INTRODUCTION

Glycosylphosphatidylinositols (GPI)¹ serve as protein anchors in most eukaryotes (reviewed in Refs. 1 and 2), although the biological function of the majority of GPI-anchored proteins and protein-free GPIs (i.e. unattached to protein or carbohydrate) is unknown. Addressing the physiological functions of GPIs requires the construction of mutants categorically deficient in the GPI pathway; in most biological systems, such cells are not available. In murine T lymphomas, however, several viable mutants exist (3), some of whose GPI-negative phenotype has been reversed with cloned genes (4). In contrast, in the yeast Saccharomyces cerevisiae, some temperature-sensitive GPI mutants are nonviable (5, 6).

Protozoan parasites, which as a group cause about 5 million human deaths a year worldwide, have developmental stages in which proteins on their plasma membrane are overwhelmingly GPI-anchored. Examples include variant surface glycoprotein (VSG) of Trypanosoma brucei, gp63 of Leishmania spp., circumsporozoite protein of Plasmodium spp., and Ssp-4 of Trypanosoma cruzi (1, 2). Several hypotheses accounting for the overproduction of GPI-anchored proteins in these parasites have been advanced but none tested (7). Indirect evidence that GPI anchors might be critical for the success of these protozoans as parasites lies in the fact that viable mutants in the "conserved core" of protein-GPIs (EtN-phospho-Man₃-GlcN-PI) are unknown.

Trypanosoma cruzi is the causative agent of Chagas' disease, which afflicts an estimated 18 million people in south and central America. Metacyclic trypomastigotes transmitted to humans by Reduviid bugs enter mammalian cells where they differentiate into and replicate as amastigotes. Proliferation of amastigotes is critical to propagation of an infection because trypomastigotes cannot replicate.

We sought to delineate possible functions of GPIs in T. cruzi. Since genes for targeted mutagenesis of the GPI pathway in these parasites are not available, we engineered a phenotypic GPI mutant of T. cruzi by heterologous expression of a GPI-phospholipase C (GPI-PLC) from the related kinetoplastid T. brucei (8-10). GPI-PLC cleaves GPI anchor intermediates in vivo, causing the biochemical equivalent of a dominant-negative loss of function mutation in the GPI pathway (11).

Cell surface expression of GPI-anchored proteins in GPI-deficient T. cruzi was reduced as compared with wild-type cells. With increased GPI-PLC expression by the parasite, amastigotes inside mammalian cells replicated their single mitochondrion but could not sustain mitosis. These studies establish a role for GPIs in intracellular replication of a protozoan parasite of humans. Since GPI-deficient mammalian cells are viable, drugs targeted at the GPI biosynthetic pathway of trypanosomes are likely to be of chemotherapeutic value.

EXPERIMENTAL PROCEDURES

Parasites

Epimastigotes of T. cruzi (Brazil strain) were cultured at 26 °C in liver infusion tryptose (LIT) medium supplemented with 5% heat-inactivated fetal bovine serum (FBS). Infective metacyclic trypomastigotes were obtained from 4-week-old stationary phase cultures of epimastigotes (12). Amastigotes were converted extracellularly in LIT medium from Vero (ATCC-CRL-1586)-derived trypomastigotes (13).

Construction of pTEX.GPI-PLC and Transfection of T. cruzi

An EcoRI fragment of T. brucei GPI-PLC cDNA from plasmid pDH4 (8) was cloned into the EcoRI site of a T. cruzi episomal expression vector pTEX (14). Epimastigotes were transfected (15) with 25 µg of pTEX.GPI-PLC. Following 48 h of incubation at 28 °C, G418 was added to a final concentration of 100 µg/ml for selection of stable transformants, which were eventually subcultured in medium containing 200-800 µg/ml G418. G418-resistant cells were studied 3 months after drug selection.

Cell Lysis, Partial Fractionation, and GPI-PLC Assay

A pellet of 10⁸ cells was lysed on ice in 1 ml of hypotonic buffer (10 mM Na₂HPO₄, 2 mM KH₂PO₄, 13.7 mM NaCl, 8 mM KCl, pH 7.4) containing a protease inhibitor mixture (11). The cell suspension was incubated on ice for 30 min and centrifuged at 14,000 × g for 15 min at 4 °C. The membranous pellet was washed with PBS (10 mM Na₂HPO₄, 2 mM KH₂PO₄, 137 mM NaCl, 8 mM KCl, pH 7.4), and extracted with 500 µl of 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1% Nonidet P-40 (1 × AB). The detergent extract (1 × AB extract) was assayed for GPI-PLC activity using [³H]myristate-labeled variant surface glycoprotein (VSG) of T. brucei as substrate (16).

Metabolic Labeling, Immunoprecipitation, and "Pulse-Chase" Analysis

Amastigotes were washed thrice with PBS and once with methionine-free RPMI 1640 (Life Technologies, Inc.). Parasites (5 × 10⁸) were resuspended in 5 ml of methionine-free RPMI 1640 containing 10% FBS (dialyzed against PBS overnight at 4 °C), 50 mM HEPES, pH 7.0, and incubated for 1 h at 37 °C. Cells were labeled with 250 µCi of [³⁵S]methionine (1322 Ci/mmol; Amersham) for 2 h, washed twice with PBS, and resuspended in RPMI 1640 supplemented with 100 µg/ml nonradioactive methionine (chase medium) at 37 °C, 10% CO₂. Resuspended cells (1 ml) were withdrawn at 0, 6, and 20 h into the "chase," and harvested by centrifugation at 12,000 × g for 5 min at 4 °C. The medium and cell pellet were stored separately at 20 °C until use.

For ethanolamine labeling, washed amastigotes were resuspended in labeling medium (RPMI 1640, 10% FBS, 40 mM HEPES (pH 7.5), 0.2 N NaOH, 20 mM L-glutamine, and 1 × nonessential amino acids (Life Technologies, Inc.) and labeled with 100 µCi/ml [1-³H]ethanolamine hydrochloride (50 Ci/mmol, American Radiolabeled Chemicals Inc.) for 16 h at 37 °C (11). Cells were harvested and stored as described above.

[³⁵S]Methionine or [1-³H]ethanolamine-labeled cells (5 × 10⁷) were lysed in 1 ml of ice-cold immunoadsorption buffer (25 mM Tris-HCl, pH 7.5, 5 mM EDTA, pH 7.5, 250 mM NaCl, 1% Triton X-100) and immunoprecipitated as described (17). Radiolabeled protein was eluted from the protein A-Sepharose-antibody-antigen complex by heating at 95 °C for 5 min in 50 µl of 2.5 × SDS-PAGE buffer and analyzed by 10% SDS-PAGE/fluorography with preflashed X-Omat AR film (Eastman Kodak Co.). [¹⁴C]Methylated proteins (Sigma) were used as molecular weight standards. Quantitation of the fluorograms was performed on an IS-1000 Digital Imaging System (Alpha Innotech. Corp.).

Triton X-114 Phase Partition of GPI-anchored Proteins

[³⁵S]Methionine-labeled wild-type T. cruzi amastigotes (5 × 10⁷ cells) were lysed at 4 °C by incubation for 30 min in 100 µl of Tris-buffered saline (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl) containing 2% precondensed Triton X-114 (18) and protease inhibitor mixture (11). Before lysis, parasites, when indicated, were treated with PI-PLC as described above. The lysate was incubated at 37 °C for 5 min and centrifuged at 14,000 × g for 1 min at room temperature to separate the aqueous (detergent-depleted) and detergent-enriched phases. Triton X-114 (12%) was added to the aqueous phase, while Tris-buffered saline was added to detergent phase to finally have 2% Triton X-114 concentration in both phases. The phase separation was repeated. Aqueous and detergent-enriched phases were pooled separately, and proteins therein precipitated (19) and resolved by SDS-PAGE/fluorography.

Isolation of Glycolipids, and Thin Layer Chromatography

[³H]Ethanolamine-labeled amastigotes (2 × 10⁹ cells) were delipidated and extracted with chloroform/methanol/water (CMW, 10:10:3; Ref. 11). Glycolipids (2 × 10⁷ cell eq) were resolved by thin layer chromatography (TLC) on Silica Gel 60 in CMW, and detected by fluorography.

To isolate GPIs, [³H]EtN-labeled glycolipids were resolved by high performance thin layer chromatography (HPTLC) in CMW on Silica Gel 60 plates. TcAmLP-3 (2 × 10⁹ cell eq) was scraped from the TLC plate and extracted thrice each time with 1 ml of CMW. The eluates were pooled, dried under a stream of nitrogen and redissolved in 100 µl of n-butanol. Samples were stored at 20 °C until use. [³H]Man_1-3GlcN-PI from T. brucei (ILTat at 1.3) bloodstream form were generated in a cell-free system in presence of 0.25 mM phenylmethylsulfonyl fluoride (20).

Partial Structural Analysis of TcAmLP-3

[³H]EtN-labeled T. cruzi glycolipids (2 × 10⁷ cell eq), TLC-purified [³H]TcAmLP-3 (10,000 cpm), or standard [³H]Man_1-3 GlcN-PI (10,000 cpm) were digested in a final volume of 100 µl as follows. To cleave with recombinant GPI-PLC (16), the sample was resuspended in 100 µl of 1 × AB and digested with 100 units (~13 ng) of enzyme at 37 °C for 5 h. For digestion with Bacillus cereus PI-PLC (Boehringer Mannheim), substrate was resuspended in 25 mM EtN-HCl (pH 7.5), 0.27 M sucrose, 1 mg/ml bovine serum albumin, and 0.002% sodium azide containing 1 × 10² units of PI-PLC (17 ng), and incubated for 5 h at 37 °C (21). Phospholipase A₂ (PLA₂, 24 units, from bee venom (Apis melifera), Sigma) was incubated with substrate in 1 × PLA₂ buffer (25 mM HEPES, pH 7.4, 1 mM CaCl₂) for 5 h at room temperature. For mild base treatment, dried TcAmLP-3 or Man_1-3GlcN-PI resuspended in 100 µl of 40% propan-1-ol, 13 M ammonia (1:1) was incubated at 37 °C overnight and then dried under a nitrogen stream at 40 °C. The dried product was resuspended in 15 µl of n-butanol for TLC analysis.

TcAmLP-3 or Man_1-3GlcN-PI were treated with 2 units of jack bean -mannosidase (Oxford Glycosystems, Rosedale, NY) according to manufacturer's instructions at 37 °C overnight, in 200 µl of sodium acetate, pH 5.0, 2 mM zinc acetate containing 0.3% sodium taurodeoxycholate (22).

Products of cleavage were extracted with 300 µl of water-saturated n-butanol, followed by two back extractions of the butanol phase with 300 µl of water. Butanol-soluble products were dried under nitrogen and resuspended in 10 µl of n-butanol. Products from various digestions were resolved by TLC on Silica Gel 60 in CMW (10:10:3) or CHCl₃:MeOH:0.25% KCl (11:9:2) as indicated in figure legends and detected by fluorography.

Microsequencing of GPI-glycans

[³H]EtN-labeled GPIs (from 1-3 × 10⁹ parasites) were scraped after HPTLC in CMW from Silica Gel 60 plates (EM Separations, Gibbstown, NJ). Following dephosphorylation with hydrofluoric acid, the resulting neutral glycan was deaminated with nitrous acid and radiolabeled with sodium [³H]borohydride (DuPont NEN) (23). The [³H]glycans were separated from impurities present in the sodium [³H]borohydride by HPTLC in propanol:acetone:water (PAW, 9:6:5) and elution of the appropriate carbohydrates with PAW. The eluates were dried down, resuspended in 100 µl of 40% propanol, and stored at 20 °C until use. Along with the TcAmLP-3, GPI-glycans Man₃-GlcN and Man₄-GlcN were isolated from T. brucei (24, 25) and Leishmania mexicana (2), respectively, for use as standards.

The reduced neutral [³H]glycan (~20,000 cpm) was resuspended in 100 µl of 100 mM trifluoroacetic acid and heated at 100 °C for 4 h. When needed, complete hydrolysis was carried out in 300 mM trifluoroacetic acid for 8 h. Additionally, the sealed reaction tube was monitored so that liquid which evaporated on to the cap was recovered repeatedly by microcentrifugation. After drying in a SpeedVac, the products were analyzed by HPTLC/fluorography.

Reduced neutral [³H]glycans were digested with jack bean -mannosidase (JBAM; 2.5 units/ml, Boehringer Mannheim) in 100 µl of 100 mM NaOAc, pH 5.0, 1.5 mM ZnSO₄ for 16 h at 37 °C. The reaction was terminated and analyzed as described previously (23).

Dried, deaminated, reduced [³H]glycans were subjected to acetolysis and processed for HPTLC as described (23).

Glycans were resolved on aluminum-backed Silica Gel 60 HPTLC plates, which were developed sequentially in (i) 1-propanol/acetone/water (9:6:5, v/v/v), (ii) 1-propanol/acetone/water (5:4:1, v/v/v), and (iii) 1-propanol/acetone/water (9:6:5, v/v/v) (23).

Flow Cytometric Analysis

T. cruzi cells were washed in PBS containing 0.1% bovine serum albumin (Sigma) and 0.1% sodium azide (PAB). For each assay, 1 × 10⁶ parasites were resuspended in 50 µl of PAB containing purified antibody (100 µg/ml) at either 1:1000 dilution or in undiluted hybridoma supernatants and kept for 30 min at 4 °C. The parasites were washed with 1 ml of PAB and incubated for 30 min at 4 °C in the dark in 50 µl of PAB containing a 1:50 dilution of fluorescein isothiocyanate-labeled affinity-purified goat F(Ab)₂ anti-mouse or anti-rabbit Ig (IgG+IgM) antibody (Southern Biotechnology Associates, Birmingham, AL). Parasites were washed, resuspended in 1 ml of PAB, and analyzed by flow cytometry on an EPICS 753 Elite cytofluorimeter (Coulter Electronics, Hialeah, FL). In some experiments, before staining, parasites (2 × 10⁷) were washed with PBS and then treated with 2 × 10² units of B. cereus PI-PLC.

Growth of T. cruzi Amastigotes in Mammalian Cells

CSWAE1A cells (a neomycin-resistant mouse fibroblast cell line transfected with E1A gene of adenovirus) were irradiated with 7500 Rad to stop their division, and allowed to attach to coverslips in 24-well flat-bottomed culture plates for 24 h at 37 °C, 5% CO₂ in RPMI, 5% FBS containing 400 µg/ml G418. The fibroblasts were infected with metacyclic trypomastigotes (20:1, parasites:CSWAE1A cells) of pTEX/T. cruzi or pTEX.GPI-PLC/T. cruzi previously cultured in 400 µg/ml G418. Infected cells were stained with 1 mM SYTO 11 nucleic acid stain (Molecular Probes Inc., Eugene, OR) at 37 °C for 10 min and visualized with a laser scanning confocal microscope (Bio-Rad, MRC-600).

RESULTS

Epimastigotes (extracellular insect stage form) of T. cruzi were transfected with pTEX.GPI-PLC (see "Experimental Procedures") and selected in G418 to obtain stable transfectants, which were then converted to the different life cycle stages. Epimastigotes harboring pTEX.GPI-PLC and adapted to grow in 200 µg/ml G418 expressed approximately 370 units of GPI-PLC activity/10⁸ cells. Adapting the cells to grow in higher concentrations of G418 (400 or 800 µg/ml) raised the level of GPI-PLC expression to 560 and 750 units (per 10⁸ cells), respectively. The background level of VSG cleavage activity in control pTEX/T. cruzi epimastigotes was approximately 1.1 units/10⁸ cells, representing 336-682-fold less activity than that present in pTEX.GPI-PLC transfectants.

GPI anchors of proteins contain EtN in a conserved core composed of EtN-phospho-6Man1-2Man1-6Man1-4GlcN1-6myo-inositol. To investigate the effect of GPI-PLC expression on free GPIs in vivo, we analyzed [³H]EtN-labeled glycolipids from wild-type and pTEX.GPI-PLC/T. cruzi amastigotes.

GPI-PLC expressing amastigotes were deficient in an [³H]EtN-labeled glycolipid, termed TcAmLP-3 (Fig. 1). As compared with the amount detected in wild-type cells (Fig. 1A, compare lanes 1 and 2), TcAmLP-3 was reduced 5-fold in pTEX.GPI-PLC/T. cruzi (Fig. 1, A and B). A less polar [³H]EtN-labeled glycolipid TcAmLP-4 was decreased 3-fold in pTEX.GPI-PLC/T. cruzi (Fig. 1, A and B). In vitro treatment of glycolipids from wild-type T. cruzi with purified recombinant GPI-PLC resulted in the cleavage of TcAmLP-3 and TcAmLP-4 (Fig. 1C) as well as the molecules labeled as TcAmLP-1 and TcAmLP-2. These results suggested that TcAmLP-1 through TcAmLP-4 are GPIs.

2

The possibility that decreased TcAmLP-3 and TcAmLP-4 in pTEX.GPI-PLC/T. cruzi is the result of a generalized reduction in cellular metabolism can be ruled out, because (i) the lipids migrating close to the solvent front (marked with asterisks) are present in approximately equal quantities in both wild-type and in pTEX.GPI-PLC/T. cruzi (Fig. 1A, compare lanes 1 and 2); (ii) no differences were observed between the wild-type and GPI-PLC transfectants in their [³H]EtN-labeled neutral lipids extracted with chloroform/methanol (Fig. 1A, lanes 3 and 4); and (iii) approximately equal quantities of TcAmLP-5, TcAmLP-6, and TcAmLP-7, none of which was cleaved in vitro by GPI-PLC (data not shown), were detected in wild-type T. cruzi and pTEX.GPI-PLC/T. cruzi (Fig. 1). TcAmLP-7 is not identical to TcAmLP-3, because the former molecule is not cleaved by GPI-PLC in vitro under conditions when the latter is cleaved (data not presented). These observations suggest that pTEX.GPI-PLC/T. cruzi and pTEX/T. cruzi are equally competent in general utilization of [³H]EtN. (We note that on occasion EtN labeling of TcAmLP-1 and TcAmLP-2 was greater than observed for TcAmLP-3 and TcAmLP-4 (see profiles in Fig. 1A and 1C). The data presented in Fig. 1A are more typical, and the occasional variability in the pattern of EtN-labeling does not alter our conclusions.)

Information on the structure of TcAmLP-3 (Fig. 1) was obtained by a combination of enzymatic and chemical cleavages. [³H]EtN-labeled TcAmLP-3 was cleaved in vitro with purified PI-PLC from B. cereus and T. brucei GPI-PLC (Fig. 1D, lanes 1 and 5, respectively). TcAmLP-3 is resistant to JBAM digestion (Fig. 1D, lanes 7 and 8), indicating that the terminal mannosyl (see next sections) was blocked: Under identical conditions, the mannosyl residues of the control GPIs (Man_1-3GlcN-PI) were cleaved by JBAM (Fig. 1D, lane 12). Treatment with phospholipase A₂ did not affect the mobility of TcAmLP-3 (Fig. 1D, lane 4). As a positive control, [³H]mannose-labeled Man_1-3GlcN-PI from T. brucei was digested with PLA₂ resulting in the loss of label (Fig. 1D, lane 10). The lyso species of Man_1-3GlcN-PI was detected in the aqueous phase of the butanol extraction (data not shown). TcAmLP-3, as well as Man_1-3GlcN-PI, was sensitive to base treatment (Fig. 1D, lanes 6 and 11, respectively). Thus, the inability of PLA₂ to cleave TcAmLP-3 suggests it contains a fatty acid only in the sn-1 position of a glycerolipid, or that the alkyl group is derived from ceramide. The latter possibility is excluded by the mild base sensitivity of TcAmLP-3. We infer from these properties that TcAmLP-3 is a GPI containing a phosphoglycerol backbone, which is acylated at the sn-1 position.

The neutral glycan present in TcAmLP-3 (NG-TcAmLP-3) was characterized. GPI-glycan standards Man₃-anhydromannitol (Man₃-AHM) and Man₄-AHM from T. brucei (26) and L. mexicana (2), respectively, were used as standards. The HPTLC mobility of NG-TcAmLP-3 indicated that it was smaller than Man₃-AHM (Fig. 2A, lanes 1-3). By partial acid hydrolysis, a ladder of glycans containing Man₁-AHM, Man₂-AHM, and Man₃-AHM were generated from Man₃-AHM and Man₄-AHM (Fig. 2B, lanes 2 and 5, respectively). Comparing the mobility of these glycans with NG-TcAmLP-3 (Fig. 2B, lanes 6 and 7) showed that Man₁-AHM was the major component of TcAmLP-3.

NG-TcAmLP-3 was subjected to acetolysis and JBAM treatment (Fig. 2C, lanes 7-9). After acetolysis, no change in the mobility of NG-TcAmLP-3 occurred, indicating the absence of an 1-6 glycosidic bond. Control Man₃-AHM and Man₄-AHM were both cleaved with production of Man₁-AHM, as expected (Fig. 2C, lanes 2 and 5). Digestion of the NG-TcAmLP-3 with JBAM resulted in cleavage only of a minor species, which comigrated with Man₃-AHM; AHM was not released from the T. cruzi Man₁-AHM species (Fig. 2C, lane 9). (AHM was produced from the Man₃-AHM and Man4-AHM standards (Fig. 2C, lanes 3 and 6).) Nevertheless, TcAmLP-3 can be metabolically labeled with [³H]mannose (data not presented) and with [³H]EtN (Fig. 1). In addition, trifluoroacetic acid released [³H]AHM from the neutral [³H]glycan of TcAmLP-3 (Fig. 2B, lane 8), denoting the existence of glycosidic bonds in that molecule. The JBAM resistance suggests that the Man residue is most likely not in an 2, 3, or 6 linkage, since the enzyme's specificity for the terminal mannoses is 2Man, 6Man > 3Man. An 4Man linkage in NG-TcAmLP-3 (see below) might exhibit some relative resistance to JBAM.

In summary, TcAmLP-3 consists predominantly of EtN-phospho-Man₁-GlcN-Ins-phospho-sn-1-glycerolipid. Although we did not specifically determine the location of the EtN, we presume that it is found on the single mannosyl substituent, since it is the only component of GPI-glycans known to accept the phospho-EtN (reviewed in Ref. 2).

The effect of a GPI deficiency (see Fig. 1A) on the expression of cell surface proteins was assessed by staining three developmental stages (epimastigotes, trypomastigotes, and amastigotes) of T. cruzi with antibodies raised against GPI-anchored proteins. Flow cytometric analysis showed that in all three stages, the cell surface expression of GPI-anchored proteins was decreased in the GPI-deficient strain (pTEX.GPI-PLC/T. cruzi) relative to control wild-type or pTEX/T. cruzi parasites (Fig. 3). In GPI-deficient epimastigotes stained with anti-gp50/55 (Fig. 3A) and in amastigotes stained with anti-Ssp-4 or anti-gp50/55 (Fig. 3, C and D, respectively), the decrease in expression of these GPI-anchored proteins was similar to that obtained when wild-type parasites were treated with an excess of extracellularly added phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus. Trypomastigotes of pTEX.GPI-PLC/T. cruzi also showed less cell surface expression of a trans-sialidase (evident by anti-SAPA (shed acute phase antigen) staining (Fig. 3B)). The general decrease in cell surface expression of proteins in pTEX.GPI-PLC/T. cruzi was specific for GPI-anchored molecules, as shown by staining with the anti-amastigote antibody IC10B²; expression of the molecule recognized by this monoclonal antibody was similar in wild-type and GPI-deficient amastigotes (Fig. 3E).

To investigate the fate of GPI-anchored proteins, amastigotes were metabolically labeled with [³⁵S]methionine and chased. For both cells and "chase medium," a profile of [³⁵S]methionine-labeled proteins was obtained after SDS-PAGE and fluorography.

Significant differences were observed between the major [³⁵S]methionine-labeled amastigote proteins of pTEX.GPI-PLC/T. cruzi and the wild-type control cells. Two proteins of  60 kDa (p60 doublet, marked with arrows; Fig. 4A) present in wild-type cells at the beginning of the chase period, remained unchanged relative to other proteins (in the same lysate) during a 20-h chase (Fig. 4A, compare lanes 1, 3 and 5). The p60 doublet was absent from pTEX.GPI-PLC/T. cruzi even at the beginning of the chase (Fig. 4A, compare lanes 2, 4, and 6), and was not detected in amastigotes labeled for 15 min with [³⁵S]methionine (data not shown). Apparently, p60 is either not expressed at all, or is degraded very rapidly in the GPI-deficient cells. A 50-kDa protein, p50 (marked with asterisk, Fig. 4A) was expressed in wild-type T. cruzi at levels apparently similar to p60 and remained relatively unchanged during a 20-h chase. In pTEX.GPI-PLC/T. cruzi, p50 was present in slightly higher amount at 0 h than in wild-type cells (Fig. 4A, lane 1 and 2). The level of this protein remained unchanged within the first 6 h (Fig. 3A, lane 4), but only 30% of the total remained after 20 h (Fig. 4A, compare lanes 2 and 6).

Since the identities of p60 (Fig. 4A) and p50 were unknown and we have no antibodies specific to either protein, the possibility that they are GPI-anchored was checked by Triton X-114 phase partition before and after PI-PLC digestion. Membrane fractions from a hypotonic lysate of the parasites were studied for this purpose. The [³⁵S]methionine-labeled p60 doublet, similar to p50, was in the detergent phase prior to PI-PLC digestion (Fig. 4B, lanes 1 and 2). In contrast, both p60 and p50 partitioned into the aqueous phase after treatment with PI-PLC (Fig. 4B, lanes 3 and 4), indicating that p60 and p50 are GPI-anchored.

The fate of a known GPI-anchored protease, gp50/55, was monitored by immunoprecipitation of total cell lysate and chase medium from [³⁵S]methionine-labeled amastigotes with monoclonal antibody C10 (21). In pTEX/T. cruzi, 55% of gp50/55 was retained in the cell after a 24-h chase (Fig. 4, C, lanes 4-6, and D). In contrast, only 10% of gp50/55 is retained in GPI-deficient amastigotes at 24 h (Fig. 4, C, lanes 1-3, and D). Most likely, gp50/55 is degraded within the cell, since it was never detected in the chase medium of either pTEX/T. cruzi or pTEX.GPI-PLC/T. cruzi. Thus, GPI-deficient amastigotes degrade gp50/55 approximately 5 times faster than wild-type T. cruzi.

We examined the basis of the decreased cell surface expression of Ssp-4 (Fig. 4) by studying the kinetics of Ssp-4 cell association. Release of Ssp-4 was monitored by immunoprecipitation from chase medium of [³⁵S]methionine-labeled amastigotes with an Ssp-4-specific monoclonal antibody 2C2. In wild-type or pTEX/T. cruzi, significant release is noticeable after 20 h of chase (Fig. 5A, lanes 1-3). In pTEX.GPI-PLC/T. cruzi, however, an amount of Ssp-4 equivalent to that released in 20 h from wild-type cells is detectable only 6 h into the chase (Fig. 5A, compare lane 3 with lane 5). Furthermore, at the 6-h time point, 5-fold more Ssp-4 ( 180 kDa) was released by the GPI-deficient T. cruzi as compared with wild-type parasites (Fig. 5, A, compare lanes 2 and 5, and B).

Rapid and increased release of Ssp-4. A, GPI deficiency increases the rate and extent of Ssp-4 secretion. Amastigotes were metabolically labeled with [³⁵S]methionine and chased for 0 h (lanes 1 and 4), 6 h (lanes 2 and 5), and 20 h (lanes 3 and 6). ³⁵S incorporation into macromolecules was determined by trichloroacetic acid precipitation of a lysate of 10⁶ cells to be 27,696 cpm and 29,323 cpm for wild-type and pTEX.GPI-PLC/T. cruzi, respectively.) Ssp-4 was immunoprecipitated from chase medium (5 × 10⁷ cell eq) with mAb 2C2. Lanes 1-3, T. cruzi; lanes 4-6, pTEX.GPI-PLC/T. cruzi. B, quantitation of secreted Ssp-4. Fluorographic images of Ssp-4 secreted into culture media (A) were quantitated on an IS-1000 Digital Imaging System. Open bars, T. cruzi; hatched bars, pTEX.GPI-PLC/T. cruzi. C, other proteins released into the chase medium. [³⁵S]Methionine-labeled amastigotes (2 × 10⁷ parasites) were chased for 0, 6, and 20 h. Proteins in the corresponding culture medium from chases at 0 h (lane 1, T. cruzi; lane 2, pTEX.GPI-PLC/T. cruzi), 6 h (lane 3, T. cruzi; lane 4, pTEX.GPI-PLC/T. cruzi), and 20 h (lane 5, T. cruzi; lane 6, pTEX.GPI-PLC/T. cruzi) were analyzed by SDS-PAGE and fluorography.

The chased medium was also examined for proteins not detected inside GPI-deficient T. cruzi (i.e. as compared with wild-type cells; Fig. 4A). The predominant radiolabeled proteins in the medium were 50, 56, 75, and 180 kDa (p50, p56, p75, and p180, respectively) (Fig. 5C). Interestingly, the predominant secreted proteins p75 and p180 were not the major polypeptides in the total cell lysate (compare Figs. 4A and 5C). The latter observation indicates that secretion of proteins did not occur en mass. Several other proteins of less than 45 kDa were secreted in the GPI-deficient cells (Fig. 5C, lanes 4 and 6).

In conclusion, two effects of a GPI deficiency are observed on amastigote proteins that are normally GPI-anchored, as exemplified by Ssp-4 and gp50/55: (i) a 5-fold acceleration of secretion (Fig. 5, A and B), and (ii) a 5-fold increase in the rate of intracellular degradation (Fig. 4, B and C).

The GPI deficiency in T. cruzi was associated with striking phenotypic changes. Metacyclic trypomastigotes obtained from epimastigotes that had been cultured in 200 µg/ml G418 infected various mammalian fibroblast cell lines, differentiated into and replicated slowly as amastigotes, but still completed the life cycle.³ However, transfectants cultured at 400 µg/ml G418, thereby increasing the level of expression of GPI-PLC, infected mammalian cells and differentiated into amastigotes but divided only once.

GPI-deficient amastigotes in most cases replicated the kinetoplast, but failed to sustain replication of the cell nucleus, and were di-kinetoplastid (Fig. 6). Hence, GPI-deficient T. cruzi amastigotes are arrested in the cell cycle apparently in anaphase. In agreement with these observations, replication of GPI-deficient T. cruzi was severely inhibited such that a lawn of CSWAE1A cells, which is normally lysed in 4 days if infected with wild-type T. cruzi, remained intact after 10 days of infection with GPI-deficient parasites.³

DISCUSSION

One of our major interests was to examine the effect of a GPI deficiency on T. cruzi. However, studies of GPI biosynthesis in T. cruzi have only recently been initiated (27-29). No genes involved in this pathway have been cloned. In the absence of genes for targeted mutagenesis of the GPI pathway, we generated cells with this desired deficiency by stable expression in T. cruzi of a GPI-PLC gene from the related protozoan parasite T. brucei. This approach was used previously to explore the topography of the protein and polysaccharide-GPI pathways in Leishmania (11).

GPI-PLC requires GlcN(1-6)Ins for efficient substrate recognition (30) and is therefore highly specific for GPIs.⁴ The enzyme cleaves most GPI biosynthetic intermediates in vivo (Fig. 1), thereby conferring on pTEX.GPI-PLC/T. cruzi a phenotype equivalent to a dominant-negative loss of function mutation in the GPI pathway. The severity of the phenotype was dependent upon the amount of GPI-PLC expressed, which was in turn driven by the concentration of G418 used in the culture medium. At a permissive G418 concentration (i.e. <200 µg/ml), biochemical studies were possible (Figs. 1, 2, 3, 4, 5), since the parasite completed its life cycle. However, at a nonpermissive level of G418 (400 µg/ml), replication and differentiation of pTEX.GPI-PLC/T. cruzi amastigotes was inhibited.³ These observations are reminiscent of the temperature-sensitive yeast mutants in GPI biosynthesis, which fail to grow at the nonpermissive temperature (5, 6).

TcAmLP-3 is the first GPI to be characterized from the amastigote stage of T. cruzi (Figs. 1, 2). Its depletion in GPI-PLC expressing cells (Fig. 1, A and B) is diagnostic of a GPI deficiency. The structure of TcAmLP-3 is EtN-P-Man-GlcN-Ins-phosphoglycero-sn-1-lipid (reminiscent of H5, a mammalian GPI; Refs. 22 and 31). This situation contrasts with GPIs from the epimastigote stage of T. cruzi, where only the Man₄ species contains EtN-P (32, 33). Apparently, the pathway for GPI biosynthesis is developmentally regulated in T. cruzi (or stage-specific), as documented for T. brucei (2, 34).

The GPI deficiency in T. cruzi causes a decrease in cell surface expression of proteins that are normally GPI-anchored (Fig. 3). Some of these proteins are secreted constitutively (e.g. Ssp-4 (Fig. 5, A and B)), while others are degraded intracellularly (e.g. p60 (Fig. 4A) and protease gp50/55 (Fig. 4, B and C)).

In murine Thy-1-negative T cell lymphomas, degradation of proteins during a GPI deficiency occurs in class A, C, and H complementation groups (3). Secretion of Thy-1 occurs in class E and B GPI-deficient Thy-1-negative cells (3). However, our work with T. cruzi provides the first evidence that both secretion and degradation of proteins with GPI signal sequences can occur in an identical genetic background during a GPI deficiency.

How could proteins with functional GPI signal sequences acquire different fates although present in the same cell? We hypothesize entry into either route (i.e. intracellular degradation or secretion) is determined by the primary structure of the protein under consideration.

A protein-GPI transamidase (PGTase) normally transfers prefabricated GPI anchors to the cleavage/addition site of the COOH-terminal GPI signal sequences in the endoplasmic reticulum (ER) (reviewed in Ref. 35). During a GPI shortage, the absence of one substrate (i.e. GPIs) could affect the interaction of PGTase with its second substrate (i.e. COOH-terminal GPI signal sequence). For example, a protein with a high affinity GPI signal sequence might outcompete another protein with a weak GPI signal sequence for binding to PGTase. Binding to PGTase in the absence of GPIs can result in cleavage of the COOH-terminal GPI signal sequence, in a half-reaction of the normal transamidation, possibly followed by secretion of the cleaved protein. Cleavage of a GPI signal sequence without attachment of a GPI has been documented (36, 37). Proteins with lower affinity GPI signal sequences (i.e. with regard to binding the PGTase), being outcompeted for binding to the transamidase, might not be cleaved and could be retained in the cell initially. Extended lingering in the ER, presumably attached to the lumenal membrane by a hydrophobic COOH terminus, coupled with exposure of degradation signals (38), might result in proteolysis. In effect, during a GPI shortage, proteins with low affinity GPI signal sequences might be analogous to proteins with "uncleavable GPI signal sequences," which are degraded in an ER/post-ER compartment (39, 40), while proteins with high affinity GPI signal sequence are secreted.

Unlike most eukaryotic cells, which contain numerous mitochondria, the Trypanosomatidae contain a single mitochondrion (kinetoplast) whose replication is coordinated with nuclear division. In T. brucei, initiation of kinetoplast replication precedes mitosis in S phase with completion of nuclear division after successful kinetoplast replication (41). That this temporal order of kinetoplast and nuclear division occurs in T. cruzi as well is supported by our data. In GPI-deficient T. cruzi kinetoplast division can be uncoupled from division of the cell nucleus.

We speculate that GPIs might act by one of the two general mechanisms to influence nuclear division. First, lack of some GPI-anchored proteins or free GPIs unattached to proteins might lead to the inhibition of mitosis. Both these classes of putative mediators (i.e. free GPIs and GPI-anchored proteins), however, are expected to be found mainly on the plasma membrane of these intracellular amastigotes, so how could they function? First, they could be receptors for signals originating from the cytoplasm of the mammalian cells to trigger amastigote proliferation. Second, it is possible that free GPIs (or a GPI-anchored protein) control nuclear division in T. cruzi. Here, it is worth recalling that the ER, where GPIs are synthesized (11, 42), is continuous with the outer membrane of the cell nucleus. This implies that free GPIs and/or GPI-anchored proteins may be present on nuclear membranes from where they could influence mitosis.