Báo cáo khoa học: Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes potx

Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes Maria A. Juliano 1 , Darren R. Brooks 2 , Paul M. Selzer 3 , Hector L. Pandolfo 1 , Wagner A. S. Judice 1 , Luiz Juliano 1 , Morten Meldal 4 , Sanya J. Sanderson 5 , Jeremy C. Mottram 2 and Graham H. Coombs 5 1 Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sa ˜ o Paulo, Brazil; 2 Wellcome Centre for Molecular Parasitology, The Anderson College, University of Glasgow, UK; 3 Akzo Nobel, Intervet Innovation GmbH, BioChemInformatics, Schwabenheim, Germany; 4 Center for Solid-Phase Organic Combinatorial Chemistry, Department of Chemistry, Carlsberg Laboratory, Valby, Denmark; 5 Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, UK The CPB genes of the protozoan parasite Leishmania mex- icana encode stage -regulated cathepsin L-like cysteine pro- teases that are important virulence factors and are in a tandem a rray o f 1 9 genes. In this study, w e h ave c ompared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, a nd a H 84Y muta nt of the latter e nzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues ( N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S 1 -S 3 and S 1 ¢-S 3 ¢ subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P 3 to P 3 ¢ by natural amino acids. Carboxydipep- tidase act ivities of CPB3 and H 84Y were also observed u sing the peptide Abz-FRAK(Dnp)-OH and some of its ana- logues. The kinet ic parameters of hydrolysis by C PB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S 3 to S 3 ¢ subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference fo r Pro in the P 2 ¢ position for the hydrolytic a ctivity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs. Keywords: carboxydipeptidase; cysteine protease; fluoro- genic p eptides; Leishmania ;parasite. Cysteine proteases (CPs) are p resent in almost all organisms and are associated with numerous physiological and pathological conditions [1,2]. Cysteine proteases of the papain supe rfamily, designated Clan CA, family C1 [3], are synthesized as zymogens that are activated by cleavage of the pro-domain to generate mature enzymes located predominantly within lysosomes. The m ature protease folds into an ellipsoid conformation with the a ctive site cleft located between two structural domains. One domain consists predominantly o f b-barrel folds, while a prominent central helix of th e second domain is adjacent t o, and helps define, the opposite side of the active site cleft [ 4]. Leishmania mexicana possesses three CPs of the papain superfamily, d esignated CPA and CPB, both of which are cathepsin L -like, and CPC, w hich is cathepsin B -like [ 5]. The CPB proteases exist as multiple isoenzymes, which are encodedbyatandemarrayof19similarCPB genes l ocated in a single locus [6,7]. L. mexicana CPB i soenzymes are expressed as inactive zymogens c omprising an 18 amino acid pre-region that is thought to be rapidly removed by a signal peptidase upon transfer into the endoplasmic reticu- lum,a106aminoacidpro-region,a218aminoacidmature domain that includes the active site, and a C-terminal domain of either 16 or 100 amino acids [7]. The first two genes of t he array, CPB1 and CPB2, are atypical because they encode enzymes with a C-terminal domain of just 16 amino a cids [7]. Furthermore, CPB1 and CPB2 are expressed almost exclusively in the infective metacyclic stage, whereas the remaining isogenes, namely CPB3– CPB17 (which include CPB2.8 and CPB3)andCPB18 are expressed p redominantly i n a mastigotes, w hereas CPB19 is a pseudogene [8]. The role of the C-terminal domain remains unc ertain. Roles in intracellular targeting to the megasomes, immune evasion and modulation of the Correspondence to G. H. Coombs, Division of Infection & Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow, G12 8QQ, UK. Fax: +44 141 330 3516, Tel.: +44 141 330 4777, E-mail: G.Coombs @bio.gla.ac.uk Abbreviations:Abz,ortho-amino-benzoyl; AMC, 7-amino-4-methyl- coumarin; CTE, C-terminal extension; DMF, dimethylformamide; EDDnp, N-[2,4-dinitrophenyl]-ethylenediamine; K(Dnp), (2,4 di- nitrophenyl)-e-NH 2 -lysine; MCA, 4-methylcoumarin-7-amide; rCPB2.8, recombinant Leishmania mexicana cysteine protease CPB2.8 lacking the C-terminal extension, originally designated CPB2.8DCTE; Suc-LY-MCA, N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin; t-Boc, tert-butyloxycarbonyl. (Received 2 5 May 2004, revised 12 July 2004, accepted 28 J uly 2004) Eur. J. Biochem. 271, 3704–3714 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04311.x enzyme’s activity have all been postulated, although defin- itive data are lacking [5]. Information about the functions and importance of the Leishmania enzymes in host–parasite interactions has been obtained by the generation of mutants deficient in the multicopy CPB gene array (Dcpb). L. mexicana Dcpb mutants have reduced virulence w ith poor lesion growth in BALB/c m ice and induce a pr otective Th1 response [ 6,9]. Reinsertion of the amastigote-specific CPB2.8 or meta- cyclic-specific CPB2 into Dcpb mutantsfailedtorestore either a Th2 response or sustained virulence, and only the re-expression of multiple CPB genes from a cosmid significantly restored virulence [10]. A recombinant form of the enzyme encoded by CPB2.8 but lacking the C-terminal extension, originally designated CPB2.8DCTE but herein nam ed r CPB2.8 (Table 1 lists nomenclature of all proteins analysed), was expressed [11], and its substrate specificity h as been studied extensively [12–15] and several peptide inhibitors have also been reported for it [16–18]. The CPB3 gene, originally designa- ted cDNA C PB as it was isolated from a cDNA library [19], is another CPB gene from the central region of the array [7]. The corresponding protein, CPB3, when expressed in Dcpb mutants was devoid of the gelatinase activity in nondenat- urating g el electrophoresis that was o bserved f or CPB2.8 [7]. These two CPB isoforms d iffer from e ach other in the mature enzyme domain in only t hree positions, CPB2.8 h as Asn60, Asp61 and Asp64 whereas CPB3 has Asp60, A sn61 and Ser64. Interestingly, CPB18, which also has Asp60, Asn61 and Ser64 but also Tyr84 and Asn18 instead of the His84 and Asp18 in CPB2.8, is active towards gelatin but differs f rom CP B2.8 i n i ts a ctivity towards s ome s hort peptidyl-7-amido-4-methylcoumarin substrates [7]. Thus the substrate preferences of some CPB isoenzymes seem to be determined by just a few amino acids. In order to explore the effects on substrate utilization of the r estricted l ocal amino acid variations of the CPB is oen- zymes of L. mexicana, the recombinant CPB3 ( rCPB3), and arecombinantH84YmutantofCPB3thatwasgenerated, were expressed in Escherichia coli and their S 1 -S 3 and S 1 ¢-S 3 ¢ subsite (based o n t he Schechter & Berger nomenclatu re [20]) specificities i nvestigated in a systematic w ay u sing intramole- cularly quenched fluorescence substrates derived f rom Abz- KLRFSKQ-EDDnp ( where Abz is ortho-amino-benzoyl and EDDnp is N-[2,4-dinitrophenyl]-ethylenediamine), which were previously used to study the specificity of rCPB2.8 [13]. Moreover, t h e locations of the varyi ng residues were analyzed via molecular modeling to gain insight into how the c hanges may impinge upon enzyme activity. TherecombinantcysteineproteasecruzainfromTrypan- osoma cruzi and r CPB2.8 of L. mexicana are both c athepsin L-like and characteristically endopeptidases. However, we have shown that these enzymes have carboxydipeptidase activities and have compared them with those of human recombinant cathepsin B and cathepsin L [21]. Therefore we also comparatively a nalyzed the carboxydipeptidase activit- ies of rCPB3 and r H84Y using the internally quenched fluorescent peptide Abz-FRFK(Dnp)-OH and some of its analogues, where K(Dnp) is (2,4 dinitrophenyl)-e-NH 2 - lysine, in order to characterize further the importance of the varying amino acids. Materials and methods Parasites Leishmania mexicana (MNYC/BZ/62/M379) promastigotes were grown in modified Eagle’s medium (designated com- plete H OMEM medium when supplemented with 10% (v/v) heat-inactivated fetal bovine serum, pH 7 .5) at 25 °Cas described p reviously [7]. The required a ntibiotics were added as follows: hygromycin B (Sigma) at 50 lgÆmL )1 , phleomy- cin (Cayla 1 , Toulouse, France) at 1 0 lgÆmL )1 , and neo mycin (G418, Geneticin, Life T echnologies Inc.) at 25 lgÆmL )1 . Molecular modeling A homology-based protein model o f a Leishmania majorcath- epsinL-likecysteineprotease(GenBanklocusU43706,PDB identification code 1bmj) was built using INSIGHTII [22] soft- ware (Accelrys Inc, San Diego, CA, USA) and the crystal structures of papain [23] and cruzain [24] as reference proteins. The L. mexicana CPB isoforms w ere then modeled by s uper- imposition using MIDAS PLUS (Computer Graphics L aboratory, University of California San Francisco, CA, U SA) [25,26]. Mutagenesis, constructs, transfections and production of recombinant enzymes Mutations were incorporated into pGL27, a pBluescript SK– plasmid containing the S WB1a CPB cDNA gene [19] now designated CPB3, using the QuikChange Site-Directed mutagenesis kit (Stratagene) and the following reverse- phase-purified oligonucleotides (only the sense strand Table 1. Plasmids, L. mexicana cell lines, and proteins used in this study. CTE, C-terminal extension ; r, recombinant. Plasmid Cell lines (plasmids expressed in Dcpb) Abbreviation Expressed cysteine protease Abbreviation Comments pGL37 Dcpb [pXCPB3] DcpbCPB3 CPB3 CPB3 native CPB3 pGL43 Dcpb [pXCPB3 D18N ] DcpbCPB3D18N CPB3(D18N) D18N CPB3 mutated D18N pGL44 Dcpb [pXCPB3 H84Y ] DcpbCPB3H84Y CPB3(H84Y) H84Y CPB3 mutated H84Y pGL45 Dcpb [pXCPB3 D60N, N61D, S64D ] DcpbCPB3M3 CPB3(D60N, N61D, S64D) M3 CPB mutated D60N, N61D, S64D pGL46 Dcpb [pXCPB2.8] DcpbCPB2.8 CPB2.8 CPB2.8 native CPB2.8 pGL180 not applicable CPB2.8DCTE rCPB2.8 rCPB2.8 lacking CTE [11] 6 pGL400 not applicable CPB3DCTE rCPB3 rCPB3 lacking CTE pGL401 not applicable CPB3(H84Y)DCTE rH84Y rCPB3(H84Y) lacking CTE Ó FEBS 2004 Cysteine protease isoforms of Leishmania (Eur. J. Biochem. 271) 3705 primers are shown and the mutated sites are given in bold): OL416 to generate pGL40: 5¢-GACGCCGGTGA AGAATCAGGGTGCGTG-3¢, OL418 to generate pGL41: 5¢-CGAACGGGCACCTGTACACGGAGGACAGC-3¢ and OL420 to generate pGL42: 5 ¢-GCTGCGATGACA TGAAC GATGGTTGCGACGGCGGGCTGATGC-3¢. These mutant constructs were verified by sequence analysis using an ABI 373 a utomated D NA sequencer (PerkinElmer). The native and mutant CPB3 genes were excised from pBluescript SK– with XbaI–XhoI. Blunt ends were created using Klenow fragment (NEB) and these were ligated to the SmaI site of the pX episomal shuttle vector [27] to generate the pGL plasmids detailed in Table 1. The CPB2.8 gene [6] was excised from p Bluescript SK– ( pGL28) as a 2.0 kb Eco RV fragment and ligated to the SmaIsiteof pX to generate pGL46 (Table 1). All pX-based constructs were then used to transfect Dcpb [6]. Transfection of L. mexicana promastigotes was as des- cribed previously [6]. Briefly, pX-based constructs were prepared using Qiagen Tip100 columns as outlined by the manufacturer. Transfection u tilized 10 lg o f DNA and 4 · 10 7 late-log phase Dcpb promastigotes. Following electroporation, cells were allowed t o recover in 10 mL complete HOMEM medium for 24 h at 25 °Candthen transfectants were selected in complete HOMEM medium containing 25 lgG418ÆmL )1 . To generate recombinant L. mexic ana CPBs, t he 203 bp KpnI–SacIfragmentofpGL180(pQE-30CPB2.8DCTE) [11] was replaced with the corresponding fragments from pGL27 and pGL41 to give e xpression constructs pGL400 (pQE-30 CPB3) and pGL401 (pQE-30 H84Y) (Table 1). These e ncode proteins comprising the pro- and mature domains of the enzymes, and which lack the pre- and C-terminal domains. The recombinant enzymes were pro- duced without the C-terminal extension (CTE) to aid refolding f rom t he insoluble inclusion body phase. The production of active, mature recombinant enzyme using E. coli was essentially as described previously for isoenzyme CPB2.8 [11]. The concentration of the enzyme stock solutions ( 11 l M ) were determined b y active site titration with human cystatin C, which was a generous gift from M. Abrahamson (University of Lund, Sweden), using Z-FR-7-amido-4-methylcoumarin (Sigma) a s the substrate. Activity analyses of cysteine proteases using gelatin SDS/PAGE Parasite cysteine protease activities were analysed using substrate SDS/PAGE a s d escribed previously [7,28]. Parasite cell lysates (10 7 cells) were subjected to electro- phoresis under nonreducing conditions using 12% (w/v) acrylamide gels containing 0.2% (w/v) gelatin. Following electrophoresis, the gel w as washed for 1 h w ith 2.5% (v/v)TritonX-100andthenincubatedfor2hin0.1 M sodium acetate, pH 5.5, containing 1 m M dithiothreitol. Gelatin hydrolysis w as detected by staining with Coomas- sie Blue R-250 (0.25% w/v). When analysing activities towards N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin (Suc-LY-MCA; 2 Sigma), the peptidyl amidomethylcou- marin fluorogenic substrate was added to 0.01 m M and, following a 10 m in incubation, fluorescence was detected by exposure of the gel to low intensity UV light [28]. Western blotting Western blotting utilized polyclonal a nti-CPB serum (1 : 2500) raised against rCPB2.8 expressed in a nd purified from E. coli [11]. Synthesis of Abz-peptidyl-Q-EDDnp All the intramolecularly quenched fluorogenic peptides contain N-[2,4-dinitrophenyl]-ethylen ediamine ( EDDnp) attached to glutamine. This is a necessary result o f the solid-phase peptide synthesis strategy employed, t he details of which were provided elsewhere [29]. An automated bench-top simultaneous multiple solid-phase peptide syn- thesizer (PSSM 8 system; Shimadzu, Tokyo, Japan) was used for the solid-phase synthesis of all the peptides b y the Fmoc-procedure. The final de-protected peptides were purified by semipreparative HPLC using an Econosil C-18 column (10 lm, 22.5 · 250 mm) and a two-solvent system: (A) trifluoroacetic acid/H 2 O (1 : 1000) and (B) trifluoro- acetic acid/acetonitrile/H 2 O (1 : 900 : 100). The column was eluted a t a flow rate of 5 mLÆmin )1 with a 1 0 ( or 30))50 (or 6 0)% g radient of solvent B o ver 30 or 4 5 min. Analytical HPLCwasperformedusingabinaryHPLCsystemfrom Shimadzu with a SPD-10AV Shimadzu UV-vis detector and a Shimadzu RF-535 fluorescence detector, coupled to an Ultrasphere C-18 column (5 lm, 4.6 · 150 mm) which was e luted w ith s olvent systems A1 ( H 3 PO 4 /H 2 O, 1 : 1000) and B1 (acetonitrile/H 2 O/H 3 PO 4 ,900:100:1)ataflow rate of 1.7 mLÆmin )1 and a 10–80% gradient of B1 over 15 min. The H PLC column eluates were monitored b y their absorbance at 220 nm and by fluorescence emission a t 420 nm f ollowing ex citation at 320 nm. The molecula r m ass and purity of synthesized peptides were checked by MALDI-TOF mass spectrometry (TofSpec-E, Micromass, Manchester, UK) 3 and or p eptide sequencing using a protein sequencer PPSQ-23 (Shimadzu). The concentrations of the solutions of the substrates were determined by colorimetric determination of 2,4-dinitrophenyl group (extinction coef- ficient at 365 nm is 17 300Æ M )1 Æcm )1 ) 4 . Enzymatic hydrolysis of fluorescent quenched substrates Hydrolysis of the fluorogenic peptide substrates by rCPB2.8, rCPB3 and rH84Y were carried out in 0.1 M sodium acetate, 2 m M EDTA, 200 m M NaCl, pH 5.5, at 37 °C. All kinetic analyses were carried out at 37 °Cwith 5 min enzyme preincubation in 2.5 m M dithiothreitol and by measuring the fluorescence at 420 nm, following excita- tion at 320 n m, using a Hitachi F-2500 spectrofluorometer to follow the Abz-peptidyl-Q-E DDnp s ubstrate hydrolysis. The k inetic param eters were calc ulated according Wilkinson [30] as well as by using Eadie–Hofstee plots. The standard derivations of K m and k cat determinations were in no case higher than 5% of the obtained value. HPLC analysis of the enzymatic hydrolysis products of the synthetic fluorogenic substrates The c leaved p eptide bonds in each substrate w ere i dentified by isolation of the fragments by HPLC reverse-phase chromatography on a C 18 column equilibrated in 10% 3706 M. A. Juliano et al.(Eur. J. Biochem. 271) Ó FEBS 2004 solvent B (90% acetonitrile, 0.1% t rifluoroacetic acid, v/v). The column w as eluted at a flow r ate of 1 mLÆmin )1 with 10– 80% gradient of solvent B o ver 28 min. The elution profile was monitored by absorbance at 220 nm and by fluorescence at 420 nm after excitation at 320 nm. The Abz-containing fragments were compared with authentic synthetic sequences and/or by amino acid sequencing and molecular mass determination by M ALDI-TOF mass spectrometr y. Results Correlation between structure and activity of CPB isoenzymes using substrate SDS/PAGE The CPB locus of L. mexicana consists of 19 genes in a tandem array [7]. The proteins encoded by three of these genes differ in j ust a few a mino acids. The p resent study was based on the finding that CPB2.8 and CPB18 are both highly active towards gelatin as a substrate w hen a ssessed b y in situ substrate SDS/PAGE, whereas CPB3 was inactive [6,7]. CPB3 differs from CPB2.8 i n just t hree residues (60, 61 and 64) whereas t he only difference between CPB3 and CPB18 are residues 18 and 84. Thus it was reasoned that one or more of these c hanges must play a key role in modulating the enzyme activity. There appeared two possible explanations for the activity differences observed: that the amino acid substitutions modulated enzyme activity directly, or they had an indirect effect by influencing the enzyme’s folding and stability. To investigate t he role of the different amino acid residues we generated two mutants of CPB3 in which residues 18 and 84 were changed to those present in CPB18. We also mutated residues 60, 61 and 64 of CPB3 to those present in CBP2.8 – a s a positive control for the procedure. Incorporating mutations into the CPB3 gen e and intro- ducing the mutated genes into Dcpb by transfection a llowed an investigation into the functional roles of these amino acids. The activity of the enzyme expre ssed in the parasite was then analyzed towards gelatin and a small fluorogenic peptidyl substrate, in both cases using in situ substrate SDS/ PAGE. Conversion of three variant residues in the mature domain of the CPB3 isoenzyme (Asp60, Asn61 and Ser64) to those present in CPB2.8 (Asn60, Glu61 and Glu64) to give a p rotein designated M3, restored gelatinase activity to the protease as expected (Fig. 1A, lane 6). This demon- strates that one or more of these residues play an i mportant role either in modulating the activity towards gelatin or in enabling the enzyme to reactivate after the electrophoresis procedure used. Mutation of His84 to T yr84 (to g ive H 84Y) also restored gelatinase activity to the CPB3 i soenzyme (Fig. 1A, lane 5), whereas mutation of Asp18 t o Asn18 (to give D18N) did not (Fig. 1A, lane 4). The level of re-expressed CPBs was to the same order in all of the cell lines, as assessed by Western blotting (Fig. 1B, lanes 3–7), although there were differences in expression levels that may account in part for the differences in proteolytic activities apparent (for example between lanes 5–7 of Fig. 1A). Interestingly, the H84Y a ctivity appeared to be somewhat greater than that of M3. However, such in situ gel assays, although they a re useful in providing qualitative r esults, need to be interpreted with caution with respect to quantitative data. The mutated forms of CPB3 were also assessed for activity towards a small fluorogenic peptide using the gel- based a ssay, which a lso is u seful for assessing activity but is not very quantitative (Fig. 2). As observed for gelatin, the mutants expressing the CPB3 or D18N were inactive towards Suc-LY-MCA ( Fig. 2A, lanes 1 a nd 2). In contrast, the H84Y isoenzyme hydrolyzed Suc-LY-MCA well (Fig. 2A, lane 3). The M3 enzyme was a lso active towards this fluorogenic compound (Fig. 2A, lane 4). This was to be expected, a nd so served as a positive control, as the mutant has the same mature domain as CPB2.8 (Fig. 2A, lane 5), which is active towards this substrate [7]. The higher molecular mass activities towards Suc-LY-MCA (approxi- mately 35 kDa) correspond to activated p recursor forms of the isoenzymes [11]. To confirm t hat similar amounts o f the re-expressed proteases had been applied to these activity gels, a Western blot was performed on duplicate samples with the CPB-specific antiserum (Fig. 2B). These results indicated that residues 60, 61, 64 and 84 influenced the activity of CPB, thus we produced as recombinant enzymes CPB3 and also H84Y in order to carry out a fuller analysis of their substrate specificities and 123456 7 123 4 5 6 7 A B 30 - 22 - 42 - 30 - 22 - kDa kDa Fig. 1. Gelatin SDS/PAGE and West ern blot analyse s of L. mexicana CPB isoenzym es ex pressed in Dcpb. (A) Extracts from 10 7 stationary phase promastigotes were used for gelatin SDS/PAGE. Wild type parasites (lane 1), Dcpb (lane 2), Dcp bCPB3 (lane 3), DcpbCPB3D18N (lane 4), DcpbCPB3H84Y (lane 5), DcpbCPB3M3 (lane 6) and DcpbCPB2.8(lane7).Thehighermolecular mas s activities (approxi- mately 35 kD a) evident with some samples resulted from the activation in situ of precursor forms of the isoenzyme s [11]. (B) E xtracts from 5 · 10 6 stationary phase promastigotes were used for Western b lotting with anti-CPB serum (1 : 2500 ). Samples were applied to lanes 1–7 as denoted for (A). Molecular mass markers are shown in kDa. The anti- CPB serum recogn ized CPB isoenzymes tha t migrated as a major band with a molecular mass of 27 kDa and a m inor band o f molecular m ass 26 kDa in cell l ysates of stationary p hase p roma stigotes o f wild type L. mexicana (lane 1). The specificity of the antiserum was confirmed by absence of d etected proteins in Dcp b (lane 2). Ó FEBS 2004 Cysteine protease isoforms of Leishmania (Eur. J. Biochem. 271) 3707 compare them with that of CPB2.8 [13]. All enzymes were produced without the C -terminal extension as previously [11]. S 1 subsite specificity characterization of CPB3 and H84Y A series derived from the peptide Abz-KLRFSKQ-EDDnp were synthesized with systematic variation of Arg at P 1 using all natural amino acids as previously reported for the subsite specificity studies of rCPB2.8 [13]. Table 2 shows the kinetic parameters for the hydrolysis of this series of peptides by CPB3 and H84Y, and, for comparison, also the k cat /K m and K i values of rCPB2.8. The substrate i nhibition w ith t he peptides containing hydrophobic and non-charged amino acids that occurred with rCPB2.8 was not observed with rCPB3 and rH84Y, and some hydrolysis o ccurred with a ll substrates. H owever, the s pecificity c onstants ( k cat /K m ) v alues f or the se proteases were considerably lower than those obtained with r CPB2.8. The higher k cat /K m values for rCPB2.8 were due to both lower K m s and higher k cat s. For example, with X ¼ R, the rCPB2.8 values were 0.04 l M (K m ) and 2.60 s )1 (k cat ) compared with the values of 0.3 l M (K m )and0.48s )1 (k cat ) for rCPB3 (Table 2). rCPB3 hydrolyzed with highest k cat /K m values the peptides containing Ser and Thr, followed by those with Phe, Arg, Lys and Tyr. These higher c atalytic efficiencies are mainly due to the low K m value rather than the catalytic component. Similar hydrolytic behaviour was observed with rH84Y and the best substrates were those containing Met, Ser and Thr. 30 - 22 - 42 - A B kDa 21345 21345 30 - 22 - 42 - kDa Fig. 2. Fluorogenic SDS/PAGE and Western blot analyses of L. mexicana CPBs expressed i n Dcpb. (A) Extracts from 10 7 stationary phase promastigotes of Dcpb re-expressing the following proteases were an alysed for activity t owards Suc-L Y-MCA 12 . DcpbCPB3 (lane 1), DcpbCPB3D18N (lane 2), DcpbCPB3H 84Y (lane 3), DcpbCPB3M3 (lane 4) a nd DcpbCPB2.8 (lane 5). (B) Extracts from 5 · 10 6 stationary phase promastigotes were analysed with a nti-CPB serum to show that roughly equivalent protein loadings were applied for the fluo rogenic SDS/PAGE analy ses: samples we re applied t o lanes 1– 5 as d enoted for (A). Mole cular mass markers are shown i n kDa. The mature C PBs are arrowed. Table 2. Kinetic p arameters for hydrolysis, by CPB, of the p eptides derived from Abz-KLXFSKQ-EDDnpwithmodificationsinX(P 1 ). Conditions of hydrolysis: 100 m M NaOAc, 200 m M NaCl, 2 m M EDTA, pH 5.5 and 3 7 °C. The enzymes were p reactiva ted by 2.5 m M dithiothreitol 7 for 5 min. The cleavage si te is indicated b y ÔflÕ, and the numbe rs following give 8 the percentage of hydro lysis at each peptide bond. All the other hydrolyses were at the X–F bond. rCPB2.8 data from reference [13]. The units for K i values are n M ,andk cat /K m values are i n (m M Æs) )1 . X indicate s the residue varied. Modification rCPB2.8 rCPB3 rH84Y k cat /K m (m M Æs) )1 K m (l M ) k cat (s )1 ) k cat /K m (m M Æs) )1 K m (l M ) k cat (s )1 ) k cat /K m (m M Æs) )1 R 65000 0.3 0.48 1600 1.6 0.22 138 K 31395 0.1 0.12 1200 0.2 0.04 200 H 36667 0.5 0.40 800 0.5 0.09 180 D 871 0.2 0.03 150 0.1 0.01 100 E 7727 0.8 0.53 663 0.3 0.04 133 C 14060 0.6 0.47 783 4.3 0.26 61 W K i ¼ 28 1.2 0.12 100 0.3 0.04 133 Y K i ¼ 26 0.1 0.11 1100 0.2 0.06 300 F K i ¼ 18 0.1 0.21 2100 0.3 0.08 267 L K i ¼ 15 k cat /K m ¼ 600 (XflF ¼ 55, FflS ¼ 45) k cat /K m ¼ 700 (XflF ¼ 50, FflS ¼ 50) I K i ¼ 9 k cat /K m ¼ 350 (XflF ¼ 15, FflS ¼ 85) k cat /K m ¼ 100 (XflF ¼ 19, FflS ¼ 81) V K i ¼ 29 k cat /K m ¼ 250 (XflF ¼ 24, FflS ¼ 76) k cat /K m ¼ 300 (XflF ¼ 28, FflS ¼ 72) M K i ¼ 22 0.3 0.15 500 0.05 0.16 3200 A K i ¼ 27 0.2 0.14 700 0.1 0.12 1200 P K i ¼ 400 0.8 0.04 50 3.8 0.05 13 S K i ¼ 24 0.1 0.28 2800 0.04 0.09 2250 T K i ¼ 16 0.03 0.08 2667 0.04 0.09 2250 N K i ¼ 32 k cat /K m ¼ 300 (XflF ¼ 67, FflS ¼ 33) k cat /K m ¼ 133 (XflF ¼ 67, FflS ¼ 33) Q K i ¼ 43 0.1 0.08 800 0.2 0.15 750 3708 M. A. Juliano et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Most of the peptides were hydrolyzed by rCPB3 and rH84Y at the X–F peptide bond, wher e X represents all the substitutions of Arg. A second cleavage, a t the F–S bond, was observed for the peptides with X ¼ Leu, Ile, Val and Asn. Like cathepsin L, t he S 2 –P 2 interaction i s d eterminant in defining the cleavage point and these cysteine proteases prefer hydrophobic amino acids at P 2 position [31,32]. Thus the cleavage of the F–S bond with Asn at the S 2 subsite by rCPB3 and rH84Y is surprising, a lthough this cleavage corresponded to only 33% of the total (Table 2). S 2 and S 3 subsite specificity characterization of rCPB3 and rH84Y The kinetic parameters for hydrolysis of the peptides modified at positions P 2 and P 3 areshowninTable3.S 2 specificity is considered critical for the activity of clan CA 5 cysteine proteases [31]. The r CPB3 preferred L eu at P 2 position of the substrate, while Pro is the worst amino acid of those t ested for activity. O n the other h and, rH84Y hydrolyzed the peptide with Arg at P 2 with higher k cat /K m ; this was mainly due to k cat contribution because the K m was relatively high. The extended binding site of rC PB3 and rH84Y also included the S 3 subsite, as the m odifications at P 3 position of the substrates r esulted in significant variations on the k cat /K m values (Table 3). rCPB3 hydrolyzed with better efficiency the peptides with Lys and Leu at P 3 , whereas rH84Y was most efficient with the peptides containing Leu and Ala. S 1 ¢ to S 3 ¢ subsite specificity characterization of rCPB3 and rH84Y The kinetic parameters for hydrolysis of the peptides modified at positions P 1 ¢ to P 3 ¢ are shown in Table 4. The trend of t he k cat /K m values obtained w ith t he substrates with modifications at P 1 ¢ position were similar between rCPB3 and rCPB2.8, however, t he latter enzyme h ydrolyzed all the peptides of this series with k cat /K m values a t l east one order of magnitude higher. In contrast to rCPB3 and rCPB2. 8, the mutant enzyme rH84Y hydrolyzed the peptide with Phe only poorly. This enzyme hydrolyzed best the peptide containing Ala at P 1 ¢, f ollowed b y those with L eu and Arg. The peptide with Pro w as almos t resistant to all three proteases, although a low r ate of hydrolysis w as observed at the P–S bond. The modification at the P 2 ¢ position of the substrates revealed the very significant effect of Pro, resulting in considerably higher k cat /K m values for the h ydrolysis by rCPB3 and rH84Y – even above those with rCPB2.8. These high values mainly reflected a marked decrease in the K m value. The k cat /K m values for the hydrolysis of Abz- KLRFPKQ-EDDnp by these two proteases were the highest found. Such preference for Pro in the S 2 ¢ subsite is a peculiarity of cruzain (of T. cruzi)andLeishmania CPB. These enzymes accept P ro in this position in synthetic substrates very well [13,1 5,33,34] and a lso in t he auto- processing of their pro-enzymes to active enzymes – Pro is the second amino acid in the mature form of the proteases [11,35]. The importance of S 2 ¢–P 2 ¢ interaction was further evidenced by the variations in the k cat /K m values for the hydrolysis of various substrates with modifications at P 2 ¢ position by rH84Y – changing the Ser favoured by rCPB2.8 in each case resulted in increased activity. Table 3. Kinetic parameters for hydrolysis, by CPB, of the peptides derived from Abz-KLRFSKQ-EDDnp, containing modifications at the Leu (P 2 ) and Lys (P 3 )residues.Conditions of hydrolysis: 100 m M NaOAc, 200 m M NaCl, 2 m M EDTA, pH 5.5 and 37 °C. The enzymes were preactivated by 2.5 m M dithiothreitol for 5 min. rCPB2.8 data from reference [13]. X indicates the residue varied. The c leavage s ite is indicated by ÔflÕ. 9 Substrate rCPB2.8 rCPB3 rH84Y k cat /K m (m M Æs) )1 K m (l M ) k cat (s )1 ) k cat /K m (m M Æs) )1 K m (l M ) k cat (s )1 ) k cat /K m (m M Æs) )1 Abz-KXRflFSKQ-EDDnp L 65000 0.3 0.48 1600 1.6 0.22 138 F 15517 0.9 0.13 144 0.8 0.17 212 A 2204 0.2 0.02 100 0.4 0.04 100 R 3295 0.3 0.04 133 1.4 0.66 471 P 471 0.5 0.01 20 4.8 0.39 81 Abz- XLRflFSKQ-EDDnp K 65000 0.3 0.48 1600 1.6 0.22 138 L 6167 0.05 0.07 1400 0.05 0.13 2600 A 16769 0.1 0.08 800 0.1 0.25 2500 H 6320 0.7 0.28 400 2.2 0.46 209 R 10421 0.4 0.12 300 0.5 0.29 630 Table 4. Kinetic parameters for hydrolysis, by CPB, of the peptides derived from Abz-KLRFSKQ-EDDnp, containing modifications at the Phe (P 1 ¢), Ser (P 2 ¢) and Lys (P 3 ¢)residues.Conditions of hydrolysis: 100 m M NaOAc, 200 m M NaCl, 2 m M EDTA, p H 5 .5 and 3 7 °C. The enzymes were preactivated by 2.5 m M dithiothreitol for 5 m in. rCPB2.8 data from reference [13]. The units for K i values are n M . X indicates the residue varie d. 10 The cleavage site is indicated by ‘ fl’. 11 Substrates rCPB2.8 rCPB3 rH84Y k cat /K m (m M Æs) )1 K m (l M ) k cat (s )1 ) k cat /K m (m M Æs) )1 K m (l M ) k cat (s )1 ) k cat /K m (m M Æs) )1 Abz-KLRflXSKQ-EDDnp F 65000 0.3 0.48 1600 1.6 0.22 138 L 14117 0.7 0.28 400 0.06 0.03 500 A 14882 0.2 0.09 450 0.3 0.28 933 R 5438 0.4 0.11 275 0.4 0.17 453 P a K i ¼ 490 0.5 0.01 20 0.3 0.03 93 Abz-KLRflF XKQ-EDDnp S 65000 0.3 0.48 1600 1.6 0.22 138 F 27778 0.2 0.18 977 0.3 0.19 633 A 8909 0.1 0.10 774 0.06 0.11 1833 R 15833 0.3 0.32 944 0.3 0.50 1667 P 11875 0.01 0.18 18000 0.01 0.16 16000 Abz-KLRflFS XQ-EDDnp K 65000 0.3 0.48 1600 1.6 0.22 138 F K i ¼ 40 0.5 0.04 80 0.1 0.23 2300 A 12727 0.6 0.20 333 0.6 0.12 200 R 24286 0.4 0.15 375 0.2 0.31 1550 P 11000 0.2 0.02 100 0.6 0.27 450 a Cleavage at P–S bond. Ó FEBS 2004 Cysteine protease isoforms of Leishmania (Eur. J. Biochem. 271) 3709 The S 3 ¢ subsite a lso influenced the binding of rCPB3 and rH84Y. Significant variations in the k cat /K m values were observed with t he amino acid substitutions at P 3 ¢ position of the substrates (Table 4). T he peptide with Lys was hydro- lyzed best by rCPB3 (it is also favoured by rCPB2.8), whereas r H84Y was more efficient on the peptides contain- ing Phe and Arg. Carboxydipeptidase activity of rCPB3 and rH84Y The kinetic parameters for the carboxydipeptidase activit- ies of rCPB3 and rH84Y on the internally quenched fluorescent peptide Abz-FRFK(Dnp)-OH and some of its analogues are shown in T able 5. The k cat /K m values for human recombinant c athepsin L and rCPB2.8 [ 21] are a lso shown for comparison. The carboxydipeptidase activities of rCPB3, and rH84Y were lower than those of cathepsin L and rCPB2.8, although relative activities t owards the different substrates were rather similar and in each c ase Abz-FRAK(Dnp)-NH 2 was the best substrate. The sub- strates with free C-terminal carboxyl group were hydro- lyzed with K m values t hat are a n order of magnitude higher than those presented in Tables 2–4. The unfavorable effects of the C-terminal negatively charged carboxyl group on the protease activity of rCPB3 and rH84Y is demonstrated by the pH-profiles of the carboxydipeptidase activities of these enzymes on the peptides Abz- FRAK(Dnp)-OH and Abz-FRAK(Dnp)-NH 2 (Fig. 3 ). The pH optima of t he carboxydipeptidase activities towards Abz-FRAK(Dnp)-OH of rCPB3 and rH84Y are displaced to 4.0–4.5 and the activity decreases greatly by pH 5.5. This pH range corresponds to the pK of carboxylate g roup formation from the substrate, indicating that the protonated carboxyl group fits better to the enzymes than its carboxylate form. This is confirmed by the pH-profiles for the hydrolysis of Abz-FRAK(Dnp)- NH 2 . I n this c ase, the pH optimum is in t he range 6–8. It is noteworthy that the pH-profile of carboxydipeptidase activity of rCPB2.8 on Abz-FRAK(Dnp)-OH contrasts greatly with tho se of rCPB3 and rH84Y. It has optimal activity at pH 6–8, showing that rCPB2.8 accommodates much better the negatively charged carboxylate group. Interestingly, the pH-profile o f h yd rolysis o f A bz- FRAK(Dnp)-OH by rH84Y presents a second small but significant peak of activity around pH 7.5. It is difficult to assign the group responsible for this effect because there are several groups that change their ionization status around this pH. Analysis of amino acid locations via modelling The location of the amino acids that differ between the CPB isoenzymes at positions 18, 60, 61, 64 and 84 was determined by constructing a model of the L. mexicana CPB isoenzymes by superimposing upon that obtained for the enzymes’ homologue in L. major [22] (Fig. 4). The crystal structure of papain [23] and of a recombinant cysteine protease of T. cruzi, cruzain [ 24], w hich has a high degree of sequence identity to CPB, was used as a template for the leishmanial CPB model. The mature regions of the L. mexicana and L. major enzymes used for the modelling have an overall 80% amino acid sequence identity, which reaches even higher values within the structurally conserved regions and especially within the active site (Table 6). Therefore, their protein structures appear to be very similar as determined by secondary structure alignments. The polypep tide backbone of these cysteine proteases folds into a series of a-helices and b-she ets and the active site cleft is located between two structural domains. The C-terminal extension is not shown on the model, as the structure of this d omain has not been solved. The p rotease activity of all pap ain-like cysteine protease s is associated with the catalytic triad (L. mexicana residues Cys25, His163 and Asn183; Fig. 4) but the substrate specificity is defined by the bindin g affinities of the subsites. The c atalytic residues and t he S 1 and S 1 ¢ subsites are highly conserved between the three parasite species, and key residues a t the three major subsites are completely conserved between the L. me xican a CPB isoenzymes and the L. major homologue (Table 6). Consequently, differences in activitie s between the L. mexicana CPB isoenzymes must be associ- ated with amino acid var iations in more peripheral positions of the molecule. The model revealed the location of the amino acids that differ between the CPB isoenzymes under study and so mediate the ob served activity changes (the residues are highlighted in Fig. 4B). Residues 60, 61 and 64 are located above the a-helix that forms a wall of the active site cleft. Amino acid 18 is relatively close to the active site cleft and also near to one disulphide bridge (Cys22–Cys63), whereas residue 84 is sited on a surface loop of one domain of the Table 5. Kinetic constant parameters for ca rboxydipeptidase activities. Conditio ns o f hydrolysis: 100 m M NaOAc, 20 0 m M NaCl, 2 m M EDTA, pH 5.5 and 37 °C. The enzymes were preactivated by 2.5 m M dithiothreitol for 5 min. Cath L a nd rCPB2.8 data a re from reference [21]. Substrates Cath L rCPB2.8 rCPB3 rH84Y k cat /K m (m M Æs) )1 k cat /K m (m M Æs) )1 K m (m M ) k cat (s )1 ) k cat /K m (m M Æs) )1 K m (m M ) k cat (s )1 ) k cat /K m (m M Æs) )1 Abz-FRFK(Dnp)-OH 256 306 3.3 0.23 70 3.9 0.51 130 Abz-RRFK(Dnp)-OH 2.2 4.5 K i ¼ 4.3 l M Resistant Abz-ARFK(Dnp)-OH 0.5 1.1 K i ¼ 2.3 l M 4.7 0.004 0.85 Abz-FRK(Dnp)W-OH 477 625 0.7 0.14 210 1.9 0.24 121 Abz-FRAK(Dnp)-OH 667 389 2.9 0.35 118 2.2 0.46 203 Abz-FRAK(Dnp)-NH 2 5739 4909 1.2 0.90 780 0.85 1.14 1340 3710 M. A. Juliano et al.(Eur. J. Biochem. 271) Ó FEBS 2004 enzyme but adjacent to another disulphide bridge (Cys56– Cys101). Discussion The CPB cysteine proteases of L. mexicana have been shown t o be v irulence factors [ 6], therefore it is important to understand the relative contributions that different isoenzymes play in the pathogenicity of the parasite. Those isoen zymes that have been characterized so far are highly conserved (98% identical) and yet some activity differences were apparent [7]. The aim of this study was to determine the extent to which these amino acid variations generated activity differences. The CPB sequences obtained to d ate all possess identical residues aligning the substrate subsites so it seemed that other residues outside of the active s ite cleft must affect activity. Abz-FRAK(Dnp)-NH 2 Abz-FRAK(Dnp)-OH pH 345678 0 400 800 1200 1600 2000 2400 2800 109 pH 34567891 0 200 400 600 800 1000 1200 1400 1600 1800 2000 0 pH 345678910 0 200 400 600 800 1000 pH 345678910 0 20 40 60 80 100 120 140 160 180 pH 345678910 0 40 80 120 160 200 240 pH 345678910 kcat/Km (mM -1 .s -1 )kcat/Km (mM -1 .s -1 )kcat/Km (mM -1 .s -1 ) kcat/Km (mM -1 .s -1 ) kcat/Km (mM -1 .s -1 ) kcat/Km (mM -1 .s -1 ) 0 2000 4000 6000 8000 10000 12000 14000 16000 CPB3H84Y CPB2.8 Fig. 3. pH-profile a ctivity (k cat /K m ) for the h ydrolysis of Abz-FRAK(Dnp)-OH a nd Abz- FRAK(Dnp)-NH 2 by rCPB2.8, rCPB3 and rH84Y. The reactions were carried out in standard buffer containing 25 m M acetic acid, 25 m M Mes, 75 m M Tris base, 2 5 m M glycine, and 2 m M EDTA. The pH range w as 3.5–10 a nd adjusted with 2 M NaOH or HCl. The enzymes wer e preactivated by 2.5 m M dithiothreitol for 5 min a t 37 °C. Ó FEBS 2004 Cysteine protease isoforms of Leishmania (Eur. J. Biochem. 271) 3711 The activity results presented show that the few amino acid variations known to exist between some isoenzymes of CPB of L. mexicana are i ndeed important in modifying the substrate specificities of the CPB isoenzymes. Both rCPB3 and r H84Y have lower activity towards some substrates than does rCPB2.8, but they are able to accommodate a wider r ange of amino acids at P 1 (Table 2). The kinetic parameters of hydrolysis by rCPB3, rH84Y and rCPB2.8 of the substrates with variations at P 1 position indicated that the s pecificity of S 1 subsite i s greatly influenced by the modifications at 60, 61, 64 and 84. However, the enzymes have an extended binding site that goes at least from S 3 to S 3 ¢ and importantly each CPB isoform also shows signifi- cant differences in the specificity of each subsite (Tables 3 and 4). Thus, the overall conclusion is that the different isoenzymes do indeed have different hydrolytic c apabilities and presumably t his is important for the parasite’s survival. This view is supported by the recent observation that the expression of multiple CPB genes encoding cysteine proteases, rather than just one, is required for L. mexicana virulence in vivo [10]. A particularly noteworthy finding was t he effect of Pro in the P 2 ¢ position in decreasing considerably the K m value with rCPB3 and rH84Y. Clearly these isoenzymes greatly favour Pro at this s ite. This may reflect a n important role for the enzymes in the activation o f CPB in the parasite, as Pro isthesecondaminoacidofthematuredomaininallofthe CPB isoenzymes for which the structure i s known. The variation of a mino a cid residues 6 0, 61 and 64 between the CPB isoenzymes examined in this work involve amino acids with charged side chains and this necessarily results in significant modifications on the electrostatic potential on the s urface of the e nzymes. The modeling analysis (Fig. 4) shows clearly that residues 60, 61 and 64 are located near the c atalytic g roove of the e nzyme and so it is likely that the localized charge variations resulting from the introduction or removal of residues with charged side groups to these positions could be the basis for the differences observed with respect to the utilization of substrates. The pH-profile differences for the carboxydi- peptidase activities of rCPB3 and rCPB2.8 clearly suggest that the latter isoform accommodates the substrate carb- oxylate group better than the former isoform, perhaps indicating that re sidue 60 (Asn i n CPB2.8 but Asp i n CPB3) is particularly important for this binding. These findings agree well w ith a study of the p H-activity profile of c ruzain, a related cathepsin L -like c ysteine pro tease of T. cruzi, which highlighted the importance of several ionizable groups and suggested that Asn60 is potentially involved in substrate recognition [36]. Thus the data obtained provide further evidence for the role of electrostatic potential in defining the substrate specificity of the CPB isoforms. 84 60 61 64 18 SS 63 22 25 163 183 SS 204 156 SS 101 56 A B Fig. 4. Homology-based protein model of the mature domain of L. mexicana CPB. The protein i s shown as a ribbon s tructure ( spi- rals, a-helices ; arrows, b-sheets). (A) High- lighted in yellow are the location of the active site triad ( Cys25, His163 an d Asn183) and disulphide bonds (Cys22–Cys63, Cys56– Cys101 and Cys156–Cys204). (B) Highlighted in red a re the residues that d iffer between CPB2.8, CPB3 a nd CPB18. Table 6. Key active site res idues of cathepsin L -likecysteineproteases.The major variations between the active sites of parasite CPs and papain occur between the S subsit es. Additional S and SÕ subsites are n ot listed because they are so far not identified by the cocrystallizat ion of a peptide substrate and the proteases o f the p arasites. Protease S2 subsite S1 subsite Catalytic triad S1¢ subsite Papain W69, S205, F207 Y67, P68, V133, A160 C25, H159, N175 Q19, W177 Cruzain N69, E208, S210 L67, M68, A138, G163 C25, H162, N182 Q19, W177 L. mexicana CPB L69, Y209, V211 L67, M68, A139, G164 C25, H163, N183 Q19, W185 L. major CPB L69, Y209, V211 L67, M68, A139, G164 C25, H163, N183 Q19, W185 3712 M. A. Juliano et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Residue 84 is located near t he surface of the mature domain s tructure and so it is less easy to understan d how it plays a role in affecting t he binding of substrates and their hydrolysis. However, it is positioned near to a disulphide bridge (Cys56–Cys101) and it is conceivable that the replacement of His with Tyr may impinge upon this structure a nd so change the active site i s s ome w ay. Clearly the findings on the activity of this mutant compared with rCPB3 suggest that mutation had some effects, although more minor than those resulting from the three changes (N60D, D61N and D64S) between CPB2.8 and the other isoenzymes. The results show that rCPB3 has activity towards p eptide substrates and yet the enzyme showed no activity in substrate SDS/PAGE analysis, whereas both CPB2.8 and CPB18 were both highly active in similar analyses (Figs 1 and 2). This suggests not only that one or more of amino acid changes H60D, D61N and D64S plays a key role in modulating the enzyme activity directly but may also be able to do so indirectly by affecting the enzyme’s refolding and/or stability under the conditions e mployed for the gelatin SDS/PAGE. Moreover, H84Y but not D18N can counteract this effect. It is too early to be able to interpret the way is which this is achieved, but the results show the complexity of the interactions that occur both within the mature protein and in the acquisition of its tertiary structure. In conclusion, the data reported suggest that the set of CPB isoenzymes with only a few sequence modifications have the modifications at strategic positions such that the enzyme’s substrate specificity i s changed and that t hese variations between isoenzymes provide the parasite with an array o f h ydrolytic activity that is needed for its interac tion with the mammalian host, and ensure its survival and success as a parasite. Acknowledgements This work was supported by F undac¸ a ˜ odeAmparoPesquisadoEstado de Sa ˜ o Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientı ´ fico e Tecnolo ´ gico (CNPq) and Human Frontiers for Science Progress (RG 0 0043/2000-M). J.C.M. a nd G.H.C. are supported by the Medical Research Council. References 1. Bromme, D. & Kaleta, J. (2002) Thio l-dependent cathepsins: pathophysiological implicatio ns and recent advances i n inhibitor design. Curr. P harm. Des. 8, 163 9–1658. 2. Sajid, M. & McKerrow, J.H. (2002) Cysteine prote ases of parasitic organisms. Mol. Biochem. Parasitol. 120, 1–21. 3. Barrett, A.J. & Rawlings, N .D. (2001) Evolutionary lines of cysteine peptidases. Biol. C hem. 382, 7 27–733. 4. McGrath, M.E. (1999) The lysosomal cysteine proteases. Annu. Rev. Bio phys. Biomol. Struc t. 28, 1 81–204. 5. Mottram, J.C., Brooks, D.R. & Coombs, G.H. (1998) Roles of cysteine proteinases of trypanosomes and Leishmania in host– parasite interactions. Curr. Opin. Microbiol. 1, 455–460. 6. Mottram, J .C., Souza, A.E., H utchison, J .E., Carter, R., Frame, M.J. & Coombs, G.H. (1996) Evidence from disruption of the lmcpb gene array of Le ishmania mexicana that cysteine proteinases are virulence fac tors. Proc.NatlAcad.Sci.USA93, 6008–6013. 7. Mottram, J.C., Frame, M.J., Brooks, D.R., Tetley, L., Hutchison, J.E., Sou za, A.E. & Coombs, G.H. (1997) The m ultiple cpb cysteine proteinase genes of Leishmania mexicana encode iso- enzymes which differ in their stage-regulation and substrate pre- ferences. J. Biol. Chem. 272, 14285–14293. 8. Brooks, D.R., Denise, H., Westrop, G.D., Coombs, G .H. & Mottram, J.C. (2001) The stage-regulated expression of Leish- mania mexicana CPB cysteine proteases is mediated by an intercistronic sequence element. J. Biol. Chem. 276, 47081– 47089. 9. Alexander, J., Coombs, G .H. & M ottram, J .C. ( 1998) Leishmania mexicana cysteine proteinase-deficient mutants have attenuated virulence for mice and potentiate a T Helper 1 (TH1) response. J. Immunol. 161 , 6794–6801. 10. Denise, H., McNeil, K.S ., Brooks, D.R., Alexander, J ., Coombs, G.H. & Mottram, J.C. (2003) The expression of multiple CPB cysteine protease genes is r eq uired for Leishmania me xicana viru- lence in vivo . Infect. Immun. 71 , 3190–3195. 11. Sanderson, S.J., Pollock, K.G., Hilley, J.D., Meldal, M ., St. Hil- aire, P.M., Juliano, M.A., Juliano, L., Mot tram, J.C. & Coom bs, G.H. (2000) Expression and characterisation of a recombinant cysteine proteinase of Leishmania mexicana. Biochem. J. 347, 383–388. 12. Alves, L .C., Melo, R.L ., Sanderson, S. J., Mottram, J .C., Coombs, G.H.,Caliendo,G.,Santagada,V., Juliano, L. & Juliano, M.A. (2001) S 1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids. Eur. J. Biochem. 268 , 1206–1212. 13. Alves, L.C., Judice, W .A.S., S t. Hi laire, P.M., Meldal, M., Sanderson, S.J., Mottram, J.C., Coombs, G.H., Juliano, L. & Juliano, M.A. (2001) Substrate speci ficity of recombin ant cysteine proteinase, CPB, of Leishmania mexicana. Mol. Biochem. Para- sitol. 116, 1 –9. 14. Alves, L.C., Melo, R.L., Cezari, M.H., S anderson, S.J ., Mottram, J.C., Coombs, G.H., Juliano, L. & Juliano, M.A. (2001) Analysis of the S 2 subsite specificities of the recombinant cysteine protei- nases CPB of Leishmania mexicana, a nd cruzain of Trypanosoma cruzi, using fluorescent substrates containing non-natural basic amino acids. Mol. Biochem. Parasitol. 117, 137–143. 15. St. Hilaire, P.M., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs,G.H.&Meldal,M.(2000) The substrate specificity of a recombinant cysteine protease from Leishmania mexicana: application of a combinatorial peptide library a pproach. Chembiochem. 1, 115–122. 16. Alves, L.C., St. Hilaire, P.M., M eldal, M., Sanderson, S.J., Mot- tram, J . C., Coombs, G .H ., Juliano, L. & Juliano, M.A. (2001) Identification o f peptides inhibitory to recombinant c ysteine pr o- teinase, CPB, of Leishmania mexicana. Mol. Biochem. Parasitol. 114, 81–88. 17. St. Hilaire, P.M., Alves, L.C., Herrera, F ., Renil, M., S anderson, S.J., Mottram, J.C., Coombs, G.H., Juliano, M.A., Juliano, L., Are ´ valo, J. & Meldal, M. (2002) Solid-Phase library synthesis, screening and selection of tight-binding reduced peptide bond inhibitors of a recombinant Leishmania mexicana cysteine pro- tease B. J. Med. Chem. 45 , 1971–1982. 18. Graven, A., St. Hilaire, P.M., Sanderson, S.J., Mottram, J.C., Coombs, G.H. & Meldal, M. (2001) Combinatorial library of peptide isosters based on D iels-Alder reac tions: Identifi cation of novel inhibitors against a recombinant cysteine protease from Leishmania mexicana. J. Com b. Chem. 3, 441–452. 19. Souza, A.E., Waugh, S ., Coombs, G.H. & Mottram, J .C. (1992) Characterization of a multicopy ge ne fo r a major stage-specific cysteine prote inase of Leishmania mexicana. FEBS Lett. 311, 124–127. Ó FEBS 2004 Cysteine protease isoforms of Leishmania (Eur. J. Biochem. 271) 3713 [...]... P3¢ variations in extended substrates Biochem J 347, 123–129 Serveau, C., Lalmanach, G., Hirata, I., Scharfstein, J., Juliano, M .A & Gauthier, F (1999) Discrimination of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, and mammalian cathepsins B and L, by a pH-inducible fluorogenic substrate of trypanosomal cysteine proteinases Eur J Biochem 259, 275–280 Serveau, C., Lalmanach, G., Juliano,... C.M., McMahon-Pratt, D & Beverley, S.M (1990) Development of a stable Leishmania expression vector and application to the study of parasite surface-antigen genes Proc Natl Acad Sci USA 87, 9736–9740 28 Robertson, C.D & Coombs, G.H (1990) Characterization of 3 groups of cysteine proteinases in the amastigotes of Leishmania mexicana mexicana Mol Biochem Parasitol 42, 269–276 29 Hirata, I.Y., Cezari, M.H.S.,... M .A. , Scharfstein, J., Juliano, L & Gauthier, F (1996) Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors Biochem J 313, 951–956 Eakin, A. E., Mills, A. A., Harth, G., McKerrow, J.H & Craik, C.S (1992) The sequence, organization, and expression of the major cysteine protease (cruzain)... Tam, W., Storer, A. C., Krupa, J.C., Mort, J.S & Menard, R (1999) Interdependency of sequence and positional specificities for cysteine proteases of the papain family Biochemistry 38, 4868–4874 Portaro, F.C.V., Santos, A. B.F., Cezari, M.H.S., Juliano, M .A. , Juliano, L & Carmona, E (2000) Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2¢ and... Kuntz, I.D., Sakanari, J .A. , Cohen, F.E & McKerrow, J.H (1997) Leishmania major: Molecular modeling of cysteine proteases and prediction of new nonpeptide inhibitors Exp Parasitol 87, 212–221 23 Kamphuis, I.G., Kalk, K.H., Swarte, M .A & Drenth, J (1984) ˚ Structure of papain refined at 1.65 A resolution J Mol Biol 179, 233–256 24 McGrath, M.E., Eakin, A. E., Engel, J.C., McKerrow, J.H., Craik, C.S & Fletterick,... M.H.S., Nakaie, C., Boschcov, P., Ito, A. S., Juliano, M .A & Juliano, L (1994) Internally quenched 30 31 32 33 34 35 36 fluorogenic protease substrates: solid-phase synthesis and fluorescence spectroscopy of peptides containing ortho-aminobenzoyl/ dinitrophenyl groups as donor-acceptor pairs Lett Peptide Sci 1, 299–308 Wilkinson, G.N (1961) Statistical estimations in enzyme kinetics Biochem J 80, 324–332 Nagler,... 3714 M A Juliano et al (Eur J Biochem 271) 20 Schechter, I & Berger, A (1967) On the size of the active site in proteases I Papain Biochem Biophys Res Commun 27, 157–162 21 Judice, W .A. , Puzer, L., Cotrin, S.S., Carmona, A. K., Coombs, G.H., Juliano, L & Juliano, M .A (2004) Carboxydipeptidase activities of recombinant cysteine peptidases Eur J Biochem 271, 1046–1053 22 Selzer, P.M., Chen, X.W., Chan, V.J.,... Fletterick, R.J (1995) The crystal structure of cruzain: a therapeutic target for Chagas’ disease J Mol Biol 247, 251–259 25 Ferrin, T.E., Huang, C.C., Jarvis, L.E & Langridge, R (1988) The midas display system J Mol Graphics 6, 13–27 26 Huang, C.C., Pettersen, E.F., Klein, T.E., Ferrin, T.E & Langridge, R (1991) Conic – a fast renderer for space-filling molecules with shadows J Mol Graphics 9, 230–236 27 LeBowitz,... J.H & Craik, C.S (1992) The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi J Biol Chem 267, 7411–7420 Stoka, V., McKerrow, J.H., Cazzulo, J.J & Turk, V (1998) Substrate inhibition of cruzipain is not a ected by the C-terminal domain FEBS Lett 429, 129–133 . Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes Maria A. . proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids. Eur.