Vol. 59, No. 4INFECTION AND IMMUNITY, Apr. 1991, p. 1409-14160019-9567/91/041409-08$02.00/0Copyright C) 1991, American Society for Microbiology

Characterization of a Glycosyl-Phosphatidylinositol-AnchoredMembrane Protein from Trypanosoma cruzi

CRISTINA HERNANDEZ-MUNAIN, MONICA A. FERNANDEZ, ANTONIO ALCINA,AND MANUEL FRESNO*

Centro de Biologia Molecular Consejo Superior de Investigaciones Cientfficas-Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 4 October 1990/Accepted 18 January 1991

Four monoclonal antibodies (MAbs) specific for Trypanosoma cruzi were obtained. Flow cytometry analysisshowed that these four MAbs stained the membranes of the three main morphological forms of T. cruzi:amastigotes, trypomastigotes, and epimastigotes. The four MAbs seemed to recognize the same 50- to 55-kDaantigen that was revealed by immunoblotting. Competition experiments revealed that they defined at least twodifferent epitopes on the molecule. The antigen was detected on the external surface of the membrane byimmunoelectron microscopy. Several experiments indicated that the 50- to 55-kDa antigen recognized by thesefour MAbs was a glycosyl-phosphatidylinositol-anchored membrane protein. (i) The antigen could be removedfrom the cell surface by treatment with proteases, NaOH, HNO2, and phosphatidylinositol-specific phospho-lipase C (PI-PLC). (ii) The phase distribution of the antigen in Triton X-114 solutions changed drastically upontreatment with PI-PLC. The antigen was found mainly in the detergent phase in nontreated samples and inthe aqueous phase in PI-PLC-digested samples. (iii) A cross-reacting determinant that was found in otherglycosyl-phosphatidylinositol-anchored membrane proteins appeared after PI-PLC treatment.

The protozoan flagellate Trypanosoma cruzi is the caus-ative agent of Chagas' disease, which affects several millionpeople in Central America and South America (6, 15). Thisprotozoan has a complex life cycle and exists in at least threemorphologically distinct forms: infective (metacyclic orblood trypomastigotes), insect (epimastigotes), and intracel-lular (amastigotes). Epimastigotes multiply in the insect gutand differentiate into infective metacyclic trypomastigotes asthey move through the digestive tract. Once in the vertebratehost, they enter susceptible cells, in which they replicateintracellularly as amastigotes (6, 15).

Several alterations of the immune response have beendescribed in this disease (6, 15); these include a severeimmunosuppression of the humoral and cellular responses tounrelated antigens during the acute phase of the infectionand functional defects in the responding cell population.However, the mechanisms underlying these defects arepoorly understood (14, 18, 25). Furthermore, massive lym-phocyte polyclonal activation (21) resulting in the generationof autoantibodies cross-reacting with the parasites and hostcells and tissues has also been described (8, 17, 33).

Analyses of T. cruzi cell surface antigens have beencarried out to achieve a better understanding of this complexhost-parasite relationship (31). Several cell surface proteinshave been identified with the help of monoclonal antibodies(MAbs). Among them are the glycoproteins GP 72 (32), GP90 (24), and recently GP 57/51 (27). GP 72 has been shown topartially protect the host from infection with metacyclictrypomastigotes (29) and seems to be involved in parasitedifferentiation (30). GP 90 has been claimed to be trypo-mastigote specific (24), and GP 25, which recently has beenshown to be derived from GP 57/51 (27), is a good serologicalmarker for the disease (26). In addition, some not-so-wellcharacterized MAbs have been reported to be stage specific

* Corresponding author.t Present address: Instituto L6pez-Neyra, Granada, Spain.

(4, 34) or strain specific (12). Recently, several MAbs havebeen used to characterize an amastigote-specific 70- to84-kDa protein (3).Many eukaryotic proteins are anchored to the membrane

by a glycosyl-phosphatidylinositol (GPI) linkage (9, 19).Among them are several proteins from members of thefamily Trypanosomatidae, such as the variant surface glyco-protein from Trypanosoma brucei and the major surfaceprotease of Leishmania spp. (5, 7, 11). Recently, two pro-teins from T. cruzi, GP 90 (28) and the amastigote-specific70- to 84-kDa protein, also have been shown to be GPI-anchored membrane proteins (3).We describe here four MAbs against a T. cruzi 50- to

55-kDa surface protein which is present in all differentiationstages and which seems to be anchored to the membrane byGPI.

MATERIALS AND METHODS

Parasites. The strain of T. cruzi used was originally ob-tained from a patient with Chagas' disease in the InstitutoNacional de la Salud, Madrid, Spain. It was cloned andnamed strain G (2). Strains Y and Tulahuen were kindlysupplied by John David (Harvard University, Boston,Mass.).The trypanosomes were continuously cultured in liver

infusion-tryptose medium supplemented with 10% fetal calfserum as described previously (13). Metacyclic trypomasti-gotes were prepared by differential centrifugation of parasitecultures at 150 x g for 5 min, and this centrifugation wasrepeated several times with the resulting supernatant. Thefinal supernatant contained >95% metacyclic trypomasti-gote forms. Epimastigotes were obtained during exponentialgrowth from parasites cultures which contained more than95% epimastigotes. The cultures were centrifuged for 15 minat 1,000 x g and washed twice with phosphate-bufferedsaline (PBS). Amastigotes were obtained from infected cul-tures of J774 cells as described previously (1) and separated

1409

1410 HERNANDEZ-MUNAiN ET AL.

from epimastigotes and cells by metrizamide (Nyegaard,Oslo, Norway) discontinuous gradient (8 and 16%) centrifu-gation.

Preparation of MAbs. Female BALB/c mice were infectedby injection with cultures which contained a mixture ofmorphologies of live T. cruzi (106 trypanosomes per mouse)on the first (intraperitoneal), 15th (intraperitoneal), and 27th(intramuscular) days. Five days after the last injection,spleen cells were obtained and fused with P3/X63-Ag 8.653myeloma cells as described previously (1). Successful hy-brids were selected and screened for antibody production byan enzyme-linked immunoabsorbent assay (ELISA) and anindirect immunofluorescence assay. The selected hybridswere cloned three times. Immunoglobulin subclasses of theMAbs were determined by double immunodiffusion withantimouse subclass-specific antibodies (Nordik Laborato-ries, Tilburg, The Netherlands). MAb C10 (immunoglobulinGl [IgGl) was purified from the peritoneal fluid of ascitictumors in BALB/c mice by affinity chromatography in pro-tein A-Sepharose CL-4B (Sigma Chemical Co., St. Louis,Mo.). The other MAbs were of the IgM subclass.Antibody to the cross-reacting determinant (anti-CRD)

was a generous gift from M. L. Cardoso de Almeida (EscolaPaulista de Medicina, Sao Paulo, Brazil).

Localization of the 50- to 55-kDa antigen by immunogoldlabeling. T. cruzi parasites from a liver infusion-tryptoseculture at 27°C were washed three times with PBS andincubated with MAb CIO ascitic fluid diluted 1:50 in PBS for1 h at room temperature. After the washes, the pellets wereincubated with rabbit antimouse immunoglobulin (RMIG)(50 mg/ml in PBS) for 1 h at room temperature. After threewashes with PBS, protein A complexed to 5-nm colloidalgold particles (Janssen, Beerse, Belgium) diluted 1:50 in PBSwas added and the mixture was incubated for 2 h at roomtemperature. Parasites were washed with PBS, and thepellets were fixed with 4% formaldehyde-2% glutaralde-hyde-2% tannic acid in PBS for 1 h at 4°C. After three PBSrinses, the pellets were treated with 1% osmium tetraoxide inPBS for 30 min at 4°C. After another three rinses of 20 mineach with PBS, the pellets were progressively dehydrated byincubation with different dilutions of acetone: 30% for 30min, 50% for 1 h, and 70% for 1 h. The samples werecompletely dehydrated by incubation with 70% acetone inPBS for 2 days at 4°C and incubation with 100% acetone for1 h. The samples were embedded in vegetal resin (Serva,Heidelberg, Federal Republic of Germany), and thin sec-tions of 50 to 80 nm were picked up, stained with 1%aqueous uranyl acetate-2% lead citrate, and examined in anelectron microscope.Flow cytometry analysis. T. cruzi parasites were centri-

fuged twice in PBS with 2% bovine serum albumin (BSA)-0.1% sodium azide. For each assay, 1 x 106 to 2 x 106parasites were resuspended and incubated in 100 p. of MAbhybridoma supernatant for 30 min at 4°C. The parasites werewashed and incubated for 30 min at 4°C in the dark with 50p.1 of fluorescein isothiocyanate-labeled F(ab')2 RMIG(Southern Biotechnology Associates Inc., Birmingham,Ala.). After three rinses, the parasites were resuspended inthe same buffer containing 1% paraformaldehyde and thefluorescence was analyzed in an EPICS cytofluorimeter.Treatment of intact parasites. To study the biochemical

nature of the antigen recognized by the MAbs, we subjectedthe parasites to several treatments. (i) Before the blockingsolution was added to ELISA wells, the parasites weretreated with a 1 M solution of sodium hydroxide for 30 minat 37°C or subjected to nitrous acid deamination by incu-

bation with 0.25 M sodium acetate (pH 3.5) plus 0.2 Mfresh sodium nitrite overnight at room temperature. (ii) Forthe phosphatidylinositol-specific phospholipase C (PI-PLC)treatment, a suspension of 108 inactivated (by heating at56°C for 15 min) parasites was treated with 20 to 200 U ofPI-PLC of Bacillus cereus (Boehringer, Mannheim, FederalRepublic of Germany) per ml in 100 p.l of 0.27 M sucrose-25mM ethanolamine-NaOH (pH 7.5)-l mg of BSA per ml-0.002% sodium azide for 1 to 2 h at 37°C. (iii) For theprotease (Sigma) treatment, a suspension of live parasiteswas incubated with 200 p.g of each protease per ml for 16 hat 37°C.

Reactivity with antibodies was determined by ELISA orflow cytometry analysis.ELISA. In brief, parasites were washed three times with

PBS, heated at 56°C for 15 min, and resuspended to 106/ml inPBS. They were added to a 96-well flat-bottom polyvinylchloride plate (Titertek; Flow Laboratories, Irvine, Scot-land) and incubated overnight at 4°C. The wells were satu-rated by incubation with 5% BSA-0.05% Tween 20 (Sigma)in PBS for 1 to 2 h at room temperature. The wells wereincubated with 100 p.l of serial dilutions of hybridomasupernatants or serum in PBS containing 0.1% BSA and0.05% Tween 20 for 1 h at room temperature, and ELISAwas carried out as described previously (2).

Immunoblotting. Parasites were washed with PBS anddisrupted with lysis buffer (1% Nonidet P-40 [Fluka Chemie,Buchs, Switzerland]), 150 mM NaCl, 20 mM Tris-HCI [pH8], and a cocktail of protease inhibitors [Boehringer] con-taining 1 mM phenylmethylsulfonyl fluoride, 1 p.g of aproti-nin per ml, 1 p.g of pepstatin per ml, 1 p.g of leupeptin per ml,and 2 mM EDTA) for 30 min at 4°C. They were electro-phoresed on 10 to 12% acrylamide gels and blotted tonitrocellulose paper (BioRad, Richmond, Calif.) as de-scribed previously (2). The paper was saturated with PBScontaining 0.05% Tween 20, 5% skim milk, and 0.1% sodiumazide, and the strips were treated with the hybridomasupernatants of the different MAbs and 1% skim milk for 4 hat room temperature. The strips were washed with PBS-0.05% Tween 20 and incubated with 125I-RMIG (specificactivity, S x 106 cpm/p.g; 106 cpm/ml diluted in PBS-0.05%Tween 20) for 1 h at room temperature.

Phase separation of glycolipid-anchored membrane proteinsin TX-114 solution. After treatment with PI-PLC, the para-sites were washed two times with 1 ml of Tris-HCI (pH7.4)-140 mM NaCl (TBS) containing the cocktail of proteaseinhibitors described above. The supernatants obtained werefiltered through 0.2-p.m-pore filters to eliminate possiblecontaminating parasites and were kept at 4°C to be mixedtogether with the aqueous phase resulting from Triton X-114(TX-114) partition of the parasites. The parasites in thepellets were lysed by 30 min of incubation at 4°C with 2%precondensed TX-114 (Serva) in TBS with protease inhibi-tors. The lysates were centrifuged at 100,000 x g for 30 minat 4°C to remove the insoluble material, incubated at 37°C for5 min, and centrifuged at 17,000 x g in a minicentrifuge atroom temperature for 1 min. The resulting aqueous (deter-gent-depleted) and detergent-enriched phases were sepa-rated, and a volume of TBS containing protease inhibitors orprecondensed TX-114 was added to the opposite phase torestore the initial detergent/H20 ratio to start a new phasepartitioning. The two samples were incubated at 4°C for 10min with occasional stirring, warmed in a 37°C bath, andcentrifuged at 17,000 x g in a minicentrifuge at roomtemperature for 1 min. New phases were separated. Thecorresponding aqueous and detergent-enriched phases were

INFECT. IMMUN.

NEW GPI-LINKED T. CRUZI MEMBRANE PROTEIN 1411

oKTcG

MAb C2

MAb C4

MAb C7

MAb C10

Epimastigotes Amastigotes Tripomastigotes

Fluorescence IntensityFIG. 1. Flow cytometry analysis of T. cruzi with specific MAbs. The reactivities of MAb C10, C2, C4, and C7 culture supernatants with

purified epimastigotes, amastigotes, or trypomastigotes were determined. Shown is the number of cells versus the logarithm of fluorescenceintensity. The percentage of positive cells in each case is also indicated. The blank profiles represent staining with an irrelevant antibody.aTcG, Antisera from immune mice.

mixed, and the supernatant obtained in the first centrifuga-tion was added to the aqueous phase obtained. The finalvolume of each phase was adjusted to 2.5 ml with TBScontaining protease inhibitors. These samples were used inELISAs or immunoprecipitations.

Immunoprecipitation. Parasites (2 x 108 epimastigotes)were washed with PBS, resuspended in 150 ,ul of the same

buffer, and incubated for 20 min at room temperature with 1mCi of 125I-Na (16.4 mCi/,ug; Amersham, Buckinghamshire,United Kingdom)-30 p.l of lactoperoxidase (140 U/ml; Sig-ma)-10 ,ul of H202 (0.06%). Every 5 min, 10 ,ul of H202 was

added. The cells were washed once with 10 ml of PBScontaining 20 mM Nal and 0.5% BSA. 125I-labeled T. cruziepimastigotes (108) were either treated or not treated withPI-PLC and extracted with a TX-114 solution. The phasesobtained after the TX-114 partitioning were separated as

described above.The immunoprecipitations were done with preformed pro-

tein A-Sepharose CL-4B-antibody complexes. These com-

plexes were formed by incubating 10 p.g of RMIG and 10of 50% protein A-Sepharose CL-4B suspension for 2 h at4°C. After a wash with PBS, ternary complexes were formedby further incubation with 10 p.g of purified MAb C10 or an

irrelevant mouse IgGl MAb. The complexed beads were

washed with PBS before use, and 10 p.l of a 50% suspensionof the preformed complexes was used in each incubation.The samples were precleared three consecutive times by

incubation for 1 h at 4°C with RMIG-protein A-SepharoseCL-4B complexes. The supernatants were incubated for 4 hat 4°C with specific or control antibodies in ternary com-

plexes with RMIG and protein A-Sepharose CL-4B. Thebeads were washed five times with the lysis buffer used forthe immunoblotting. The proteins were eluted with sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) buffer and subjected to SDS-PAGE on 12% acryl-amide gels as described previously (2).

RESULTS

MAbs C10 (IgGl), C4 (IgM), C7 (IgM), and C2 (IgM) were

selected by ELISA and indirect immunofluorescence assayfrom a pool of hybridomas obtained from T. cruzi-infectedmice. These MAbs reacted very strongly with epimastigotesand trypomastigotes and somewhat more weakly with amas-

tigotes, as determined by flow cytometry analysis (Fig. 1).To characterize the antigens recognized by the different T.

cruzi-specific MAbs, we used the immunoblotting technique.For this purpose, epimastigote extracts were subjected toSDS-PAGE, blotted, and incubated with the four MAbs. Thesera from infected mice but not from normal mice reactedwith 72-kDa, 50-kDa, and other, lower-molecular-mass an-

tigens, as well as with the lipopeptidophosphoglycan (LPPG)antigen (Fig. 2). However, the four MAbs detected the same

broad band in the 50- to 55-kDa region of the gel. Therelationship among all of the MAbs was studied by MAb C10binding competition experiments with an ELISA. C4 and C7partially inhibited MAb C10 binding, whereas C2 was inef-fective (Fig. 3).The four MAbs were specific for T. cruzi, since they did

not react with other members of the family Trypanosoma-tidae, such as Leishmania mexicana, L. donovani, L. infan-tum, or T. brucei. However, no significant differences were

observed in their reactivities with several strains of T. cruzi(data not shown).MAb C10 recognized in the indirect immunofluorescence

assay a determinant that was present in the membranes ofboth living and fixed parasites. The staining was still visibleafter 24 h of incubation of live parasites in serum-free

84.79% 96.84% 97.54%

92.04% 89.64% 95.39%

93.99% 90.94% 96.09 %

94.94% 91.54% 96.64%

95.74%/* 86.39% 94.54%

_ _tm~

VOL. 59, 1991

1412 HERNANDEZ-MUNAIN ET AL.

I-

aLi

c: On: Lj

CD

L)4 U- U- U

4:iL- L-iz<M <0 <0z71 x: z M

- 200

gp 72-

gp 55 -

LPPG -

c

0

._

:_C

- 97

*

- 68

- 43

31

FIG. 2. Immunoblot analysis of hybridoma supernatants with T.cruzi epimastigote extracts. anti TcG, Mouse anti-T. cruzi G serum.An irrelevant antibody was used as a negative control. M, Molecularmasses in kilodaltons.

medium, suggesting that the antigen-antibody complex wasvery stable. No capping was observed with MAb C10 (datanot shown). This result was further confirmed by immuno-electron microscopy. The staining was clearly detectable allalong the extracellular membrane of the parasite (Fig. 4).The structure of the antigen detected by the MAbs was

studied in greater detail. The antigen could be extracted fromthe membrane by chloroform-methanol mixtures and recov-ered in the aqueous phase, suggesting an amphipatic struc-ture (data not shown). Intact parasites were subjected tovarious treatments to further study the nature of the epitopesrecognized by the MAbs. Treatment of the intact parasiteswith pronase, proteinase K, or bromelain (Table 1) but notwith V8 protease, pepsin, chymotrypsin, or trypsin (data notshown) destroyed the epitope recognized by MAb C10.Furthermore, the antigen was liberated from the epimasti-gote surface by treatment with PI-PLC (Fig. 5), suggestingthat the antigen was anchored via a GPI linkage to themembrane. Mild treatment of intact epimastigotes withNaOH or HNO2, each of which also cleaves the GPI-anchored membrane proteins (11, 19), resulted in a completeloss of MAb C10 reactivity (Table 1). Similar results wereobtained with the other three MAbs, C2, C4, and C7 (datanot shown). Furthermore, the reactivity was recovered inthe supernatants from PI-PLC-, sodium hydroxide-, or ni-trous acid-treated T. cruzi epimastigotes (data not shown).Treatment with sodium periodate also destroyed the MAbC10-binding site. Abrogation of the binding of the MAbsafter such treatments of the parasites seemed to be due to thespecific removal of the 50- to 55-kDa membrane antigen,since the binding of the polyclonal anti-T. cruzi sera wasunaffected.

In addition, the antigen had a different distribution inphases obtained after the TX-114 partitioning, depending onthe PI-PLC treatment. 125I-labeled T. cruzi epimastigoteswere either treated or not treated with PI-PLC and extracted

Supernatant dilutionFIG. 3. Competition of the binding of purified MAb C10 to intact

epimastigotes. The binding of biotin-labeled MAb C10 was detectedby ELISA and was titrated after incubation with different dilutionsof MAb C2, C4, C7, and C10 hybridoma supernatants. Thesesupernatants were found to contain approximately the same anti-body concentrations by ELISA titration.

with a TX-114 solution as described in Material and Meth-ods. The resulting aqueous and detergent phases were usedfor the immunoprecipitations. MAb C10 immunoprecipitateda 125I-labeled 50- to 55-kDa antigen from the detergent phasebut not from the aqueous phase in samples not treated withPI-PLC. By contrast, the antigen was immunoprecipitatedfrom the aqueous phase but not from the detergent phase inPI-PLC-treated samples (Fig. 6).

Several of the GPI-anchored membrane proteins frommembers of the order Kinetoplastida present an immunolog-ically cross-reacting determinant (CRD) detectable afterPI-PLC treatment (9, 11). To investigate the presence of aCRD in the 50- to 55-kDa antigen, we allowed the same fourphases that were used for the immunoprecipitations to bindto MAb C10-coated plates. The plates were developed withanti-CRD or normal rabbit serum. Reactivity with anti-CRDwas observed only in the MAb C10-bound antigen from theaqueous phase obtained after PI-PLC treatment (Fig. 7).However, the 50- to 55-kDa antigen was also present in thedetergent phase from nontreated parasites and boundequally well to the plates, as detected by the binding of thenonoverlapping MAb C2 (data not shown). Taken together,the above-mentioned results suggested that the antigen rec-ognized by these MAbs is a GPI-anchored membrane pro-tein.

DISCUSSION

Most of the mammalian membrane proteins are anchoredthrough a short stretch of hydrophobic amino acids. Re-cently, a new class of membrane molecules attached to themembrane by means of a GPI anchor has been described (9,19); the functional significance of the GPI anchor is still

INFECT. IMMUN.

NEW GPI-LINKED T. CRUZI MEMBRANE PROTEIN 1413

bf~~~~~~~~~~~~~

FIG. 4. Immunoelectron microscopy of MAb C10. (a) T. cruzi epimastigotes stained with MAb C10 ascitic fluid at a 1:50 dilution(magnification, x10,000). Insert from panel a shown at a higher magnification (x30,000). Parasites stained with an irrelevant antibody(magnification, x20,000).

unknown, but this anchor may facilitate the lateral mobilityof the proteins (16, 35). Interest in these phosphatidylinosi-tol-containing molecules has increased lately, not only be-cause they serve as an anchor structure but also becausethey may constitute precursors of inositol phosphate glycan

TABLE 1. Effect of various treatments on the reactivity ofT. cruzi with MAb C10 culture supernatant or

mouse anti-T. cruzi serum'

Titer of:Treatment

MAb CIO Anti-T. cruzi serum

None 105 105Heating (80°C, 20 min) 105 105Sodium periodate 103 105NaOH <1 105HNO2 <1 105PI-PLC <1 105Pronase <1 103Proteinase K 102 103Proteinase K + heating 1 103Bromelain 102 103Bromelain + heating 102 103

a Intact T. cruzi epimastigotes bound to plates were treated as described inMaterials and Methods, and the titers of the antibodies were determined byELISA. The titer is the inverse of the dilution of antibody giving 50%maximum binding.

second messengers (22, 23). These classes of GPI glycolipidsare abundant in the protozoa, and the first description ofsuch an unusual membrane anchor was made for the variantsurface glycoprotein of T. brucei (7). In addition, severalclasses of GPI-anchored membrane antigens have beendescribed in Leishmania spp.: lipopeptidoglycan (LPG),glycosylinositol phospholipid (GIPL), and the major surfaceprotein (10).We describe here four MAbs which were obtained from T.

cruzi-infected mice and which seemed to recognize a previ-ously undescribed GPI-anchored membrane antigen of 50 to55 kDa. This antigen was present in all three developmentalforms: epimastigotes, amastigotes, and trypomastigotes.The antigen recognized by MAbs C4, C7, C10, and C2seemed to be of a proteic nature and anchored to themembrane by GPI, as determined by its susceptibility toprotease treatment; its release to the supernatant from intactparasites by PI-PLC; its amphipatic structure; its differentialpartitioning in TX-114, depending on PI-PLC treatment; andthe concomitant appearance of the CRD. The release of theantigen by mild NaOH or HNO2 treatment further supportedthe notion that the 50- to 55-kDa antigen is a GPI-linkedmembrane protein.

Recently, GPI-anchored proteins have been described inthe membrane of T. cruzi as well. GP 90 present in trypo-mastigotes (28) has been shown to be anchored by GPI, asshown by the appearance of a CRD that is also present in the

VOL. 59, 1991

1414 HERNANDEZ-MUNAIN ET AL.

-PI-PLC

+PI-PLC

o4TcG MAb C2 MAb C4 MAb C7 MAb C10

FIG. 5. Effect of PI-PLC treatment on the binding of the MAbs to intact T. cruzi epimastigotes. Shown is a flow cytometry analysis ofnontreated (-PI-PLC) or PI-PLC-treated (+PI-PLC) T. cruzi stained with antisera from immune mice (aTcG) or MAb supernatants. Theblank profiles represent staining with an irrelevant antibody.

variant surface glycoprotein of T. brucei and by partitioningin TX-114 solution after PI-PLC treatment (28). Andrews etal. have identified an amastigote-specific 70- to 84-kDaprotein which is released by endogenous PI-PLC duringparasite differentiation (3). In addition, it has been suggestedthat the GPI anchor may serve to facilitate its release byphospholipases and generate a second messenger which mayin turn be used as a differentiation signal (3, 22, 23).

In a recent study on the GPI-anchored proteins from thetrypomastigotes of T. cruzi, Schenkman et al. did not detectany CRD-containing protein in the 50- to 60-kDa range afterPI-PLC treatment (28). The reason for this discrepancy withour results is unknown, since the MAb C10-reacting antigenis present in high amounts in trypomastigotes. However, itcould be explained by the difficulty in labeling this proteineither metabolically or externally with 1251. Alternatively,we found that the 50- to 55-kDa antigen was easily degrad-

able to lower-molecular-mass products (2). Such sensitivityto degradation may in turn make the detection of intact CRDreactivity in the 50- to 55-kDa protein much more difficult,since it is very likely that during the PI-PLC treatmentendogenous proteases may also be functioning.

In Leishmania spp., two different structures have beenshown to be linked to the membrane by GPI: the majorsurface glycoprotein and several glycoinositol phospholipidsthought to serve as donors of GPI to proteins (20). Inaddition, Leishmania LPG and T. cruzi LPPG are anchoredto the membrane through a ceramide-inositol-phosphatelinkage. This type of linkage would also be susceptible toNaOH or HNO2 treatment. However, our results suggestthat the 50- to 55-kDa antigen recognized by the MAbs isindeed a glycoprotein and not LPPG. This suggestion isbased on the following: (i) the higher molecular mass of our

-Pi-PLC -PI-PLC

PHASE aqueous detergent aqueoius

r r10 C [1irir CLt _. _,_WI

66 --

1.5aQet ercen t

Ir

a4S -'-

31 --

FIG. 6. Immunoprecipitation with MAb C10 after partitioningwith TX-114 and with (+PI-PLC) or without (-PI-PLC) previousPI-PLC treatment. Shown is SDS-PAGE of 125I-labeled materialfrom the detergent or aqueous phases of nontreated or PI-PLC-treated parasites immunoprecipitated with MAb C10 or an irrelevantIgGl MAb (C). Numbers at left are molecular masses in kilodaltons.

UN.

rI E\120 det "20 det

Pi-PLC- Pi-PLC+FIG. 7. Recognition by anti-CRD of MAb C10-bound antigen

after P1-PLC treatment. Intact parasites were either treated (Pl-PLC+) or not treated (PI-PLC-) with PI-PLC and subjected to a

partitioning assay in TX-114. The resulting aqueous or detergent(det) extracts were bound to MAb C10-coated ELISA plates anddeveloped with nonimmune rabbit serum (O) or rabbit anti-CRDserum (0).

I ~~~~~~~~~~~~~~~~~I

I ~ ~~~~~~~I

INFECT. IMMUN.

NEW GPI-LINKED T. CRUZI MEMBRANE PROTEIN 1415

antigen than of LPPG (31, 36); (ii) the susceptibility toprotease treatment; (iii) the presence of the antigen in allthree morphological forms, in contrast to LPPG, which ismainly present in epimastigotes (36); and (iv) the ability ofthe 50- to 55-kDa antigen to be labeled with [35S]methionineand [35S]cysteine (2).The exact relationship of this GPI-anchored 50- to 55-kDa

protein to GP 57/51 previously described by others is notclear (27). Preliminary evidence, such as molecular mass andprotease degradation profile, may indicate that the protein isthe same. However, more studies are needed to confirm thispoint.We do not know the biological significance of this protein

yet. The 50- to 55-kDa protein was expressed in the mem-branes of all developmental parasite forms. Interestingly, thefour MAbs seemed to identify two populations of amasti-gotes on the basis of the level of expression of this antigen inthe parasite membrane. Preliminary evidence suggests thatthe poorly expressed amastigotes are the intracellular ones,whereas the highly expressed ones are those already re-leased into the medium after the disruption of the macro-phage membrane.Andrews et al. (3) have reported the existence of a

developmentally regulated amastigote-specific protein re-leased by PI-PLC. Similarly, our results may indicate thatthe release of the 50- to 55-kDa protein by PI-PLC plays arole at the intracellular stage either for itself or as source ofintracellular signals (22, 23). However, further work isneeded to characterize the biological role of this protein.

In summary, our results add a new protein, the 50- to55-kDa protein, present on the membrane of the differentstages of T. cruzi, to the growing list of GPI-anchoredproteins.

ACKNOWLEDGMENTS

This work was supported by grants from the Direcci6n General deInvestigaci6n Cientffica y Tecnica and from the Fundation Ram6nAreces. C. Hemrndez-Munafn is the recipient of a PFPI fellowship,and M. A. Fernandez is the recipient of an MEC fellowship.We thank Pedro Lastres and Marfa Jesus Serramia for excellent

technical assistance.

REFERENCES1. Alcina, A., and M. Fresno. 1987. Activation by synergism

between endotoxin and lymphokines of the mouse macrophagecell line J774 against infection by Trypanosoma cruzi. ParasiteImmunol. 9:175-186.

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