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Golgi UDP-GlcNAc:Polypeptide O-α-N-Acetyl-d-Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi

ABSTRACT All life cycle stages of the protozoan parasite Trypanosoma cruzi are enveloped by mucin-like glycoproteins which, despite major changes in their polypeptide cores, are extensively and similarly O-glycosylated. O-Glycan biosynthesis is initiated by the addition of αGlcNAc to Thr in a reaction catalyzed by Golgi UDP-GlcNAc:polypeptide O-α-N-acetyl-d-glucosaminyltransferases (ppαGlcNAcTs), which are encoded by TcOGNT1 and TcOGNT2. We now directly show that TcOGNT2 is associated with the Golgi apparatus of the epimastigote stage and is markedly downregulated in both differentiated metacyclic trypomastigotes (MCTs) and cell culture-derived trypomastigotes (TCTs). The significance of downregulation was examined by forced continued expression of TcOGNT2, which resulted in a substantial increase of TcOGNT2 protein levels but only modestly increased ppαGlcNAcT activity in extracts and altered cell surface glycosylation in TCTs. Constitutive TcOGNT2 overexpression had no discernible effect on proliferating epimastigotes but negatively affected production of both types of trypomastigotes. MCTs differentiated from epimastigotes at a low frequency, though they were apparently normal based on morphological and biochemical criteria. However, these MCTs exhibited an impaired ability to produce amastigotes and TCTs in cell culture monolayers, most likely due to a reduced infection frequency. Remarkably, inhibition of MCT production did not depend on TcOGNT2 catalytic activity, whereas TCT production was inhibited only by active TcOGNT2. These findings indicate that TcOGNT2 downregulation is important for proper differentiation of MCTs and functioning of TCTs and that TcOGNT2 regulates these functions by using both catalytic and noncatalytic mechanisms.

T rypanosoma cruzi is a protozoan parasite that causes Chagas disease, a debilitating condition that affects millions of humans in the American continents (1). T. cruzi traverses a complex life cycle that involves extracellular proliferation and differentiation in the digestive tract of an insect vector (2) and intracellular proliferation and differentiation in a wide range of vertebrate hosts (3). In the insect, noninfective epimastigote forms colonize the digestive tract and Malpighian tubes, attach to the cuticle of the rectal epithelium (4), and differentiate into nondividing infective metacyclic trypomastigotes (MCTs), in a process known as metacyclogenesis (5). During a blood meal, MCTs detach and are expelled together with epimastigotes within feces and urine, where they can gain access to internal body fluids via a skin discontinuity or a mucosal surface and invade host cells (6). In addition, because MCTs can also invade the gastric mucosal epithelium, T. cruzi is an emerging food-borne parasite responsible for frequent outbreaks of acute, orally contracted cases of Chagas disease that are characterized by high mortality (7).
Metacyclogenesis is usually analyzed in vitro by using axenic media that allow initial epimastigote proliferation (28) followed by transformation into MCTs (29,30). Nutritional stress is a major factor both in vitro and in vivo and prompts initial substratum adhesion of epimastigotes, followed by release and morphological transformation associated with nuclear structure changes and migration of the kinetoplast to the anterior end of the cell (14,31,32). Pharmacological and biochemical studies indicate the involvement of increased cyclic AMP (cAMP) and activation of protein kinase A (PKA) (33)(34)(35). Cruzipain expression initially increases upon adhesion (36), followed by a decrease and reduced reservosome volume as metacyclogenesis proceeds (37). Additional changes include a sharp decrease in the amount of glycoinositolphospholipids (38) and induction of stage-specific antigens, such as gp90 and gp82, that mediate adhesion to and infection of vertebrate host cells (7). MCTs do not divide and are resistant to antibody-independent complement-mediated lysis (39), owing to a surface glycoprotein that inhibits the formation of the alternative and classical C3 convertase (40). MCTs are infective toward mammalian cells, following which they proliferate intracellularly as amastigotes prior to emergence as infective tissue culture-derived trypomastigotes (TCTs).
We previously identified two genes (TcOGNT1 and TcOGNT2) predicted to encode Golgi pp␣GlcNAcTs, as well as a third, divergent gene, that are conserved throughout the trypanosomatids. TcOGNT2 was heterologously expressed in Leishmania tarentolae as an N-terminally truncated secreted protein, purified to near homogeneity, and found to possess peptide-dependent pp␣GlcNAcT activity and peptide-independent UDP-GlcNAc hydrolytic activity (21). TcOGNT2 evolved from an ancient lineage of polypeptide ␣HexNAc transferases that also provided cytoplasmic hydroxyproline pp␣GlcNAcTs in other protists and the well-known pp␣GalNAcT family in animals (41) but was specific for Thr as the amino acid acceptor and UDP-GlcNAc as the donor. As expected for this lineage, both activities depended on a DxD-like DxH sequence motif and a divalent cation (21). In comparison to the single Golgi pp␣GlcNAcT (DdGnt2) from Dictyostelium (42,43), however, the T. cruzi enzyme was not expressed well and exhibited low in vitro specific activity and high hydrolysis activity. These differences, observed using several peptide substrates, imply that other factors may be important for TcOGNT2 function in the cell, such as its transmembrane anchor, posttranslational modifications, or availability of a limiting factor, such as TcOGNT1 or TcOGNTL (21).
Little is known about the regulation of O-glycosylation in T. cruzi. The prediction that the initial step involves only two enzyme isoforms, in contrast to the myriad subsequent processing glycosyltransferases (GTs), implies a potential gatekeeper role which can now be analyzed owing to identification of the genes (21). Here we show that in comparison to proliferating insect-form epimastigotes, differentiated infective MCTs and TCTs downregulate their Golgi-associated pp␣GlcNAcT activities, which is explained at least in part by reduced levels of the TcOGNT2 protein. Forced overexpression of TcOGNT2myc overcame the na-tive enzyme downregulation and impaired formation of MCTs and TCTs, indicating an importance of downregulation for both pathways of trypomastigote production. Detailed analysis indicated that MCT formation was inhibited by sustained TcOGNT2 expression after substratum adhesion and downstream of cAMP signaling, did not involve cruzipain inhibition, and did not depend on the catalytic activity of TcOGNT2. TCT formation appeared to be inhibited at the cell invasion step by a mechanism that, in contrast, did depend on the catalytic activity of TcOGNT2 and O-glycan remodeling. These findings imply multiple functions for TcOGNT2 and highlight an important role of the Golgi apparatus in developmental transitions critical for T. cruzi pathogenesis.
Proliferation studies were initiated with 10 5 parasites ml Ϫ1 in standard medium (with or without G418) in 24-well plates, and cell density was quantitated daily in a Neubauer chamber. Three independent experiments were performed in triplicate. Metacyclogenesis was induced as previously described (29). Briefly, epimastigotes in transition from logarithmic to stationary phase were adjusted to 5 ϫ 10 8 parasites ml Ϫ1 in triatomine artificial urine (TAU) medium (190 mM NaCl, 17 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 0.035% sodium bicarbonate, 8 mM phosphate, pH 6.0). After 2 h at 28°C, cultures were diluted 100-fold in 10 ml TAU medium supplemented with 10 mM L-proline (TAU-P) and 500 g ml Ϫ1 G418 and transferred to a T25 flask kept on its side at 28°C. As indicated, the TAU-P medium was supplemented with 25 mM or 250 mM glucose (Glc), glucosamine (GlcN), or N-acetylglucosamine (GlcNAc) or 2 mM dibutyryl-cAMP (db-cAMP) (Sigma-Aldrich) to promote metacyclogenesis. After 3 to 5 days, parasite numbers were quantified in a Neubauer chamber, and the percentage of MCTs was estimated by morphology after Giemsa staining and/or resistance to lysis by the alternative pathway of complement (39). At least three independent experiments were performed in duplicate, and the results were analyzed in GraphPad Prism.
TCTs. TCTs were obtained from infected monkey kidney epithelial (LLCMK 2 ) cells as described previously (44). Prior to infection, LLCMK 2 cells were plated at 5 ϫ 10 4 cells ml Ϫ1 in RPMI 1640 medium containing 2% FBS in T25 flasks (Corning) and grown for 18 h at 37°C in a humidified atmosphere of 7% CO 2 . Cells were initially infected with MCTs obtained from TAU-P medium (see below) at a ratio of 40 cell Ϫ1 by incubation for 18 h at 37°C. Cultures were then washed with RPMI 1640, and after about 2 weeks, TCTs were collected from culture supernatants and used to infect new LLCMK 2 cell cultures (4 h of interaction at a ratio of 20 TCTs cell Ϫ1 ). TCTs first appeared in the culture supernatant after 4 days, and their continuous release was quantified by direct counting. TCT attachment/invasion was assayed as previously described (45). Briefly, LLCMK 2 cells attached to glass coverslips in 24-well plates were incubated with TCTs (at a ratio of 50 cell Ϫ1 ) for 1 h at 37°C, washed 5 times with phosphate-buffered saline (PBS) to remove extracellular parasites, fixed with Bouin's solution (71.4% saturated picric acid, 23.8% formaldehyde, and 4.8% acetic acid), washed with 70% ethanol (EtOH), and stained with InstantProv hematological stain (NewProv, Brazil). The number of cells per field and the number of infected cells per field were estimated by direct counting of at least 200 fields. Assays comparing the proliferation of intracellular amastigotes were performed similarly, after an interaction of 4 h (20 parasites cell Ϫ1 ) followed by 48 h of culture before fixation and staining (46). The numbers of total and infected LLCMK 2 cells per field and the number of amastigotes per infected cell were estimated by direct counting of at least 300 fields.
Rhodnius prolixus midgut binding and in vivo assays. R. prolixus insects were reared as described previously (47), following the guidelines of the CCS-UFRJ Animal Care Ethics Committee. Adult females in their third feeding cycle were used at 2 weeks postfeeding. Binding assays were performed as previously described (48). In summary, epimastigotes were washed 3 times with BHI medium without FBS and suspended in the same medium at 10 7 parasites ml Ϫ1 . Posterior midguts dissected and longitudinally sectioned to expose their luminal surfaces (5 group Ϫ1 ) were placed in excavated slides together with 200 l of the above-described suspension of parasites and incubated for 1 h at room temperature in a humidified chamber. Midguts were washed 5 times with PBS, individually transferred to Eppendorf microcentrifuge tubes containing 50 l of PBS, and homogenized. Released epimastigotes were counted in a Neubauer chamber.
The in vivo infection assay was modified from the work of Mello et al. (49), using fifth-instar larvae starved for 30 days after the last ecdysis. Briefly, groups of 30 insects were infected orally with epimastigotes diluted in complement-inactivated rabbit blood (1.5 ϫ 10 8 parasites ml Ϫ1 ) by use of an artificial apparatus (50). After intervals of 3, 10, 17, and 24 days, 5 to 7 insects were dissected, the whole digestive tract was removed and homogenized in 0.2 to 1 ml PBS, and the number of parasites was quantified by phase-contrast optical microscopy using a Neubauer chamber.
Antibodies. Antibodies against the bacterially expressed catalytic domain of TcOGNT2 were raised in mice. Escherichia coli Rosetta(DE3) harboring pET15TEVTcOGNT2cat (21) was induced with 0.75 mM isopropyl-1-thio-␤-D-galactopyranoside for 18 h at 18°C, centrifuged at 4,000 ϫ g for 10 min at 4°C, suspended in 300 g ml Ϫ1 lysozyme in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5% (wt/vol) glycerol, 1 mM dithiothreitol (DTT), and protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF] and 1 g ml Ϫ1 leupeptin), and stirred for 30 min on ice. The suspension was brought to 1 g ml Ϫ1 DNase and 5 mM MgCl 2 for 30 min on ice, lysed by probe sonication, and centrifuged at 30,000 ϫ g for 40 min at 4°C. The TcOGNT2cat-rich inclusion bodies were washed 4 times with 2% (wt/vol) Triton X-100, 2 M urea, 100 mM Tris-HCl (pH 7.5), 5 mM Na 2 EDTA, 5 mM DTT, 1 mM PMSF, and 1 g ml Ϫ1 leupeptin, centrifuged at 22,000 ϫ g for 30 min at 4°C, washed a final time in the same buffer lacking Triton X-100, and resuspended in 8 M urea in 100 mM Tris-HCl (pH 7.5) and 0.5 M NaCl (solubilization buffer). After centrifugation at 100,000 ϫ g for 1 h at 10°C, the supernatant was adjusted to a final concentration of 5 mM imidazole and applied twice to a 1-ml His-Trap HP column (GE Healthcare) preequilibrated with the same buffer. TcOGNT2cat, which eluted in the 0.25 M and 0.5 M imidazole fractions, was precipitated with 9 vol of ice-cold EtOH, collected by centrifugation, and suspended in phosphate-buffered saline. Five female BALB/c mice (10 to 12 weeks old) were immunized by subcutaneous injection with 100 g of TcOGNT2cat in a 1:1 emulsion with Freund's complete adjuvant (Sigma) twice within a 1-week interval and then boosted by intraperitoneal injection at 2-week intervals with a similar preparation using Freund's incomplete adjuvant. The immunization protocol was approved by the Ethics Committee of the Centro de Ciências da Saúde of UFRJ (protocol number IBCCF-085). Crude antisera were affinity purified by adsorption to recombinant TcOGNT2cat purified from the culture supernatant of L. tarentolae (21) and coupled to CNBr-activated Sepharose-4B (Sigma) according to the manufacturer's instructions. Anti-TcOGNT2 was eluted with 0.1 M glycine, pH 2.4, neutralized with 1 M Tris-HCl, and frozen at Ϫ80°C until use.
Immunofluorescence microscopy. Three-to four-day epimastigotes or metacyclics from TAU-P medium were washed 3 times with PBS, and the suspension was fixed in 4% paraformaldehyde in PBS for 1 h at room temperature, washed twice with PBS, allowed to adhere to poly-L-lysinecoated coverslips for 15 min, permeabilized using 0.4% saponin for 15 min in PGN (PBS, pH 7.2, 0.2% gelatin, and 0.1% NaN 3 ), and blocked using 50 mM NH 4 Cl for 30 min, followed by PBS-3% bovine serum albumin (BSA) for 30 min. Samples were probed with preimmune or immune murine anti-TcOGNT2cat (1:50), affinity-purified anti-TcOGNT2 (1:100), anti-gp90 (1:100), or anti-cruzipain (1:200) in PGN with 0.1% saponin and incubated overnight at 4°C. After washing with PGN, samples were incubated with Alexa Fluor 546-or 488-conjugated goat antimouse and/or Alexa Fluor 568-conjugated goat anti-rabbit (Molecular Probes) diluted 1:500 in PGN for 1 h at room temperature. For Golgi complex labeling (55), 1 ϫ 10 7 (4-day) epimastigotes were washed and resuspended in 150 l of cold BHI medium without FBS. BODIPY FL C 5 -ceramide (Avanti Polar Lipids, Inc.) complexed to fatty acid-free BSA (5 M) was added. After 1 h of incubation on ice, the cells were washed with cold BHI medium without FBS and suspended in 150 l of the same 37°C prewarmed medium, followed by 30 min of incubation at 37°C to allow dye uptake. Cells were then washed, fixed, and processed for fluorescence microscopy as described above. Coverslips were washed in PBS and mounted on slides by use of Vectashield mounting medium with 4=,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Images were obtained using a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Jena, Germany) coupled to a charge-coupled device (CCD) color video camera or a Leica TCS NT confocal microscope and processed with Leica confocal software.
Flow cytometry. Flow cytometry was performed as described by Bourguignon et al. (56). Briefly, 5 ϫ 10 6 epimastigotes or 2 ϫ 10 6 TCTs were washed 3 times in PBS and incubated in a final volume of 200 l of PBS plus 0.1 mM CaCl 2 and 0.1% sodium azide, with or without 5 g ml Ϫ1 wheat germ agglutinin (WGA), 5 g ml Ϫ1 Canavalia ensiformis agglutinin (ConA), or 30 g ml Ϫ1 isolectin GS-IB 4 from Griffonia simplicifolia, each conjugated with Alexa 488 (Molecular Probes, Invitrogen), 20 g/ml Ϫ1 Maackia amurensis agglutinin (MAA) conjugated with fluorescein isothiocyanate (FITC) (E-Y Laboratories), or 10 g/ml Ϫ1 Arachis hypogaea agglutinin (PNA) conjugated with tetramethyl rhodamine isocyanate (TRITC) (E-Y Laboratories), for 30 min on ice. After washing twice in PBS, parasites were fixed in 4% paraformaldehyde in PBS for 15 min at 4°C and kept on ice until data acquisition with a FACSCalibur flow cytometer (Becton-Dickinson Biosciences) and analysis with Summit (Dako Cytomation). Five thousand events were acquired in the region previously established to correspond to the parasites. Data were obtained from 3 independent experiments.
Site-directed mutagenesis of TcOGNT2. The amino acid sequence of TcOGNT2 was altered by site-directed mutagenesis of pTEX-TcOGNT2(DSH)myc as previously described (21). The D234A and D234N substitutions were generated as described before, and the H236D mutation was generated using primers TcE5H236Dfor (5=-GTGATTGA CAGCGATTCTAGATTTTGTGCCTGAGTGG) and TcE5H236Drev (5=-CACAAATCTAGAATCGCTGTCAATCACCATATAGTAATCC), which yielded a C706G mutation and a novel XbaI restriction site.
Subcellular fractionation of epimastigotes. Subcellular fractionation was performed by sucrose differential centrifugation as described previously (61). Briefly, epimastigotes (ϳ4 ϫ 10 10 ) were collected at 5,860 ϫ g for 20 min at 4°C, washed twice in TES buffer (50 mM Tris-HCl, pH 7.4, 5 mM Na 2 EDTA, and 250 mM sucrose), and lysed to Ͼ98% (based on microscopy) by abrasion in a chilled mortar with an equal volume of Carborundum (Fluka). The homogenate was suspended in 5 ml TES buffer with a 1:50 dilution of protease inhibitors (GE Healthcare), and unbroken cells and nuclei were removed by 3 cycles of centrifugation at 1,500 ϫ g for 10 min at 4°C and resuspension. The supernatants (S 1.5 ) were combined and diluted to 20 ml in the same buffer. The S 1.5 sample was centrifuged at 5,000 ϫ g for 10 min at 4°C to produce a large granular fraction pellet (P 5.0 ), and the resulting supernatant was centrifuged at 12,350 ϫ g for 10 min at 4°C to produce a small granular pellet (P 12 ). A final centrifugation at 105,000 ϫ g for 1 h at 4°C yielded the microsomal fraction (P 100 ) and the cytosolic fraction, or S 100 . Each pellet was resuspended in 3 ml of TES buffer with protease inhibitors and stored at Ϫ80°C. The amount of protein was quantified using the Bradford method (62), with BSA as the standard.
Enzymatic deglycosylation. Microsomal fractions (100 g of protein) were adjusted to 0.5% SDS, 40 mM DTT, in a final volume of 10 l and heated at 100°C for 10 min. For endo-␤-N-acetylglucosaminidase H (endo H) treatment, samples were adjusted to 100 l containing 5 mM citrate buffer (pH 5.5) and 250 mU of active or heat-inactivated (75°C for 10 min) endo H (New England BioLabs Inc.). For peptide N-glycosidase F (PNGase F) treatment, samples were adjusted to 100 l with 5 mM sodium phosphate buffer (pH 7.5) containing 1% NP-40 and 150 mU of active or heat-inactivated peptide N-glycosidase F (New England BioLabs Inc.). After 1 h at 37°C, proteins were precipitated with 90% cold ethanol and centrifuged at 10,000 ϫ g for 10 min at 4°C, and the pellets were dried in a SpeedVac concentrator.
TCTs (2 ϫ 10 6 ) from the culture supernatant of LLCMK 2 cells were collected by centrifugation, washed 3 times with PBS, and suspended in 100 l of PBS containing 100 U of recombinant neuraminidase from Clostridium perfringens (New England BioLabs). After 1 h at 37°C, cells were washed twice with PBS at 4°C and directly subjected to flow cytometry as described above.
Extraction of mucin-like glycoproteins. Glycoconjugates were extracted based on the method of Almeida et al. (17). Briefly, 5-day epimastigotes (1 ϫ 10 9 ) grown in BHI without FBS for 1 day were washed 5 times in PBS and freeze-dried. Samples were extracted 3 times by suspension in 1.9 ml of chloroform-methanol-water (1:2:0.8 [vol/vol/vol]) for 4 h at room temperature, followed by centrifugation (12,000 ϫ g for 20 min). The supernatants were pooled, concentrated in a Speed-Vac, and freezedried to produce fraction F1. The pellet was dried under N 2 and extracted 3 times with 1 ml of water saturated with butan-1-ol for 4 h at room temperature. Fraction F1 was dissolved in 500 l of water saturated with butan-1-ol and extracted 3 times with 1 ml of butan-1-ol saturated with water. The mucin-like glycoprotein-containing aqueous phases were pooled and dried under N 2 .
Glycomic studies. N-Glycans were released from mucin-like glycoproteins from 5 ϫ 10 8 cells by use of PNGase A according to the method of Feasley et al. (64). O-Glycans were released by reductive ␤-elimination as described by Dell et al. (65). The mucin-like glycoprotein sample was suspended in 100 l of 0.1 M NaOH before addition of 200 l freshly made 2 M NaBH 4 . After incubation for 18 h at 45°C, samples were neutralized with acetic acid, desalted in 0.5 ml of Dowex-50 (H ϩ ) resin (Sigma), and subjected to borate removal. N-and O-glycans were analyzed in their native state or after permethylation with iodomethane by use of a solid-phase spin column as described by Kang et al. (66) and were then analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) on an Ultraflex II mass spectrometer (Bruker Daltonics, Billerica, MA) operated in reflector positiveion mode, using 2,5-dihydroxybenzoic acid as the matrix (64).
Statistical analyses. Data significance was evaluated by one-way analysis of variance (ANOVA) in GraphPad Prism 5.0, using Bonferroni's posttest to compare all pairs of columns. P values are indicated in the figure legends.

Overexpression of TcOGNT2 in epimastigotes.
To investigate the function of TcOGNT2, a C-terminally myc-tagged version was overexpressed using the pTEX expression system. The scheme is outlined in Fig. S1 in the supplemental material, and all transformants were verified by PCR (data not shown). Using a newly pre-pared affinity-purified murine anti-TcOGTN2 antibody, substantial expression of a novel protein was observed in Western blots of whole-cell extracts of epimastigotes, at the expected apparent M r position of 56,500 (Fig. 1A). This band was not seen using preimmune serum (not shown). The expected endogenous protein was not detected under these conditions (Fig. 1A, lane 3). The 56.5-kDa band was labeled by MAb 9E10 against the myc epitope, confirming it to be encoded directly by the transgene. Strains were also prepared that expressed similar levels of mutant TcOGNT2myc proteins (Fig. 1A) expected to be catalytically inactive owing to a conservative point mutation in the DSH motif: D234A (ASH), D234N (NSH), or H236D (DSD) (see Fig. S1). The canonical DXD motif characteristic of most superfamily A glycosyltransferases changed to DXH in CAZy GT27 and GT60 family members, and similar point mutations in this motif inactivated other GT60 (pp␣GalNAcTs) and GT27 (pp␣GlcNAcTs) family members (42,67).
Endogenous TcOGNT2. Cell fractionation revealed that TcOGNT2myc was enriched in the P 100 (microsomal) fraction (Fig. 1B), as previously described for pp␣GlcNAcT activity (20,23). A direct comparison of the microsomal fractions from normal TcOGNT2myc (DSH) and control (empty pTEX) strains revealed a slightly more rapidly migrating band corresponding to endogenous TcOGNT2 (Fig. 1C). To validate its identity as TcOGNT2, its N-glycosylation status was analyzed by treatment with PNGase F or endo H, each of which is expected to release the high-mannose N-glycans typically formed by T. cruzi. TcOGNT2 possesses 2 predicted N-glycosylation sites (21), and as expected, treatment with either enzyme shifted TcOGNT2myc to a slightly more rapidly migrating position (Fig. 1D). The indistinguishable effects of these enzyme treatments on the endogenous band strongly support its identity as TcOGNT2. Densitometric studies showed that TcOGNT2 is overexpressed 7-fold in the transgenic strain.
Enzymatic activity of overexpressed TcOGNT2. Microsomes were analyzed as a means to assess the impact of TcOGNT2myc protein expression on catalytic activity in cells. As shown in Fig. 2A, control (empty pTEX) cells exhibited substantial pp␣GlcNAcT activity using the previously described T16 peptide substrate. Cells expressing TcOGNT2myc exhibited 50% in- TcOGNT2myc were subjected to SDS-PAGE, Western blotted using affinity-purified anti-TcOGNT2, MAb 9E10 against the myc tag, or anti-tubulin (as a loading control), and detected using alkaline phosphatase-coupled secondary antibodies. A previously characterized (21) culture supernatant from L. tarentolae secreting ⌬TcOGNT2, which is subject to partial proteolytic cleavage, and a naive culture supernatant are shown in lanes 2 and 1, respectively. (B) Subcellular fractionation. TcOGNT2myc-expressing epimastigotes were mechanically disrupted, fractionated by differential centrifugation, and Western blotted using anti-TcOGNT2 and anti-myc antibodies. Fractions are labeled according to the supernatant (S) or pellet (P) formed at the indicated centrifugation speed (ϫ g [ϫ 10 Ϫ3 ]). ⌬TcOGNT2 was included as a control. (C) P 100 fractions from TcOGNT2myc-expressing and pTEX control epimastigotes were compared using affinity-purified anti-TcOGNT2. (D) N-Glycosylation analysis. P 100 fractions (100 g of protein) from TcOGNT2myc-expressing and pTEX control epimastigotes were analyzed after treatment with active (ϩ) or heat-inactivated (Ϫ) PNGase F or endo H, using anti-TcOGNT2 as described above. ⌬TcOGNT2 (lanes 5) was included as a control. creased activity, and as expected, cells expressing the mutant proteins did not exhibit any change. Enzyme activity was distributed among the cell fractions (Fig. 1B) in proportion to the TcOGNT2myc levels (data not shown). The TcOGNT2myc extracts also exhibited a 5-fold increase in peptide-independent UDP-GlcNAc hydrolysis activity relative to pTEX extracts (Fig.  2B), an activity that was previously associated with ⌬TcOGNT2 (N-terminally truncated to remove the membrane anchor) expressed in L. tarentolae (21). As expected, extracts of mutant TcOGNT2-expressing cells did not exhibit increased hydrolysis activity. Thus, 7-fold-increased protein expression of TcOGNT2 was accompanied by 5-fold-increased hydrolysis activity but only 1.5-fold-increased transferase activity, indicating that some other factor is limiting for pp␣GlcNAcT activity in these extracts.
Compartmentalization of endogenous and overexpressed TcOGNT2. Affinity-purified antiserum was used to compare the localization of TcOGNT2 and overexpressed TcOGNT2myc in cells. Analysis of pTEX control cells revealed a single major locus of accumulation of TcOGNT2 (Fig. 3A) that always colocalized with the Golgi apparatus (Fig. 3B), based on labeling with a BODIPY-labeled C 5 -ceramide-BSA complex (55). This punctate labeling was not observed using preimmune serum (Fig. 3A). Sim-ilar findings were observed for overexpressed TcOGNT2myc, though most of the cells (ϳ70%; n ϭ 200) exhibited multiple sites of accumulation (Fig. 3C) that were dissimilar to the rough endoplasmic reticulum (rER), whose resident proteins, such as BiP and calreticulin, exhibit a punctate pattern throughout the cell body and around the nucleus (68,69). Simultaneous localization of cruzipain, a marker for the reservosome, shows that these compartments also appear independent of this organelle. The localization pattern of the ASH mutant was similar (data not shown). The unexpectedly low transferase activity of microsomes from TcOGNT2myc-overexpressing cells (Fig. 2) might be due to the extra-Golgi localization of some of the protein. The predominant Golgi association of the endogenous protein is consistent with previous cell fractionation results (Fig. 1B) (20,23).
Developmental regulation of TcOGNT2 expression in insect-stage parasites. Epimastigotes were subjected to nutritional stress in TAU-P medium to induce metacyclogenesis. Efficient metacyclogenesis was confirmed by a reduction in the total cruzipain level (Fig. 4A, bottom panel, compare lanes 3 and 4). The resulting MCT preparation was analyzed for TcOGNT2 expression and total pp␣GlcNAcT activity in comparison with epimastigotes and TcOGNT2myc-expressing epimastigotes. As shown by Western blotting of total cell membranes using the affinity-purified antiserum, TcOGNT2 protein levels decreased to undetectable levels in the MCT preparation (Fig. 4A, top panel, compare  lanes 3 and 4). Analysis of crude membranes from these preparations showed a 40% reduction in pp␣GlcNAcT activity in the MCT preparation compared to that in epimastigotes, in contrast to the increased activity in the TcOGNT2-overexpressing cells (Fig. 4B). The remaining activity may have been contributed by non-MCTs remaining in the preparation and by the other predicted pp␣GlcNAcT, TcOGNT1. In addition, TcOGNT2 was not observed in MCTs by immunofluorescence assay (data not shown). Thus, in vitro metacyclogenesis is associated with a major downregulation of the TcOGNT2 protein and associated pp␣GlcNAcT activity.
Inhibition of metacyclogenesis by TcOGNT2 overexpression. The proliferation of TcOGNT2myc-expressing cells was indistinguishable from that of wild-type or pTEX cells in standard medium (Fig. 5A). In contrast, these cells were highly deficient in the ability to undergo metacyclogenesis in aged cultures, based on cell morphology (Fig. 5B). To examine the generality of this finding, metacyclogenesis was also induced in TAU-P medium (29) and monitored not only by morphology but also by resistance to complement killing at multiple times and by MCT stage-specific marker expression. As shown in Fig. 5C to F, similar inhibitions of metacyclogenesis were recorded at two times in TAU-P medium. In addition, the metacyclic stage-specific surface antigens gp90 and gp82 (70) failed to be induced detectably in the population (Fig. 6A). However, immunofluorescence analysis showed that the MCTs that did differentiate from cells expressing either active or mutant TcOGNT2myc expressed gp90 normally (data not shown). Furthermore, as also presented in Fig. 5 and 6, similar findings were observed for the mutant TcOGNT2myc-expressing strains, indicating that the effect of TcOGNT2 overexpression was independent of its enzymatic activities. Parallel cultures transfected with the empty pTEX vector differentiated normally.
Metacyclogenesis is a multistep process in which initial substratum adhesion is followed by release and subsequent morphological and biochemical differentiation (see the introduction). Adhesion was monitored using both tissue culture flask plastic (29) and preparations of insect midgut mucosa (27). As shown in Fig. 6B, no significant change in the capacity for attachment to plastic was observed among the different transfected parasites, since the same numbers of unattached epimastigotes were found in the supernatants. Furthermore, all strains were equally able to attach to freshly dissected posterior midgut epithelia from the vector R. prolixus (Fig. 6C), whereas only the overexpression strains were unable to transform into MCTs after contact with R. prolixus in vivo (see Fig. S2B in the supplemental material). Since high intracellular cAMP levels inhibit cell proliferation (14) and, after adhesion, increased cAMP is accompanied by increased transformation of epimastigotes into MCTs (33), we considered whether cAMP production might be inhibited. Thus, we supple-mented the TAU-P medium with a stable and cell-permeative cAMP analogue (dibutyryl-cAMP) that is a well-known stimulator of metacyclogenesis (33). Metacyclogenesis was not stimulated in the TcOGNT2-overexpressing strains (see Fig. S2A), indicating that the blockade occurred downstream of cAMP signaling. Interestingly, the overexpressing strains showed a modest metacyclogenic response to a high concentration of GlcNAc (250 mM), which was not observed with Glc. Thus, the overexpressors retained a hidden ability to transform, and the blockade appeared to occur upstream of the intersection of GlcNAc signaling with intrinsic regulation, though the nature of this intersection is unknown. proaches. Probing of Western blots of whole epimastigotes with MAbs 2B10 and 10D2, directed toward highly O-glycosylated major 35/50-kDa mucin populations (24), failed to detect a change in either mucin glycoprotein mobility during SDS-PAGE or labeling intensity (Fig. 7A). These MAbs preferentially react with galactopyranose (7) and galactofuranose (53) residues, respectively, consistent with little or no change in natively Gal-rich O-glycans. Similarly, no effect of MAbs J01 and 212 was observed on cruzipain (52) (Fig. 7B), which is alternatively compartmentalized in the reservosome, flagellar pocket, and plasma membrane (71,72) and expresses truncated O-glycans (see the introduction).

Impact of TcOGNT2 overexpression on O-glycosylation. Cellular O-glycosylation levels were examined by multiple ap-
To test for more subtle changes in O-glycome profiles, O-glycans were released from a mucin-enriched preparation by ␤-elimination and derivatized by permethylation to facilitate analysis by MALDI-TOF MS. Except for the larger amount of sialic acidcontaining oligosaccharides, O-glycans detected here matched those described previously for the Dm28c strain (73). As shown in Fig. 7B and in Fig. S3 in the supplemental material, quantitation failed to detect a statistically different change in O-glycan class compositions between TcOGNT2-overexpression and normal strains.
Finally, normal and TcOGNT2-overexpressing epimastigotes were incubated with fluorescent lectins, and labeling was quantitated by flow cytometry. Initial correlation of forward-and sidescattered light measurements recognized a single, though hetero-dispersed, population of parasites, based on their cell surface area, size, or cell granularity (not shown). However, plots of forward versus side scatter did not show any significant change in comparing populations of parasites transfected with the different pTEX versions (not shown). After proper gating around the population of interest, flow cytometric analyses were performed with fixed epimastigotes that were labeled with fluorescent WGA, ConA, and GS-IB 4 lectins. The percentage of labeled cells and the mean values of peak fluorescence intensities were indistinguishable among the strains (see Fig. S4 in the supplemental material). Taken together, the data show no evidence that the presence of ectopic TcOGNT2 proteins in the Golgi apparatus or other compartments had an impact on Golgi glycosylation functions in epimastigotes.
Cruzipain status. Previous studies showed that inhibition of cruzipain pharmacologically (74) or by overexpression of the natural endogenous inhibitor chagasin (75) arrested parasite differentiation in vitro. To address the potential that TcOGNT2myc might act by such a mechanism, the localization of TcOGNT2myc was investigated by confocal immunomicroscopy. As shown in Fig. 3C, little colocalization with cruzipain, which normally accumulates in the reservosome, was observed. To address this further, whole-parasite extracts (see Fig. S5 in the supplemental material) and P 100 fractions (not shown) were assayed to compare cysteinespecific protease activities by using the fluorogenic protease substrate CBZ-FR-AMC and the E-64 inhibitor (51). However, no evidence of an effect of TcOGNT2 overexpression on cruzipain activity was detected (see Fig. S5).
Impact of TcOGNT2 overexpression in cell-derived trypomastigote forms of T. cruzi. Although metacyclogenesis was inhibited in the TcOGNT2-overexpressing strains, occasional MCTs differentiated, based on morphology and resistance to lysis by the alternative pathway of complement ( Fig. 5; see Fig. S2A in the supplemental material). Immunofluorescence observations using MAb 1G7 confirmed gp90 surface labeling equivalent to that of wt MCTs (not shown). To test their infectivity, scaled-up preparations were presented to cultured LLCMK 2 cells at a multiplicity of infection (MOI) of 40. After 4 h of infection, TcOGNT2(DSH)myc TCTs infected fewer LLCMK 2 cells than TcOGNT2(DSD)myc and control TCTs (Fig. 8A and B), but once they were infected, cells harbored similar numbers of amastigotes (Fig. 8C). Over time, this difference was reflected in a substantial delay in TCT accumulation and reduced overall numbers of TCTs in cells infected with TcOGNT2(DSH)myc relative to the other strains (Fig. 8D). When the TCTs that appeared were allowed to revert to proliferative epimastigotes and retested for metacyclogenesis, the frequency of conversion of TcOGNT2(DSH)myc-overexpressing cells remained low ( Fig. 8E and F), showing that impaired MCT differentiation did not simply reflect selection for previously competent cells.
TCTs downregulated endogenous TcOGNT2 protein (Fig. 9A) and activity (Fig. 9B) levels relative to those in epimastigotes, as observed above for MCTs (Fig. 4). In contrast, TcOGNT2myc expression was not reduced, suggesting that downregulation of endogenous TcOGNT2 was not posttranslational. To test the impact of forced TcOGNT2-myc overexpression on surface glycosylation, TCTstransfectedwithpTEX,pTEX-TcOGNT2(DSH)myc,orpTEX-TcOGNT2(DSD)myc were incubated with fluorescent WGA, ConA, GS-IB 4 , and MAA lectins and analyzed by flow cytometry (see Fig. S6 in the supplemental material). Notably, TCTs overexpressing active TcOGNT2 presented significantly more labeling with WGA, based on the percentage of labeled cells and the mean values of fluorescence intensities ( Fig. 9C and D). No difference was seen in cells overexpressing mutant TcOGNT2(DSD)myc. Treatment of cells with neuraminidase caused only a slight decrease in WGA labeling and an increase in PNA labeling (not shown), suggesting that increased labeling was due to the other WGA ligand, GlcNAc, rather than sialic acid. No labeling difference was observed for the other lectins, including MAA, specific for ␣2,3-linked sialic acid. The cell surface remodeling of O-glycans as a result of forced overexpression of active TcOGNT2 may explain the reduced infectivity of these cells toward epithelial cells.

DISCUSSION
We report here that as T. cruzi differentiates into either metacyclic or tissue culture trypomastigotes, endogenous TcOGNT2, which initiates O-glycosylation, is downregulated (at least 3-fold) to undetectable levels ( Fig. 4 and 9A). To address the significance of this change, TcOGNT2myc was constitutively expressed from an alternative promoter. As a result, epimastigote differentiation into MCTs was dramatically inhibited and host cell invasion by TCTs was reduced, resulting in substantially reduced TCT production by monolayers. Mechanistic studies showed that inhibition of TCT production depended on the O-glycosylation function of TcOGNT2, whereas inhibition of metacyclogenesis was, in contrast, independent of its pp␣GlcNAcT activity and any evidence of perturbed O-glycosylation. The findings suggest that TCT O-glycosylation is regulated by changes in the protein level of this gatekeeper pp␣GlcNAcT and that the protein has important enzymatic and nonenzymatic roles in TCT differentiation and MCT function, respectively.
TcOGNT2 protein downregulation. The TcOGNT2 protein was monitored by use of a new affinity-purified murine antiserum generated against recombinant TcOGNT2, because a previous rabbit antiserum proved too insensitive. The specificity of the new antibody was established based on (i) predominant reactivity with a cellular protein that migrates slightly more rapidly than transgenic TcOGNT2myc expressed in the same cells (Fig. 1C), near the expected 55-kDa position; (ii) N-glycosylation that is very similar to that of TcOGNT2myc (Fig. 1D); (iii) enrichment in a biochem- (C to F) Metacyclogenesis was induced in TAU-P medium, and the percentage of MCTs was evaluated by morphology (C and D) or resistance to lysis by the alternative pathway of complement (E and F) after 3 (C and E) or 5 (D and F) days of culture. Results are means and SEM from three independent experiments performed in triplicate. Asterisks indicate statistical significance compared to controls, as follows: *, P Ͻ 0.05; **, P Ͻ 0.01; and ***, P Ͻ 0.001. ically generated microsomal fraction; and (iv) microscopic colocalization with the Golgi apparatus ( Fig. 3A and B), the compartment of its function, based on previous pp␣GlcNAcT activity studies (23). This antiserum showed reductions of the native TcOGNT2 protein to near undetectable levels, which was corroborated by reduced pp␣GlcNAcT activity in extracts ( Fig. 4 and 9). The decline in TcOGNT2 level was not, however, observed in a recent study employing a different rabbit antiserum (76). That antiserum was raised against a TcOGNT2 fragment representing Ͻ30% of the full protein and was not affinity purified. Other than its apparent M r and microsomal enrichment, the reactive protein antigen was not validated and, in fact, accumulated more in the reservosome than in the Golgi apparatus. Our target antigen, as expected, was observed to be strictly Golgi associated and N-glycosylated, like transgenic TcOGNT2myc, and it was observed to migrate slightly behind TcOGNT2, as expected based on the myc tag. We concluded from this comparison that TcOGNT2 is a Golgi apparatus resident that declines during trypomastigote differentiation and that an alternative protein recognized by the other antiserum (76) increases during differentiation and preferentially resides in the reservosome.
The decline in MCT TcOGNT2 observed with our murine Ab does not correlate with expression of the core proteins (69) but is consistent with reduced biosynthetic needs of noncycling MCTs, whose O-glycosylation needs might instead be supported by TcOGNT1, the only other predicted pp␣GlcNAcT (21). The existence of only two pp␣GlcNAcT proteins contrasts with the multitudes of mucin protein cores (10) and predicted ␤GalT and trans-sialidase enzymes that modify them (77) and implies that TcOGNT2 and TcOGNT1 might together serve a gatekeeper function to regulate the extent of mucin-type O-glycosylation, especially in TCTs (Fig. 9). Further studies on TcOGNT1 are needed to investigate this possibility.
Constitutive overexpression of TcOGNT2. Expression from the pTEX construct yielded constitutive TcOGNT2myc levels in epimastigotes that were 7-fold higher than endogenous protein levels (Fig. 1C). These values are comparable to those from other studies using pTEX or pTEX-derived vectors to overexpress proteins in the cytosol (78,79), mitochondria (80), glycosomes (81), endosomal/lysosomal system and reservosomes (75,82), ER (68), flagellar pocket (83), or plasma membrane (69,(84)(85)(86) of T. cruzi. The protein was stable, with no indication of a C-terminal proteolytic cleavage event observed when expressed in L. tarentolae (21), and it appeared to be inserted into the rER and N-glycosylated normally (Fig. 1D). A catalytically active status of the expressed enzyme was indicated by the corresponding 5-fold increase in UDP-[ 3 H]GlcNAc hydrolysis activity relative to that of controls (Fig. 2). However, pp␣GlcNAcT activity was increased only 1.5-fold (Fig. 2). These increases were directly attributed to TcOGNT2, since they were not induced by the catalytically dead variants tested. The difference between activity and protein levels suggests a normally tight physiological regulation of transferase activity. This was consistent with the failure to detect increased O-glycosylation based on analysis of mucins by use of anti-mucin or anti-cruzipain antibodies or mass spectrometry (Fig. 7), or by lectin flow cytometry of cells (see Fig. S4 in the supplemental material), though the alternative explanation, that O-glycosylation is normally already saturated, cannot be excluded. Immunofluorescence studies indicated that though the majority of overexpressed TcOGNT2 was localized normally in the Golgi apparatus, some protein was distributed in nearby compartments other than the ER and reservosomes (Fig. 3B), which might not support activity. Alternatively, TcOGNT2 transferase activity might be regulated by another factor. For example, the glycosyltransferases EXT1 and EXT2 must be coexpressed for heparan sulfate biosynthesis (87), and similarly, POMT1 and POMT2 are mutually required for mammalian protein O-mannosylation (88). In other cases, less closely related proteins are required, such as fukutin for POMGnT1 to glycosylate ␣-dystroglycan (89), Cosmc for the mammalian core 1 ␤3-GalT (90), and a Drosophila DHHC protein family-related protein for ␤4GalNAcTB (91). Interestingly, Cosmc and GnT1IP, an inhibitor of murine GnT1 (92), are distantly related to the glycosyltransferases that they regulate, which is reminiscent of the distant similarity of TcOGNTL, which lacks important catalytic residues, to TcOGNT2 (and TcOGNT1).
Thus, the potential roles of TcOGNTL and TcOGNT1 in controlling TcOGNT2 activity deserve further investigation. The possibility of a positive rather than a negative regulator is implied by the inactivity of TcOGNT2 when expressed in another protist, i.e., Dictyostelium (21).
Constitutive TcOGNT2 expression inhibits metacyclogenesis. Cells that constitutively overexpressed TcOGNT2myc were severely deficient in the ability to differentiate into MCTs, using either of three methods of induction, including introduction into the insect host (Fig. 5B to D; see Fig. S2B in the supplemental material). Further studies showed that inhibition occurred at a stage after initial adhesion to the insect vector midgut substratum ( Fig. 6B and C) but prior to induction of complement resistance ( Fig. 5E and F) or the metacyclic mucins gp90 and gp82 (Fig. 6A). Interestingly, metacyclogenesis was enhanced by high concentra- tions of free GlcNAc, in a sugar-specific fashion (see Fig. S2A), suggesting that signaling is involved. cAMP is involved in the induction of metacyclogenesis through activation of adenylate cyclase and PKA (14,33), but db-cAMP was unable to overcome inhibition (see Fig. S2A), suggesting that TcOGNT2 renders its effects downstream. There was no evidence for effects on cruz-ipain ( Fig. 3B; see Fig. S5), whose inhibition can also inhibit metacyclogenesis. The inhibition of metacyclogenesis by TcOGNT2 overexpression was far stronger than in the case of cruzipain inhibition by chagasin overexpression (75), and it did not involve intermediary forms or death as observed with overexpression of the Ras-related GTP-binding protein TcRjl (83). The reduced After 4 h, each culture flask was washed 5 times, followed by incubation for 11 days. TCT density was determined by direct counting. (E and F) Epimastigotes derived from TCT forms were transferred to TAU-P and incubated at 28°C to induce metacyclogenesis. After 3 days, parasite density (E) was determined by direct counting, and the percentage of MCTs was estimated by resistance to lysis by the alternative pathway of complement (F). Results are means Ϯ SD from three independent experiments (n ϭ 2). Asterisks indicate statistical significance (***, P Ͻ 0.001). ability of cells to differentiate in the presence of sustained levels of TcOGNT2 suggests that TcOGNT2 is an important regulator of metacyclogenesis.
Remarkably, inhibition did not depend on the catalytic activities of TcOGNT2myc. This was most conclusively indicated by introduction of either of 3 different inactivating point mutations (21) into the active site of the TcOGNT2myc transgene ( Fig. 5 and  6). Thus, the inhibitory effect of the overexpressed proteins depended on a second function that is apparently unrelated to even indirect effects on protein glycosylation, based on the analysis of mucin O-glycosylation and cell level lectin flow cytometry ( Fig. 7; see Fig. S4 in the supplemental material). Although it is difficult to exclude the possibility that the 7-fold overexpression of a single protein, such as TcOGNT2, inhibits metacyclogenesis by a nonspecific stress effect, it is notable that proliferation and TCT production were not affected ( Fig. 8 and 9) and that overexpression of other proteins failed to inhibit this process (58,68,69,(78)(79)(80)(81)(82)(84)(85)(86). The protein burden of 7-fold accumulation of a single enzyme is not anticipated to be great, and indeed, overexpression of a truncated N-terminal fragment of the TcOGNT2 homolog TbOGNT2 in Trypanosoma brucei did not affect Golgi replication (93). However, overexpressed TcOGNT2s were also ectopically partially localized to an unknown compartment, which must be considered an alternative site of action of the overexpressed protein. The Golgi apparatus is an organelle of many functions, some of which are likely still undiscovered.
Constitutive TcOGNT2 expression inhibits TCT production in host cells. Metacyclogenesis was not abolished by TcOGNT2, and the MCTs that appeared were normal, based on morphology, resistance to lysis by the alternative pathway of complement, gp90 stage-specific surface expression ( Fig. 5; see Fig. S2 in the supplemental material; data not shown), and the general ability to infect cultured epithelial (LLCMK 2 ) cells ( Fig. 8A to C). Epimastigotes derived from these TCTs continued to overexpress TcOGNT2myc (Fig. 9A) and, again inefficiently, differentiated into MCTs (Fig.  8F). Therefore, TcOGNT2myc overexpression did not block metacyclogenesis, and breakthrough cells did not represent a resistant subpopulation.
However, the TcOGNT2 cells were deficient in generating TCTs, which manifested both as a 2-day delay in TCT production and a 3-fold reduction in maximal numbers produced (Fig. 8D). Analysis of 4-h postinfection monolayers revealed a lower percentage of infected cells but nearly normal numbers of proliferating amastigotes per infected cell (Fig. 8A to C), suggesting that one deficit is initial cell infection. Cell surface glycosylation was affected in the TcOGNT2-overexpressing cells (Fig. 9C), which might inhibit the parasite-host interaction. A necessity for altered O-glycosylation was indicated by the lack of inhibition of TCT production by catalytically inactive TcOGNT2. Further studies are needed to determine the role of parasite cell surface glycosylation in infection.
Recently, an independent strain of T. cruzi Dm28c was modified to overexpress TcOGNT2 by use of the pTEX plasmid system (76). This strain was described to yield increased amastigote numbers in Vero cells at a single time point of 3 days. The consequence of this on TCT production was not examined, and no reference was made to a deficit in metacyclogenesis. It is not clear why the impact of TcOGNT2 overexpression was so different in our strain. In our study, we validated that active TcOGNT2myc was overexpressed in TCTs by M r determination relative to that of the en-dogenous untagged protein, similar N-glycosylation, predominant Golgi localization, increased enzyme activity in extracts, and altered lectin reactivity of cells. The protein detected in the other strain, whose M r was not described, by an antiserum with uncertain specificity (see above) may not have been active or might represent another protein induced by the transformation.
In conclusion, TcOGNT2 overexpression was found to be inhibitory to T. cruzi cells only at those stages where its expression is normally downregulated-MCTs and TCTs. But the mechanisms were distinct: nonenzymatic for MCT production and enzymatic for TCT production. TcOGNT2 has thus far been intractable to gene disruption studies to extend these findings, but inducible expression approaches directed toward TcOGNT2, TcOGNT1, and TcOGNTL are expected to provide additional insights into the underlying mechanisms of control.