supported by the University Grants Commission (UGC) (F no.-11-91/2008/BSR) /RFSMS and Council of Scientific and Industrial Research (CSIR), Government of India (37(1537)/12-EMR-II). The authors are grateful to the Indian Institute of Chemical Biology (IICB), Kolkata, and Sandor Proteomics, Hyderabad, for providing the cell lines and mass spectrometry facilities, respectively. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.lfs.2015.12.004.
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