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A novel extracellular calcium-dependent cysteine proteinase from Crithidia deanei

A novel extracellular calcium-dependent cysteine proteinase from Crithidia deanei
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  A novel extracellular calcium-dependent cysteine proteinasefrom  Crithidia deanei  Claudia M. d  Avila-Levy, Rodrigo F. Souza, Rosana C. Gomes,Alane B. Vermelho, and Marta H. Branquinha * Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de G   oo es, Universidade Federal do Rio de Janeiro, CCS,Bl I, 21941-590, Rio de Janeiro, RJ, Brazil  Received 15 April 2003, and in revised form 25 September 2003 Abstract An extracellular cysteine proteinase from an aposymbiotic strain of   Crithidia deanei   was purified 39-fold by a combination of anion-exchange and gel filtration chromatographies. The native molecular mass of this proteinase was estimated to be 225kDa bygel filtration chromatography and it migrates in SDS–PAGE as a single band of 80kDa. The optimal enzymatic activity on gelatinwas found to occur in the presence of calcium at a neutral pH and at 28  C. The enzyme was completely blocked by E-64 and EGTA,and partially inhibited by iodoacetamide, leupeptin, and EDTA. Compounds such as PMSF, aprotinin, and pepstatin weakly in-hibited the enzyme. The protein purified in the present work shares some features with those of the family of neutral calcium-dependent cysteine proteinases named calpains, previously detected in the family Trypanosomatidae as cell-associated enzymes in Leishmania donovani   and  Trypanosoma brucei  . The cysteine proteinase from  C. deanei   is distinct from the well-characterizedmammalian calpains, but some degree of similarity is displayed to invertebrate calpain-related enzymes.   2003 Elsevier Inc. All rights reserved. Keywords:  Endosymbiont; Trypanosomatid; Purification; Extracellular proteinase; Cysteine proteinase; Calpain The medical and economic importance of trypano-somatids that infect vertebrates (e.g.,  Trypanosomabrucei  ,  T. congolense ,  T. cruzi  , and  Leishmania  spp.) andplants ( Phytomonas  spp.), together with their unusualcellular and antigenic characteristics, has resulted in thepreferential investigation of these taxa by researchers,whereas trypanosomatids that infect insects have largelybeen overlooked. Nevertheless, the lower trypanoso-matids have been used as laboratory models for bio-chemical and molecular studies because they are easilycultured under axenic conditions [1].The genus  Crithidia  comprises monoxenic parasitesof insects that present amastigote forms and a barley-corn form, named choanomastigote, in their life cycle[1]. In this genus, the trypanosomatids  C. deanei  ,  C.desouzai  , and  C. oncopelti   contain bacterial symbionts inthe cytoplasm, known as endosymbionts. The availabledata in the literature indicate that the presence of theendosymbiont interferes in many aspects with the me-tabolism of the trypanosomatid, suggesting that severalmetabolites important for the eukaryotic cell growth aresynthesized by the bacterium (reviewed in [2]). Thepossibility of elimination of the endosymbiont by theusage of antibiotics (cure) has increased the interest inthe study of endosymbiont-harboring species, sinceseveral bacteria–protozoa interactions can be analyzedby the comparison of cured and normal strains.Our group has been examining the role of proteolyticactivities in trypanosomatids. Proteases are enzymesthat catalyze the hydrolysis of peptide bonds. They aredivided into four major families by virtue of the specificchemistry of their active site: aspartic-, serine-, metallo-,and cysteine proteinases. These enzymes have been im-plicated in various facets of host–parasite relationship,including parasite survival and pathogenesis as well asthe breakdown of host proteins to provide amino acidsfor nutrition (reviewed in [3]). * Corresponding author. Fax: +55-21-2560-8344. E-mail address: (M.H. Branquinha).0003-9861/$ - see front matter    2003 Elsevier Inc. All rights reserved.doi:10.1016/ of Biochemistry and Biophysics 420 (2003) 1–8  ABB  The detection of cysteine proteinases in the culturesupernatant of monoxenic trypanosomatids was re-cently reported in  C. deanei   (cured and normalstrains) and  C. desouzai   [4]. Up to now, these are theonly known monoxenic trypanosomatids that secretethis proteolytic class. Released cysteine proteinaseshave also been detected in the heteroxenic species  T.cruzi   [5] and  T. brucei   [6]. In the former, the majorcell-associated cysteine proteinase, named cruzipain, isalso detected in the culture supernatant [7]. Metallo-proteinases have been widely detected extracellularlyin monoxenic parasites of the genera  Crithidia  [4,8,9], Herpetomonas  [10],  Blastocrithidia  [11], and also in theheteroxenic genus  Phytomonas  [12,13]. The secretionof cysteine proteinases by a restricted group of monoxenic trypanosomatids may reflect some para-site–host interactions or differences in the overallmetabolism of these parasites that were not studiedyet [4].Here, we report the purification and partial charac-terization of an extracellular calcium-dependent cysteineproteinase from an aposymbiotic strain of   C. deanei  .The cured strain was used because a proteolytic assayusing gelatin as substrate demonstrated a twofold en-hancement of the overall extracellular proteolytic ac-tivity, in comparison to the wild strain [4]. Thisapproach is a way for the characterization of similarenzymes in monoxenic and heteroxenic species to de-termine the importance of the extracellular proteinasesin the Trypanosomatidae family. Materials and methods Parasite The bacterium-free strain of the trypanosomatid  C.deanei   was kindly provided by Dr. Maria Cristina M.Motta (Instituto de Biof   ıısica Carlos Chagas Filho,Universidade Federal do Rio de Janeiro, Brazil). Preparation of the cell-free supernatantCrithidia deanei   was maintained in yeast extract– peptone–KCl–sucrose medium supplemented with2mg% hemin (w/v) and 1% (v/v) heat-inactivated fetalbovine serum. Cells (10 11 ) were harvested at the logphase (36h) by centrifugation (2500  g   for 15min at4  C) and washed three times with cold PBS. The intactcells were resuspended in 1L of isotonic phosphatebuffer and incubated for 6h at 28  C. After this inter-val, the cells were removed by centrifugation and thesupernatant was passed over a 0.22- l m filtration unit(Millipore). The supernatant was concentrated 250times in an Amicon Diaflo membrane (cut-off 10,000MW). Cellular viability The survivability of the organisms along the incuba-tion in the isotonic phosphate buffer was assessed bymobility, trypan blue cell dye exclusion [14], and bycounting(withhemocytometer)thenumberoforganismsthroughout the incubation period. Cellular viability wasalso determined by monitoring malate dehydrogenase(MDH), an intracellular enzyme, in the supernatant [15].Briefly, the MDH activity was determined by followingphotometricallytheoxidationofNADHat340nminthepresence of oxalacetate; the production of NAD þ is ac-companied by observing the lack of absorbance at340nm. At each hour of incubation, 10-ml aliquots werecollected, and the protein content, the proteolytic andMDH activities were measured in the cell-free superna-tant. A positive control of the MDH activity was done inthe culture by lysing cells with 20% Triton X-100. Proteinase purification The chromatographic separations were carried out atroom temperature (about 22  C) using an FPLC system(Pharmacia). Briefly, the sample was applied to a Mono-QHR5/5anion-exchangecolumn(Pharmacia)witha40-mingradientfrom0to1MNaClin10mMTris–HCl(pH8.9) at a flow rate of 1mlmin  1 . Fractions with proteo-lytic activity towards gelatin were pooled and concen-tratedwithanAmiconultrafiltrationcellequippedwithaYM-10 membrane. The concentrate was subjected to gelfiltration using a Superose 12 HR10/30 column (Phar-macia), previously equilibrated in 100mM Tris–HCl, pH8.9,andthechromatographywasconductedataflowrateof 0.5mlmin  1 for 40min. The column was calibrated inthe same buffer with the following markers: catalase(232kDa),  b -amylase (200kDa), alcohol dehydrogenase(150 kDa), bovine serum albumin (66kDa), and cyto-chrome  c  (12.4kDa) (Sigma). The enzyme was storedat ) 20  C and used throughout this study. Enzyme assay The proteinase activity at each purification step wasmeasured spectrophotometrically using the substrategelatin (Merck) according to the method of Jones et al.[16]. Briefly, 5 l l of the enzyme solution and 395 l l of 0.1M sodium phosphate buffer, pH 7.0, were added to600 l l of the substrate solution (1% (w/v) gelatin indistilled water) and the mixture was incubated at 28  Cfor 30min. A 300- l l sample was removed from the re-action mixture and added to 400 l l of isopropanol. Aftera 16,000  g  , 10-min centrifugation, the supernatant wasremoved and the absorbance was measured at 280nm.One unit of enzyme activity was defined as the amountof enzyme that caused an increase of 0.001 in absor-bance unit, under standard assay conditions. 2  C. M. d’Avila-Levy et al. / Archives of Biochemistry and Biophysics 420 (2003) 1–8  Protein determination For protein determination, the Bradford method [17]was employed using bovine serum albumin as standard. Electrophoresis analysis SDS–PAGE was performed at each purification stepusing 10% polyacrylamide gels in Laemmli  s buffers [18].The fractions from the Superose 12 column with pro-teolytic activity were analyzed in two different ways: (a)a non-reduced sample was mixed with SDS–PAGEbuffer and applied to the gel and (b) a reduced samplewas prepared by boiling for 5min in SDS–PAGE buffercontaining 2-mercaptoethanol. The protein bands wereidentified by silver staining [19]. Optima pH and temperature determination The effect of pH on the proteolytic activity was de-termined using the standard proteinase assay with gel-atin described above, with the following buffer systems:sodium citrate (0.1M, pH 4.0–5.0), sodium phosphate(0.1M, pH 6.0–8.0), and glycine–NaOH (0.2M, pH 9.0– 10.0). The proteolytic activity of the purified enzyme ongelatin was analyzed at different temperatures (20, 28,37, 50, and 60  C) as described before. Heat stability This assay was determined by pre-incubating thepurified enzyme in 0.1M sodium phosphate buffer, pH7.0, for 30min at 20, 28, 37, 50, and 60  C, and the re-maining enzymatic activity was measured under thestandard assay conditions described previously. Proteinase class determination The following proteinase inhibitors (Sigma) wereused: 2 l g/ml aprotinin, 2 l g/ml STI, 1mM PMSF,20 l M E-64, 10 l M iodoacetamide, 70 l M leupeptin,250 l M of 1,10-phenanthroline, 5mM EDTA, 5mMEGTA, and 1mM pepstatin. DTT was used at a finalconcentration of 2.5mM. The purified enzyme was pre-incubated for 1h at 28  C with each compound at thefinal concentrations cited above. Following this incu-bation, the substrate gelatin was added and the re-maining activity was measured under standard assayconditions. Results are expressed as the relative per-centage of activity with inhibitors subtracted from theactivity without inhibitors. Determination of calcium sensitivity The purified enzyme was dialyzed overnight at 4  Cagainst 100mM Tris–HCl, pH 8.9, containing 5mMEDTA, and 5mM EGTA. For the proteolytic assay, thereaction mixtures contained the apoenzyme, 100mMTris–HCl, pH 7.0, and varying amounts of CaCl 2  toobtain free Ca 2 þ concentrations from 0.01 to 20mM. Indifferent reaction mixtures, 5mM inorganic salt solu-tions (ZnSO 4 , MgSO 4 , MnCl 2 , and BaCl 2 ) replacedCaCl 2 . The apoenzyme was incubated for 1h at 28  Cwith each metal ion at the final concentrations citedabove. Following incubation, the substrate gelatin wasadded and the proteolytic activity measured understandard assay conditions. The results are expressed aspercentage of total activity prior to dialysis. Protein immunoblotting  The purified enzyme was electrophoretically trans-ferred to nitrocellulose. The blots were blocked with10% nonfat dried milk in PBS containing 0.05% Tween20 for 1h at room temperature and incubated with theprimary antibodies at 1:500 dilution overnight at 4  C.The membranes were incubated with the secondary an-tibody conjugated to peroxidase and visualized withdiaminobenzidine (DAB) as described by the manufac-turer (Sigma, St. Louis, MO, USA). The primary anti-bodies used were C21, C23, and C24 (raised against thewhole molecule, the cysteine active site, and the histidineactive site, respectively, of human brain m-calpain) [20],anti-Dm-calpain (raised against the  Drosophila melano- gaster  calpain) [21], and anti-cruzipain (raised againstthe major cysteine protease of   Trypanosoma cruzi  ) [22]. Results Cellular viability The cellular viability was assessed throughout the in-cubationperiodinPBSbymonitoringthemobilityofcellsand trypan blue cell dye exclusion. Non-motile or deadcells were not detected along the 6-h incubation in PBS.Besides,MDH,anintracellularenzyme,wasnotfoundinthe supernatant, indicating that the extracellular pro-teinase detected in this study was not released by autoly-sis.Fig.1showsthetimecourseofreleaseofproteinsandproteinases by the bacterium-free strain of   C. deanei   andthe absence of MDH activity in its culture supernatant. Proteinase purification An extracellular proteinase from  C. deanei   was pu-rified by a combination of anion-exchange and gel fil-tration chromatographies. The proteinases releasedalong the 6-h incubation in PBS were concentrated 250times by ultrafiltration and submitted to a Mono-Qanion-exchange column. Six major protein peaks wereeluted from this column, according to their absorbance C. M. d’Avila-Levy et al. / Archives of Biochemistry and Biophysics 420 (2003) 1–8  3  at 280nm (Fig. 2A). The fifth and sixth peaks displayedproteolytic activity and were pooled, concentrated, andseparated further by a Superose 12 HR10/30 gel filtra-tion column (Fig. 2B). This column yielded two pro-teinase peaks, and the first one (fractions 7–9) wasgathered and characterized further. The result of the  C.deanei   proteinase purification protocol, starting with2.6mg protein extract, is summarized in Table 1. Thesecond peak with proteinase activity corresponds to a60-kDa metalloproteinase found in the culture super-natant of   C. deanei  , and previously identified by ourgroup by SDS–PAGE-gelatin analysis [4]. This fractionwas not further analyzed in the present work. Estimation of molecular mass The enzyme was eluted in a fraction corresponding toa molecular mass of approximately 225kDa by gel fil-tration (Fig. 2B). SDS–PAGE analysis of the purifiedenzyme under denaturing and reducing conditions re-vealed a major band with a molecular mass of 80kDaand a lower molecular mass band at 68kDa. The non-reduced protein was also applied into SDS–PAGE tocheck the purity of the enzyme and showed a single bandat approximately 80kDa (Fig. 3). Optima pH and temperature determination The pH dependence of the purified proteinase activitywas determined by an enzymatic assay using gelatin.Under the conditions of this experiment, the enzymeexhibited maximal activity at pH 7.0. The enzyme ac-tivity decreased markedly at pH values below 6.0 andabove 9.0 (Fig. 4A). The optimum temperature of thepurified enzyme was 28  C, retaining less than 74% of itsmaximum activity at 50  C (Fig. 4B). Heat stability The stability of the proteinase was tested by pre-in-cubating the enzyme at various temperatures (20–60  C)for 30min before assaying the activity at 28  C, pH 7.0,using gelatin as substrate (Table 2). The purified enzymeretained 37 and 26% of its maximum activity at 20 and50  C, respectively. Proteinase class determination The extracellular proteinase of bacterium-free strainof   C. deanei   was completely blocked by E-64 andEGTA, and partially inhibited by iodoacetamide, leu-peptin, and EDTA. The activity of the purified pro- Fig. 2. Elution profiles of an extracellular proteinase from  C. deanei   oncolumn chromatographs. (A) Mono-Q anion-exchange column. Thecell-free extract was applied on a Mono-Q column; proteins wereeluted with a 0–1M NaCl linear gradient at a flow rate of 1ml min  1 for 40min. (B) Superose 12 gel filtration column. The fractions withproteinase activity from Mono-Q were applied to a Superose 12 gelfiltration column and elution was performed at a flow rate of 0.5mlmin  1 for 40min. The arrows indicate the calibration withprotein standards.Fig. 1. Detection of proteins and proteinases from the aposymbioticstrain of   C. deanei   in the cell-free supernatant of PBS cultivation. Themalate dehydrogenase activity was assessed throughout the incubationperiod; a positive control was done at the 6h in a culture aliquot bylysing cells with 20% Triton X-100. The values represent means of three independent experiments, which were performed in triplicate.4  C. M. d’Avila-Levy et al. / Archives of Biochemistry and Biophysics 420 (2003) 1–8  teinase against gelatin was greatly stimulated in thepresence of DTT. Compounds such as PMSF, aprotinin,STI, and pepstatin weakly inhibited the enzyme. 1,10-Phenanthroline was found to be an ineffective inhibitorof the enzyme and actually the proteolytic activity wasslightly enhanced (Table 3). Ion requirement for enzyme activity The activity of the cysteine proteinase was absolutelycalcium-dependent. The ions Zn 2 þ , Mg 2 þ , Mn 2 þ , andBa 2 þ could replace Ca 2 þ , but with less efficiency(Table 4). Western blot analysis To test if mammalian and/or Dm calpain antibodiescross-react with the calcium-dependent cysteine pro-teinase from  C. deanei  , blots were probed with differentpolyclonal antibodies. As shown in Fig. 5, only the anti-Dm calpain cross-reacted with the enzyme. No commonepitopes were found between mammalian calpains and C. deanei   cysteine proteinase. Additionally, the blotprobed with anti-cruzipain showed no cross-reactivitywith the well-characterized cysteine proteinase from  T.cruzi  . Lysates of mouse liver were recognized by C21, Table 1Purification of an extracellular cysteine proteinase from  Crithidia deanei  PurificationstepTotal protein( l g)Total activity(U)Specific activity(U l g  1 )Purification factor(fold)Activity yield(%)PBS supernatant 2613.45 3508 1.34 1 100Mono-Q 33.86 1444 42.65 31.83 41.16Superose 12 13.08 700 53.52 39.94 19.95Fig. 3. SDS–PAGE analysis of the purification steps of the extracel-lular proteinase from the bacterium-free strain of   C. deanei  . Aliquotsof samples containing proteolytic activity were taken from the fol-lowing steps: concentrated crude supernatant (lane A), anion-exchangecolumn (lane B), gel filtration column (lane C), and a non-reducedsample from gel filtration column (D). About 5 l g of protein was an-alyzed in lane A, 1.3 l g in lane B, and 0.5 l g in lanes C and D. Thenumbers on the left indicate molecular mass markers (in kDa).Fig. 4. Effect of pH (A) and temperature (B) on  C. deanei   extracellularproteinase activity. The proteolytic activity was determined at pH 4.0– 10.0 and at 20–60  C as described under Materials and methods.Maximal proteolytic activity is shown as 100%. The values representmeans of three independent experiments, which were performed intriplicate.Table 2Effect of temperature on the stability of the purified extracellularproteinaseTemperature (  C) Residual activity (%)20 3728 10037 5050 2660 0 Note.  The values represent means of three independent experi-ments, which were performed in triplicate. C. M. d’Avila-Levy et al. / Archives of Biochemistry and Biophysics 420 (2003) 1–8  5
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