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• W1975581657 abstract "Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites, in the host-parasite relationship, and in the pathogenesis of parasitic diseases. Furthermore, proteases are druggable targets for the development of new anti-parasitic therapy. The subtilisin protease (SUB; Clan SB, family S8) of Leishmania donovani was cloned and found to possess a unique catalytic triad. This gene was then deleted by gene knock-out, which resulted in reduced ability by the parasite to undergo promastigote to amastigote differentiation in vitro. Electron microscopy of SUB knock-out amastigotes revealed abnormal membrane structures, retained flagella, and increased binucleation. SUB-deficient Leishmania displayed reduced virulence in both hamster and murine infection models. Histology of spleens from SUB knock-out-infected hamsters revealed the absence of psammoma body calcifications indicative of the granulomatous lesions that occur during Leishmania infection. To delineate the specific role of SUB in parasite physiology, two-dimensional gel electrophoresis was carried out on SUB−/− versus wild-type parasites. SUB knock-out parasites showed altered regulation of the terminal peroxidases of the trypanothione reductase system. Leishmania and other trypanosomatids lack glutathione reductase, and therefore rely on the novel trypanothione reductase system to detoxify reactive oxygen intermediates and to maintain redox homeostasis. The predominant tryparedoxin peroxidases were decreased in SUB−/− parasites, and higher molecular weight isoforms were present, indicating altered processing. In addition, knock-out parasites showed increased sensitivity to hydroperoxide. These data suggest that subtilisin is the maturase for tryparedoxin peroxidases and is necessary for full virulence. Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites, in the host-parasite relationship, and in the pathogenesis of parasitic diseases. Furthermore, proteases are druggable targets for the development of new anti-parasitic therapy. The subtilisin protease (SUB; Clan SB, family S8) of Leishmania donovani was cloned and found to possess a unique catalytic triad. This gene was then deleted by gene knock-out, which resulted in reduced ability by the parasite to undergo promastigote to amastigote differentiation in vitro. Electron microscopy of SUB knock-out amastigotes revealed abnormal membrane structures, retained flagella, and increased binucleation. SUB-deficient Leishmania displayed reduced virulence in both hamster and murine infection models. Histology of spleens from SUB knock-out-infected hamsters revealed the absence of psammoma body calcifications indicative of the granulomatous lesions that occur during Leishmania infection. To delineate the specific role of SUB in parasite physiology, two-dimensional gel electrophoresis was carried out on SUB−/− versus wild-type parasites. SUB knock-out parasites showed altered regulation of the terminal peroxidases of the trypanothione reductase system. Leishmania and other trypanosomatids lack glutathione reductase, and therefore rely on the novel trypanothione reductase system to detoxify reactive oxygen intermediates and to maintain redox homeostasis. The predominant tryparedoxin peroxidases were decreased in SUB−/− parasites, and higher molecular weight isoforms were present, indicating altered processing. In addition, knock-out parasites showed increased sensitivity to hydroperoxide. These data suggest that subtilisin is the maturase for tryparedoxin peroxidases and is necessary for full virulence. Protozoan parasites of the genus Leishmania cause a variety of vector-borne diseases in vertebrates, including cutaneous, mucocutaneous, and visceral leishmaniases in humans. Due to the lack of safe and effective treatments for this disease, leishmaniasis is classified by the World Health Organization as a Tropical Disease Research Category I disease, an emerging or uncontrolled disease (1Scientific Working Group on Leishmaniasis and UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases Report of the Scientific Working Group Meeting on Leishmaniasis, Geneva, 2–4 February, 2004. World Health Organization, Geneva2004Google Scholar). This kinetoplastid parasite has a relatively simple dimorphic life cycle consisting of promastigote and amastigote stages. Leishmania promastigotes multiply extracellularly as spindle-shaped flagellates in the midgut of the phlebotomine sandfly vector. The parasites are then transmitted to a mammalian host when an infected sandfly bites to take in a blood meal. In the naïve host the parasites infect macrophages and differentiate into amastigotes. This form of the parasite is an ovoid intracellular aflagellate. Throughout its life cycle, Leishmania is exposed to a variety of reactive oxygen species that it must detoxify to survive. Antioxidant defense is particularly important for amastigotes, because they must also survive the oxidative burst generated by the host macrophages (2Levick M.P. Tetaud E. Fairlamb A.H. Blackwell J.M. Mol. Biochem. Parasitol. 1998; 96: 125-137Crossref PubMed Scopus (99) Google Scholar). Recent advances in parasite molecular biology and bioinformatics have enabled us to strategically identify and study Leishmania proteins as therapeutic targets. Parasite proteases are viable drug targets, because many of them are required for the pathogenic life cycle of the parasite (3Sajid M. McKerrow J.H. Mol. Biochem. Parasitol. 2002; 120: 1-21Crossref PubMed Scopus (672) Google Scholar, 4McKerrow J.H. Caffrey C. Kelly B. Loke P. Sajid M. Annu. Rev. Pathol. 2006; 1: 497-536Crossref PubMed Scopus (322) Google Scholar). We have identified an unusual Clan SB, family S8 subtilisin-like serine protease in Leishmania as one of these therapeutic targets. This family of endopeptidases is conserved across all biological kingdoms (5Siezen R.J. de Vos W.M. Leunissen J.A. Dijkstra B.W. Protein Eng. 1991; 4: 719-737Crossref PubMed Scopus (306) Google Scholar). Subtilisins are protein-processing enzymes and known virulence factors for both Plasmodium and Toxoplasma parasites (6Miller S.A. Binder E.M. Blackman M.J. Carruthers V.B. Kim K. J. Biol. Chem. 2001; 276: 45341-45348Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In Plasmodium falciparum a subtilisin-like serine protease is required for erythrocyte egress by infectious merozoites (7Blackman M.J. Cell. Microbiol. 2008; 10: 1925-1934Crossref PubMed Scopus (162) Google Scholar, 8Arastu-Kapur S. Ponder E.L. Fonović U.P. Yeoh S. Yuan F. Fonović M. Grainger M. Phillips C.I. Powers J.C. Bogyo M. Nat. Chem. Biol. 2008; 4: 203-213Crossref PubMed Scopus (206) Google Scholar) and is believed to be the convertase for the maturation of merozoite surface protein 1 and SERA proteins (9Barale J.C. Blisnick T. Fujioka H. Alzari P.M. Aikawa M. Braun-Breton C. Langsley G. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 6445-6450Crossref PubMed Scopus (88) Google Scholar, 10Yeoh S. O'Donnell R.A. Koussis K. Dluzewski A.R. Ansell K.H. Osborne S.A. Hackett F. Withers-Martinez C. Mitchell G.H. Bannister L.H. Bryans J.S. Kettleborough C.A. Blackman M.J. Cell. 2007; 131: 1072-1083Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). In Toxoplasma gondii a subtilisin is involved in rhoptry organelle protein processing (11Miller S.A. Thathy V. Ajioka J.W. Blackman M.J. Kim K. Mol. Microbiol. 2003; 49: 883-894Crossref PubMed Scopus (82) Google Scholar, 12Kim K. Acta Trop. 2004; 91: 69-81Crossref PubMed Scopus (74) Google Scholar). In this study we describe the identification and phenotypic characterization of Leishmania subtilisin. This protease was found to process the terminal peroxidases of the trypanothione reductase system. This system plays an important role in Leishmania survival within host macrophages and is being intensely studied as a target for antiparasitic drug development (13Krauth-Siegel R.L. Comini M.A. Biochim. Biophys. Acta. 2008; 1780: 1236-1248Crossref PubMed Scopus (331) Google Scholar). This study has found that subtilisin is an important regulator of this system and is key for parasite infectivity and virulence. Commercially bred, 6- to 8-week-old, female BALB/c mice (Mus musculus) were used for the murine footpad infection model (Charles River Laboratories International, Inc., Davis, CA). Commercially bred, 4- to 5-week-old, male Golden Syrian hamsters (Mesocricetus auratus) were used for the visceral infection model (Simonsen Laboratories, Inc., Gilroy, CA). Leishmania donovani donovani MHOM/ET/67/HU3 cloned stock and Leishmania major LV39 MRHO/SU/59/P were used for knock-out studies and for animal infections. Leishmania promastigotes were cultured at 27 °C in M199 (Sigma) liquid medium as previously described (14Wallis A.E. McMaster W.R. J. Exp. Med. 1987; 166: 1814-1824Crossref PubMed Scopus (40) Google Scholar). Axenic amastigotes of L. donovani were cultured at 37 °C in 100% fetal bovine serum (Omega Scientific Inc., Tarzana, CA) as previously described (15Doyle P.S. Engel J.C. Pimenta P.F. da Silva P.P. Dwyer D.M. Exp. Parasitol. 1991; 73: 326-334Crossref PubMed Scopus (121) Google Scholar). Genomic DNA from L. donovani was isolated as previously described (16Medina-Acosta E. Cross G.A. Mol. Biochem. Parasitol. 1993; 59: 327-329Crossref PubMed Scopus (236) Google Scholar). The subtilisin gene was then amplified from this genomic DNA by PCR using the Expand High Fidelity PCR System (Roche Diagnostics, Indianapolis, IN) in two overlapping pieces, termed the 5′ and 3′ halves. For each half, an external primer from the non-coding flanks of the gene and an internal primer were designed based on regions of identity between the known L. infantum and L. major sequences (Sanger Institute GeneDB: LinJ13_V3.0940 and LmjF13.1040). Both halves were cloned into the pGEM-7Zf(−) vector (Promega, Madison, WI) and then spliced together using an internal HindIII site. The open reading frame was sequenced using the following primers: 5′-CAT GCA TCA GCC GGT AC-3′, 5′-GCG GCA TGG TCA TCT AC-3′, 5′-TAC TCA CAA TCT CTA CG-3′, 5′-CAC CAG TAA GAG TGC GG-3′, 5′-GAA GAG CCG CCA CCG TG-3′, 5′-CGT GCT GGC AGG ACA GC-3′, 5′-TCC TCT TTG AGG GTG CG-3′, 5′-TAG CCA TAG CCC ACC GC-3′, 5′-CTC CTC TTT GAG GGT GC-3′, 5′-CGC TCT GTC TCG AGG CG-3′, 5′-GTG TGG GGC AGC GGC AG-3′, 5′-ACC GTT GGC TGT CAG AG-3′, 5′-CGT TAG GAG ACG CCG CA-3′, 5′-CGT CGT CAG CAC AAG AG-3′, 5′-ACC CAC CTT CCG CTT CG-3′, 5′-GTG CCA GCA GAC CAC GG-3′, 5′-TAG GCA GCG GTG CCG AC-3′, 5′-AAC GGC AGC AGG CTC TC-3′, 5′-GTC GGC ACC GCT GCC TA-3′, 5′-ATC GGC TAT AGG ATT CC-3′, 5′-CAG TAG CCC GCA GGT GC-3′, 5′-CGT TGT CTG TGC CGA CC-3′, and 5′-ACT GAT CAG CCA AGG CG-3′. The amino acid sequence of L. donovani subtilisin catalytic core was identified using Pfam (accession number PF00082, Subtilase family) and was aligned with homologous sequences from L. infantum, L. major, L. braziliensis, T. cruzi (1 and 2), T. brucei (1 and 2), P. falciparum (1, 2, and 3), T. gondii (1a, 1b, and 2), B. licheniformis, B. amyloliquefaciens, B. subtilis, H. sapiens (furin and Site-1), M. musculus, S. cerevisiae, S. pombe, C. intestinalis, A. mellifera, X. laevis, and D. rerio (respectively: LinJ13_V3.0940, LmjF13.1040, LbrM13_V2.0860, Tc00.1047053511045.40, Tc00.1047053511859.60, Tb11.02.1280, Tb927.3.4230, CAD51440, XP_001348051, CAD51437, XP_002370002, XP_002368971, XP_002364650, P00780, P00782, P04189, P09958, EAW95506, P23188, P13134, Q09175, XP_002122807, XP_395754, NP_001087381, and CAK04389) using the ClustalW algorithm from MegAlign (DNASTAR, Madison, WI). Transformation constructs were generated by PCR amplification of L. donovani and L. major SUB 2The abbreviations used are: SUBsubtilisinAMC7-amino-4-methylcoumarinSite-1membrane-bound transcription factor peptidaseTRtrypanothione reductaseTS2 and T(SH)2oxidized and reduced trypanothioneTXNtryparedoxinTXNPxtryparedoxin peroxidasePrxperoxidoxinZbenzyloxycarbonyl. cores, adding a 5′ SalI site (bold) followed by a Kex2 cleavage site (underlined) and a 3′ SpeI site (underlined bold) using forward (L.d.: 5′-CTC GTC GAC AAA AGA GCA CAC CGT TCC ACA GAT GCG-3′; L.m.: CTC GTC GAC AAA AGA GCA CGC CGT TCC ACC GAT GCG) and reverse (L.d.: 5′-CTC ACT AGT TCA ACA CGG GCA AGT CGA TTC TGA C-3′; L.m.: 5′-CTC ACT AGT TCA ACA CGA GAG AGT CGA TTC TGA CG-3′) primers, and then cloned into pPICZa A (Invitrogen). The pPICZα-SUB constructs were electroporated into X-33 P. pastoris, and expression clones were isolated and induced for 96 h as per the manufacturer's protocol. For each L. donovani and L. major SUB, three clones were independently evaluated for protease activity. Supernatant from induced cultures was harvested by centrifugation at 3,000 × g for 10 min, followed by 0.2-μm filtration (Nalge Nunc). Expressed SUB protein was then buffer-exchanged with 50 mm Tris-HCl, pH 7.5, and concentrated on an Amicon Ultra-4 10,000 NMWL filter device (Millipore, Billerica, MA). This concentrate was then fractionated by hydrophobic interaction. The sample was diluted 1:1 to a final buffer concentration 30 mm Tris-HCl, pH 8.0, with 1 m ammonium sulfate and loaded onto a HiTrap Octyl-Sepharose 4FF hydrophobic interaction column (Amersham Biosciences). A 10 mm Tris-HCl, pH 8.0, buffer with 1 m ammonium sulfate was used for column equilibration, sample loading, and washing at a 1 ml/min flow rate. The ammonium sulfate concentration was decreased from 1–0 m over 40 column volumes to elute the SUB protein. The eluate was desalted by buffer exchanging with 50 mm Tris-HCl, pH 7.5, and concentrated by Amicon. SUB protein concentration was measured by inhibitor titration with PPACK (H-d-Phe-Pro-Arg-CMK) and NanoDrop 1000. subtilisin 7-amino-4-methylcoumarin membrane-bound transcription factor peptidase trypanothione reductase oxidized and reduced trypanothione tryparedoxin tryparedoxin peroxidase peroxidoxin benzyloxycarbonyl. Protease activity was measured using peptide substrates containing C-terminal 7-amino-4-carbamoylmethylcoumarin (AMC) as the fluorogenic leaving group. The synthetic substrates Z-VFRSLK-AMC, Z-RVRR-AMC, and Z-RR-AMC were used. Initial test reactions contained 20 μm substrate. For Km determination VFRSLK was serially diluted from 100–0.05 μm, and RVRR was serially diluted from 20 to 0.025 μm. Enzyme samples were mixed with substrate in 50 mm Tris-HCl, pH 7.5, with 0.2% DMSO in 150 μl of total volume. Hydrolysis of the substrates was measured at 25 °C using a FlexStation microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA). Excitation/emission for AMC and 7-amino-4-carbamoyl-methylcoumarin were 355/460 nm and 380/460 nm, respectively. Vmax values were calculated using the accompanying SoftMax Pro v4.8 software. For Southern blot analysis, genomic DNA was digested with indicated restriction endonucleases (New England Biolabs, Ipswich, MA and Roche Diagnostics, Indianapolis, IN), and fragments were separated by electrophoresis on a 0.6% agarose gel (17Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). These were then transferred to Hybond-N+ (Amersham Biosciences) nylon filters by the manufacturer's instructions for alkali transfer. Southern blot probes were 32P-labeled using a Rediprime II Random Prime Labeling System (Amersham Biosciences) as per the manufacturer's instructions. Hybridization and washing conditions were performed as previously described (18Button L.L. Russell D.G. Klein H.L. Medina-Acosta E. Karess R.E. McMaster W.R. Mol. Biochem. Parasitol. 1989; 32: 271-283Crossref PubMed Scopus (102) Google Scholar). RNA was isolated from L. donovani promastigotes and axenic amastigotes (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). RNA (5 μg/lane) was size-fractionated, and Northern blot hybridization was performed as previously described. Two knock-out cassettes (one for each allele) were created to delete the L. donovani and L. major SUB genes. These cassettes each contained an antibiotic resistance gene, hygr (conferring hygromycin B resistance) (20Gritz L. Davies J. Gene. 1983; 25: 179-188Crossref PubMed Scopus (611) Google Scholar), pacr (conferring puromycin resistance, used for L. donovani only) (21Lacalle R.A. Pulido D. Vara J. Zalacaín M. Jiménez A. Gene. 1989; 79: 375-380Crossref PubMed Scopus (49) Google Scholar), or satr (conferring nourseothricin resistance, L. major only) (22Joshi P.B. Webb J.R. Davies J.E. McMaster W.R. Gene. 1995; 156: 145-149Crossref PubMed Scopus (44) Google Scholar), followed by 1.5 kb of the 3′-untranslated region of the L. major dhfr-ts gene (23Joshi P.B. Sacks D.L. Modi G. McMaster W.R. Mol. Microbiol. 1998; 27: 519-530Crossref PubMed Scopus (102) Google Scholar). This untranslated region ensures high level expression during the life cycle of Leishmania. To target these knock-out cassettes to the SUB locus, 5′ and 3′ targeting flanks were created and ligated into their respective sides of the cassettes. These targeting flanks were generated by PCR amplification of the untranslated regions directly 5′ of the SUB ORF and 3′ of the SUB catalytic core (L. donovani) or 3′ of the ORF (L. major). PCR primers were designed based on the published L. infantum and L. major sequences (GeneDB): L. donovani 5′ flank forward (5′-CTC ACT AGT CGC CTC CTC GTC GTC GCA CTC-3′) and reverse (5′-CTC TCT AGA CAC CAC TAC CTC AAT CGG AGC G-3′) (1.3-kb fragment), 3′ flank forward (5′-CTC ACT AGT TGG TCA TCT ACG TCC GCT GTA GC-3′) and reverse (5′-CTC TCT AGA CGT GCC CTG ATC TGC GGC AGC-3′) (0.7-kb fragment); L. major 5′ flank forward (5′-CTC ACT AGT TGC GCA ACC ACA GCG GTC ATC-3′) and reverse (5′-CTC TCT AGA TAC CTC AAT GGG AGC GTG CTT G-3′) (1.5-kb fragment), 3′ flank forward (5′-CTC ACT AGT TCG TTG GAG AGG CCA ACG CGC-3′) and reverse (5′-CTC TCT AGA CGA GTA GGA AGA GGT GAC CGT C-3′) (0.8-kb fragment) primers (SpeI sites, in bold, and XbaI sites, underlined, were included for cloning). These constructs were maintained and amplified in the pGEM-9Zf(−) vector (Promega). For targeted gene deletion, 50 μg of the targeting constructs was excised from their vectors using the flanking restriction endonucleases SpeI and XbaI (New England Biolabs) and purified by electrophoresis on 0.8% agarose gels then purified using the QIAEX II Gel Extraction Kit (Qiagen Inc., Valencia, CA). Purified transfection constructs described above were used to transfect log phase Leishmania promastigotes by electroporation (2.25 kV/cm, 500 microfarads) as previously described (23Joshi P.B. Sacks D.L. Modi G. McMaster W.R. Mol. Microbiol. 1998; 27: 519-530Crossref PubMed Scopus (102) Google Scholar). After electroporation, the cells were grown and selected using hygromycin B, puromycin, or nourseothricin on both plates and in liquid media and clones were isolated as previously described (24Joshi P.B. Kelly B.L. Kamhawi S. Sacks D.L. McMaster W.R. Mol. Biochem. Parasitol. 2002; 120: 33-40Crossref PubMed Scopus (201) Google Scholar). Day 4 SUB knock-out and wild-type parasites were split in triplicate into new M199 (for promastigote replication rates) and into 37 °C fetal bovine serum (for axenic amastigote replication). Parasite culture densities were determined on days 1–4 post-split by cell counting on a Multisizer 3 Coulter Counter (Beckman Coulter, Inc., Fullerton, CA). Axenic amastigote differentiation was observed by microscopy. Approximately 108 day 4 L. donovani axenic amastigotes from wild-type or SUB−/− cultures were pelleted and washed 3× in PBS. The parasites were processed for conventional EM by freeze-substitution in 1% OsO4/0.1% uranyl acetate in acetone and embedded in Epon resin. Sections were cut with a Leica Ultracut UCT Ultramicrotome (Leica Microsystems, Bannockburn, IL) and viewed on a Tecnai T20 electron microscope (FEI Co., Hillsboro, OR) with a 4000 × 4000 UltraScan charge-coupled device camera (Gatan Inc., Pleasanton, CA). Hamsters were infected intraperitoneally with 109 day 4 SUB knock-out or wild-type L. donovani promastigotes (groups of three) (25Wyllie S. Fairlamb A.H. Acta Trop. 2006; 97: 364-369Crossref PubMed Scopus (19) Google Scholar). Animals were weighed weekly over the length of the experiment. Hamsters were culled 200 days post-infection by CO2 inhalation followed by thoracotomy. Pieces of each spleen and liver were fixed in 10% formalin in PBS and then embedded in paraffin for histology. Sections were cut at 5 μm and stained with either Wright-Giemsa or hematoxylin and eosin by the University of California at San Francisco (UCSF) Morphology Core using standard protocols. Psamomma bodies were identified by microscopy and counted. Mice were infected with metacyclic L. major promastigotes purified from day 4 SUB+/− and wild-type cultures using negative selection by binding to peanut agglutinin as has been previously described (26Sacks D.L. Hieny S. Sher A. J. Immunol. 1985; 135: 564-569PubMed Google Scholar). BALB/c mice (groups of 5) were anesthetized by isoflurane inhalation and infected subcutaneously in the left hind footpad with 5 × 106 metacyclic promastigotes in 50 μl of Hanks' balanced salt solution. Footpad swelling was measured weekly after inoculation using a Mitutoyo caliper. Parasites were recovered from infected mice by resection of the left popliteal lymph node. Three experimental replicates were prepared from separately cultured samples of both wild-type and SUB knock-out L. donovani. Approximately 109 cells were pelleted, washed 3× with PBS, and stored at −80 °C. Lysates were prepared by resuspending the cell pellets in 2 ml of native lysis buffer containing 20 mm HEPES, pH 7.5, 250 mm sucrose, 3 mm MgCl2, 0.5% Nonidet P-40, 1 mm DTT, and 1× Halt EDTA-free protease inhibitor mixture (Pierce). Cells were then broken by mechanical lysis using 70 strokes of a Dounce homogenizer. The lysates were centrifuged at 12,000 × g for 20 min at 4 °C, and the clarified supernatants were dialyzed overnight against 50 mm Tris-HCl, pH 7.5, 100 mm NaCl using 8-kDa molecular weight cut-off dialysis membranes. The following day the protein samples were concentrated and washed by precipitation using the ReadyPrep 2-D Cleanup Kit (Bio-Rad Laboratories, Inc., Hercules, CA). Approximately 300 μg of protein per gel was brought up to 300 μl in Bio-Rad Rehydration/Sample buffer and was passively loaded onto 17-cm, 3–10 pH immobilized pH gradient isoelectric focusing strips. Isoelectric focusing was performed using slow increases in voltage over multiple steps up to 10 kV for a total of 60–65 kVh focusing time. Next, the strips were reduced and alkylated using sequential 10-min incubations in 2% DTT then 2.5% iodoacetamide and dissolved in sample equilibration buffer. The isoelectric focusing strips were run in the second dimension on 17 × 17 cm, 12.5% acrylamide Tris-glycine, SDS-PAGE gels. Gels were stained with SYPRO Ruby and imaged using a Typhoon Trio Variable Mode Imager (Amersham Biosciences). These images were utilized for spot intensity analysis using Bio-Rad PDQuest software (v. 7.4). For proteomic analysis, gels were silver-stained (27Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7832) Google Scholar), and selected protein spots were excised and in-gel-digested with trypsin (28Hellman U. Wernstedt C. Góñez J. Heldin C.H. Anal. Biochem. 1995; 224: 451-455Crossref PubMed Scopus (687) Google Scholar, 29Rosenfeld J. Capdevielle J. Guillemot J.C. Ferrara P. Anal. Biochem. 1992; 203: 173-179Crossref PubMed Scopus (1132) Google Scholar). The resulting peptides were extracted and analyzed by on-line liquid chromatography/mass spectrometry using an Eksigent nanoflow pump (Dublin, CA) coupled to a QStar Pulsar quadrupole orthogonal acceleration, time-of-flight hybrid instrument (Applied Biosystems, Foster City, CA). The reversed-phase chromatographic column was controlled with a Famos autoinjector (Sunnyvale, CA) and Eksigent software to run at a 5–50% acetonitrile gradient in 0.1% formic acid with a 350 nl/min flow rate. Data were analyzed in Analyst 2.0 software (Applied Biosystems) with the Mascot script 1.6b20 (Matrix Science, London, UK). Analyst-processing options for peak finding in spectrum were 0.5% default threshold, 400 Gaussian filter, and a Gaussian filter limit of 10; for TOF auto-centroiding: 20 ppm merge distance, 10 ppm minimum width, 50% percentage height, and 100 ppm maximum width. Default parameters were used except that “no de-isotoping” was selected and precursor mass tolerance for grouping was set to 0.2. Database searches were performed using ProteinProspector v. 5.3.0 (available on-line) using the Batch-Tag and Search Compare modules (30Chalkley R.J. Baker P.R. Medzihradszky K.F. Lynn A.J. Burlingame A.L. Mol. Cell. Proteomics. 2008; 7: 2386-2398Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Searches were performed on the SwissProt databank (December 16, 2008) to evaluate sample purity followed by searching TriTrypDB v. 1.0 beta (available on-line, January, 2009). These assays were performed as was previously described (31Castro H. Sousa C. Santos M. Cordeiro-da-Silva A. Flohé L. Tomas A.M. Free Radic. Biol. Med. 2002; 33: 1552-1562Crossref PubMed Scopus (88) Google Scholar). Stationary phase Leishmania promastigotes were split into 2-ml M199 at 2 × 106 per ml in the presence of different concentrations tert-butylhydroperoxide (Sigma). Culture densities were determined after 5–6 days by using the Coulter Counter. Relative density was calculated by normalizing to untreated controls. The gene encoding L. donovani SUB was cloned as described above. Sequencing yielded a 5,235-bp gene. This sequence was submitted to GenBankTM with accession number ADA81891. The resultant 1,744-amino acid protein has an estimated molecular mass of 184.7 kDa. This protein has a predicted signal peptide (SignalP V2.0 HMM probability of 0.995) with a cleavage site between amino acids 38–39 (0.421 probability), and a probable C-terminal transmembrane helix between amino acids 1709–1731 (TMHMM v. 2.0). The published L. major SUB (GeneDB: LmjF13.1040) shares this general layout; however, SUB from the more closely related L. infantum (a subspecies from within the L. donovani complex) has a C-terminal truncation after amino acid 1192 (LinJ13_V3.0940). The Pfam predicted Subtilase family core for L. donovani SUB is between amino acids 86–414. Comparisons of L. donovani SUB to other trypanosomatid SUBs are summarized in Table 1. Interestingly, L. donovani SUB has a non-canonical catalytic triad with the catalytic Glu in place of the standard Asp due to a single C to G base pare change. L. infantum SUB also has Glu in place of the Asp, indicating that this adaptation may be specific to parasites in the L. donovani complex. The SUB catalytic core amino acid sequences are relatively conserved within the Leishmania species; however, these sequences have diverged considerably from those of the trypanosomes, with only a 40% identity between the genera.TABLE 1Comparison of predicted SUB proteins from trypanosomatidsSpeciesGeneaa identityCore identityLengthMolecular massCatalytic triad%%aakDaL. donovaniSUB100.0100.01744184.7Glu-97, His-130, Ser-395L. infantumSUB99.699.71192126.2Glu-97, His-130, Ser-395L. majorSUB89.094.51722182.7D99, His-132, Ser-397L. braziliensisSUB70.083.01785190.7D97, His-130, Ser-405T. cruziSUB123.140.31430160.5D219, His-269, Ser-502T. cruziSUB223.440.11408158.0D196, His-246, Ser-480T. bruceiSUB124.439.51487161.5D191, His-238, Ser-476 Open table in a new tab To determine the subfamily of Leishmania subtilisin, the catalytic core sequence was compared with the cores of other Clan SB, family S8 family members. Core sequences were aligned using ClustalW2 (EMBL-EBI), and a phylogenetic tree was generated (Fig. 1). The Leishmania SUBs group with the subfamily S8A proteases, which include the eukaryotic Site-1 peptidases and the bacterial subtilisins. This distinguishes Leishmania SUB from the Toxoplasma and Plasmodium SUBs and from the subfamily S8B kexins and furins. Site-1 peptidases are restricted to metazoan organisms and are known to process sterol regulatory element binding proteins, which are not found in trypanosomatids (32Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. 2nd Ed. Elsevier Academic Press, Amsterdam2004Google Scholar). The catalytic cores of the subtilisin proteins from L. donovani and L. major were successfully recombinantly expressed. Site-1 proteases, to which Leishmania SUB is most similar, have a requirement for a lysine or arginine in the P4 position. For this reason SUB activity was evaluated using synthetic substrates with and without P4 Arg. Cleavage of the synthetic substrates RVRR and VFRSLK was detected in all of the SUB-expressing Pichia supernatants compared with the X-33 background strain. Slight activity against the RR substrate was only detected in one L. major clone. In this clone the RVRR Vmax was over six times that of RR. Poor cleavage of RR and the lack of detected protease activity in the X-33 control strain indicate that the cleavage is not due to endogenous KEX2. These results show that, like the Site-1 proteases, Leishmania SUB prefers a basic P4 residue. L. donovani and L. major SUB was isolated from the Pichia supernatants, and kcat and Km values were determined for both RVRR and VFRSLK substrates (Table 2). Both enzymes catalyzed the RVRR substrate at about a 10-fold faster rate than VFRSLK. The L. donovani SUB had a similar affinity for both substrates, whereas L. major SUB had a 10-fold higher affinity for RVRR. Interestingly, L. donovani SUB had a much lowe" @default.
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• W1975581657 title "Leishmania Subtilisin Is a Maturase for the Trypanothione Reductase System and Contributes to Disease Pathology" @default.
• W1975581657 cites W1507630962 @default.
• W1975581657 cites W1553527521 @default.
• W1975581657 cites W1958092593 @default.
• W1975581657 cites W1982903538 @default.
• W1975581657 cites W1998728525 @default.
• W1975581657 cites W2008317513 @default.
• W1975581657 cites W2009916885 @default.
• W1975581657 cites W2009972285 @default.
• W1975581657 cites W2017500476 @default.
• W1975581657 cites W2021746796 @default.
• W1975581657 cites W2032045884 @default.
• W1975581657 cites W2032980181 @default.
• W1975581657 cites W2034720263 @default.
• W1975581657 cites W2046223523 @default.
• W1975581657 cites W2047246462 @default.
• W1975581657 cites W2054484042 @default.
• W1975581657 cites W2054934898 @default.
• W1975581657 cites W2060290397 @default.
• W1975581657 cites W2064859244 @default.
• W1975581657 cites W2065503544 @default.
• W1975581657 cites W2072843074 @default.
• W1975581657 cites W2074089440 @default.
• W1975581657 cites W2078594804 @default.
• W1975581657 cites W2084696890 @default.
• W1975581657 cites W2087677809 @default.
• W1975581657 cites W2090150963 @default.
• W1975581657 cites W2093020034 @default.
• W1975581657 cites W2097184060 @default.
• W1975581657 cites W2107328613 @default.
• W1975581657 cites W2111176992 @default.
• W1975581657 cites W2136393989 @default.
• W1975581657 cites W2138859411 @default.
• W1975581657 cites W2142327485 @default.
• W1975581657 cites W2153290883 @default.
• W1975581657 cites W2170302951 @default.
• W1975581657 cites W2171240084 @default.
• W1975581657 cites W2175401589 @default.
• W1975581657 cites W2325498096 @default.
• W1975581657 cites W2397674508 @default.
• W1975581657 cites W290744757 @default.
• W1975581657 cites W4211211149 @default.
• W1975581657 cites W4251174927 @default.
• W1975581657 cites W4294216491 @default.
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