Cellular localization and expression of gp63 homologous metalloproteases in Leishmania ( Viannia) braziliensis strains

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Acta Tropica 106 (2008) 143–148

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Acta Tropica

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Cellular localization and expression of gp63 homologous metalloproteasesin Leishmania (Viannia) braziliensis strains

Patricia Cuervoa,b,c, Andre L.S. Santosd, Carlos R. Alvese, Gustavo C. Menezes f, Bianca A. Silvad,Constanca Brittoe, Octavio Fernandesb, Elisa Cupolilloa, Jose Batista De Jesus e,g,∗

a Laboratorio de Pesquisa em Leishmanioses, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazilb Laboratorio de Epidemiologia Molecular de Doencas Infecciosas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazilc Centro Internacional de Entrenamiento e Investigaciones Medicas, CIDEIM, Cali, Colombia

d Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Goes, UFRJ, Rio de Janeiro, Brazile Laboratorio de Biologia Molecular e Doencas Endemicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazilf Laboratorio de Biologia da Superfıcie Celular, Instituto de Biofısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazilg Departamento de Ciencias Naturais, Universidade Federal de Sao Joao del Rei, Sao Joao del Rei, MG, Brazil

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Article history:Received 19 July 2007Received in revised form 12 February 2008Accepted 3 March 2008Available online 20 March 2008

Keywords:TrypanosomatidsLeishmania (Viannia) braziliensisMetalloproteases localization

a b s t r a c t

Leishmania (Viannia) brazdisease that encompassesthat Brazilian and Colombtations, display differentthe cellular localization oLeishmania. Comparative aas an evaluation of the efproteases were also perforpatterns with a similar pro

gp63 microscopy analyses corroborain the parasites and revealed dicellular distribution of metallocellular metalloproteases withpocket. Finally, it was observed(V.) braziliensis isolates remain uactivity pattern is a stable phen

1. Introduction

The flagellate protozoan Leishmania (Viannia) braziliensis is themajor causative agent of American tegumentary leishmaniasis inmany endemic regions of Central and South America (Grimaldi etal., 1989). This parasite has been associated with a broad range ofclinical manifestations from simple cutaneous ulcers to dissemi-nated lesions and mucosal involvement, a very destructive form ofleishmaniasis (Marsden, 1986).

The Leishmania life cycle alternates between sand fly and mam-malian hosts, and involves two principal developmental stages: the

∗ Corresponding author at: Laboratorio de Biologia Molecular e DoencasEndemicas, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil, 4365, Manguinhos, Pav,Leonidas Deane, sala 209, CEP: 21045-900, Rio de Janeiro, RJ, Brazil.Tel.: +55 21 38658153; fax: +55 21 25903495.

E-mail address: [email protected] (J.B. De Jesus).

0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.actatropica.2008.03.005

s is the major causative agent of American tegumentary leishmaniasis, aoad spectrum of clinical manifestations. In a previous study, we showed. braziliensis strains, isolated from patients with distinct clinical manifes-rn of metalloprotease activities. Following these results, we investigatedse molecules and their relation to the major surface protease (gp63) ofes of metalloprotease expression among different clinical isolates as wellf long-term in vitro passage on the expression pattern of these metallo-

. Western blot analysis, using an anti-gp63 antibody, revealed polypeptideo that observed in zymographic analysis. Flow cytometry and fluorescenceted the presence of metalloproteases with homologous domains to gp63fferences in the expression level of such molecules among the isolates. Theproteases, assessed by confocal analysis, showed the existence of intra-homologous domains to gp63, predominantly located near the flagellarthat differential zymographic profiles of metalloproteases exhibited by L.naltered during prolonged in vitro culture, suggesting that the proteolyticotypic characteristic of these parasites.

© 2008 Elsevier B.V. All rights reserved.

amastigote form that lives and replicates in the phagolysosomalcompartment of mammalian macrophages, and the metacyclic pro-mastigote, an infective form that is inoculated into vertebrate hostsby the insect vector. Proteases produced by pathogenic microor-ganisms play central roles during the parasite–host interaction,including cytoadherence, tissue invasion, survival, proliferation anddifferentiation (Klemba and Goldberg, 2002). Leishmanolysin orgp63, the major metalloprotease of Leishmania (reviewed by Yaoet al., 2003), has been identified and implicated as a virulence fac-tor in several species of this genus (Chang and Chang, 1986; Russelland Wilhelm, 1986; Brittingham et al., 1995). This enzyme is themost abundant surface glycoprotein of promastigotes (Bouvier etal., 1985), but is also found in large lysosomes of amastigote forms(Medina-Acosta et al., 1989). Internal localization of gp63 has beendemonstrated in promastigotes of L. (L.) mexicana and L. (L.) chagasi(Weise et al., 2000; Yao et al., 2004, 2005).

In a recent work, our group showed by gelatin-SDS-PAGEanalyses that different clinical isolates of L. (V.) braziliensis

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presented cell-associated and released metallo-type protease activ-ities (Cuervo et al., 2006). In the present study, we have investigatedthe cellular localization of these metalloproteases in promastig-otes of L. (V.) braziliensis and whether these enzymes are relatedto the major surface protease of Leishmania spp., gp63. In addi-tion, we report here that L. braziliensis strains isolated from patientspresenting distinct clinical manifestations of tegumentary leishma-niasis exhibited different subpopulations of metalloproteases withhomologous domains to gp63, whose expression patterns remainunaltered during prolonged in vitro culture.

2. Materials and methods

2.1. Chemicals

All chemicals were purchased from Sigma Chemical Co. (USA).The inhibitor 1,10-phenanthroline was dissolved in ethanol to reacha concentration of 200 mM (stock solution) and maintained at−20 ◦C.

2.2. L. (V.) braziliensis strains and culture conditions

Five L. (V.) braziliensis strains were used: (i) two were isolatedfrom both cutaneous and mucosal lesions of a Colombian patient(International Center for Training and Medical Research, CIDEIM,Cali, Colombia, gently provided by Dr. Nancy Saravia) and (ii) threefrom cutaneous, disseminated, and mucosal lesions of differentpatients (University Hospital of Federal University of Bahia, Brazil,strains deposited in the Leishmania Type Culture Collection, Insti-tuto Oswaldo Cruz by Dr. Edgar Carvalho). Clinical presentation,geographical origin, and strain identity were previously reported(Cuervo et al., 2006). For zymographic assays, both recently thawedparasites (maintained for up 2 weeks in in vitro culture) and long-term parasites (maintained in in vitro culture during 1 year withweekly passages) were used. Promastigote forms of the parasitewere grown at 25 ◦C in Schneider’s medium supplemented with20% (v/v) heat-inactivated fetal bovine serum. All strains were har-vested at late log phase of growth by centrifugation at 3000 × g for5 min and washed twice in 0.01 M phosphate-buffered 0.9% NaCl(PBS), pH 7.2. Parasite density was estimated by counts in a hemo-cytometer, and the viability was assessed using the Trypan blue dyeexclusion test (Freshney, 1987).

2.3. Western blotting

Protein extracts of late log phase parasites were separated by10% SDS-PAGE and the polypeptides electrophoretically transferredat 4 ◦C at 100 V/300 mA for 1 h to a nitrocellulose membrane.The membrane was blocked in 5% (w/v) low-fat dried milk inTBS (150 mM NaCl; 10 mM Tris, pH 7.4) containing 0.5% Tween20 (TBS/Tween) for 1 h at room temperature. Then, membraneswere washed three times (10 min each) with the blocking solu-tion and incubated with rabbit anti-gp63 antibody raised againstL. (L.) mexicana (kindly provided by Dr. Peter Overath fromMax-Planck-Institut fur Biologie, Abteilung Membranbiochemie,Germany), at 1:2500 dilution for 1 h. The secondary antibodyused was peroxidase-conjugated goat anti-rabbit IgG at 1:2500.Immunoblots were exposed to X-ray film after reaction with ECLreagents for chemiluminescence (Brittingham et al., 1995).

2.4. Zymographic and inhibition assays

Protease activities present in late log phase parasites wereinvestigated as previously described (Cuervo et al., 2006). Briefly,parasites were disrupted in lysis buffer containing 10% glycerol,

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0.6% Triton X-100, 100 mM Tris–HCl at pH 6.8, and 150 mM NaCl.The resulting extracts from 1 × 107 cells were fractioned on SDS-PAGE (10%) copolymerized with 0.1% fish gelatin. The gels wereloaded with 30 mg of protein per slot. After electrophoresis, theresulting gels were washed twice for 30 min at 4 ◦C in 0.1 M sodiumacetate buffer at pH 5.5, containing 2.5% Triton X-100. Proteaseactivity was detected by incubating the gels in the reaction buffercontaining 0.1 M sodium acetate, pH 5.5, at 37 ◦C for 4 and 24 h.Bands of gelatin degradation were visualized by staining the gelswith 0.2% (w/v) CBB R-250, 40% (v/v) methanol, 10% (v/v) aceticacid, and destaining them with 10% (v/v) acetic acid. For inhibitionassays, the lysates of promastigotes were prepared as previouslydescribed and the metalloprotease inhibitor 1,10-phenanthroline(10 mM) was added to the reaction at 4 and 24 h. Samples werethen resolved as mentioned above.

2.5. Confocal fluorescence microscopy and flow cytometry for cellsurface gp63

Late log phase parasites (5 × 106 cells) used for these experi-ments were fixed at 4 ◦C with 4% paraformaldehyde in PBS (pH7.2) for 5 min, followed by extensive washing in the same buffer.Fixed cells maintained their morphological integrity, as verified bymicroscopic observation. To assess gp63 localization in the cells byconfocal microscopy, parasites were permeabilized with acetonefor 15 min at −20 ◦C. They were further incubated for 1 h with a1:500 dilution of anti-gp63 antibody and then incubated for anadditional hour with a 1:250 dilution of fluorescein isothiocyanate(FITC)-labeled goat anti-rabbit immunoglobulin G (IgG). These cellswere washed three times in PBS and observed with a Zeiss LSM510 Meta Confocal microscope. The same protocol was employedfor flow cytometry, with the exception that cells were not perme-abilized. Control images for flow cytometry were taken in a Zeissepifluorescence microscope (Axioplan). For flow cytometric analy-sis, cells were examined in an EPICS ELITE flow cytometer (CoulterElectronics, Hialeah, FL) equipped with a 15 mW argon laser emit-ting at 488 nm. Untreated cells and those treated only with thesecondary antibody were used as controls. Each experimental pop-ulation was then mapped using a two-parameter histogram offorward-angle light scatter versus side scatter. The mapped pop-ulation (n = 10,000) was then analyzed for log green fluorescenceusing a single-parameter histogram (Brittingham et al., 1995).

3. Results and discussion

The proteolytic activities of both whole-promastigote extractsand extracellular secretions of five strains of L. (V.) brazilien-sis isolated from patients with distinct clinical manifestations ofAmerican Tegumentary Leishmaniasis were recently characterized(Cuervo et al., 2006). Zymographic assays demonstrated that thesestrains exhibit distinct profiles of enzymatic activities ranging from50 to 125 kDa. Biochemical characterization showed that theseenzymes belong to the metalloprotease class, presenting optimalactivity in the pH range between 5.5 and 9.0 for all the analyzedstrains. In the present work, we continue such studies investigat-ing the cell localization, identity, and the effect of continued invitro passage on the expression pattern of metalloproteases in L.(V.) braziliensis strains.

To determine whether these enzymes are related to the gp63metalloprotease, late log phase whole-promastigote extracts wereprobed using a polyclonal antibody raised against gp63 of L. (L.)mexicana. This antibody strongly recognized polypeptide bandsbetween 35 kDa and 120 kDa (Fig. 1). The lysates of the Colombianstrains, 1407 cutaneous (1407 C) and 1407 mucosal (1407 M)

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Fig. 1. Western blot showing polypeptides with homologous domains to gp63detected in the cellular extract of late log phase promastigotes of Leishmania (V.)braziliensis isolated from patients with distinct clinical manifestations of tegumen-tary leishmaniasis. The numbers on the right indicate the apparent molecular massof the reactive polypeptides.

Fig. 2. Comparative zimographic analysis of proteases profile from late log phase whole-continuous in vitro culture, and (B) parasites maintained few weeks in in vitro culture. Pro24 h of reaction in the presence of 10 mM 1,10-phenanthroline. The numbers on the right

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isolated from the same patient, presented a similar reactivity pro-file, with two bands of apparent molecular mass of 64 and 82 kDa(Fig. 1). The Brazilian strain, IOC-L 2463 isolated from a patient with

mucosal leishmaniasis, displayed reactivity with four polypeptidebands of approximately 35, 64, 82, and 120 kDa. The strains isolatedfrom disseminated (IOC-L 2483) and cutaneous (IOC-L 2481) casesfrom the same locality in Brazil exhibited identical reactivityprofile to the aforementioned IOC-L 2463, with an extra bandof 100 kDa (Fig. 1). Band profiles revealed by the anti-gp63 anti-body are similar to those previously obtained using zymographicanalysis (Cuervo et al., 2006). The close similarity between theband pattern detected in the western blot and the metalloproteaseactivity profile observed in the zymograms strongly indicates thatL. (V.) braziliensis strains express several metalloproteases withhomologue domains to gp63. However, in that previous work noproteolytic activity band with apparent molecular mass of 35 kDawas observed in the zymogram. The polypeptide band of 35 kDadetected in the western blot analysis could be related to: (i) anadditional intracellular metalloprotease synthesized as inactiveprecursor; (ii) a same enzyme in different state of post-translationalmodification; (iii) different isoforms of metalloproteases codedby different genes; (iv) an inactive proteolytic fragment or similardegradation product. In fact, such possibilities should be consideredsince it has been reported that gp63 is synthesized as an inactive

promastigote extracts of L. (V.) braziliensis strains (A) maintained during 1 year inteolytic profiles were observed after (a) 4 h of reaction, (b) 24 h of reaction, and (c)indicate the apparent molecular mass of the proteolysis bands.

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precursor and undergoes several post-translational modificationsbefore to be an active enzyme (Kink and Chang, 1988; Olafson etal., 1990; Macdonald et al., 1995; McGwire and Chang, 1996; Yaoet al., 2003). On other hand, the high polymorphism observed ingene sequences coding for gp63 in Leishmania spp. (Medina-Acostaet al., 1993; Yao et al., 2004) could result in different isoformsof this protease. Actually, it has been reported that species of L.(Viannia) subgenus present a larger number of gp63 coding geneswhen compared to species of L. (Leishmania) subgenus (Steinkrauset al., 1993; Voth et al., 1998; Victoir et al., 1998, 2005). In L. (V.)guyanensis, 50 gp63 coding genes were described (Steinkraus etal., 1993). In addition, recently was reported that L. (V.) braziliensispresent a minimum of 37 gp63 genes distributed in 8 isogenes withhigh polymorphism among them (Victoir et al., 2005). Such studyalso showed that the gp63 regions coding for domains involved inadhesion and internalization of the parasites to the macrophages(Puentes et al., 1999) exhibit the highest heterogeneity among theL. (V.) braziliensis gp63 coding sequences (Victoir et al., 2005). Onother hand, according to data available in the GeneDB databases ofthe Wellcome Trust Sanger Centre (http://www.sanger.ac.uk/), theL. (V.) braziliensis genome present 33 entries for gp63 sequences(http://www.genedb.org/genedb/lbraziliensis) whereas L. (L.)major present only 5 entries (http://www.genedb.org/genedb/leish/).

Furthermore, we tested the hypothesis that proteolytic profilesof the late log phase promastigotes of L. (V.) braziliensis strainsmight have changed during prolonged in vitro culture. Comparativezymographic assays were carried out using extracts from para-sites maintained few weeks in in vitro culture (time zero samples),and extracts from long-term cultivated parasites (samples after12 months of continuous in vitro passages). Proteolytic activitiesperformed at 4 and 24 h of reaction (Fig. 2a and b, respectively)showed that the enzymatic profile of parasites maintained by 12months in continuous in vitro culture (Fig. 2A) was identical to theprofile displayed by time zero samples (Fig. 2B). Additionally, dur-ing 12 months, samples of the parasites were checked monthlyfor their proteolytic activities and identical profile was observed.The inhibition assays using 1,10-phenanthroline, a potent inhibitorof metalloproteases, abrogated enzymatic activities in all strains(Fig. 2c), corroborating our previous observations (Cuervo et al.,2006). Together, these data suggest that proteolytic activity profileis a stable phenotypic characteristic of L. (V.) braziliensis strains.Finally, since (i) zymographic assay allows detecting variabilityamong strains, and (ii) the proteolytic profiles of different isolates

do not suffer modifications during prolonged in vitro culture, zymo-graphic analysis could be used as an additional tool for Leishmaniacharacterization, as suggested for other trypanosomatids (Santoset al., 2005).

Several groups have used fluorescence microscopy and flowcytometry analyses to locate gp63-like molecules in trypanoso-matids (Elias et al., 2006; Nogueira de Melo et al., 2006). Usingflow cytometry, we provided measurements for the relative levelsof metalloproteases with homologous domains to gp63 expressedby late log phase promastigotes of the five strains of L. (V.) brazilien-sis (Fig. 3A). These data showed that Brazilian L. (V.) braziliensisstrains IOC-L 2463, IOC-L 2483, and IOC-L 2481 presented, amongthem, different levels of proteins with homologous domains togp63, while Colombian strains 1407 C and 1407 M have similarlevels to IOC-L 2463. Fluorescence microscopy analysis corrobo-rated these results: proteases with homologous domains to gp63were recognized in L. (V.) braziliensis strains IOC-L 2463, IOC-L2483 and IOC-L 2481 at different levels of intensity (Fig. 3B). Theseresults suggest that distinct L. (V.) braziliensis strains display differ-ent affinities for the anti-gp63 antibody, and indicate that proteinswith homologous domains to gp63 could not be equally expressed

Fig. 3. Differential expression of metalloproteases with homologous domains togp63, in late log phase promastigotes of L. (V.) braziliensis strains. (A) Flow cytometryanalysis showing the anti-gp63 antibody binding to the parasites; (B) differen-

tial interferential contrast microscopy (a–c) and the corresponding fluorescencemicroscopy (a′–c′) of IOC-L 2483 (a and a′), IOC-L 2481 (b and b′) and IOC-L 2463 (cand c′) strains sequentially incubated with anti-gp63 and FITC-secondary antibodies.

by the parasites. Although the three Brazilian strains belong tothe same zymodeme (Z-27), and are from the same geographicalregion, the results here indicate that these strains exhibit consider-able heterogeneity in the expression of molecules with homologousdomains to gp63. Whether such heterogeneity is related to the inva-sive and immunomodulatory roles attributed to metalloproteases(Brittingham et al., 1999; McGwire et al., 2003), and whether itcould be reflected in the clinical outcome of tegumentary leishma-niasis by L. (V.) braziliensis remain to be elucidated.

Finally, to verify the cellular distribution of metalloproteaseswith homologous domains to gp63, permeabilized promastigotes oflate log phase were probed with anti-gp63 antibody and observedby confocal fluorescence microscopy. It was possible to detectimmunolabeling in different regions of the promastigote body,specifically located at the posterior (Fig. 4a1 and a2) and anteriorregions (Fig. 4b). The strongest labeling was predominantly located

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63 inas seeling l

he flag

Fig. 4. Cellular localization of metalloproteases with homologous domains to gpincubated with anti-gp63 and FITC-secondary antibodies. The cells are presentedmicroscopy (a2), and as an overlay of both images (a1); arrows show an intense labeanterior localization of metalloproteases with homologous domains to gp63, near t

in a concentrated area of the anterior region of promastigotes, nearthe flagellar pocket (Fig. 4b). This observation indicates the exis-tence of subcellular metalloproteases with homologous domainsto gp63, probably present in vesicular structures of exocytic path-ways. Accordingly, it has been shown that gp63 of L. (L.) mexicanais located intracellularly, mainly in the flagellar pocket, but also in

the endoplasmic reticulum and Golgi complex as well as in vesiclesinvolved in endo- and exocytosis (Weise et al., 2000). It was alsodemonstrated that promastigotes of L. (L.) chagasi contain internalsubpopulations of gp63, of which a fraction remains internally sta-ble without a detectable decrease in abundance several days afterbiosynthesis, and another fraction that is released into extracellu-lar medium (Yao et al., 2005). Moreover, it has been shown thatgp63 is released into extracellular medium in both membrane-associated and -free forms in several Leishmania species (McGwireet al., 2002; Yao et al., 2002). In this protozoan, different classesof internally located proteases, such as cysteine and serine pro-teases, were also found to be released into extracellular medium(Silva-Lopez et al., 2004; Alves et al., 2005). In conclusion, ourfindings show that late log phase L. (V.) braziliensis promastigotespresent internal metalloproteases with homologous domains togp63, which could be secreted into extracellular medium. However,future studies are necessary to (i) determine the precise cellular fateof these internal metalloproteases and (ii) to understand the role ofL. (V.) braziliensis metalloproteases in the different clinical manifes-tations of American tegumentary leishmaniasis and the associatedmolecular mechanisms.

permeabilized promastigotes of L. (V.) braziliensis IOC-L 2483 strain sequentiallyn under differential interferential contrast microscopy (a3), immunofluorescenceocated in the posterior region of the promastigotes. Confocal microscopy (b) showsellar pocket (arrow).

Acknowledgements

We are grateful to Bruno Eschenazi (Laboratorio de Producao eTratamento de Imagem-FIOCRUZ) for their precious technical assis-tance. This work has been supported by the following Brazilianagencies: FIOCRUZ/CNPq-PAPES IV (J.B.J. grant #400218/2006-2),

FIOCRUZ-IOC, FAPERJ (E.C.), CNPq, and CAPES. P.C. is a CNPq-FIOCRUZ (P.V. Program) fellowship recipient.

References

Alves, C.R., Corte-Real, S., Bourguignon, S.C., Chaves, C.S., Saraiva, E.M., 2005. Leish-mania amazonensis: early proteinase activities during promastigote-amastigotedifferentiation in vitro. Exp. Parasitol. 109, 38–48.

Bouvier, J., Etges, R.J., Bordier, C., 1985. Identification and purification of membraneand soluble forms of the major surface protein of Leishmania promastigotes. J.Biol. Chem. 260, 15504–15509.

Brittingham, A., Morrison, C.J., McMaster, W.R., McGwire, B.S., Chang, K.P., Mosser,D.M., 1995. Role of the Leishmania surface protease gp63 in complement fixationcell adhesion and resistance to complement-mediated lysis. J. Immunol. 155,3102–3111.

Brittingham, A., Chen, G., McGwire, B.S., Chang, K.P., Mosser, D.M., 1999. Interactionof Leishmania gp63 with cellular receptors for fibronectin. Infect. Immun. 67,4477–4484.

Chang, C.S., Chang, K.P., 1986. Monoclonal antibody affinity purification of a Leish-mania membrane glycoprotein and its inhibition of leishmania-macrophagebinding. Proc. Natl. Acad. Sci. U. S. A. 83, 100–104.

Cuervo, P., Saboia-Vahia, L., Costa, Silva-Filho, F., Fernandes, O., Cupolillo, E., JesusDE, J.B., 2006. A zymographic study of metalloprotease activities in extracts andextracellular secretions of Leishmania (Viannia) braziliensis strains. Parasitology132, 177–185.

Page 6

ropica

148 P. Cuervo et al. / Acta T

Elias, C.G., Pereira, F.M., Silva, B.A., Alviano, C.S., Soares, R.M., Santos, A.L., 2006.Leishmanolysin (gp63 metallopeptidase)-like activity extracellularly releasedby Herpetomonas samuelpessoai. Parasitology 132, 37–47.

Freshney, R., 1987. Culture of Animal Cells. A Manual of Basic Technique, second ed.Wiley-Liss, New York, p. 397.

Grimaldi Jr., G., Tesh, R.B., McMahon-Pratt, D., 1989. A review of the geographicdistribution and epidemiology of leishmaniasis in the New World. Am. J. Trop.Med. Hyg. 41, 687–725.

Kink, J.A., Chang, K.P., 1988. N-glycosylation as a biochemical basis for virulence inLeishmania mexicana amazonensis. Mol. Biochem. Parasitol. 27, 181–190.

Klemba, M., Goldberg, D.E., 2002. Biological roles of proteases in parasitic protozoa.Annu. Rev. Biochem. 71, 275–305.

Macdonald, M.H., Morrison, C.J., McMaster, W.R., 1995. Analysis of the active siteand activation mechanism of the Leishmania surface metalloproteinase GP63.Biochim. Biophys. Acta 1253, 199–207.

Marsden, P.D., 1986. Mucosal leishmaniasis (“espundia” Escomel 1911). Trans. R. Soc.Trop. Med. Hyg. 80, 859–876.

McGwire, B.S., Chang, K.P., 1996. Posttranslational regulation of a Leishmania HEXXHmetalloprotease (gp63). The effects of site-specific mutagenesis of catalytic zincbinding N-glycosylation and glycosyl phosphatidylinositol addition sites on N-terminal end cleavage intracellular stability and extracellular exit. J. Biol. Chem.271, 7903–7909.

McGwire, B.S., O’Connell, W.A., Chang, K.P., Engman, D.M., 2002. Extracellular releaseof the glycosylphosphatidylinositol (GPI)-linked Leishmania surface metallopro-tease gp63 is independent of GPI phospholipolysis: implications for parasitevirulence. J. Biol. Chem. 277, 8802–8809.

McGwire, B.S., Chang, K.P., Engman, D.M., 2003. Migration through the extracellularmatrix by the parasitic protozoan Leishmania is enhanced by surface metallo-protease gp63. Infect. Immun. 71, 1008–1010.

Medina-Acosta, E., Karess, R.E., Russell, D.G., 1993. Structurally distinct genes for thesurface protease of Leishmania mexicana are developmentally regulated. Mol.Biochem. Parasitol. 57, 31–45.

Medina-Acosta, E., Karess, R.E., Schwartz, H., Russell, D.G., 1989. The promastigotesurface protease (gp63) of Leishmania is expressed but differentially processedand localized in the amastigote stage. Mol. Biochem. Parasitol. 37,263–273.

Nogueira de Melo, A.C., d’Avila-Levy, C.M., Dias, F.A., Armada, J.L., Silva, H.D., Lopes,A.H., Santos, A.L., Branquinha, M.H., Vermelho, A.B., 2006. Peptidases and gp63-like proteins in Herpetomonas megaseliae: possible involvement in the adhesionto the invertebrate host. Int. J. Parasitol. 36, 415–422.

Olafson, R.W., Thomas, J.R., Ferguson, M.A., Dwek, R.A., Chaudhuri, M., Chang, K.P.,Rademacher, T.W., 1990. Structures of the N-linked oligosaccharides of Gp63 themajor surface glycoprotein from Leishmania mexicana amazonensis. J. Biol. Chem.265, 12240–12247.

106 (2008) 143–148

Puentes, F., Guzman, F., Marın, V., Alonso, C., Patarroyo, M.E., Moreno, A., 1999. Leish-mania: fine mapping of the Leishmanolysin molecule’s conserved core domainsinvolved in binding and internalization. Exp. Parasitol. 93, 7–22.

Russell, D.G., Wilhelm, H., 1986. The involvement of the major surface glycoprotein(gp63) of Leishmania promastigotes in attachment to macrophages. J. Immunol.136, 2613–2620.

Santos, A.L., Abreu, C.M., Alviano, C.S., Soares, R.M., 2005. Use of proteolytic enzymesas an additional tool for trypanosomatid identification. Parasitology 130,79–88.

Silva-Lopez, R.E., Morgado-Diaz, J.A., Alves, C.R., Corte-Real, S., Giovanni-De-Simone,S., 2004. Subcellular localization of an extracellular serine protease in Leishmania(Leishmania) amazonensis. Parasitol. Res. 93, 328–331.

Steinkraus, H.B., Greer, J.M., Stephenson, D.C., Langer, P.J., 1993. Sequence hetero-geneity and polymorphic gene arrangements of the Leishmania guyanensis gp63genes. Mol. Biochem. Parasitol. 62, 173–185.

Victoir, K., Banuls, A.L., Arevalo, J., Llanos-Cuentas, A., Hamers, R., Noel, S., De Don-cker, S., Le Ray, D., Tibayrenc, M., Dujardin, J.C., 1998. The gp63 gene locus, atarget for genetic characterization of Leishmania belonging to subgenus Viannia.Parasitology 117, 1–13.

Victoir, K., Arevalo, J., De Doncker, S., Barker, D.C., Laurent, T., Godfroid, E., Bollen,A., Le Ray, D., Dujardin, J.C., 2005. Complexity of the major surface protease(msp) gene organization in Leishmania (Viannia) braziliensis: evolutionary andfunctional implications. Parasitology 131, 207–214.

Voth, B.R., Kelly, B.L., Joshi, P.B., Ivens, A.C., McMaster, W.R., 1998. Differentiallyexpressed Leishmania major gp63 genes encode cell surface leishmanolysin withdistinct signals for glycosylphosphatidylinositol attachment. Mol. Biochem. Par-asitol. 93, 31–41.

Weise, F., Stierhof, Y.D., Kuhn, C., Wiese, M., Overath, P., 2000. Distribution of GPI-anchored proteins in the protozoan parasite Leishmania based on an improvedultrastructural description using high-pressure frozen cells. J. Cell. Sci. 113,2603–4587.

Yao, C., Leidal, K.G., Brittingham, A., Tarr, D.E., Donelson, J.E., Wilson, M.E., 2002.Biosynthesis of the major surface protease GP63 of Leishmania chagasi. Mol.Biochem. Parasitol. 121, 119–128.

Yao, C., Donelson, J.E., Wilson, M.E., 2003. The major surface protease (MSP orGP63) of Leishmania sp. Biosynthesis regulation of expression and function. Mol.Biochem. Parasitol. 132, 1–16.

Yao, C., Luo, J., Storlie, P., Donelson, J.E., Wilson, M.E., 2004. Multiple products ofthe Leishmania chagasi major surface protease (MSP or GP63) gene family. Mol.Biochem. Parasitol. 135, 171–183.

Yao, C., Luo, J., Hsiao, C., Donelson, J.E., Wilson, M.E., 2005. Internal and surfacesubpopulations of the major surface protease (MSP) of Leishmania chagasi. Mol.Biochem. Parasitol. 139, 173–183.