Experimental Parasitology 127 (2011) 752–761

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Experimental Parasitology

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Leishmania mexicana: LACK ( homolog of receptors for activated C-kinase) is a plasminogen binding protein

Amaranta Gómez-Arreaza a, Héctor Acosta a, Ximena Barros-Álvarez b, Juan L. Concepción b, ⇑ Fernando Albericio c, Luisana Avilan a, a Laboratorio de Fisiología, Facultad de Ciencias, Universidad de Los Andes, La Hechicera, Mérida 5101, Venezuela b Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, La Hechicera, Mérida 5101, Venezuela c Institute for Research in Biomedicine and CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain article info abstract

Article history: Leishmania mexicana is able to interact with the fibrinolytic system through its component plasminogen, Received 3 September 2010 the zymogenic form of the protease plasmin. In this study a new plasminogen binding protein of this par- Received in revised form 8 December 2010 asite was identified: LACK, the Leishmania homolog of receptors for activated C-kinase. Plasminogen Accepted 17 January 2011 binds recombinant LACK with a K value of 1.6 ± 0.4 lM, and binding is lysine-dependent since it is inhib- Available online 25 January 2011 d ited by the lysine analog e-aminocaproic acid. Inhibition studies with specific peptides and plasminogen

binding activity of a mutated recombinant LACK have highlighted the internal motif 260VYDLESKAV268, Keywords: similar to those found in several enolases, as involved in plasminogen binding. Recombinant LACK and LACK secreted proteins, in medium conditioned by parasites, enhance plasminogen activation to plasmin by Plasminogen Leishmania mexicana the tissue plasminogen activator (t-PA). In addition to its localization in the cytosol, in the microsomal fraction and as secreted protein in conditioned medium, LACK was also localized on the external surface of the membrane. The results presented here suggest that LACK might bind and enhance plasminogen activation in vivo promoting the formation of plasmin. Plasminogen binding of LACK represents a new function for this protein and might contribute to the invasiveness of the parasite. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction tion of plasminogen is linked to invasiveness and pathogenecity (Lähteenmäki et al., 2005; Walker et al., 2005; Sun, 2006) and this Leishmania parasites are the causal agents of a group of clinical recruitment of plasminogen is generally due to the presence of sev- manifestations known collectively as . During their eral plasminogen binding receptors (Miles et al., 2005). On the sur- life cycle, these parasites alternate between invertebrate host (vec- face of the pathogen, plasminogen is transformed into plasmin tor) and mammalian host invading macrophages and reproducing either by host plasminogen activators or by the pathogen’s own inside phagolysosomes. Infection by Leishmania and establishment activator. This acquired protease can degrade extracellular matrix of the parasite in the mammalian host is known to depend on mul- proteins and fibrin. This latter is part of the host defense against tiple factors, of which invasive/evasive molecular determinants are infections (Sun, 2006). Parasites are also among the pathogens that key elements (Chang and McGwire, 2002). These invasive/evasive interact with plasminogen (Avilan et al., 2000; Jolodar et al., 2003; determinants are usually found on the cell surface of the parasite Bernal et al., 2004; Almeida et al., 2004; Ramajo-Hernández et al., and/or are secreted (Chang and McGwire, 2002; Naderer et al., 2007; Marcilla et al., 2007; Mundodi et al., 2008). In the case of 2004). Possible molecular determinants in Leishmania for invasion Leishmania mexicana this interaction has been previously charac- and/or establishment in mammalian hosts could be plasminogen terized (Avilan et al., 2000; Calcagno et al., 2002) and contributes binding proteins that allow interaction of the parasite with the to virulence of the parasite (Maldonado et al., 2006). In addition, fibrinolytic system. enolase was identified as plasminogen binding protein on the sur- Plasminogen is the zymogenic form of the serine-protease plas- face of L. mexicana (Vanegas et al., 2007). min and is found in plasma and extracellular fluids (Vassalli et al., In this study we report that LACK (Leishmania homolog of recep- 1991). In several bacterial pathogens, the acquisition and activa- tors for Activated C-kinase) is another plasminogen binding protein in L. mexicana. LACK is an analog of the RACK (receptor for activated C-kinase) proteins present in that are ⇑ Corresponding author. Fax: +58 274 2401286. members of the family of WD40 repeat proteins (Neer et al., E-mail address: [email protected] (L. Avilan). 1994). RACKs are known for their function as stabilizer of the

0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.01.008 A. Gómez-Arreaza et al. / Experimental Parasitology 127 (2011) 752–761 753 active form of protein kinase C. In addition these proteins mediate 50_GCCATATGAACTACGAGGGTCACCTGAA_30 (NdeI site in bold) protein–protein interactions serving as adaptors for several multi- and the reverse primer was 50_CGGAATTCTTACTCGGCGTCGGA complex proteins involved in signaling pathways (Schechtman and GATG_30 (EcoRI site in bold). The amplification mixture (50 ll) con- Mochly-Rosen, 2001). The molecular function of LACK in Leish- tained 1 lg of genomic DNA, 1 lM of each primer, 200 lM of each mania is not clear although the immunological response to this of the four deoxynucleotides, 1.5 mM MgCl2 and 1.5 U of DNA molecule has been well studied (Launois et al., 2007) and used polymerase (Go Taq Flexi DNA polymerasa, Promega, USA) with for experimental vaccine studies in the mouse model (Coler and the corresponding PCR buffer. PCR was performed as follows: an Reed, 2005). It has been clearly demonstrated that LACK is essen- initial incubation at 94 °C for 2 min, 36 cycles of denaturation at tial for the viability of the parasite and to establish the parasite 94 °C for 45 s, annealing at 60 °C for 45 s, and extension at 72 °C in the host. LACK mutants of with diminished lev- for 90 s; the final incubation was at 72 °C for 10 min. The PCR prod- els of this protein fail to develop lesions in susceptible mice and uct was ligated into the pGEM-T vector (Promega), cloned and se- have reduced capacity to reproduce in macrophages in vitro (Kelly quenced using the T7 and SP6 primers in an automated sequencer. et al., 2003). In , the RACK1 homolog is required The gene was then transferred to the pET28a vector (Novagen, for cytokinesis (Rothberg et al., 2006). In addition to its cytoplas- Germany) and used to transform the Escherichia coli strain mic localization (Gonzalez-Aseguinolaza et al., 1999; Taladriz BL21(DE3)pLys for the production of the recombinant proteins et al., 1999; Kelly et al., 2003), LACK was recently found to also with an N-terminal 20 residue-long extension containing a poly- be actively secreted (Silverman et al., 2008, 2010; Cuervo et al., His-tag. The strain harboring the expression plasmids was grown 2009), this secretion occurring via exosomes (Silverman et al., at 25 °C for 48 h in ZYM-5052 autoinduction medium (Studier, 2010). All these findings suggest that LACK could have several 2005), supplemented with 33 lg/ml kanamycin and 34 lg/ml functions, its plasminogen binding capacity representing a novel chloramphenicol, to O.D600nm values of 10. The bacteria from function of this protein. This function could be important in 50 ml culture were harvested by centrifugation, washed with Leishmania–host interaction. PBS, resuspended in 20 ml lysis buffer (50 mM potassium phos-

phate K2HPO4:KH2PO4, pH 8 and 0.5 M NaCl) in the presence of a cocktail of protease inhibitors (Sigma) and broken by sonication. 2. Materials and methods The lysate was then centrifuged at 12,000g for 15 min at 4 °C and the supernatant was applied onto a HisLink resin (Promega) col- 2.1. Parasites and culture conditions umn equilibrated with lysis buffer. After washings, LACK was eluted with 50 mM potassium phosphate buffer pH 6 and 0.5 M The AZV strain of L. mexicana (Pérez et al., 1979) was used in NaCl containing 200 mM imidazole. The imidazole was removed this study. Promastigotes were cultured at 28 °C with gentle shak- from the samples using a PD-10 column (Amersham Biosciences). ing in Schneider’s medium supplemented with 20% heat-inacti- The purity of LACK was checked by sodium dodecyl sulfate (SDS) vated fetal bovine serum. Promastigotes were harvested by polyacrylamide gel electrophoresis (PAGE) followed by Coomassie centrifugation (2000g for 15 min) and washed in phosphate buf- blue R-250 staining. For molecular mass determination of the re- fered saline (PBS). In all cases, promastigotes in the logarithmic combinant protein, gel filtration was performed on a Sephadex phase of their growth were used. G-75 column (76 1.4 cm) equilibrated with PBS. Site-directed mutagenesis of the LACK gene was performed using 2.2. Proteins, peptides and antibodies the QuikChange II site-directed mutagenesis kit (Agilent Technolo- gies, USA) following the manufacturer instructions. The gene cloned Human plasminogen was purified to homogeneity from human in the pET28a plasmid was used as template with the individual blood according to the method of Deutsch and Mertz (1970). Tissue mutagenic primers. Two recombinant mutated LACK proteins were plasminogen activator (t-PA) and urokinase were obtained from constructed: LACKseq1 and LACKseq2 using one pair of complemen- Chromogenix (Italy) and Choay Laboratory (France), respectively. tary oligonucleotide primers for each mutant protein. LACKseq1 The peptides H-VYDLESKAV-NH2, H-SWDNTIKVW-NH2, and containing substitutions at positions 262(Asp ? Ala) and 266(Ly- H-VYALGSLAV-NH2 were synthesized by a solid-phase approach s ? Leu) (forward primer: 50_CTGTCCGTGTACGCCCTCGAGAGCCT using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (t-Bu) GGCCGTGATTGC_30) and LACKseq2 containing substitutions at strategy using a Fmoc-Rink-resin (Lloyd-Williams et al., 1997). positions 173(Asp ? Ala) and 177(Lys ? Leu) (forward primer: The purity of the peptides (95%, 93% and 93%, respectively) was 50_GCCAGCTGGGCCAACACCATCCTGGTATGGAATGTGAACG_30). DNA established by analytical high-performance liquid chromatography sequence analyses were performed to confirm the mutations intro- (HPLC). These peptides were dissolved in dimethyl sulfoxide duced using the T7term primer. The mutated recombinant proteins (DMSO). The commercial antibodies used were mouse anti-human were expressed in E. coli strain BL21(DE3)pLys by cell growth in a-tubulin (Sigma, USA), goat anti-rabbit IgG conjugated with either autoinduction medium as specified above. alkaline phosphatase (Sigma), peroxidase (Sigma) or Cy3 dye (Amersham Biosciences, Sweden) and goat anti-mouse IgG conju- gated with fluorescein isothiocyanate (FITC) (Sigma). The prepara- 2.4. Anti-L. mexicana LACK antibodies tion of rabbit anti-human plasminogen and anti-L. mexicana enolase has been described (Vanegas et al., 2007; Quiñones et al., Polyclonal antiserum against recombinant LACK was raised in a 2007). Mouse anti-L. mexicana gp63 was obtained previously by rabbit by intradermic injection with 200 lg of purified protein in intraperitoneal injection with 50 lg of recombinant gp63 using complete Freund’s adjuvant for the first inoculation and for four aluminum (Rehydragel HPA, USA) as adjuvant. The serum was booster injections, at 2-week intervals, using incomplete Freund’s collected after five booster injections. Anti-T. cruzi PGK (phospho- adjuvant. The rabbit was bled by cardiac puncture 2 weeks after glycerate kinase C) was used to detect this protein in L. mexicana. the last injection. The anti-LACK antibodies were purified using affinity chromatography. Recombinant LACK was coupled to Affigel 2.3. Recombinant LACK, site-directed mutagenesis and purification 10 resin (Bio-Rad, USA) according to manufacturer’s instructions and chromatography was carried out using the serum proteins pre- The LACK gene (GenBank accession number AF363976) was cipitated with ammonium sulfate (50% saturation). After incuba- amplified by PCR from genomic DNA. The forward primer was tion with the resin and washing steps in PBS, the antibodies 754 A. Gómez-Arreaza et al. / Experimental Parasitology 127 (2011) 752–761 were eluted with 50 mM glycine, pH 2, 10% ethyleneglycol, (Walker, 2002). The proteins were transferred to nitrocellulose 150 mM NaCl, and neutralized with 1 M Tris–HCl pH 8. by passive diffusion and visualized using anti-L. mexicana LACK antiserum and alkaline phosphatase-conjugated second antibod- 2.5. Differential centrifugation ies. The proteins contained, in the sample buffer, 100 mM NaCl and 1 mM DTT. Leishmania mexicana promastigotes (2 L of culture) were homogenized by grinding with silicon carbide and submitted to 2.7. Plasminogen binding and activation assays differential centrifugation following the procedure described pre- viously (Pabón et al., 2007). This method yielded a nuclear, a large The interaction of plasminogen with recombinant LACK was granular, a small granular, a microsomal and finally a supernatant determined by an ELISA system immobilizing the recombinant (cytosolic) fraction. In each fraction the marker enzymes malate protein (100 ng) in a microtiter plate (MaxiSorp surface, Nalge dehydrogenase, hexokinase and alkaline phosphatase were deter- Nunc International, USA) and incubating with different plasmino- mined as described (Bergmeyer, 1983). The microsomal fraction gen concentrations as described (Vanegas et al., 2007). In some was resuspended in 25 mM Tris–HCl, pH 7.6, 1 mM EDTA, and experiments e-aminocaporic acid or the peptides were included. 250 mM sucrose and centrifuged at 105,000g for 1.5 h. The pres- In the experiments performed with peptides, all assays contained ence of LACK in the different cell fractions was monitored by Wes- the same amount of DMSO. The plasminogen bound was detected tern blotting using anti-L. mexicana LACK antiserum. with anti-human plasminogen antibodies and peroxidase-conju- gated secondary antibodies as described (Vanegas et al., 2007). 2.6. 2D gel electrophoresis, ligand blotting, Western blotting Plasminogen activation assays were performed by incubating 200 nM plasminogen in PBS with 0.6 mM chromogenic substrate The pellet of the microsomal fraction obtained after differential D-val-Leu-Lys-p-nitroanilide dihydrochloride in a total volume of centrifugation, was resuspended in solubilization buffer (15 mM 100 ll, in the presence of different concentrations of recombinant Tris–HCl, pH 7.2, 7 M urea, 2 M thiourea, 4% nonidet P-40 and 2% LACK. Activation of plasminogen was initiated by adding 10 nM b-mercaptoethanol); the final protein concentration was 6.5 mg/ t-PA or 2 nM urokinase. The absorbance at 405 nm was monitored ml. After homogenization and centrifugation, an aliquot (118 ll) and the activation value was estimated from the slope of the absor- was mixed with 6.25 ll carrier ampholytes (Bio-lyte pH 3/10, bance plot versus time2 (Longstaff, 2002). Bio-Rad) and traces of bromophenol blue. The proteins were first separated according to their isoelectric point along a linear immo- 2.8. Immunofluorescence assays bilized pH gradient (IPG strip, pH 3–10, 7 cm long). After the first dimension, the IPG strips were reduced in 4% dithiothreitol (DTT) Promastigotes were washed with PBS and processed in two dif- for 15 min and then alkylated in 2.5% iodoacetamide for 15 min ferent ways to obtain non-permeabilized and permeabilized para- in equilibration buffer containing 6 M urea, 375 mM Tris–HCl, pH sites. (i) For non-permeabilized cells, living parasites were 8.8, 30% glycerol, 5% SDS and bromophenol blue. In the second incubated in a microcentrifuge tube with rabbit anti-LACK antise- dimension, the strips were loaded onto SDS–polyacrylamide gradi- rum (dilution 1:80) and mouse anti-human a-tubulin antibodies ent gels (Mini-Protean 3 Cell System, Bio-Rad). In one gel, the pro- (dilution 1:40) in PBS containing 1% BSA at room temperature for teins were detected by staining with Coomassie brilliant blue 60 min. The anti-LACK antiserum was previously heated to inacti- R250. The proteins in the replica gel were transferred to a polyvi- vate complement, at 56 °C for 30 min. After five washings with PBS nylidene difluoride (PVDF) membrane and used for ligand blotting the cells were treated with 4% formaldehyde in PBS for 3 min. After assays. The membrane was first blocked with PBS containing 10% additional washings, they were allowed to adhere to poly-(L-ly- casein and 0.1% Tween 20 for 2 h and then incubated with 1 lM sine)-coated slides and blocked with PBS containing 3% BSA and human plasminogen in PBS containing 1% casein for 90 min at 50 mM ammonium chloride for 60 min. (ii) For permeabilized cells, room temperature. After washings with PBS containing 0.1% the parasites were directly treated with 4% formaldehyde as spec- Tween, the membrane was incubated with anti-human plasmino- ified above, fixed to poly-(L-lysine)-coated slides, permeabilized gen antibodies (dilution 1:2000) for 1 h and revealed using perox- with 0.2% Triton X-100 for 10 min and finally washed with PBS. idase-conjugated second antibodies (dilution 1:10.000) and 0.05% After blocking with BSA and ammonium chloride, the cells were diaminobenzidine, 0.0025% CoCl2 and 0.015% H2O2, in PBS. The incubated with the primary antibodies at the same dilution used protein spot excised from the gel stained with Coomassie blue, for non-permeabilized cells. In both cases the cells were then incu- was digested with trypsin (12.5 lg/ml in 20 mM NH4HCO3) and bated for 60 min with anti-mouse IgG conjugated with FITC and analyzed by mass spectrometry (MALDI-TOF) (kindly performed anti-rabbit IgG conjugated with Cy3. The slides were examined in the Fingerprints Proteomics Facility of Dundee University, UK). using a Nikon fluorescence microscope. The resulting peak list was searched in the National Center for Bio- technology nonredundant (NCBInr) database using the Mascot 2.9. Biotin cell surface labeling software (www.matrixscience.com). To reprobe the membrane used for ligand blotting, with other antibodies, stripping was per- Promastigotes (8 108 parasites) washed with PBS, were cell- formed by submerging the PDVF membrane in methanol for surface labeled by incubation with 3.2 mg EZ-link sulfo-NHS-LC- 3 min and then in PBS for 10 min. The membrane was incubated biotin (Pierce, USA) in a final volume of 1.3 ml, for 1 h at 4 °C. After in 2.5 mM glycine, pH 2, 1% SDS for 30 min, washed with PBS one washing with PBS containing 100 mM glycine and two addi- and blocked again with 5% casein in PBS. The stripped membrane tional washings with PBS, the cells were treated with lysis buffer was reprobed with anti-L. mexicana enolase antibodies (dilution (PBS pH 7.4, 0.1% Triton X-100, 2 mM phenylmethylsulfonyl 1:5000) and revealed with alkaline phosphatase-conjugated sec- fluoride (PMSF) and protease inhibitors) for 10 min and briefly ond antibodies (dilution 1:10.000) and 5-bromo-4-chloro-3-indo- sonicated. The mixture was centrifuged at 3000g for 10 min. lyl phosphate/nitroblue tetrazolium (BCIP/NBT) as substrate. The Biotinylated proteins in the supernantant were separated from membrane was dried and stripped again to be reprobed with unbiotinylated proteins by rotation and adsorption onto Streptavi- anti-L. mexicana LACK antiserum (dilution 1:1000). din-agarose resin (Thermo Scientific, USA) equilibrated with lysis LACK form the cytosol and recombinant proteins were submit- buffer for 90 min at 4 °C. The resin was then washed with lysis buf- ted to native gel electrophoresis (7.5% acrylamide) as described fer containing 0.5 M NaCl, and the proteins bound were eluted by A. Gómez-Arreaza et al. / Experimental Parasitology 127 (2011) 752–761 755 boiling the resin in SDS sample buffer. Aliquots were subjected to established that other plasminogen receptors were present on the SDS–PAGE and Western blotting. In control experiments, biotin surface of the parasite. In the present study attempts were made to was omitted. identify other plasminogen binding proteins. A microsomal frac- tion obtained after differential centrifugation was submitted to 2.10. Production and analysis of promastigote-conditioned medium 2D electrophoresis (Fig. 1A). A gel run in parallel was transferred to a PVDF membrane and incubated with plasminogen to perform Promastigotes (1.5 109 parasites) were centrifuged at 1400g ligand blotting. As shown in Fig. 1B, several spots appeared in the for 10 min, washed twice with PBS and the final pellet was care- ligand blotting experiment suggesting the presence of several plas- fully resuspended in 1 ml PBS containing 1% glucose. This suspen- minogen binding proteins in the microsomal fraction of L. mexica- sion was incubated for 4 h at 25 °C. Parasite integrity was assessed na. No staining was observed when plasminogen was omitted by the exclusion test using trypan blue. Secreted proteins in the (data not shown). After stripping, the same membrane used for conditioned medium were obtained by centrifugation. They were ligand blotting was reprobed with anti-L. mexicana enolase anti- precipitated with 10% trichloroacetic acid and the precipitate was bodies, and revealed that one of the spots (N° 1inFig. 1A) corre- solubilized in sample buffer and submitted to SDS–PAGE. LACK sponded to enolase (Fig. 1C). Another spot (N° 2inFig. 1A) was was visualized by Western blotting. Glucose 6-phosphate dehydro- excised from the gel and submitted to mass spectrometry for iden- genase activity was measured spectrophotometrically as described tification. This led to the unambiguous identification in the spot (Silverman et al., 2008) in conditioned medium and in parasites (Mascot score 105) of the protein LACK, an analog of the mamma- lysed by adding 150 mM NaCl and 0.1% Triton X-100. lian receptor for activated protein kinase C (RACK) (Schechtman and Mochly-Rosen, 2001). This membrane was stored, stripped la- 2.11. Sequence analysis and modeling ter and assayed with anti-LACK antiserum to confirm that LACK was present in this spot (Fig. 1D). Since this spot could correspond Alignment of sequences was performed using the Muscle align- to a mixture of different proteins, we cloned and over-expressed ment program (Edgar, 2004). A three-dimensional model of L. mex- LACK to test its plasminogen binding capacity. Recombinant LACK icana LACK was built using the Swiss-Model (Arnold et al., 2006) was expressed as N-terminal fusion proteins with a poly-His-tag and as template the structure of the RACK 1 of Arabidopsis thaliana in E. coli. LACK formed inclusion bodies upon induction with iso- (PDB entry 3DM0A; Ullah et al., 2008) which has 48.2% sequence propylthio-b-D-galactoside (IPTG). The formation of inclusion identity with L. mexicana LACK at the protein level. bodies, when LACK is expressed in E. coli, has been reported previ- ously (Salay et al., 2007). However, we could obtain this protein 2.12. Ethics partially soluble by cell growth in an autoinduction medium. The protein was purified by metal affinity chromatography and Experiments involving animals were approved by the ethics the yield of purified protein was about 2 mg from about 100 mg committee of animal experimentation of the Universidad de Los protein of bacterial cell-free extract, corresponding to 50 ml cul- Andes (BIOULA). ture. Recombinant LACK was visualized as only one band in native gel (data not shown). This band had lower mobility that natural 3. Results LACK from the cytosol in agreement with the increase in isolectric point and molecular mass introduced by the N-terminal extension 3.1. Identification of LACK as plasminogen binding protein of the recombinant protein. The native molecular mass of the recombinant protein, as determined by gel filtration, was approx- In a previous study it was shown that enolase is a plasminogen imately 33 kDa, consistent with the monomeric form of the receptor in L. mexicana (Vanegas et al., 2007). However, it was also protein.

Fig. 1. 2D electrophoresis of a microsomal fraction of L. mexicana. (A) Proteins from the microsomal fraction were submitted to 2D electrophoresis and stained with Coomassie blue. 1 and 2 are spots identified. (B) Ligand blotting with plasminogen of a replica gel. (C) Western blotting, using anti-L. mexicana enolase antiserum, after stripping of the membrane used for ligand blotting. (D) Western blotting, using anti-L. mexicana LACK antiserum, after a second stripping of the same membrane. 756 A. Gómez-Arreaza et al. / Experimental Parasitology 127 (2011) 752–761

A B 100 100

80 80

60 60 /min

450nm 40 40 mA

20 20

0 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 20406080100120 [PLG] (μM) [ε-ACA] (mM)

Fig. 2. Plasminogen binding on immobilized recombinant LACK. (A) Plasminogen (PLG) at different concentrations was added to immobilized recombinant LACK (d), gelatin (s) or another recombinant protein containing a His-link [malic enzyme from P. infestans (j)]. Bound plasminogen was detected with anti-human plasminogen antibodies and peroxidase-conjugated secondary antibodies. (B) Immobilized recombinant LACK (d) or gelatin (s) were incubated with 1 lM plasminogen and different concentrations of e-aminocaproic acid (e-ACA).

3.2. Characterization of plasminogen binding of recombinant LACK Many plasminogen binding proteins bind plasminogen through ex- posed C-terminal lysines (Miles et al., 2005). Since this protein To determine the plasminogen binding capacity of LACK, the re- lacks a C-terminal lysine, internal residues must be responsible combinant protein was immobilized on microtiter plates and incu- for binding with plasminogen. An internal motif consisting of nine bated with increasing concentrations of plasminogen. As shown in amino acids (248FYDKERKVY256) in enolase from Streptococcus Fig. 2A, plasminogen bound LACK in a concentration-dependent pneumoniae was previously identified as responsible for plasmino- and saturable manner. The binding of plasminogen to gelatin and gen binding in this molecule (Bergmann et al., 2003). A similar mo- other recombinant proteins with a His-tag (malic enzyme from tif is also responsible for the binding of plasminogen in the enolase

Phytophthora infestans) was minimal. The Kd estimated from this of L. mexicana (Vanegas et al., 2007) and Aeromonas hydrophila (Sha curve was 1.6 ± 0.4 lM; this value is compatible with in vivo recog- et al., 2009). A search for such a motif in LACK, revealed two similar nition since the physiological plasminogen concentration is about internal sequences that contain a lysine residue: Seq. 1: 260VYDLE- 2 lM(Vassalli et al., 1991). The binding was inhibited by e-amino- SKAV268 and Seq. 2: 171SWDNTIKVW179,(Fig. 3A). As suggested by caproic acid (Fig. 2B) indicating that lysine binding sites are prob- a model of LACK of L. mexicana, these internal sequences contain ably involved in the interaction between LACK and plasminogen. loops that are exposed on the surface of the protein (Fig. 3B).

A B Seq. 1

S. pneumoniae enolase 248F Y D K E R K V Y 256 A. hydrophila enolase 252F Y D A E K K E Y 260 L. mexicana enolase 249A Y D A E R K M Y 257

Seq. 1 LACK 260 V Y D L E S K A V268 Seq. 2 LACK S W D N T I K V W 171 179 Seq. 2

50 70 C D 60 40 50 30 /min /min 40 450nm 450nm 20 30 mA mA 20 10 10

0 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 [Peptide] (mM) [PLG] (μM)

Fig. 3. Plasminogen binding motif in LACK of L. mexicana. (A) Alignment of the plasminogen binding motif in enolases from S. pneumoniae, A. hydrophila and L. mexicana with similar motifs found in LACK of L. mexicana. Shading in black indicates identity; shading in gray indicates hydrophobic residues at that position. (B) Modeling of the structure of L. mexicana LACK. In black are shown the sequences Seq. 1 and Seq. 2 presented in A. (C) Inhibition of plasminogen binding to immobilized recombinant LACK by the peptides Seq. 1 (d) and Seq. 2 (s) presented in A. The peptide VYALGSLAV was used as control (j). Plasminogen concentration was 1 lM. (D) Plasminogen binding to the immobilized mutant recombinant LACKseq1 with amino acid substitutions at positions 262(Asp ? Ala) and 266(Lys ? Leu) (j), compared to wild-type recombinant LACK (d) and gelatin (s). A. Gómez-Arreaza et al. / Experimental Parasitology 127 (2011) 752–761 757

0.25 However, one of them (Seq. 2) seems to have the lysine residue 7 outside of the loop in contrast to that of the plasminogen binding A 6 5 motif of S. pneumoniae (Ehinger et al., 2004). To assess the role of 5 0.20 these two internal sequences in the LACK molecule as plasminogen x 10 4 2 binding motifs, these two peptides were tested for inhibition in as- 3 says of plasminogen binding on immobilized LACK. As shown in 2 0.15 A/min Fig. 3C both peptides inhibited plasminogen binding to LACK (from 1 0

39% to 59% inhibition at 1.25 mM peptide) when compared to 405nm 020406080100120 A binding in the absence of the peptides. A control peptide, VYALG- 0.10 SLAV, did not inhibit plasminogen binding (Fig. 3C). The peptide [LACK] (nM) VYDLESKAV produced more inhibition at lower peptide concentra- tions. This suggests that these internal sequences might be respon- 0.05 sible for binding of plasminogen in LACK similarly to what was found in enolase from several species. 0.00 To asses whether these proposed internal sites are involved in 0 10203040506070 plasminogen binding, recombinant mutated LACK proteins were constructed: LACKseq1 with amino acid substitutions at the posi- time (min) tions 262(Asp ? Ala) and 266(Lys ? Leu) and LACKseq2 with the B kDa 1 2 3 C 0.14 amino acid substitutions at the positions 173(Asp ? Ala) and 0.12 177(Lys ? Leu), two critical amino acids for plasminogen binding 74.3 - 0.10 (Bergmann et al., 2003). LACKseq1 was obtained soluble as was - 0.08 the wild-type recombinant protein with the same mobility in a 48.6 405nm 0.06 native gel (data not shown). However, LACKseq2 was completely A 0.04 insoluble indicating that these mutations destabilize the native 30 - 0.02 state and expedite aggregation. The plasminogen binding activity 23.1 - 0.00 was thus compared between the wild-type recombinant LACK 0 10203040506070 and the mutated protein LACKseq1. Both proteins were immobi- time (min) lized in a microtitre plate and incubated with increasing plasmin- Fig. 4. Activation of plasminogen by t-PA. (A) t-PA (10 nM) was added to a mixture ogen concentrations. Compared to the wild-type recombinant containing 200 nM plasminogen, 0.6 mM chromogenic substrate (D-val-Leu-Lys-p- LACK, the plasminogen binding capacity of LACKseq1 was almost nitroanilide dihydrochloride) for plasmin and increasing concentrations of recom- abolished (Fig. 3D). This result indicates that this site 260VYDLE- binant LACK, in 100 ll. The absorbance at 405 nm was determined at different times. (d) 0 nM, (s) 5 nM, (.) 10 nM, (5) 25 nM, (j) 50 nM, (h) 75 nM, (Ç) 100 nM SKAV268 (Seq. 1, Fig. 3A) is responsible for plasminogen recognition of recombinant LACK. Inset, plasminogen activation at different LACK concentra- by LACK. Although the peptide SWDNTIKVW (Seq. 2, Fig. 3A) was tions was estimated from the plot Absorbance at 405 nm versus time2. (B) Western able to produce some inhibition in plasminogen binding of LACK, blotting using anti-LACK antiserum of conditioned medium (lane 1), parasite the result obtained with the mutated protein LACKseq1 suggests extract (lane 2) and crude extract of the parasites used to produce the conditioned medium (lane 3). In each lane, 56 lg proteins were loaded. (C) Activation of that the motif 171SWDNTIKVW179 (Seq. 2) is not fully exposed or d s does not have the appropriate conformation for binding. plasminogen by t-PA in the absence ( ) or presence ( )of35lg/ml of parasite conditioned medium.

3.3. Enhancement of plasminogen activation by LACK and secreted proteins from L. mexicana ity of the parasite and indicates that the conditioned medium con- tains mainly secreted proteins. This conditioned medium also We examined the ability of recombinant LACK to enhance enhanced plasminogen activation into plasmin by t-PA (Fig. 4C) plasminogen activation. Fig. 4A shows that t-PA-mediated plas- indicating that some secreted proteins bind plasminogen. No plas- minogen activation by LACK was concentration dependant. The min activity was detected in the absence of plasminogen activator. maximum activation rate expressed as absorbance at 405 nm/ Other plasminogen binding proteins can produce the same effect. 2 min was increased 2.9-fold in the presence of 100 nM LACK. When Enolase is also present in the secretoma of L. donovani and L. bra- the same assays were performed with urokinase, no enhancement ziliensis (Silverman et al., 2008; Cuervo et al., 2009). of plasminogen activation was observed (data not shown). The enhancement of plasminogen activation by t-PA confirms the plas- minogen binding capacity of this protein. 3.4. Presence of LACK on the surface of the cell membrane Previous studies have demonstrated that LACK is secreted in Leishmania parasites (Silverman et al., 2008,2010; Cuervo et al., Analysis by Western blotting showed that the anti-LACK antise- 2009) through exosomes (Silverman et al., 2010). L. mexicana also rum recognized only one band, at the expected molecular mass, in secretes proteins since exosomes have been detected and analyzed the crude extracts of the parasites (Fig. 4B). Thus this antiserum from an ultrastructural point of view (Silverman et al., 2010). In was used to compare each fraction obtained after subcellular frac- our conditions, LACK was also secreted. As shown in Fig. 4B, LACK tionating of the parasites. This analysis showed that LACK is pres- was present in the conditioned medium that contains secreted pro- ent primarily in the microsomal fraction; as expected, it is also teins, and in crude extracts of parasites that were used to generate observed in the cytosol corresponding to the final supernatant of the conditioned medium as well in extracts of intact parasites. The the fractionating process (Fig. 5A). The microsomal fraction con- trypan blue exclusion assay showed parasite mortality to be tains plasma membrane as assessed by the enzyme marker alka- around 0.8% before and after incubation in PBS-glucose that was line phosphatase (Urbina et al., 2002)(Fig. 5B). The presence of used to obtain the secreted proteins. The glucose 6-phosphate LACK associated to membranes agrees with the function of RACK dehydrogenase, mainly a cytosolic enzyme (Mottram and Coombs, proteins in anchoring signaling proteins (Schechtman and Moc- 1985), was present in the conditioned medium (0.009 U), this hly-Rosen, 2001). In this case, LACK would be associated with the activity being 1.4% of the total units of the parasites (0.65 U) used internal side of cell membranes or other inner membranes through to generate this medium. This value is consistent with the mortal- protein–protein interactions. To examine if LACK is present on the 758 A. Gómez-Arreaza et al. / Experimental Parasitology 127 (2011) 752–761

0.5 A B MDH S 0.4 N LG SG M S 0.3 SG 0.2 LG 0.1 N M 0.0 2.0 HK 1.5

1.0

0.5

0.0 0.010 Specific Activity (U/mg) ALP 0.008 0.006 0.004 0.002 0.000 0204 06 0 8 0100 Cumulative Protein (%)

Fig. 5. Subcellular distribution of L. mexicana LACK. (A) Western blot analysis, using the anti-LACK antiserum of each fraction obtained after differential centrifugation. The different fractions (30 lg protein) are: N, nuclear; LG, large granular; SG, small granular; M, microsomal and S, final supernatant. (B) Enzyme marker activity (U/mg) in each fraction (MDH, malate dehydrogenase; HK, hexokinase and ALP, alkaline phosphatase). external side of the cell, immunofluorescence analyses were per- antiserum was performed as positive controls since these proteins formed. In these experiments non-permeabilized and permeabili- are known to be on the cell surface. As negative control anti-PGK zed parasites were compared. For the non-permeabilized cells, antiserum was used since PGK is mainly a cytosolic protein (Mot- living parasites were incubated with anti-LACK antiserum and tram and Coombs, 1985). As shown in Fig. 7, these antibodies re- anti-tubulin antibodies. The anti-tubulin antibodies were used as acted with a band in the case of gp63, enolase and LACK. No control since tubulin is only present inside the cell. For the perme- band was detected when biotin was omitted or when anti-PGK abilized cells, the cells were previously fixed, adhered to slides and was used. This result further documents the presence of LACK on permeabilized with detergent before being treated with the same the cell surface. LACK on the surface might contribute to plasmin- antibodies. As shown in Fig. 6A, staining was detected in permeabi- ogen binding on the surface of the parasite. lized cells with both antibodies indicating that LACK is present in the cytoplasm. In non-permeabilized cells (Fig. 6B), staining was 4. Discussion only observed with anti-LACK antibodies. No staining was detected when the primary antibodies were omitted (data not shown). This The importance of the interaction between pathogens and the indicates that LACK is present at the cell surface, although this does fibrinolytic system, through plasminogen binding and activation, not allow us to determine how much LACK from the microsomal in the infection process and in pathogenesis has been well estab- fraction is exposed to the cell surface. To verify this result, para- lished in several bacteria (Lähteenmäki et al., 2001, 2005). This sites were surface-labeled with EZ-link sulfo-NHS-LC-biotin, lysed, interaction provides a proteolytic activity that can function for and the biotin-labeled proteins were separated by absortion onto fibrin and matrix extracellular degradation, procollagenase activa- streptavidin-agarose followed by Western blotting with anti-LACK tion and release of peptide for nutrition (Lähteenmäki et al., 2001). antiserum. Western blotting with anti-enolase and anti-gp63 In the case of parasites and other eukaryotic pathogens, the

Fig. 6. Immunofluorescence of permeabilized and non-permeabilized L. mexicana promastigotes. (A) Cells treated with formaldehyde and permeabilized with Triton X-100 were incubated with both anti-LACK antiserum (1) and anti-a-tubulin antibodies (2). (B) Living cells were incubated with both anti-LACK antiserum (1) and anti-a-tubulin antibodies (2). After washing, the cells were fixed with formaldehyde, adhered to slides and blocked with BSA. Anti-a-tubulin antibodies were detected with anti-mouse IgG coupled to FITC. Anti-LACK antibodies were detected with anti-rabbit IgG coupled to Cy3. In A and B, panels (3) correspond to single optical images of the cells. A. Gómez-Arreaza et al. / Experimental Parasitology 127 (2011) 752–761 759

A B C D E 1 2 1 2 1 2 1 2 M kDa

- 98.6 - 74.4

- 48.6

- 30

Fig. 7. Cell-surface protein biotinylation. Living promastigotes were labeled with EZ-link sulfo-NHS-LC-biotin and lysed. The biotin-labeled proteins were then separated by absorption onto streptavidin-agarose and analyzed by Western blotting with anti-L. mexicana enolase (A), anti-L. mexicana gp63 (B), anti-L. mexicana LACK (C) and anti-T. cruzi PGK (D) antisera. Lanes 1, controls using cells from which biotin was omitted. Lanes 2, experiment with biotinylated cells. M, molecular mass marker. (E) Western blotting of parasite cytosol (final supernatant) using anti-PGK antisera. function of plasminogen binding in host–pathogen interaction is fraction. In addition, LACK was also secreted by the parasite in not entirely clear particularly in Leishmania parasites that thrive agreement with previous studies (Silverman et al., 2008, 2010; in macrophages of the vertebrate host. As a step to understanding Cuervo et al., 2009). The secretion of LACK and of other proteins the role of this interaction in Leishmania, we first identified enolase is through exosomes visible as budding vesicles on the surface as one of the receptors of plasminogen in the promastigote surface, (Silverman et al., 2010). These vesicles, if still associated to the cell a feature shared by other parasites (Jolodar et al., 2003; Bernal surface could be part of the microsomal fraction in the subcellular et al., 2004; Marcilla et al., 2007; Mundodi et al., 2008; de la Tor- fractionating procedure and their content could enrich the amount re-Escudero et al., 2010; Yang et al., 2010). Ligand blotting and pro- of LACK visualized in this fraction. In addition to being secreted, teomic analysis, using a microsomal fraction of L. mexicana, LACK was also found attached to the external side of the surface permitted the identification of another plasminogen binding pro- of the parasite by two independent approaches (immunofluores- tein, LACK, the homolog of receptors for the activated C-kinase cence analysis, and surface biotinylation and streptavidin affinity RACK of L. mexicana. The recombinant protein binds plasminogen purification). LACK (GenBank accession number P62883) has also with a similar affinity (Kd: 1.6 lM) as several plasminogen recep- been visualized previously in proteomic analyses of the plasma tors in other cells and organisms including the value for Kd for membrane fraction, from Leishmania chagasi, prepared by surface plasminogen binding in L. mexicana (Avilan et al., 2000). Inhibition biotinylation and affinity purification (Yao et al., 2010). One expla- of plasminogen binding to recombinant LACK by peptides sug- nation for this surface localization is the reassociation of secreted gested that this binding could be through internal motifs LACK. This could also be occurring with enolase that is also se-

(171SWDNTIKVW179 and/or 260VYDLESKAV268) similar to that of creted by exosomes (Silverman et al., 2010) and found on the enolase of S. pneumoniae, A. hidrophyla and L. mexicana. These inter- external side of the membrane (Quiñones et al., 2007). The reasso- nal sequences in LACK contain both positively and negatively ciation of secreted enolase was demonstrated for S. pneumoniae charged residues flanked by hydrophobic amino acids, a feature (Bergmann et al., 2001). By its capacity to induce protein–protein that seems important for plasminogen binding (Ehinger et al., interactions, LACK could be interacting with other proteins of the 2004), although the model of the structure of LACK suggested that surface membrane. Since Leishmania parasites have an ecto-protein the motif 171SWDNTIKVW179, is not fully exposed in a loop. The kinase C-like (Alvarez-Rueda et al., 2009), one may speculate that strongly reduced plasminogen binding activity of the mutated this protein might interact with secreted LACK on the cell surface. LACK, containing substitutions at positions 262(Asp ? Ala) and In summary, LACK is found in the cytoplasm, associated to the

266(Lys ? Leu), suggests that the motif 260VYDLESKAV268 is external side of the membrane and secreted, these different local- mainly responsible for plasminogen binding. This result implies izations being also observed for other plasminogen-binding pro- that such internal motif, responsible for plasminogen binding in teins such as enolase (Bergmann et al., 2001; Bernal et al., 2004; certain enolases, can be found in other proteins, such as LACK, Hurmalainen et al., 2007; Jones and Holt, 2007; Mundodi et al., and thus could be markers of possible plasminogen binding pro- 2008) and glyceraldehyde 3-phosphate dehydrogenase (Hurmalai- teins in addition to C-terminal lysines that mediate plasminogen nen et al., 2007; Egea et al., 2007; Marcilla et al., 2007) of several binding in several plasminogen binding proteins (Miles et al., prokaryotic and eukaryotic pathogens. 2005). Recombinant LACK facilitated t-PA-mediated activation of plas- LACK is localized in the cytoplasm of Leishmania parasites minogen to plasmin as does the conditioned medium obtained (Gonzalez-Aseguinolaza et al., 1999; Taladriz et al., 1999; Kelly from Leishmania parasites. Enhancement of plasminogen activation et al., 2003). In addition, it is associated with kinetoplasts (Gonz- in this latter context could be due not only to LACK but also to alez-Aseguinolaza et al., 1999; Taladriz et al., 1999) and mem- other plasminogen binding proteins. Enolase, another plasminogen branes (Gonzalez-Aseguinolaza et al., 1999; Kamoun-Essghaier binding protein is found in the secretoma of Leishmania parasites et al., 2005). In other trypanosomatids, LACK homologs have been (Silverman et al., 2008; Cuervo et al., 2009). One may propose that identified in the cytoplasm but also associated to membranes. Such in vivo, these secreted plasminogen binding proteins enhance the is the case of CACK of Crithidia fasciculata (Taladriz et al., 1999) and conversion from plasminogen to plasmin and therefore contribute TRACK of T. brucei (Rothberg et al., 2006). RACKs in mammalian to the degradation of fibrin and extracellular matrix proteins; this cells can associate with the internal side of the surface membrane function could complement that of its own proteases such as gp63 interacting with protein kinase C (Besson et al., 2002). In the pres- which is also secreted (McGwire et al., 2002). Thus the encounter ent study, after subcellular fractionation by differential centrifuga- between parasites and macrophages would be facilitated through tion, LACK was detected in the cytosol and in the microsomal the production of plasmin in the environment of the parasite. This 760 A. Gómez-Arreaza et al. / Experimental Parasitology 127 (2011) 752–761 function might be a strategy used by promastigotes once transmit- parasite Onchocerca volvulus that binds human plasminogen. Biochimica et ted into the host by the vector, or by amastigotes released from Biophysica Acta 1627, 111–120. Jones, M.N., Holt, R.G., 2007. Cloning and characterization of an alpha-enolase of the macrophages, to infect other cells. oral pathogen Streptococcus mutans that binds human plasminogen. Biochemical and Biophysical Research Communications 364, 924–929. Kamoun-Essghaier, S., Guizani, I., Strub, J.M., Van Dorsselaer, A., Mabrouk, K., Acknowledgments Ouelhazi, L., Dellagi, K., 2005. Proteomic approach for characterization of immunodominant membrane-associated 30- to 36-kiloDalton fraction antigens We thank to Dr. B. Kelly for providing us with anti-LACK anti- of promastigotes, reacting with sera from Mediterranean bodies used in preliminary experiments. We also thank Sol Gibson patients. Clinical and Diagnostic Laboratory Immunology 2, 310–320. and Alvaro Acosta-Serrano for proteomics analyses and Maria Kelly, B.L., Stetson, D.B., Locksley, R.M., 2003. Leishmania major LACK antigen is Alexandra Pérez for her technical help in 2D electrophoresis. We required for efficient vertebrate parasitization. The Journal of Experimental wish to thank Dr. Anne-Lise Haenni for revision of the manuscript. Medicine 198, 1689–1698. Lähteenmäki, K., Edelman, S., Korhonen, T.K., 2005. Bacterial metastasis: the host This work was supported by CDCHT-ULA Grant C-1552-08-03-B, plasminogen system in bacterial invasion. Trends in Microbiology 13, 79–85. and by Fondo Nacional de Ciencia, Tecnología e Investigación Lähteenmäki, K., Kuusela, P., Korhonen, T.K., 2001. Bacterial plasminogen activators (Fonacit) Project 2007000960. and receptors. FEMS Microbiology Reviews 25, 531–552. Launois, P., Pingel, S., Himmelrich, H., Locksley, R., Louis, J., 2007. 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