Trypanosoma cruzi: cruzipain and membrane-bound cysteineproteinase isoform(s) interacts with human a2-macroglobulin

and pregnancy zone protein

Adri�aan M. Ramos,a,* Vilma G. Duschak,b Nelia M. Gerez de Burgos,c Mariana Barboza,b

Mar�ııa S. Remedi,c Miguel A. Vides,a and Gustavo A. Chiabrandoa

a Departamento de Bioqu�ıımica Cl�ıınica, Facultad de Ciencias Qu�ıımicas, Universidad Nacional de C�oordoba, (5000) C�oordoba, Argentinab Instituto de Investigaciones Biotecnol�oogicas, Universidad Nacional de General San Mart�ıın (IIB, UNSAM),

CC30 (1650) San Martin, Prov. Buenos Aires, Argentinac C�aatedra de Qu�ıımica Biol�oogica, Facultad de Ciencias M�eedicas, Universidad Nacional de C�oordoba, (5000) C�oordoba, Argentina

Received 20 November 2001; accepted 15 February 2002

Abstract

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as a2-macro-

globulin (a2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of a2-M and PZP with cruzipain, the

major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both a-M inhibitors under reaction with

cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native a2-M and PZP, respectively; (ii) limited proteolysis of bait region in a2-M and PZP, and (iii) covalent binding of cruzipain to PZP and

a2-M. Conformational and structural changes experimented by a-Ms correlate with modifications of the enzyme electrophoretic

mobility and activity. Cruzipain–a-M complexes were also detected by gelatin SDS–PAGE and immunoblotting using polyclonal

anti-cruzipain antibodies. Concomitantly, a2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA sub-

strate. In addition, a-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with

cruzipain. The present study suggests that both human a-macroglobulin inhibitors could prevent or minimize harmful action of

cruzipain on host’s molecules and hypothetically regulate parasite functions controlled by cruzipain.

Index Descriptors and Abbreviations: Human a-macroglobulins (a-Ms); a2-Macroglobulin (a2-M); pregnancy zone protein (PZP);

Chagas’ disease; T. cruzi; cysteine proteinase (CP); cruzipain; Hydrophobic membrane proteins (HMP); Isoforms of cysteine

proteinases; Polyacrylamide gel electrophoresis (PAGE); NADP-linked glutamate dehydrogenase (NADP-GDH); Phenylme-

thanesulphonyl fluoride (PMSF); N-Benzoyl-Pro-Phe-Arg-p-nitroanilide hydrochloride (Bz-Pro-Phe-Arg-pNA); Low-density lipo-

protein receptor-related protein (LRP). � 2002 Elsevier Science (USA). All rights reserved.

1. Introduction

Early studies of Trypanosoma cruzi–host cell interac-tion were focused on the role played by parasite pro-teinases. Moreover, the use of synthetic inhibitors ofproteinases and genetic manipulations demonstrated theneed for cruzipain, the main cysteine proteinase presentin all life-cycle stages of T. cruzi, for parasite viability,

differentiation and cell invasion (Bonaldo et al., 1991;Engel et al., 1998; Meirelles et al., 1992). This cathepsinL-like proteinase is encoded by a large family of poly-morphic genes arranged in tandem arrays, and as manyas 130 copies may be present in some strains of T. cruzi(Campetella et al., 1992). Such polymorphism of genesencoding cruzipain is responsible for the synthesis ofcruzipain isoenzymes with probably different cellularlocalization and functions. In fact, cruzipain was pri-marily associated with lysosomal vesicles (Bontempiet al., 1989), although there is growing evidence forthe occurrence of cruzipain (Souto-Padron et al., 1990),

Experimental Parasitology 100 (2002) 121–130

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*Corresponding author. Fax: +54-351-4334187.

E-mail address: amramos@bioclin.fcq.unc.edu.ar (A.M. Ramos).

0014-4894/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.

PII: S0014 -4894 (02 )00007-3

or cruzipain-like isoforms of immunologically cross-re-active CPs, and with an apparent molecular mass similarto that of cruzipain, bound to parasitic surface (Parussiniet al., 1998). More recently, the participation of severalsecreted cruzipain isoforms in the invasion of mamma-lian cells, linked to a novel mechanism involving its ki-nins releasing activity, has been firmly established(Scharfstein, 2000).

a2-Macroglobulin (a2-M) and pregnancy zone pro-tein (PZP) are proteinase-inhibiting glycoproteins ofhuman plasma. a2-M remains at approximately constantlevels (2–4 mg/ml) throughout life, while PZP is normallypresent in plasma of males and non-pregnant females atlow concentrations ð<10 lg=mlÞ, and rises significantlyin pregnant women and in some inflammatory states (upto 300 lg/ml) (Munk Petersen, 1993). These a-Ms canbind covalently to proteinases without affecting theiractivity (Barret and Starkey, 1973). This mechanism istriggered when the proteinase cleaves a peptide bondlocalized within the bait region placed in each subunit ofthe inhibitor. This event leads to a conformationalchange in the a-Ms, resulting in the entrapment of theproteinase (via e-lysyl-c-glutamyl bonds), and exposureof receptor recognition sites for the a2-M receptor,known as low-density lipoprotein receptor-related pro-tein (LRP). This endocytic receptor is mainly expressedin hepatocytes and macrophages, and participates in therapid clearance of a-Ms–proteinases complexes fromcirculation and tissues (Gliemann et al., 1986; Herz et al.,1988; Moestrup and Gliemann, 1989).

In humans, numerous in vitro studies have shownthat a2-M functions by binding to a broad spectrum ofproteinases of human and microbial origin, and so, aputative role as a back-up proteinase inhibitor is widelyaccepted. Comparatively to a2-M, few investigationsdealing with the proteinase inhibition by PZP have beenperformed, so its role as back-up or more specific in-hibitor remains to be elucidated (Chu and Pizzo, 1994;Munk Petersen, 1993). Studies about the role of a2-M inT. cruzi infection have been carried out (Ara�uujo-Jorgeet al., 1994), and the involvement of a2-M as an inhib-itor of parasite proteinases and host cell invasion byT. cruzi has been earlier suggested (Araujo-Jorge et al.,1986). In addition, our previous studies demonstratedthat a2-M is really able to inhibit the total activity ofproteinases of T. cruzi, suggesting that cruzipain couldbe one of the enzymes involved in such interactions(Ramos et al., 1997). The possible interaction of PZPwith T. cruzi proteinases has not been studied so far.

In this work our current goal was to investigate ifPZP, like a2-M, could also interact with T. cruzi pro-teinases, as well as to study the ability of isolated a2-Mand PZP to complex cruzipain and cruzipain-like iso-forms of CPs. The possible biological implications ofsuch interactions in the parasite–host relationship arealso discussed.

2. Materials and methods

Proteins. Human a2-M was isolated from pooledhealthy males’ plasmas according to Imber and Pizzo(1981), and human PZP was purified from pooled latepregnancy plasma as described earlier (Chiabrandoet al., 1997). The concentration of a-Ms was assessed byoptical density at 280 nm using an extinction coefficientof 8.9 and 8.2 for a2-M and PZP, respectively (Sandet al., 1985). Cruzipain was purified from parasite ex-tracts by affinity chromatography on concanavalinA–Sepharose, as previously reported (Labriola et al.,1993). Protein concentration was estimated by themethod of Bradford (1976) using bovine serum albuminas standard. The molecular weight of cruzipain wasconsidered to be 40 kDa, as previously deduced fromsequence studies, and considering two high-mannoseoligosaccharide chains (Mart�ıınez and Cazzulo, 1992).

Parasites, cytosolic cell-free extracts, and parasite-membrane proteins preparations. T. cruzi epimastigotes(Tulahuen strain, Tul 0 stock) were grown and harvestedas previously reported (Cazzulo et al., 1985). Epimasti-gote homogenates were obtained by disrupting theparasites through several cycles of freezing and thawing,followed by centrifugation (Ramos et al., 1997). Mem-brane proteins were extracted with 2% Triton X-114from parasite pellets obtained after freezing and thaw-ing, and next partitioned in either aqueous or detergentphases as mentioned (Parussini et al., 1998). HMPcontaining isoforms of CPs were obtained by precipi-tation from the detergent phase with methanol–chloro-form–water mixture (Wessel and Flugge, 1984). Fromthis pool of HMP, isoforms of CPs were also purified onconcanavalin A–Sepharose (Parussini et al., 1998).

Human patient plasma and detection of PZP. Venousblood samples were obtained from 30 patients (aged 6months to 12 years) with acute T. cruzi infection wholived in an endemic area of Argentina. All patientsshowed inoculation chagoma and detectable parasitemiaby classical test (microhematocrit, Strout test or xen-odiagnosis). Samples were collected before the begin-ning of parasiticidal drug treatment, and the plasmaseparated in the presence of PMSF (1 mM, final con-centration). Control sera ðn ¼ 6Þ were obtained fromseronegative and healthy individuals from the samegeographic area. A sandwich ELISA previously devel-oped in our laboratory was used to determine PZP, asdescribed in detail elsewhere (Zalazar et al., 1992). Sta-tistical significance was evaluated by the Mann–Whit-ney–Wilcoxon non-parametric test.

Electrophoretic and immunoblotting assays. Tetra-merization of PZP (10.0 lg) under reaction with T. cruziproteinases contained in cell-free extracts (5.7–69.0 lg)was evaluated in samples diluted in 0.1 M sodiumphosphate buffer, pH 8.0, and incubated at 37 �C atdifferent time intervals (30 min to 24 h). Mixtures of

122 A.M. Ramos et al. / Experimental Parasitology 100 (2002) 121–130

a2-M (14 pmol) or PZP (28 pmol) and different amountsof cruzipain (7–112 pmol) diluted in Tris saline buffer,pH 7.4, solution (TBS) were incubated at different timeintervals up to 24 h, at 37 �C. The reaction productswere analyzed by PAGE under different conditions andimmunoblotting assays. Non-denaturing PAGE fora2-M and a2-M–cruzipain complexes was performedaccording to Van Leuven et al. (1981), and for PZP andPZP–cruzipain complexes according to Davis (1964).Denaturing or reduced SDS–PAGE was carried out asdescribed by Laemmli (1970). Samples were diluted inbuffer containing 2% SDS, either with or without dithio-threitol and incubated for 60 min at 37 �C before elec-trophoresis. Proteins were visualized by Coomassiebrilliant blue R-250 or with silver staining methods(Bloom et al., 1987).

For immunoblotting, proteins were separated by de-naturing SDS–PAGE, electroblotted to nitrocellulosemembrane and blocked with 5% non-fat dried milk.Then, the membranes were incubated with polyclonalrabbit anti-cruzipain serum (Campetella et al., 1990) orpolyclonal rabbit anti-a2-M or PZP antibody (DAKO,Denmark) and finally with HRP-conjugated goatanti-rabbit IgG antibody (Sigma Chemicals, USA).Immunoblotting assays were revealed using 4-chloro-1-naphthol/H2O2, with the exception of assays of HMP(24–36 lg) or purified isoform(s) of CPs (2 lg) witha-Ms (10 lg), which were revealed by chemolumini-scence using ECL reagent (NEN, Boston, MA, USA).

In gel enzymatic activity assay. The inhibition ofproteinases by PZP was carried out in samples con-taining T. cruzi extracts (6.2 lg) and PZP (15.0 lg) in-cubated for 2–24 h at 37 �C, and in aliquots of cruzipain(12.5 pmol) with increasing amounts of PZP (6.2–50.0pmol) incubated for 120 min at 37 �C. Mixtures werediluted in sample buffer containing 2% SDS, incubatedfor 60 min at 37 �C and then subjected to gelatin SDS–PAGE as previously described (Campetella et al., 1990).

Assays of enzyme inhibition. To analyze the enzymeactivity on peptidic substrate, assays of enzyme inhib-ition were developed. Forty-four picomoles of cruzipainwas incubated with increasing amounts of a2-M or PZP(11–88 pmoles) at 37 �C, from 5 to 60 min. The enzymeactivity of cruzipain in the presence or absence of a-Msinhibitors was assayed with the synthetic chromogenicsubstrate Bz-Pro-Phe-Arg-pNA (Sigma Chemicals,USA) at pH 7.4, and the reaction was followed spec-trophotometrically at 410 nm (Cazzulo et al., 1990).

3. Results

Plasmatic PZP levels in acute chagasic children. Inseveral species, some members of the a-macroglobulinfamily behave as acute-phase response protein. Inhumans, PZP exhibits characteristics similar to acute-

phase proteins, while plasma concentration of a2-M isnot modified during inflammation (Zalazar et al., 1992;Zarzur, 1986). Our determinations of PZP levels inchildren with acute Chagas’ disease showed that themean concentration of PZP was 2.6-fold higher(18:2� 2:2 lg, P ¼ 0:0281) than that of healthy indi-viduals (6:6� 1:1 lg) from the same endemic area (Fig.1). In addition, in the same figure, it can be observedthat 66% of infected children had PZP levels higher thanthe upper limit of the reference interval (10 lg/ml)(Zalazar et al., 1992), displaying in some cases increasedlevels approximately 10-fold higher than the mean of thecontrol group.

Interaction analysis of PZP with T. cruzi proteinases.The results showing plasmatic PZP increases in the acutephase of Chagas’ disease encouraged us to investigate ifPZP, like a2-M (Ramos et al., 1997), is also able to in-teract with T. cruzi proteinases. In this sense, the non-denaturing PAGE assays showed that native dimericPZP (360 kDa) converts to tetramers (720 kDa) bytreatment with T. cruzi extract (Fig. 2A). Since thistetramerization is a common behavior of PZP uponproteinase binding (Sand et al., 1985), we assumed thatT. cruzi extracts contained proteolytic enzymes able tobe bound by PZP. Furthermore, when the proteinaseprofile of T. cruzi extracts was analyzed on gelatin SDS–PAGE, a modification was observed in the presence ofPZP, since the activity-band of 44 kDa that was previ-ously identified as cruzipain (Campetella et al., 1990)disappeared (Fig. 2B), suggesting that PZP could inter-act with the main CP of T. cruzi. This decreased pro-teolytic activity reflects lower amounts of free enzyme(s),which could be trapped inside high molecular weightPZP–proteinase complexes.

Fig. 1. Levels of PZP in healthy and acute T. cruzi-infected children.

The mean concentration of PZP levels in the infected population (I)

was compared with that of a healthy control group (HC). Horizontal

bars represent the mean of each group, and the significance (P) is in-

dicated. PZP was assayed by ELISA as described in Section 2.

A.M. Ramos et al. / Experimental Parasitology 100 (2002) 121–130 123

Interaction analysis of PZP and a2-M with cruzipain.Previous work carried out in our laboratory, as well asthe present results, strongly suggests that a2-M and PZPcould interact with cruzipain. In order to investigatethese possible interactions, we first analyzed by PAGEthe existence of conformational and structural changes inthe a-Ms in the presence of cruzipain. It is well knownthat a2-M–proteinase complexes form molecular struc-tures more compact (fast form) than native a2-M (slowform), which can be visualized using native PAGE assays(Barret et al., 1979). However, when a2-M was incubatedwith cruzipain (molar ratios from 1:0.5 up to 1:8) atdifferent times, a progressive but only partial mobilitychange was observed after 24 h of incubation, in com-

parison to both untreated a2-M and a2-M–chymotry-psin complexes used as control (Fig. 3A). In addition,when PZP was incubated with cruzipain in similar molarratios, a tetrameric form of PZP was detected in samplesincubated for only 1 h at 37 �C (Fig. 3B).

Since it is well known that the first and essential stepof proteinase inhibition by a-Ms is a proteolytic attackat the bait region (Barret and Starkey, 1973; MunkPetersen, 1993), we investigated by reduced SDS–PAGEif such proteolytic cleavage occurred in cruzipain–in-hibitor mixtures showing a partial mobility change ofa2-M and tetramerization of PZP. The occurrence ofcharacteristic fragments of approximately 90 kDa, asign indicative of bait region cleavage, and a concomi-tant reduction in the monomeric subunit of 180 kDawere observed in mixtures containing a2-M and cruzi-pain incubated for 30 min at molar ratios of 1:2 and 1:4(Fig. 4A). On the other hand, similar proteolytic frag-ments of approximately 90 kDa were also detected whenPZP was incubated with cruzipain for 5 min in molarratios of 1:2 and 1:4 (Fig. 4B).

Following the proteolytic cleavage of the bait region,both a-Ms undergo a hydrolysis in the internal thiol–ester bond, allowing the covalent attachment ofproteinases that gives rise to distinctive complexes of

Fig. 3. Non-denaturing PAGE analysis of the interactions between a-Ms and cruzipain. (A) a2-M was incubated with cruzipain at 37 �C for

24 h, diluted with sample buffer and then subjected to electrophoresis

on a 5% gel. Lanes 1–5: native a2-M (10.0 lg) incubated with cruzipain

(0.3–4.4 lg) (molar ratios of 1:0.5, 1:1, 1.2, 1:4, and 1:8, respectively);

lane 6: native a2-M (10.0 lg); lane 7: chymotrypsin-treated a2-M (10.0

lg) (molar ratio 1:2). After electrophoresis the proteins were stained

with Coomassie brilliant blue R-250. (B) PZP was incubated with

cruzipain at 37 �C for 1 h, diluted in sample buffer and then subjected

to electrophoresis on a 5% gel. Lanes 1–5: native PZP (1.0 lg) incu-bated with cruzipain (0.05–0.9 lg) (molar ratios of 1:0.5, 1:1, 1.2, 1:4,

and 1:8, respectively); lane 6: native PZP (1.0 lg). After electrophoresis

the proteins were silver stained.

Fig. 2. Non-denaturing PAGE and gelatin SDS–PAGE analysis of the

interactions between T. cruzi proteinases and PZP. PZP was incubated

with cruzipain at 37 �C for a variable period of time, diluted with

sample buffer and then subjected to electrophoresis. (A) Lane 1: native

PZP (10.0 lg); lanes 2 and 3: PZP (10.0 lg) incubated with T. cruzi-

extract (5.7 lg) for 30 min and 20 h, respectively. The samples were

electrophoresed through a 5% non-denaturing PAGE, and the gel was

stained with Coomassie brilliant blue R-250. (B) Lane 1: T. cruzi ex-

tract (6.2 lg); lane 2: T. cruzi extract incubated with a2-M (15.0 lg) for24 h; lane 3: T. cruzi extract (6.2 lg) incubated with PZP (15.0 lg) for24 h. The samples were electrophoresed through an 11% polyacryl-

amide gel with copolymerized gelatin as substrate (gelatin

SDS–PAGE). The gel was washed with distilled water and the enzyme

activity developed in MES buffer for 2 h at room temperature. The

staining was accomplished with Coomassie brilliant blue R-250 over-

night, and the activity bands were visualized after extensive destaining.

124 A.M. Ramos et al. / Experimental Parasitology 100 (2002) 121–130

molecular weights higher than 360 kDa when assayed bynon-reducing SDS–PAGE (Wang et al., 1984). In orderto demonstrate the formation of covalent complexes ofcruzipain with a2-M and PZP, immunoblotting assayswere performed using polyclonal rabbit anti-cruzipainserum. Assays shown in Fig. 5 revealed that cruzipainforms covalent complexes with a2-M and PZP detectedafter 5 and 30 min of incubation, respectively. In bothcases, the cruzipain immunoreactivity was detected inassociation with the 45 kDa band of native protein (Figs.5A and B, lane 4) and with aggregates (Figs. 5A and B,lanes 2–3) higher than 360 kDa dimers of a-Ms (Figs. 5Aand B, lane 5), indicating that cruzipain was covalentlytrapped by the inhibitor. In samples of a2-M and PZPrun as control of anti-cruzipain serum specificity, nocross-reactivity was detected (Figs. 5A and B, lane 1).

Activity of complexed cruzipain in solid-phase proteaseassays. The presence of the proteinase bound to PZPwas verified by gelatin SDS–PAGE. Samples of controlcruzipain, as well as those mixtures containing cruzipainand PZP, were able to degrade gelatin in bands of ap-proximately 44 kDa corresponding to the free enzyme(Fig. 6, lanes 2–4) and in others of molecular weightshigher than that of the PZP dimer (360 kDa) compatiblewith covalent PZP–cruzipain complexes (Fig. 6, lanes 2and 3). On the other hand, no gelatinolytic activity wasdetected at the position of the PZP dimer, ruling out anycontaminant activity in the PZP control (Fig. 6, lane 1).

Inhibition of cruzipain activity in the presence of a2-Mand PZP. In order to establish the catalytic activity ofcruzipain in the presence or absence of a-Ms, enzymeinhibition assays were performed. Formation ofthe complex between a-Ms and cruzipain, previously

Fig. 5. Immunoblotting analysis of a-Ms–cruzipain complexes. In-

creasing amounts of cruzipain were allowed to react with a2-M (30

min) or PZP (5 min) at 37 �C, diluted with sample buffer containing 2%

SDS without reduction and after 60 min loaded onto a 4–12% gradient

gel. (A) Lanes 1 and 5: a2-M (10.0 lg); lanes 2 and 3; a2-M (10.0 lg)incubated with cruzipain (0.55 and 1.10 lg) (molar ratios of 1:1 and

1:2); lane 4: cruzipain (1.10 lg). (B) Lanes 1 and 5: PZP (10 lg); lanes 2and 3: PZP (10.0 lg) incubated with cruzipain (1.10 and 2.20 lg)(molar ratios of 1:1 and 1:2); lane 4: cruzipain (1.10 lg). In both cases,

after electrophoresis the proteins were blotted onto nitrocellulose

membranes and incubated with polyclonal rabbit anti-cruzipain anti-

body (A and B, lanes 1–4), polyclonal rabbit anti-a2-M (A, lane 5) and

polyclonal rabbit anti-PZP antibodies (B, lane 5), and then incubated

with HRP-labeled polyclonal goat anti-rabbit IgG. The protein bands

were developed with 4-chloro-1-naphthol.

Fig. 4. Reduced SDS–PAGE analysis of interactions between a-Ms

and cruzipain. (A) a2-M was incubated with cruzipain at 37 �C for 30

min, diluted with sample buffer (containing 2% SDS and 50 mMDTT),

incubated again for 60 min, and finally subjected to electrophoresis on

a 7.5% gel. Lanes 1 and 2: native a2-M (10.0 lg) incubated with cru-

zipain (1.1 and 2.2 lg) (molar ratios of 1:2 and 1:4); lane 3: a2-M (10.0

lg); lane 4: cruzipain (1.1 lg); lane 5: high-range molecular-weight

standard markers (Bio-Rad) corresponding to myosin (205 kDa), b-galactosidase (116 kDa), phosphorylase B (97 kDa), and bovine serum

albumin (66 kDa). Proteins were stained with Coomassie brilliant blue

R-250. (B) PZP was incubated with cruzipain at 37 �C for 5 min, di-

luted with sample buffer (containing 2% SDS and 50 mM DTT), in-

cubated again for 60 min, and finally subjected to electrophoresis on a

7.5% gel. Lane 1: native PZP (1.0 lg); lanes 2 and 3: PZP (1.0 lg)incubated with cruzipain (0.22 and 0.44 lg) (molar ratios of 1:2 and

1:4), lane 4: cruzipain (0.22 lg); lane 5: high-molecular-weight stan-

dard markers. Proteins were silver stained.

A.M. Ramos et al. / Experimental Parasitology 100 (2002) 121–130 125

demonstrated by PAGE and immunoblotting assays,was also demonstrated in mixtures of cruzipain in thepresence of each a-Ms by a decrease in the velocity ofhydrolysis of the low molecular weight substrate,Bz-Pro-Phe-Arg-pNA. Fig. 7 shows the inhibition ofcruzipain catalytic activity in samples incubated witha-Ms for 60 min at different molar ratios. This signifi-cant inhibition compared to the control was alreadypresent with a cruzipain:a-Ms as low as 1:0.25. More-over, a stabilization of the reaction began to be observedfrom this point onwards. The inset to Fig. 7 shows thatcatalytic activity of cruzipain decreased within 5 min inthe presence of a2-M and PZP under our experimentalconditions (molar ratios of 1:1 and 1:0.25, respectively),reaching a progressive stabilization of inhibition atsubsequent times. In fact, a complete inhibition of en-zyme activity was not reached at any of the molar ratiosor times assayed. It is generally assumed that in thepresence of a large excess of inhibitor the remnant ac-tivity may be attributed to the only active specie presentin the sample, namely trapped cruzipain, as is seen inFig. 6. However, we noted by gelatin SDS–PAGE thatfree cruzipain, probably isoforms that are unable tointeract with a-Ms, was still present and active in mix-tures of cruzipain with up to a 12-fold molar excess ofa-Ms (data not shown).

Complex formation of isoform(s) of CPs with a-Ms.The former experiments proving the interaction betweena-Ms and cruzipain were developed with hydrophiliccruzipain purified from its lysosomal depots. In order toinvestigate whether a2-M and PZP could also interactwith membrane-bound cysteine proteinases very likely

related to cruzipain (Parussini et al., 1998), we tried todetect complexes of such enzymes with the a-Ms in-hibitors by means of Western blot assays. To test thishypothesis, HMP were obtained as described in Section2. These HMP were previously found to contain iso-forms of CPs, almost entirely confined to plasmamembrane, which showed immunoreactivity with apolyclonal anti-cruzipain serum, and to display a com-pletely E-64 abolished activity on gelatin SDS–PAGEemphasizing their thiol-proteinase character (Parussiniet al., 1998). Furthermore, the HMP only exhibitedbands of slightly different apparent isoform patternscompared to cruzipain control, additionally devoid ofhydrophylic cruzipain as indicated by the absence of thehydrophylic and cytosolic enzyme NADP-GDH (Fig.8A). Likewise, when mixtures of a2-M or PZP andHMP were subjected to electrophoretic isolation andthen blotted onto nitrocellulose membrane, the polycl-onal rabbit anti-cruzipain serum was able to recognizehydrophobic isoforms of CPs–a-Ms complexes of highermolecular mass (>360 kDa) (Fig. 8B), resembling thosepreviously formed between cruzipain and a-Ms. Signif-icantly, a2-M and PZP were able to trap isoform(s) ofCPs isolated by Con A–Sepharose affinity column, thus

Fig. 7. Spectrophotometric assay of cruzipain activity in presence of

a-Ms. Purified cruzipain was incubated with different concentrations of

a2-M ðjÞ or PZP ðdÞ at molar ratios of 1:0.25, 1:0.5, 1:1, and 1:2 for

60 min at 37 �C. Proteolytic activity of mixtures was determined by the

extent of hydrolysis of 3 mM Bz-Pro-Phe-Arg-pNA substrate. Proteo-

lytic activity in these mixtures was measured and compared to that of

cruzipain control without a-Ms (100% of activity), and expressed as a

percentage of inhibition. Inset: Time course inhibition of cruzipain

activity by a-Ms inhibitors. Cruzipain was incubated with a2-M (molar

ratio of 1:1) or PZP (molar ratio of 1:0.25) for 5, 15, 30, and 60 min. at

37 �C. Proteolytic activity in these mixtures was measured and

expressed as described above. Each plot represents the mean of three

experiments, and the standard deviation was within 10% of the mean.

Fig. 6. Gelatin SDS–PAGE analysis of PZP–cruzipain complexes.

Cruzipain was incubated with PZP at 37 �C for 2 h and then mixed

with sample buffer (2% SDS w/v final concentration); after incubation

for 60 min at room temperature, the mixtures were subjected to elec-

trophoresis on 7% SDS–PAGE. Lane 1: PZP (10.0 lg); lanes 2 and 3:

cruzipain (0.5 lg) incubated with PZP (2.2 and 4.5 lg) (molar ratios of

1:0.5 and 1:1); lane 4: cruzipain (0.5 lg). After electrophoresis, the gel

was washed with distilled water and the enzyme activity developed in

MES buffer overnight at 37 �C. Staining was accomplished with

Coomassie brilliant blue R-250 overnight, and the activity bands were

visualized after extensive destaining.

126 A.M. Ramos et al. / Experimental Parasitology 100 (2002) 121–130

confirming that the previously shown complexes formedwith HMP were really due to specific interaction of a-Ms with membrane isoforms of CPs closely related tocruzipain (Fig. 9).

4. Discussion

In a previous work, we demonstrated that a2-M in-teracts with and inhibits proteinases of the parasite T.cruzi, cruzipain probably being involved in these inter-actions (Ramos et al., 1997). On the other hand, our

present results indicate that plasma levels of PZP in-crease during the course of acute Chagas’ disease (Fig.1), suggesting a potential role as inhibitor of proteinasesreleased from immune and parasitized host cells andfrom circulating parasites. The evaluation of the latterpossibility allows us to demonstrate that PZP also in-teracts with proteinases included in T. cruzi extracts(Fig. 2A), and although we cannot rule out the presenceof several proteinases in addition to cruzipain, the originof such interactions could be really attributed to thisenzyme (Fig. 2B). In vitro regulation of the immuneresponse following the formation of complexes betweencruzipain and a2-M has been reported (Morrot et al.,1997), but the molecular characterization of such com-plexes was not achieved. We show here that cruzipaincan actually interact with a2-M and PZP, producingspecific proteolysis of their bait regions (Fig. 3), indi-cating that the first and basic step of the trappingmechanism was accomplished. In our assay conditions,the incomplete degradation of the 180 kDa subunitsuggests that the reaction probably involves a fast phaseduring which a limited number of subunits are cleaved,followed by a very slow phase up to the reaction com-pletion. A similar mechanism has been previously pro-posed for other proteinases such as thrombin andprostatic specific antigen (Feinman et al., 1985; Ottoet al., 1998). Interestingly, our present results demon-strate that the PZP bait region displays peptidic bondssusceptible to be cleaved by cysteine proteinases ofparasitic origin. Moreover, cruzipain was demonstrated

Fig. 9. Immunoblotting assays of complex formation between mem-

brane isoform(s) of CPs and a-Ms. Isoform(s) of CPs purified from

membranes of T. cruzi epimastigotes (2.0 lg) treated with 10.0 lg of

a2-M (lane 1), PZP (lane 2), or without treatment (lane 3) were in-

cubated for 90 min at 37 �C, diluted with sample buffer containing 2%

SDS without reduction, and after 60 min subjected to electrophoresis

on 4–12% gradient gels. Hydrophilic cruzipain (1.0 lg) was used as an

immunodetection control (lane 4). Immunoblotting was carried out in

the conditions detailed in Fig. 5, using the same polyclonal anti-

cruzipain serum, except that protein bands were visualized by ECL

reagent.

Fig. 8. Immunoblotting assays of complex formation between MHP

and a-Ms. (A) Lane 1: control purified NADP-GDH (1.0 lg), lane 2:

MHP (24.0 lg). Western blots were probed with a polyclonal anti-

NADP-linked glutamate dehydrogenase serum. (B) Triton X-114-ex-

tracted MHP from T. cruzi epimastigotes (24.0 lg) treated with 10.0 lgof a2-M (lane 1), PZP (lane 2), or without treatment (lane 3) were

incubated for 90 min at 37 �C, diluted with sample buffer containing

2% SDS without reduction, and after 60 min subjected to electro-

phoresis on 4–12% gradient gels. Cruzipain (1.0 lg) was used as an

immunodetection control (lane 4). Immunoblotting was carried out as

detailed in Fig. 5, using the same polyclonal anti-cruzipain serum. The

protein bands were developed with 4-chloro-1-naphthol.

A.M. Ramos et al. / Experimental Parasitology 100 (2002) 121–130 127

in SDS-stable complexes with a-Ms through both gela-tin degradation and immunodetection analysis, thusemphasizing the covalent nature of the interaction (Figs.5 and 6). However, cruzipain causes an incomplete a2-Mconformational change, yielding complexes of interme-diate mobility (Fig. 3A), similar to those reported withthrombin, prostatic specific antigen and matrix metal-loproteinases 2 and 9 (Arbel�aaez et al., 1997; Marshallet al., 1992; Otto et al., 1998; Wang et al., 1984). On theother hand, after reaction with cruzipain, native mole-cules of PZP exhibit a typical tetramerization (Fig. 3B).

In agreement with our results showing the interactionbetween a-Ms and cruzipain, the impaired proteolyticactivity of the enzyme upon the Bz-Pro-Phe-Arg-pNAsubstrate can be attributed to its trapping by a2-M andPZP (Fig. 7). Interestingly, this inhibitory capacity ofthe a-Ms could be only extensive to some cruzipainisoforms, as judged by the presence of free cruzipainswhose activity is not affected (data not shown). In thissense, cruzipain preparations constitute a microhetero-genic complex of isoforms (Cazzulo et al., 1995), whichupon isoelectrofocusing present up to 12 bands withisoelectric points ranging from 3.7 to 5.1 (Stoka et al.,1995). By the way, although cruzipain complexed to a-Ms could exhibit changes in kinetic parameters that maycontribute to changes in its enzymatic activity, as it wasdemonstrated with other proteinases (S�aanchez et al.,1998; Van der Graaf et al., 1984), in our assay condi-tions such analysis would require separation of a-Ms–cruzipain complexes from free cruzipain.

In the course of T. cruzi infection, cruzipain may bean important virulence factor by releasing pro-inflam-matory kinins from their low and high molecular weightkininogen precursor (Del Nery et al., 1997). Cruzipaindisplays a high genomic-sequence similarity to papainand cathepsin L, showing in addition catalytic proper-ties intermediate between cathepsins B and L (Cazzuloet al., 1997). Among the physiological selective inhib-itors of these lysosomal cathepsins the family of cystatinsmay be mentioned (Turk and Bode, 1991), and they arealso strong inhibitors of cruzipain (Stoka et al., 1995). Itis important to note that cathepsins B and L interact witha2-M (Mason, 1989), which as PZP, is also able tocomplex and inhibit cruzipain, as demonstrated in thiswork.

Recently, it was demonstrated that a2-M infiltratesthe hearts of mice infected with T. cruzi (Coutinho et al.,1999). In such a way, the inhibition by a2-M could occureven in the presence of more specific inhibitors that canselectively be inactivated by free radicals released by in-flammatory mononuclear infiltrates (Korolenko et al.,1992). Although some proteinases released from patho-gens cleave the a2-M bait region, forming complexeswithout loss of activity on macromolecular substrates(Rangajaran et al., 2000), or are able to degrade a2-M(Grenier, 1996), here we show that cruzipain is inhibited

within 5 min (Fig. 7) without cleavage outside the baitregion of the a2-M or PZP molecules after 30 and 5 minof incubation, respectively. In this context, we couldassume that a2-M may be crucial during the T. cruziinfection, reducing the effects of cruzipain by acting as aback-up inhibitor. If PZP permeates human tissues or itis synthesized by inflammatory or local cells of themyocardium is presently unknown. Thus, a role of a2-Mand eventually PZP, avoiding deletereous effects of cru-zipain in the circulation or local sites of infection throughits capacity to form complexes with the enzyme, followedby a rapid depuration of this complex via the endocyticLDL receptor-related protein (LRP/a2-MR) (Hussainet al., 1999), can be suggested. An increased clearance ofa2-M–proteinases complexes has been suggested in amurine model, based on the evidence that LRP/a2-MRexpression is up-regulated in macrophages during theacute phase of the infection (Coutinho et al., 1998).

Although the invasive process of mammalian cells byT. cruzi is a very complex mechanism, it has beendemonstrated that released cruzipain isoforms 1 and 2are involved in this process (Scharfstein, 2000). More-over, it is known that cruzipain could be also anchoredto the plasma membrane possibly through a GPI anchor(Fresno et al., 1994); so its release into the circulation orinterstitial spaces could also be attained from this sitepossibly by action of specific phospholipase C (Yoshidaet al., 2000). In this work, we demonstrated that a2-Mand PZP can interact with membrane-bound CPs closelysuspected as cruzipain isoforms in both hydrophobicextracts devoided of hydrophylic cruzipain and purifiedCPs samples (Figs. 8 and 9).

As a functional role, a-Ms may simply serve as ascavenger of potentially harmful hydrophilic cruzipainor membrane-bound isoform(s), passively or activelyreleased into circulation and interstitial spaces. More-over, the a-Ms could play more active roles in relationor in addition to their scavenger function. In this sense,a-Ms–proteinase complexes are suspected to have im-munoregulatory actions through well-established cyto-kine-binding activities (Chu and Pizzo, 1994; Goniaset al., 1994). In addition, it has been demonstrated thata2-M–cruzipain complexes are endocytosed via LRP/a2-MR resulting in an enhanced antigenic presentationof cruzipain peptides (Morrot et al., 1997).

Thus, a possible role of a-Ms as regulators of theactivity of cruzipain and other parasite proteinases in-volved in inflammation, cell host invasion and immunereaction events caused by T. cruzi in Chagas’ diseasecould be suggested.

Acknowledgments

This work was supported partly by grants from the theConsejo de Investigaciones Cient�ııficas y Tecnol�oogicas de

128 A.M. Ramos et al. / Experimental Parasitology 100 (2002) 121–130

la Provincia de C�oordoba (CONICOR), the AgenciaNacional de Promoci�oon Cient�iifica y Tecnol�oogica (AN-PCyT), the Secretar�ııa de Ciencia y Tecnolog�ııa de laUniversidad Nacional de C�oordoba (SECyT), and theConsejo de Investigaciones Cient�ııficas y Tecnol�oogicas dela Rep�uublica Argentina (CONICET). V.G.D. andN.M.G. de B. is career investigator of the CONICET,Argentina. We thank Dr. Antonio Blanco for valuablesuggestions and critical revision of the manuscript.

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