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Protein J (2010) 29:283–289 DOI 10.1007/s10930-010-9250-8

Partial Biochemical Characterization of a Metalloproteinase from the Bloodstream Forms of Trypanosoma brucei brucei Parasites Karina Pires de Sousa • Jorge Atouguia Marcelo Sousa Silva

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Published online: 23 May 2010 Ó Springer Science+Business Media, LLC 2010

Abstract Metalloproteinases (MMP) belong to the family of cation dependent endopeptidases that degrade matrices at physiological pH and to cleave extracellular matrix proteins. They play an important role in diverse physiological and pathological processes; not only there diverse types of MMP differ in structure and functionally, but also their enzymatic activity is regulated at multiple levels. Trying to shed some light over the processes that govern the pathology of African Trypanosomiasis, the aim of the present study was to examine the proteolytic activity of the crude trypanosome protein extract obtained from the bloodstream forms of Trypanosoma brucei brucei parasites. We hereby report the partial biochemical characterization of a neutral Trypanosoma brucei-metalloproteinase that displays marked proteolytic activities on gelatin and casein, with a molecular mass of approximately 40 kDa, whose activity is strongly dependent of pH and temperature. Furthermore, we show that this activity can be inhibited by classical MMP inhibitors such as EDTA, EGTA, phenantroline, and also by tetracycline and derivatives. This study has a relevant role in the search for new therapeutical targets, for the use of metalloproteinases inhibitors as treatment strategies, or as enhancement to trypanocidal drugs used in the treatment of the disease.

K. P. de Sousa J. Atouguia M. S. Silva (&) Unidade de Ensino e Investigaçaõ de Clıńica das Doenças Tropicais, Centro de Mala´ria e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Rua da Junqueira, 100, 1349-008 Lisbon, Portugal e-mail: [email protected]

Keywords Gelatinase Metalloproteinases Proteolysis Trypanosoma brucei Abbreviations AT African Trypanosomiasis BBB Blood-brain barrier EDTA Ethylenediamine tetraacetic acid EGTA Ethylene glycol tetraacetic acid HAT Human African Trypanosomiasis MMP Metalloproteinases PBS Phosphate buffered saline PMSF Phenylmethylsulfonyl fluoride T.b.brucei Trypanosoma brucei brucei TbMMP Trypanosoma brucei brucei metalloproteinase

1 Introduction The extracellular haemoflagellate protozoan Trypanosoma brucei is responsible for thousands of infections every year, leading to extensive damage on humanitarian, veterinarian and economical extents. African Trypanosomiasis (AT) has two variants: the animal (commonly know as Nagana) and the human (commonly know as Sleeping Sickness). Both infections are rooted on the inoculation of Trypanosoma brucei parasites, by infected tsetse flies, in the blood of its mammal hosts. The disease is widespread in both Western and Eastern Africa where, correspondingly, the sub-species Trypanosoma brucei rhodensiense and Trypanosoma brucei gambiense exist. In the case of the animal variant of AT, Trypanosoma brucei brucei is also the sub-specie responsible for the infection. In any case, AT plays a devastating role in the health and welfare of people

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throughout large areas of sub-Saharan Africa, where the ailment imposes a major setback on the development of the economy, due to the high mortality and morbility associated with these infections [21]. Clinically, Human African Trypanosomiasis (HAT) comprises two stages. The early stage, during which trypanosomes are observed in the haemolymphatic system, is often diagnosed after fever, splenomegaly, adenopathies, endocrine disarrays [20, 40], cardiac, and neurological or psychological disorders [2]. Trypanosomes in the bloodstream multiply and rapidly infect organs such as the spleen, liver, lymph nodes, skin, heart, eyes and endocrine system [2]. During the second or late stage of the disease, trypanosomes are localized in the central nervous system (CNS) where they lead to a number of disorders, including sensory, motor and psychic disturbances, neuroendocrine abnormalities and disturbed circardian rhythms, culminating in death [2, 9]. In accordance with features of its life cycle, T. b. brucei might express and secret some proteolytic enzymes in order to reach the host’s inner tissues. Previous studies have demonstrated the pivotal role of proteases such as MMP in the parasite invasion processes into the hosts, assisting migration by hydrolyzing specific proteins such as most macromolecular components of the extracelular matrix and the components of basement membranes [5, 28, 30, 34]. These membranes are made up of a network of specialized functional elements forming a matrix composed of collagen, laminin, entactin, proteoglycans, perlecan, nidogen and glycosaminoglycans [1, 27]. In general, the metalloproteases comprise a family of zinc-dependent neutral endopeptidases associated with the degradation of membranes at physiological pH [11, 16, 24, 37]. They were first described as responsible for the dissolution of the tadpole tail [14], but ensuing studies have shown that these enzymes are important for both normal and abnormal biological processes, ranged from uterine involution to calcific aortic stenosis [7, 18]. Several studies have shown that many parasites have deployed proteinases to accomplish some of the tasks necessary to a parasitic life style. In this context, MMP endow with the ability to process proteins and peptides, to provide required amino acids, to facilitate infection and dissemination through host tissues by hydrolyzing extracellular proteins, and by evading from host immune response [23]. For instance, Schistosoma mansoni and Strongyloides stercoralis secrete a MMP whose activity is associated with penetration of host skin [28]. Particularly, the paradigm for zinc MMP in kinetoplastid protozoa, like Leishmania and Trypanosoma, is GP63, which is highly conserved between species in terms of homology [3]. Procyclic insect stage trypanosomes have an uncharacterized surface MMP with endoproteolytic activity [4].

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Due to the pivotal role of MMP in physiological and pathological processes, and the biochemical pathways of proteolytic processes occurring in Trypanosoma brucei, the aim of this study was to examine the proteolytic activity of the crude trypanosome extract obtained from the bloodstream forms of T. b. brucei. We report herein the presence of a neutral cation-dependent metalloproteinase, which displays marked proteolytic activity in gelatin and casein and whose activity can be inhibited by classical MMP inhibitors such as EDTA, EGTA, and phenantroline, and also by tetracycline and derivates. Further studies may unlock new opportunities for better understanding AT and also indicate potential new pathways for drug targeting.

2 Materials and Methods 2.1 Reagents and Buffers Reagents: ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), 1,10-phenanthroline, phenylmethylsulfonyl fluoride (PMSF), tetracycline, doxycycline. All acquired from Sigma–Aldrich, USA. Buffers: zymography development buffer [0.5 M Tris– HCl, pH 7.5; 200 mM NaCl; 5 mM CaCl2; 0.02% (w/v) Brij-35; final pH 7.0]; SDS Sample Buffer [100 mM Tris– HCl, pH 6.8; 20% (v/v) glycerol; 4% (w/v) SDS; 0.2% (w/v) bromophenol blue; 2% b-mercaptoethanol]; destaining solution [10% methanol : 5% glacial acetic acid]. 2.2 Parasites and Sample Preparation All animals used in this study were obtained and maintained in approved animal facilities at the Instituto de Higiene e Medicina Tropical (IHMT), Lisbon, Portugal. Approximately 5.0 9 102 T. b. brucei parasites from the clonal isolate GVR 35-1.7 were inoculated intraperitoneally in three female BALB/c mice and used for the assessment of proteolytic activity. When the infected animals reached the first peak of parasitaemia, as assessed by optical microscopy 7 days after the initial infection, whole blood was collected by cardiac puncture. The bloodstream forms of T. b. brucei were then purified using a DEAESepharoseÒ Fast Flow (Amersham Pharmacia Biotech, USA) anionic exchange chromatography column [25]. This allowed us to obtain a purified crude trypanosome extract, with no traces of host cells. The fractions obtained from eluting the column with PBS-20 mM glucose were observed by optical microscopy in order to verify the presence of the bloodstream forms of T. b. brucei; the eluted fractions that were attested as positive for the presence of parasites were washed three times with PBS20 mM glucose in order to clean the extract from any

Partial Biochemical Characterization of a Metalloproteinase

residual blood or serum components. The resulting pellets were ressuspended in sterile PBS and aliquots were then kept at -80 °C until further use. Total protein concentration in T. b. brucei extracts was quantified using Bradford reagent (Bio-Rad, USA) and bovine albumin used as standard.

2.3 Determination of Metalloproteinase Activity by Zymography In order to assay the enzymatic activity of this extract, the samples were diluted 1:10 in Zymography Sample Buffer and tested by means of zymography assays. Protease activity was visualized by in-gel zymography assays using gelatin or casein as substrate. The substrate was co-polymerized in a 10% acrylamide gel at a final concentration of 0.1% (w/v). A total of 1,2 lg of protein was loaded per lane onto the gel and electrophoresis was carried out at 4 °C at a constant voltage of 90 V, after which the gels were briefly washed with water and twice with a solution of 2.5% Triton X-100 (Sigma, USA) for 30 min. Following this step there was an overnight incubation at 37 °C with zymography development buffer. To evaluate the catalytic efficiency and stability of the enzyme under different physical conditions, the development buffer was adjusted to different pH’s (an alkaline pH 9.0, the control pH 7.0 and an acid pH 5.0) and incubated at different temperatures (18, 37, and 50 °C). For the inhibitory assays, EDTA, EGTA, phenanthroline, PMSF, tetracycline or doxycycline, were added to the development buffer and incubated as described for the zymography procedure. Depending on the estimated inhibitory capacity, the concentrations of the inhibitors dissolved in the zymography development buffer were in the range of the mili- or the micromolar. In the case of EDTA and EGTA, concentrations ranging from zero to 400 mM were tested; in the case of tetracycline, from zero to 200 mM; for doxycycline, from zero to 5 lM; and for phenantroline, from zero to 5 mM. The concentration of PMSF, 10 mM, was chosen to be two fold the usual concentration described in the literature [22] in order for it to be absolutely conclusive that there is no inhibition of the metalloproteinase activity with PMSF, which is generally described as a serine protease inhibitor. After incubation, the gels were stained in Coomassie Brilliant Blue R-250 stain (Bio-Rad, USA) on a shaker for 3 h, followed by destaining on a shaker until clear bands were visible. Enzymatic activity was visible as clear bands (gelatin degradation) in a blue gel (intact gelatin). Molecular weight standards (Bio-Rad, USA) were used for assessment of protein mass.

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3 Results 3.1 Infection of BALB/c Mice with T. b. brucei The intraperitoneal infection of CD-1 mice with T. b. brucei produced an infection where the usual clinical manifestations of the disease were observed: anemia, thrombocytopenia, leukocytosis, hepato- and splenomegalia. This infection mimics the natural chronic infection and allowed the animals to survive up to 30 days. The infection reached its first peak of parasitaemia 7 days after the infection. 3.2 T. b. brucei Purification by Anionic Exchange Chromatography Anionic exchange chromatography is a simple and effective method of purifying T. b. brucei from whole blood. The crude extract obtained from the methodology described above guarantees that no serum components or blood cells can interfere in the proteolytic assay (Fig. 1). When the blood collected from euthanized animals was eluted from the chromatography column, the trypanosomes collected and forthwith referred to as extract were not only alive but also highly active. Optical microscopic analysis has shown that most of the parasites in the extract were in the slender, trypomastigote stage of the development cycle. 3.3 Determination of T. b. brucei’s Metalloproteinase Activity by Zymography Proteolytic activity was detected when the purified T. b. brucei extract was submitted to zymography on SDSGelatin gel (Fig. 2a) or SDS-Casein gel (Fig. 2b). These bands correspond to a molecular mass of approximately 40 kDa, and were the only visible activity on the gel (Fig. 2a, b). We have also observed a dose-dependent effect when increasing amounts of protein loaded onto the gel produced correspondently thicker activity bands (results not shown). Furthermore, we have confirmed the sensitivity of this method since levels as low as 120 nanograms of total protein produced detectable metalloproteinase activity in the gels (Fig. 2a, b). 3.4 Metaloproteinases from T. b. brucei can be Chemically Inhibited This proteolytic activity in the gelatin-containing gel was completely abolished by the addition of inhibitors to the development buffer, as seen in Fig. 3. The inhibition was

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Fig. 1 Trypanosoma brucei brucei a before; and b after the purification through the anionic exchange chromatography column. Notice the absence of any blood elements in image (b). These images were obtained by optical microscopy (409)

Fig. 2 a Proteolytic activity (signalled with the arrow) on a zymography gel by metalloproteases from Trypanosoma brucei bloodstream forms; b proteolytic activity on a zymography gel by metalloproteases from Trypanosoma brucei bloodstream forms

gelatin brucei casein brucei

attested by visual confirmation, since inhibited activity rendered no clear band against the dark-blue background of the gel. The results for EGTA and EDTA show a decrease in MMP activity at concentrations of 150 mM of EGTA or 100 mM of EDTA, respectively. In the case of tetracyclines, a decrease in the proteolytic activity was found when 50 mM of tetracycline, and as little as 1 lM of doxycycline were added to the development buffer. Phenantroline showed an inhibitory effect when the concentration of this chemical in the development buffer was of 2 mM. At lower concentrations these reagents produced only a slight, or no inhibition of the proteolytic activity.

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Fig. 3 Inhibition of the proteolytic activity on a gelatin zymography gel by metalloproteases from Trypanosoma brucei brucei bloodstream forms, incubated with a 10 mM PMSF; b 100 mM EDTA; c 150 mM EGTA; d 50 mM tetracycline; e 1 lM doxycycline; and f 2 mM phenantroline

There was no inhibition of the MMP proteolytic activity when 10 mM PMSF was included in the development buffer. This suggests that the proteolytic activity verified in the assays is due to proteases of the metalloproteinase class, and not to serinoproteases.


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3.5 Temperature and pH Requirements for T. b. brucei’s Metalloproteinases Activity Changes in pH, calcium concentration (results not shown) or temperature produced depletory effects on the enzyme activity present. Therefore, the MMP’s present in the T.b.brucei extract are very dependent on the physical conditions in the surrounding environment. More particularly, we tested the stability of the proteolytic activity in acid (pH 5), alkaline (pH9) and neutral (pH7) media. Activity was detected at pH7 but not pH5 or 9 (Fig. 4). We also assessed the enzyme activity at 18, 37, and 50 °C. Regarding the temperature, we tested a range from 18 to 50 °C. The results (Fig. 5) show that the proteases in the T. b. brucei extract are sensitive to temperature and the only visible proteolytic activity was found to be at 37 °C.

4 Discussion In the post-genomic era, the extensive scrutiny of countless biochemical pathways is inevitably confronted with the challenge of characterizing polypeptides with particular enzymatic activities. Previous work has shown that zymography can pinpoint enzymatic activity to a single

Fig. 4 Proteolytic activity on a gelatin zymography gel by metalloproteases from Trypanosoma brucei brucei bloodstream forms, in different pH conditions. a pH 5.0; b pH 7.0; c pH 9.0

Fig. 5 Proteolytic activity on a gelatin zymography gel by metalloproteases from Trypanosoma brucei brucei bloodstream forms, in three temperature conditions. a 18 °C; b 37 °C; and c 50 °C

polypeptide. The advantages of this method over conventional assays, include the ability to assess a repertoire of enzymes that have a particular activity in non-fractionated cell extracts, the potential to identify several proteases in the same gel, and the capacity to estimate the molecular weight and isoelectric point of the corresponding polypeptides and their isoforms, can be invaluable in identifying and monitoring specific and non-specific activities in complex biological and clinical samples [19, 26]. In addition, zymography has been shown to be extremely sensitive, as levels of less than 10 pg of gelatinase (MMP-2) have been detected on gelatin zymograms [22]. In this work, we have traced the proteolytic activity of the bloodstream forms of T. b. brucei to a single metalloproteinase.The substrates on which it has shown activity over, and the inhibitors that halt the enzymatic activity both suggest a metalloproteinase that might be capable of degrading proteins such as gelatin, casein, and matrix proteins such as collagen, laminin and elastin. Previous studies [31] have shown that T. brucei releases proteases extracellularly which are unstable or undetectable at pH 5.4 but active at pH 8.0, with the latter showing a faster rate of proteolysis. Our own results regarding the neutral pH environment required for the TbMMP to be fully active suggest that alterations in the enzyme (maybe structural alterations caused from a deviation in the

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conformation of the enzyme as a result of changes in the aminoacids’ protonation) damage its function and cease its activity. It seems very likely that the enzyme will work at a strict temperature and pH setting, if we take into account the fact that the parasite has evolved in accordance to its host, and therefore to the physical and biological settings that it presents; that is, human blood’s range of pH and temperature. In general, MMPs are released from cells in a proteolytically inactive pro-form (zymogen) which is about 10 kDa larger than the activated form. Since the pro-form becomes activated during the process of renaturation after gel electrophoresis, both forms can be detected on zymograms [8, 32]. However, we could not verify this fact since there was only one visible band of proteolytic activity. This might mean that the metalloproteinase in the extracts of T. b. brucei obtained could be activated early in the differentiation and infection processes, or that there is a wide predominance of the active form in the extract we used. Metalloproteases are cation-dependent proteases that require Zn2? or Ca2? to maintain activity [29]. Therefore, chelating agents with affinity for divalent cations will decrease their ability to form the active site of the MMP [12]. Our results have shown that, in a greater or lesser degree, all of the chosen inhibitors were able to successfully reduce the enzymatic activity and therefore are eligible candidates for further pharmacological in vitro and in vivo studies. Furthermore, other than being naturally inhibited by negative feedbacks or over-expression of other MMPs, other chemical molecules are capable of hindering the activity of these powerful enzymes. We have shown in our results that tetracycicline and its derivative doxycycline are capable of inhibiting the metalloproteinase activity in the conditions of the assay. Other than being recognized as metal chelating agents, these antibiotics are also ionophores, ribosomal-interfering and free radicals producers [35]. In the case of tetracyclines, it has been postulated that there are multiple mechanisms by which it can inhibit matrix-degrading cascades [15, 31, 41]. The results hereby presented show accordance to those found by Huet et al. [17], who have described and characterized the activity of trypanosomal released proteinases of low molecular weight which are active between pH 6.0 and 8.0. Although they have used synthetic substrates to investigate the activity of these proteases, it is safe to assume that the MMPs studied in this article might be the same. Interestingly, all of the above results can also be related to those of Caffrey et al. [10], who have studied a T. brucei cysteine protease at neutral pH, based on the fact that active cysteine proteases are secreted by living intact T. b. brucei in culture, and having concluded that significant activity and stability were maintained up to pH 8.0.

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All of the latter are consistent with a possible extracellular function. Although each MMP is often described as having a specific substrate, individual enzymes can act on many different proteins. In addition, and more importantly, the spectrum of substrates between enzymes is actually quite similar [33]. This would explain why the metalloproteinase in the extract from the bloodstream forms of Trypanosoma brucei share the same activity and spectra of activation in both casein and gelatin gels. Three potential hypotheses exist regarding the results of our investigation: (a) there is one MMP which has increased proteolytic efficiency under the assay conditions employed; (b) there is one MMP which has enhanced expression and production at a molecular level in the parasites isolated; or (c) there are several MMP inhibited or somehow disabled by the preeminence of a stronger MMP, which shows up in the zymography gel as the only active enzyme. These hypotheses are born from the fact that we could find only one band demonstrating proteolytic activity in the gels, regardless of the substrate provided, and that the enzyme causing that activity had in any case the same molecular mass of approximately 40 kDa. Several parasitic organisms rely on metalloproteinases in order to accomplish many of the functions necessary for their survival in the host [31]: filarial cathepsins are involved in larval molting and cuticle and eggshell remodeling [13], bloodfeeding hookworms express multiple proteases that mediate hydrolysis of ingested hemoglobin [30]. As such, it is reasonable to assume that the MMP that can be found in the bloodstream forms of Trypanosoma brucei brucei may play an important role in African Trypanosomiasis since they can mediate fundamental parasite processes, such as life cycle differentiation and regulation of intercellular communication, which will ultimately affect the pathological course of the disease. The results presented here are crucial to better grasp the host/ parasite interaction, considering the significant role of these proteinases as mediators of vital parasite processes. Further studies may unlock new opportunities for better understanding AT pathogenesis and also indicate new pathways as potential targets for new drugs. The potential of targeting proteases from T. brucei as a mean to achieve trypanocidal and therapeutical effects is not to be underestimated, as a line of potential research could be achieved by relating the results hereby presented with those demonstrated by Troeberg et al. [38], Vicik et al. [39] and Scory et al. [36]. The importance of cysteine proteinase activity for survival of T. brucei has been demonstrated by Scory et al., with the suggestion of such activity being an appropriate target for anti-trypanosomal chemotherapy. Moreover, Vicik et al. have studied a synthetic cysteine protease inhibitor which in addition to


inhibit rhodesain in the range of the low micromolar, remains non-toxic to host-derived macrophages up to concentrations of 125 lM. Furthermore, the same inhibitors display antileishmanial and anti-plasmodial activity, suggesting their potential as novel broad-spectrum antiparasitic lead compounds. Additionally, Troeberg et al. have suggested that these cysteine proteases are required for T. b. brucei viability. An intriguing feature of T. brucei is its ability to cross the blood–brain barrier (BBB) and invade the central nervous system [13], with a high degree of dependency on calcium signaling [30]. In this context, MMP’s play an important role on the degradation of the matrix proteins that constitute the BBB, namely collagen, fibronectin and laminin, all of which are critical vascular matrix components. It has been suggested before that the extracellular release of peptidases from T. brucei could contribute to the BBB disruption by hydrolysis of collagen and/or its degradation products [6]. This comes in agreement with the findings described in this paper since we have shown that the bloodstream forms of T. b. brucei express MMP’s with hydrolytic activity in proteins with similar functions of those of collagen. In summary, we report in this study the presence of and partial biochemical characterization of a neutral 40 kDa proteinase that displays marked proteolytic activities on gelatin and casein, with strong pH and temperature requirements in crude extracts from bloodstream form T. b. brucei parasites. Furthermore, we have shown that this activity can be inhibited by EDTA, EGTA, tetracycline, doxycycline and phenantroline, strongly suggesting that this enzyme belongs to the metalloproteinase family. Acknowledgments The team wishes to show appreciation to Dr. Danielle Paixaõ-Cavalcante for valuable suggestions in the zymography procedures. Marcelo S. Silva thanks Fundaçaõ para a Cieˆncia e Tecnologia (FCT)—Portugal for a post-doctoral fellowship (SFRH/BPD/26491/2006).

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