Surface Coat Remodeling during Differentiation of Trypanosoma brucei*

Amy E. Gruszynski {ddagger} §, Andrew DeMaster ¶, Nigel M. Hooper || and James D. Bangs ¶ **

From the {ddagger}Department of Biomolecular Chemistry and the Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin 53706 and the ||Proteolysis Research Group, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

Received for publication, February 11, 2003 , and in revised form, April 11, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
African trypanosomes (Trypanosoma brucei) are digenetic parasites whose lifecycle alternates between the mammalian bloodstream and the midgut of the tsetse fly vector. In mammals, proliferating long slender parasites transform into non-diving short stumpy forms, which differentiate into procyclic forms when ingested by the tsetse fly. A hallmark of differentiation is the replacement of the bloodstream stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procylin. An undefined endoprotease and endogenous glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) have been implicated in releasing the old VSG coat. However, GPI hydrolysis has been considered unimportant because (i) GPI-PLC null mutants are fully viable and (ii) cytosolic GPI-PLC is localized away from cell surface VSG. Utilizing an in vitro differentiation assay with pleomorphic strains we have investigated these modes of VSG release. Shedding is initially by GPI hydrolysis, which ultimately accounts for a substantial portion of total release. Surface biotinylation assays indicate that GPI-PLC does gain access to extracellular VSG, suggesting that this mode is primed in the starting short stumpy population. Proteolytic release is up-regulated during differentiation and is stereoselectively inhibited by peptidomimetic collagenase inhibitors, implicating a zinc metalloprotease. This protease may be related to TbMSP-B, a trypanosomal homologue of Leishmania major surface protease (MSP) described in the accompanying paper (LaCount, D. J., Gruszynski, A. E., Grandgenett, P. M., Bangs, J. D., and Donelson, J. E. (2003) J. Biol. Chem. 278, 24658–24664). Overall, our results demonstrate that surface coat remodeling during differentiation has multiple mechanisms and that GPI-PLC plays a more significant role in VSG release than previously thought.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
African trypanosomes are pathogenic kinetoplastid protozoa responsible for human and veterinary trypanosomiasis in sub-Saharan Africa. This parasite has a digenetic lifecycle that alternates between the insect vector (tsetse flies) and the bloodstream of mammalian hosts. In the mammalian bloodstream, trypanosomes transform from a replicating long slender form into a non-dividing short stumpy form that is pre-adapted for transmission to the tsetse fly. In the fly midgut, the short stumpy form differentiates into a replicating procyclic form.

Bloodstream and insect stage trypanosomes possess distinct developmentally regulated surface molecules: VSG1 in bloodstream trypanosomes (1) and procyclin in the procyclic insect stage (24). VSG, a homodimer that constitutes ~10% of total bloodstream protein, is attached to the cell surface by a glycosylphosphatidylinositol (GPI)-anchor. Antigenic variation, which is the sequential expression of distinct VSG genes, enables trypanosomes to evade the host immune response (5). Unlike VSG, procyclin is a monomeric coat protein that can be divided into two classes based on distinct C-terminal repeat motifs (6). EP procyclins contain up to 30 Glu-Pro repeats, whereas GPEET procyclin has six Gly-Pro-Glu-Glu-Thr repeats followed by three Glu-Pro repeats. Both repeat motifs are protease-resistant, and, therefore, one function of procyclins may be to protect the parasite from the hydrolytic environment of the tsetse fly midgut. Reflecting a difference in the biosynthetic capability of each stage of the lifecycle, procyclin is anchored to the cell surface of insect stage trypanosomes by a GPI moiety structurally distinct from the VSG anchor (7, 8).

Replacement of VSG with procyclin is a hallmark of the transformation of bloodstream stage trypanosomes into the procyclic form. Differentiation can be induced in vitro by a variety of conditions that mimic the environment of the tsetse fly midgut, including mild acid stress and trypsin treatment (9, 10). The most effective treatment, however, is the addition of cis-aconitate to the culture media coupled with a temperature reduction from 37 to 27 °C (11, 12). When initiated with enriched short stumpy populations, differentiation is essentially synchronous, allowing for the precise description of temporally regulated events (13). Early events include the induction of procyclin expression, the repression of VSG synthesis, and the shedding of the old VSG coat from the cell surface (1315). After VSG shedding has begun, the kinetoplast is repositioned with respect to the nucleus (16). Differentiating trypanosomes enter into their first procyclic cell cycle following these morphological changes (17).

There are two known ways that VSG is released from the cell surface, GPI hydrolysis and endoproteolysis. Bloodstream stage trypanosomes contain an endogenous glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) that is capable of hydrolyzing the GPI anchor found on VSG, but not procyclin (7, 18). GPI hydrolysis releases a soluble form of VSG (sVSG), which retains the GPI core glycan. This type of VSG release occurs upon cell lysis (19) as well as in normal growth of long slender cells where VSG is slowly shed into the medium with a half-life of ~32 h (20, 21). GPI-PLC-mediated release of VSG also occurs during differentiation of bloodstream stage trypanosomes (22). The second mode of VSG release, endoproteolysis, only occurs during differentiation as evidenced by the shedding of soluble VSG fragments (21, 22). At no other time is VSG released from bloodstream stage cells in such a manner. It has generally been accepted that the primary means of VSG release during differentiation is proteolytic, because GPI-PLC null mutant trypanosomes have been shown to be fully viable and capable of differentiation (23). Moreover, GPI-PLC has been reported to localize to the cytoplasmic face of intracellular vesicles, sequestering the enzyme away from extracellular VSG (24).

We have demonstrated previously that VSG expressed in transgenic procyclics is shed by proteolytic cleavage resulting in soluble N-terminal and membrane-bound C-terminal fragments in a manner similar to that observed during the differentiation of bloodstream cells (25). Release is inhibited by the zinc chelator bathophenanthroline and by peptidomimetic inhibitors of mammalian collagenase-type zinc metalloproteases (26). These findings indicate that fully differentiated procyclics have a potent cell surface metalloprotease activity, the function of which is unknown.

In this work, we establish that endoproteolytic release of VSG from the surface of differentiating bloodstream stage cells is also mediated by a zinc metalloprotease activity. This activity has a similar inhibitor profile as the activity that mediates release of transgenic VSG from the surface of procyclic trypanosomes. We propose that a zinc metalloprotease is up-regulated during differentiation to release VSG and is then maintained on the surface of fully differentiated procyclics. In addition, we find that GPI-PLC can be physically detected on the surface of intact short stumpy trypanosomes and that GPI hydrolysis can account for a significant portion of VSG release during differentiation. These results suggest that the role of GPI hydrolysis in surface coat remodeling should be reconsidered.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Compounds—Bathophenanthroline was obtained from Sigma. SB227961 and SB227962 were kindly supplied by SmithKline Beecham Pharmaceuticals (SB, Harlow, UK) and have been described previously (26). P27 (morpholinourea-phenylalanine homophenylalanine-benz-{alpha}-pyrone) was a generous gift from Dr. James McKerrow (University of California-San Francisco). All compounds were dissolved as 50–100x stocks in water or Me2SO as appropriate.

Trypanosomes—Lister 427 Strain procyclic cell lines were maintained in Cunningham's media supplemented with 10% inactivated fetal bovine serum (27). The pleomorphic cell lines used in the differentiation assay, Trypanosoma brucei rhodesiense (LouTat 1.0, from Dr. John Mansfield, University of Wisconsin-Madison) and T. brucei brucei (AnTat 1.1, from Dr. Peter Overath, Max Planck Institut für Biologie), were grown in Swiss Webster mice immunosuppressed with cyclophosphamide (300 mg/kg, Sigma) at the time of infection. LouTat 1.0 and AnTat 1.1 bloodstream trypanosomes were isolated from infected mice on day 14 and day 8, respectively, as described below. Both strains possessed short stumpy populations in excess of 90% as judged by morphology.

Synthetic Deoxyoligonucleotides and Construction of Expression Vectors—The following synthetic deoxyoligonucleotides were used for RT-PCR. Sequences complementary to the target template are in uppercase; added sequences are in lowercase. Restriction sites are underlined; all sequences are 5' to 3': JB196, aaaatcgatATGCAACCCAATGGCGC, 5' sense primer for LouTat 1.0 VSG (codons 1–7) with ClaI site; JB197, aaagaattcTTAGAAAAGCAAGGCCAC, 3' antisense primer for LouTat 1.0 VSG (codons 1526–1531) with EcoRI site. The RT-PCR product was cloned directly into the procyclic expression vector pXS2 (28) and inserts were sequenced to confirm identity. Stable transformation of cultured Lister 427 strain procyclic trypanosomes has been described (28).

Antibodies—Rabbit antiserum against Tbhsp70 (gene 4) has been described (29). Rabbit anti-LouTat 1.0 VSG and anti-AnTat 1.1 VSG were prepared by immunization with soluble VSG purified by the procedure of Cross (30). Anti-cross-reacting determinant (anti-CRD) (31) was affinity-purified from whole rabbit anti-VSG 117 serum by absorption to immobilized soluble VSG 221. Anti-EP procyclin antibody (monoclonal 247) was obtained from Cedarlane Laboratories, Ltd. (Ontario, Canada). Anti-GPEET procyclin antibody (K1 polyclonal rabbit {alpha}(GPEET)3 peptide) was a generous gift from Dr. Peter Bütikofer (University of Bern, Switzerland). Rabbit anti-Trypanosoma cruzi oligopeptidase B was a generous gift from Dr. Barbara Burleigh (Harvard School of Public Health). Rabbit anti-TbRab11 was a generous gift from Dr. Mark Field (Imperial College of Science, Technology and Medicine, England). Anti-GPI-PLC (monoclonal 2A6-6) was a generous gift from Dr. Kojo Mensa-Wilmot (University of Georgia). Rabbit anti-mouse IgG was obtained from Sigma.

For immunoprecipitation, rabbit antisera were covalently cross-linked to Protein A-Sepharose (PAS, Amersham Biosciences, Piscataway, NJ) beads. PAS suspension (1 ml; ~50% v/v; in TEN buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA) was rotated for 1 h at 4 °C with the individual whole rabbit antisera (0.2 ml). PAS:antibody beads were washed twice with sodium borate (100 mM, pH 9.0) and resuspended to 1.2 ml in the same buffer. Disuccinimidyl suberate (Pierce Chemical Co., Rockford, IL, 20 mM in Me2SO) was added to a final concentration of 2 mM. Following rotation at room temperature (2 h), 1 M Tris-HCl (pH 7.5) was added to a final concentration of 20 mM and rotation was continued (15 min). The PAS:antibody beads were then washed once with 100 mM glycine (pH 9.0), washed twice with TEN, and resuspended to 1.2 ml (~50% v/v slurry) in TEN buffer supplemented with 0.1% sodium azide. Rabbit anti-mouse IgG (400 µg) was pre-adsorbed to PAS (1.0 ml) as described above, but without cross-linking, for use in surface biotinylation assays of short stumpy cells.

VSG Release Assay—Proteolytic release of VSG from the surface of transgenic procyclic trypanosomes has been described previously (25, 26). Briefly, 427 strain procyclic cells stably expressing full-length LouTat 1.0 VSG were surface biotinylated with Sulfo-NHS-Biotin (Pierce) and then incubated for 4 h at 27 °C in TM-P medium (DTM, 10 mM glycerol, 5.5 mM proline (32)). VSG was then immunoprecipitated from cell and media fractions and separated by SDS-PAGE. After electroblotting to membranes, biotinyl-VSG was detected by chemiluminescence with avidin-HRP (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD). Cells displayed excellent motility and morphology throughout the assay period.

Differentiation Assay—When populations were >90% short stumpy by visual inspection, pleomorphic LouTat 1.0 or AnTat 1.1 cells were harvested from infected mice by cardiac puncture. To enrich trypanosomes from infected blood without temperature shifts that might induce differentiation, a modification of the DEAE-cellulose technique of Lanham and Godfrey (33) was employed. Heparinized blood (~1 ml) was placed directly into a sterile pre-warmed slurry (60% v/v, 13 ml) of DE52 (Whatman, Maidstone, England) equilibrated in Bicine-buffered saline (BBS: 50 mM Bicine, 5 mM KCl, 50 mM NaCl, 70 mM glucose, pH 8.0) and then rotated 2 min at 37 °C. DE52 with bound blood cells was removed by centrifugation (~1 min, 2000 x g, 37 °C). The cells in suspension were collected and washed once with 37 °C BBS. Cells were diluted to ~5 x 106 cells/ml in HMI9 media (34) and placed at 37 °C (5% CO2) for 1 h. After incubation, cells were washed once with prewarmed HMI9 media (37 °C) and resuspended at 2.5 x 106 cells/ml in 27 °C Cunningham's media supplemented with 3 mM each of citrate and cis-aconitate (11). Cells were cultured at 27 °C, and time points were taken over a 48-h period. At each time point, 1.5 x 106 cell equivalents (0.6 ml) were removed and centrifuged, and the supernatants were reserved. The cells were washed once in ice-cold phosphate-buffered saline containing 2 mg/ml glucose (PBSG), solubilized in 0.6 ml RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) and precleared by centrifugation (5 min, 12,000 x g). Detergents were added to culture supernatants to the same final concentrations as the RIPA buffer. Protease inhibitor cocktail (PIC; final concentration 4 µg/ml each of leupeptin, antipain, pepstatin, and chymostatin) was added to both cell and media samples, which were then reserved for subsequent immunoprecipitations. Cells displayed excellent motility and morphology throughout the assay period.

Trypanosomal proteins were specifically immunoprecipitated from cell and media fractions with appropriate antibodies covalently immobilized on PAS. The antibody:bead suspensions (40 µl) were added to 0.5 ml containing equivalent amounts of cell lysate or culture supernatant. The samples were mixed overnight at 4 °C and then washed three times with RIPA buffer and twice with TEN. All samples were boiled in SDS-sample buffer and fractionated by 12% SDS-PAGE. Gels were transferred electrophoretically to polyvinylidene fluoride membranes (Immobilon-P transfer membrane, Millipore Corp., Bedford, MA), and the presence of immunoprecipitated proteins was detected by immunoblotting with appropriate primary antibodies and secondary Protein A-HRP conjugate (KPL). Specific binding was visualized on x-ray film with an enhanced chemiluminescence substrate kit (Pierce Chemical Co.).

Procyclin Detection—Whole cell lysates (5 x 105 cell equivalents) were separated by 12% SDS-PAGE and transferred electrophoretically to an Immobilon-P transfer membrane (Millipore Corp.). Membranes were blocked in Tris-buffered saline (TBS: 25 mM Tris, 140 mM NaCl, 3 mM KCl) for 1 h and incubated overnight at 4 °C with either anti-EP procyclin or anti-GPEET procyclin antibody diluted in TBS containing 5% nonfat dry milk. Membranes were washed in TBS with 0.05% Tween 20 (Sigma), incubated with appropriate HRP-conjugated goat secondary antibodies (KPL), diluted in TBS containing 5% nonfat dry milk for 1 h, and then washed as before. Antibody binding was visualized as described above.

Biotinylated sVSG—LouTat 1.0 or AnTat 1.1 trypanosomes were isolated from infected mice at peak parasitemia (>90% long slender forms, days 3–4) on DE52 columns (33). Surface biotinylation was performed as described above. After the biotinylation reaction was quenched, cells were washed twice in PBSG and lysed hypotonically (5 min, 4 °C) at 109 cells/ml in dH2O supplemented with PIC. Lysates were made isotonic with 10x PBS and centrifuged (5 min, 12,000 x g, 4 °C), and the supernatant was discarded. The cell pellet was resuspended at 109 cell equivalents/ml in TEN buffer supplemented with PIC and incubated at 37 °C for 5 min. Cell ghosts were removed by centrifugation (5 min, 12,000 x g, 4 °C), and the supernatant containing biotinylated sVSG was saved (109 cell equivalents/ml).

Surface Biotinylation of Short Stumpy Cells—Surface biotinylation was performed as described above with some minor modifications. When populations were >90% short stumpy by visual inspection, AnTat 1.1 cells were isolated from infected mice on DE52 columns (33). The cells were washed once in BBS and placed in HMI9 media (107 cells/ml) at 37 °C (5% CO2) for 30 min. Cells were then harvested, washed twice at 107 cells/ml in PBSG, and treated (108 cells/ml, 30 min) on ice with Sulfo-NHS-Biotin (400 µg/ml, Pierce Chemical Co.). To quench the biotinylation reaction, cells were diluted to 107 cells/ml in PBSG and NH4Cl (1 M in PBSG) was added to a final concentration of 10 mM. After a 5-min incubation on ice, cells were washed twice at 107 cells/ml in PBSG and solubilized at 2 x 108 cells/ml in RIPA buffer supplemented with PIC. Cell lysates were pre-cleared by centrifugation (5 min, 12,000 x g). To remove contaminating mouse IgG that copurifies with short stumpy trypanosomes, cell lysates (2 x 108 cell equivalents) were cycled through two rounds of immunoprecipitation with 0.1 ml of rabbit anti-mouse IgG:PAS followed by one round of immunoprecipitation with 0.1 ml of PAS resuspended in RIPA buffer (0.08 g of beads/1 ml of RIPA). Trypanosomal proteins were then specifically immunoprecipitated from 1 ml of cleared lysate with appropriate antibodies and separated by SDS-PAGE. After electroblotting to membranes, biotinylated proteins were detected by chemiluminescence with avidin-HRP (KPL).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Metalloprotease Activity in Procyclic Trypanosomes—We have previously shown that VSG is shed from the surface of transgenic procyclic cell lines stably expressing recombinant VSG (25). The appearance of truncated VSG fragments in the culture medium derived from these cells suggests that release results from endoproteolytic cleavage upstream of the C-terminal GPI anchor. Shedding is inhibited by the chelator bathophenanthroline and by peptidomimetic compounds with known inhibitory activity against mammalian collagenases of the metzincin family (26). These inhibitor studies demonstrate that a zinc metalloprotease activity is responsible for VSG release from the surface of transgenic procyclic cells. VSG is also released by endoproteolysis from the surface of differentiating bloodstream stage trypanosomes (22). Although the nature of the protease is unknown, the manner of shedding is similar to that seen with transgenic procyclic cells. To investigate this process, we established an in vitro differentiation assay using pleomorphic LouTat 1.0 trypanosomes (described below). However, before studying VSG release during differentiation, we wished to establish that the LouTat 1.0 VSG was subject to release by endoproteolysis in the transgenic procyclic system.

A transgenic procyclic cell line constitutively expressing LouTat 1.0 VSG was established, and a standard biotinyl-VSG release assay was performed (Fig. 1). As seen previously with other transgenic VSGs (VSG221 and VSG117, (25)), LouTat 1.0 VSG is initially cell-associated as a doublet of full-length and proteolytically truncated forms (lane 1). In the absence of inhibitors, essentially all VSG is truncated and released into the medium after 4 h (lane 4). This pattern of release is due to the homodimeric structure of native VSG where sequential cleavage of each subunit will first produce a heterodimer anchored to the membrane by one full-length monomer. After the second subunit is cleaved, VSG is released into the medium as a soluble, truncated homodimer. Release of LouTat 1.0 VSG is blocked by BPS (lanes 5 and 6) and the peptidomimetic SB227961 (lanes 7 and 8), but not its diastereoisomer, SB227962 (lanes 9 and 10), as was previously demonstrated with VSG117 (26). Collectively, these results confirm that LouTat 1.0 VSG is subject to endoproteolytic release by the endogenous procyclic zinc metalloprotease activity.



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FIG. 1.
Inhibition of VSG release from transgenic procyclic trypanosomes. Surface biotinylated strain 427 procyclic cells stably expressing LouTat 1.0 VSG were incubated for 4 h at 27 °C in TM-P media in the presence of selected compounds at 5 mM (BPS, bathophenanthroline) or 500 µM (SB227961 and SB227962). An equivalent volume of Me2SO was added to the control. After 0 and 4 h, VSG polypeptides were specifically immunoprecipitated from cell (c) and media (m) fractions with anti-LouTat 1.0 VSG antibodies, separated by SDS-PAGE, and electrophoretically transferred to membrane. Biotinylated VSG was detected with avidin-HRP conjugate. All lanes contain 5 x 106 cell equivalents. Full-length (F) and truncated (T) forms of LouTat 1.0 VSG are indicated.

 

In Vitro Differentiation of LouTat 1.0 and AnTat 1.1 Trypanosomes—To investigate release of LouTat 1.0 VSG from differentiating bloodstream stage cells, we first established a reliable in vitro differentiation assay. In addition, to confirm that the manner of VSG release is common in other differentiating pleomorphic cell lines, a second strain, AnTat 1.1, was also examined. In each case, bloodstream stage cells were isolated from immunosuppressed mice when the majority of the population (>90%) was short stumpy in morphology and in vitro differentiation was induced by a temperature shift (37 to 27 °C) along with the addition of citrate and cis-aconitate (11). Cell density was monitored for 96 h (Fig. 2A), and expression of either EP-procyclin or GPEET-procyclin was assayed by immunoblotting cell extracts with the respective procyclin antibody (Fig. 2B). Results for both cell lines were similar to other synchronously differentiating pleomorphic strains (35, 36): logarithmic proliferation begins at approximately 12 h post-induction, and both types of procyclin expression are evident by 4 h post-induction. These results indicate that typical synchronous differentiation and cell cycle initiation occurs with both LouTat 1.0 and AnTat 1.1 trypanosomes.



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FIG. 2.
In vitro differentiation of LouTat 1.0 and AnTat 1.1 trypanosomes. Differentiation of enriched short stumpy populations of LouTat 1.0 and AnTat 1.1 was initiated as described under "Experimental Procedures." A, cell density of LouTat 1.0 (triangles) and AnTat 1.1 (squares) trypanosomes was monitored for 96 h after induction of differentiation in Cunningham's media. Logarithmic proliferation begins at ~12 h for both cell lines. B, procyclin expression in LouTat 1.0 and AnTat 1.1 trypanosomes at the indicated differentiation times was monitored by immunoblotting with anti-EP procyclin and anti-GPEET procyclin antibody, respectively. All lanes contain equivalent volumes of starting culture. Scales refer to relative molecular mass in kDa.

 

VSG Release during Differentiation—Both endoproteolysis and GPI hydrolysis by endogenous GPI-PLC have been implicated in VSG release during differentiation (22). GPI hydrolysis removes dimyristoyl glycerol, releasing the intact sVSG protein that retains the glycan portion of the GPI anchor (19, 37). The hydrolyzed glycan forms a unique immunological epitope known as the cross-reacting determinant (CRD) that can be detected with a specific anti-CRD antibody (38). Thus, while truncated VSG indicates release by proteolysis, a fulllength VSG polypeptide that is reactive with anti-CRD antibody indicates release by GPI hydrolysis.

Using these criteria along with the established in vitro differentiation assay, VSG release from differentiating LouTat 1.0 and AnTat 1.1 bloodstream stage trypanosomes was examined (Fig. 3). For differentiating LouTat 1.0 trypanosomes, all VSG is full-length and cell-associated at the 0-h time point (panel A, lane 1 versus lane 7). At 4 h, cell-associated VSG is reduced (panel A, lane 2), and released VSG is detected in the medium as a predominantly full-length species (panel A, lane 8). After 8 h, all VSG has been released into the medium as a mixture of the truncated and full-length forms (panel A, lane 9). Based on its reactivity with anti-CRD antibody (panel B, lanes 8–12), the full-length species contains sVSG, i.e. the product of GPI hydrolysis. VSG is released from differentiating AnTat 1.1 trypanosomes in the same manner. However, the ratio of sVSG to truncated VSG released into the medium is greater during AnTat 1.1 differentiation (panel D, lanes 9–12). Another minor difference is that small amounts of AnTat 1.1 VSG stay cell-associated up to 24 h post-induction (panel D, lane 5), whereas LouTat 1.0 VSG is fully released by 8 h post-induction (panel A, lane 3).



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FIG. 3.
Release of LouTat 1.0 and AnTat 1.1 VSG during in vitro differentiation. Differentiation assays were performed as in Fig. 2. At the indicated times post-induction, equal volumes of culture of either LouTat 1.0 (A, B, and C) or AnTat 1.1 (D, E, and F) trypanosomes were separated into cell and medium fractions and trypanosomal proteins were specifically immunoprecipitated with covalently immobilized anti-LouTat 1.0 VSG (A and B), anti-AnTat 1.1 VSG (D and E) or anti-HSP70 (C and F) antibodies. Precipitates were separated by SDS-PAGE, transferred to membranes, and immunoblotted with anti-LouTat 1.0 VSG (A), anti-AnTat 1.1 VSG (D), anti-CRD (B and E), or anti-HSP70 (C and F) antibodies. Full-length (F) and truncated (T) forms of LouTat 1.0 and AnTat 1.1 VSG are indicated along with CRD-positive sVSG (S) and HSP70 (H). All lanes contain equivalent volumes of starting culture. Lanes are numbered for the bottom panels only.

 

For both differentiating cell lines, HSP70, a cytosolic marker protein, is absent from the culture medium throughout the assay period (panels C and F, lanes 7–12). Along with microscopic examination, its absence ensures that cells remain fully viable and, therefore, that VSG release is not due to cell death. Additionally, the steady increase in cell-associated HSP70 beginning at 12 h post-induction (panels C and F, lane 4) mirrors the logarithmic growth at this time (see Fig. 2A). Overall, these results verify that both GPI hydrolysis and proteolysis play a role in VSG coat remodeling during differentiation, release being mediated first by GPI hydrolysis and then by endoproteolysis.

Release of VSG by Proteolysis—The possibility exists that truncated VSG is generated by proteolysis of sVSG after release from the cell surface by GPI hydrolysis. This scenario seems unlikely, because the ratio of sVSG to truncated VSG does not change after release is complete (Fig. 3, A and D, lanes 7–12). Nevertheless, to address this concern we added purified biotinyl sVSG to standard differentiation assays at the 0-h time point: biotinyl AnTat sVSG to LouTat cultures and biotinyl LouTat sVSG to AnTat cultures. As previously seen, release of endogenous VSG is bimodal in both cases (Fig. 4, A and C). However, in neither differentiation assay is fragmentation of exogenous biotinyl sVSG observed, and therefore, these results confirm that proteolytic cleavage occurs at the cell surface and is independent of GPI hydrolysis.



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FIG. 4.
Addition of biotinlyated soluble VSG to the media during differentiation. Differentiation of LouTat 1.0 or AnTat 1.1 trypanosomes was performed as in Fig. 2 except that biotinylated sVSG of the opposite cell line was added to the media at the time of induction. Cell and media fractions were taken at 0 and 8 h post-induction for LouTat 1.0 (A and B) or 0 and 12 h post-induction for AnTat 1.1 (C and D) differentiation. Trypanosomal proteins were specifically immunoprecipitated with anti-LouTat 1.0 (A and D) or anti-AnTat 1.1 (B and C) VSG antibodies, separated by SDS-PAGE, and transferred to membranes. To detect endogenous strain-specific VSG, immunoblotting was done with anti-LouTat 1.0 (A) and anti-AnTat 1.1 (C) VSG antibodies. Exogenous biotinylated sVSG was detected with avidin-HRP conjugate (B and D). Full-length (F) and truncated (T) forms of LouTat 1.0 and AnTat 1.1 VSG are indicated. All lanes contain equivalent volumes of starting culture. Scales refer to relative molecular mass in kDa.

 

Because proteolytic release of VSG from the surface of transgenic procyclic cells is due to a zinc metalloprotease activity, we wished to determine if endoproteolytic release of VSG during differentiation is mediated by a similar activity. Therefore, in vitro differentiation assays were performed with LouTat 1.0 trypanosomes in the presence of the inhibitory compounds BPS, SB227961, SB227962, and P27 (Fig. 5). P27 is a selective inhibitor of the major lysosomal protease trypanopain (39, 40) and serves as a negative control. Due to solubility problems with the inhibitory compounds in our standard differentiation medium, a different medium (TM-P (32)) was used for these experiments. TM-P had no effect on the differentiation of LouTat 1.0 cells.



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FIG. 5.
Inhibition of proteolytic VSG release during differentiation. Differentiation of LouTat 1.0 trypanosomes was performed as in Fig. 2 except that cells were cultured in TM-P media supplemented with various compounds. Compounds were included at 5 mM (BPS, bathophenanthroline), 250 µM (SB227961 and SB227962), or 2 µM (P27). Cell (c) and media (m) fractions were taken 0 and 8 h post-induction and specifically immunoprecipitated with anti-LouTat 1.0 VSG antibodies. Precipitates were separated by SDS-PAGE, transferred to membranes, and immunoblotted with the respective anti-VSG antibody. Full-length (F) and truncated (T) forms of both LouTat 1.0 VSG are indicated. All lanes contain equivalent volumes of starting culture.

 

VSG is initially full-length and cell-associated (panel A, lane 1). In the absence of inhibitors, typical full-length and truncated forms of VSG are released into the medium by the 8-h time point (panel A, lane 4). BPS blocks the release of truncated VSG, as does SB227961 (panel A, lanes 6 and 8). However, the diastereoisomer, SB227962, does not block release (panel A, lane 10). P27, although used at a concentration that completely blocks normal turnover of the endogenous lysosomal membrane protein p67 (41), had a minimal effect on proteolytic release of VSG (panel A, lane 12). Similar results were obtained with BPS when examining AnTat 1.1 VSG release during differentiation.2 These results demonstrate that VSG is subject to endoproteolytic release by a zinc metalloprotease activity during in vitro transformation of bloodstream cells to the procyclic form.

Release of VSG by GPI Hydrolysis—The appearance of sVSG in the culture media is most likely not due to cell lysis and subsequent GPI-PLC activation, because cells remain fully intact during the differentiation assay, as evidenced by the absence of HSP70 from culture media (Fig. 3, C and F). In addition, it is unlikely that VSG is released from intact cells by the action of soluble GPI-PLC derived from sub-detectable levels of dead trypanosomes, because: (i) GPI-PLC is tightly membrane-associated and requires detergent for solubilization (42, 43); (ii) in the absence of detergent GPI-PLC must be in the same membrane as its substrate to achieve GPI hydrolysis (42); and (iii) soluble GPI-PLC, should it exist, would likely be unable to penetrate the intact VSG surface coat to gain access to the membrane proximal GPI anchors. However, our data do not prove that all full-length VSG is released by the action of GPI-PLC. Indeed, quantitative immunoprecipitations with anti-CRD and anti-VSG antibodies suggest that a portion of the full-length VSG is not CRD+ and therefore, may have been released by yet another mechanism.2 What this mechanism might be is currently unclear but a possible explanation is discussed below.

It is also striking that some CRD+ sVSG is found to be cell-associated at time zero of induction (Fig. 3, B and E, lane 1), suggesting that GPI-PLC is active prior to differentiation. It is possible to detect cell-associated sVSG, because sequential hydrolysis of each subunit of homodimeric VSG first produces a heterodimer attached to the membrane by one GPI-anchored full-length monomer. The trivial possibility exists, however, that cell-associated sVSG is due to GPI-PLC activation during the cell lysis step of sample preparation. To address this concern, cell lysates were prepared by rapid boiling in the presence of SDS to inactivate GPI-PLC. Cell-associated VSG was still CRD reactive at the 0-h time point, indicating that the sVSG seen is present before sample preparation.2 The implications of these results are 2-fold: GPI-PLC is capable of gaining access to its VSG substrate in fully intact cells, and because cell-associated sVSG is present at the 0-h time point, the enzyme may be pre-activated in the starting short stumpy population.

GPI-PLC has previously been shown to localize to the cytoplasmic face of intracellular vesicles (24), thereby sequestering the enzyme from extracellular VSG, yet our results strongly indicate that VSG can be released by GPI hydrolysis during differentiation. To determine if GPI-PLC is localized extracellularly, surface biotinylation assays were performed with enriched short stumpy populations of AnTat 1.1 (Fig. 6). Cells remained fully viable throughout the biotinylation procedure, and the cytosolic markers oligopeptidase B (lane 1), Rab11 (lane 2), and HSP70 (lane 3) were used as controls for cell integrity, each of which has been shown to be specifically immunoprecipitated by their respective antibodies in pulse label analyses.2 Rab11 is particularly relevant as a control, because it is localized to the cytosolic face of internal membranes (44), as is GPI-PLC. The cytosolic markers did not become biotinylated indicating that plasma membrane integrity was maintained throughout the assay. VSG was used as a positive control for surface biotinylation of proteins, and a robust signal is evident (lane 5). Strikingly, a signal corresponding to biotinyl GPI-PLC is consistently detected (lane 4) indicating that at least a portion of the total cellular pool is present on the cell surface. Attempts to independently verify the presence of GPI-PLC on the cell surface by immunofluorescence were not successful,2 presumably due to the lower sensitivity of this assay and the relatively low abundance of GPI-PLC (24). Nevertheless, these results are consistent with the direct release of VSG from the cell surface by activated, extracellular GPI-PLC during differentiation.



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FIG. 6.
Surface biotinylation of GPI-PLC. Enriched short stumpy populations of AnTat 1.1 were surface-biotinylated, and cell lysates were subjected to specific immunoprecipitation with anti-oligopeptidase B(O), anti-Rab11 (R), anti-HSP70 (H), anti-GPI-PLC (G), or anti-AnTat 1.1 VSG (V) antibodies. Immunoprecipitates were separated by SDS-PAGE, electrotransferred to membranes, blotted with HRP-avidin, and developed by chemiluminescence. The predicted electrophoretic mobility of each protein is indicated on the right with the corresponding abbreviation. Lanes contain the indicated cell equivalents. The scale represents molecular mass in kilodaltons.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the hallmarks of differentiation of bloodstream stage cells to the procyclic insect form is exchange of the major surface coat glycoproteins. VSG, which is essential to survival in the immunocompetent mammalian host, is replaced with procyclin, which is an essential adaptation for survival in the tsetse fly. Ziegelbauer et al. (22) documented that both GPI-PLC and proteolysis mediate VSG release during differentiation but were unable to identify the nature of the proteolytic activity. Nevertheless, proteolysis has been considered to be the dominant mode of VSG release, because GPI-PLC null mutants are fully viable and can complete an entire life cycle, including surface coat exchange (23). Furthermore, GPI-PLC has been reported to be located on the cytoplasmic face of internal organellar membranes, thereby presenting a topological barrier to VSG release by GPI hydrolysis (24).

We have reinvestigated these issues using two pleomorphic cell lines capable of synchronous differentiation when enriched for short stumpy trypanosomes. Similar to Ziegelbauer et al., we have found that VSG is released by two primary mechanisms, being initially mediated by GPI hydrolysis and at later time points, being mediated by proteolysis. These two modes of release are apparently independent, with each occurring at the cell surface. Our work builds on previous studies in two major ways. First, the proteolytic activity seen during differentiation can be confidently ascribed to a zinc metalloprotease. We have previously shown that fully differentiated procyclic trypanosomes possess a surface zinc metalloprotease activity that releases transgenic VSGs (25, 26). Our hypothesis is that a specific zinc metalloprotease is up-regulated during differentiation to release VSG and is then maintained on the surface of fully differentiated procyclic cells (discussed below). Second, we have demonstrated for the first time that GPI-PLC is present on the extracellular side of the plasma membrane in intact short stumpy cells. Extracellular localization is not due to classic secretion, because the enzyme does not have an N-terminal signal sequence or any other overt hydrophobic domains (43). However, GPI-PLC is reversibly thioacylated, and this modification has been proposed to regulate access of the enzyme to its VSG substrate in intact long slender cells (45). Our findings raise the possibility that thioacylation regulates topological translocation in a manner similar to that demonstrated for Leishmania hydrophilic acylated surface protein B (46).

In addition to the two major modes of release, preliminary data indicates that some full-length VSG may be shed without apparent GPI-hydrolysis or proteolysis.2 One possible explanation is that, following removal of one GPI anchor by either GPI hydrolysis or by proteolysis, the remaining GPI anchor may not efficiently retain the resulting mixed VSG dimer in the plasma membrane. The trypanosomal transferrin receptor may provide a precedent for this behavior. Immunoelectron microscopy in GPI-PLC null cells reveals that transferrin receptor, which is a single GPI anchor heterodimer, is found predominantly in the lumen of the flagellar pocket, whereas VSG is discretely localized along the flagellar pocket membrane (47, 48). Because GPI anchors cannot be cleaved in GPI-PLC null cells, this suggests that one anchor would not be sufficient to fully prevent shedding of VSG in differentiating bloodstream trypanosomes. Additional support for this mechanism comes from the observation that VSG can spontaneously transfer from live bloodstream trypanosomes into the membranes of neighboring erythrocytes (49). The ability of VSG to leave the plasma membrane in such systems is likely related to the exclusive use of short chain (C14) myristate in the GPI anchor of bloodstream trypanosomes (8). However, there are other possible explanations for this observation, such as loss of anti-CRD reactivity following release by GPI-hydrolysis, and additional studies are underway to clarify this issue.

The paradigm for zinc metalloproteases in kinetoplastid protozoa is MSP (major surface protease, previously called GP63), the major GPI-anchored surface protein of the promastigote insect stage of Leishmania (50, 51). Similar to trypanosomes, Leishmania have a digenetic lifecycle that alternates between the sandfly vector and vertebrate macrophage cells. With a strong structural resemblance to the metzincin class of zinc metalloproteases, MSP has a conserved HEXXH zinc-binding site and positionally conserved cysteine residues throughout its catalytic domain (52). Although highly conserved between Leishmania species, individual MSPs can be placed into distinct subclasses based on differences in sequence and developmental expression (5355). Classified as a parasite virulence factor, the importance of MSP to Leishmania is underscored by its proposed multiple functions: resistance to complement-mediated lysis and participation in the attachment and entry of metacyclic promastigotes into host macrophages (5658).

Homologues of Leishmania MSP have been identified in African trypanosomes by genomic sequencing (59). Based on differences in sequence and developmental expression, T. brucei MSP genes can also be placed into three distinct classes: TbMSP-A, -B, and -C (60). With our colleagues at the University of Iowa, we have used RNA interference to demonstrate that the TbMSP-B class mediates most, if not all, of the release of transgenic VSG from procyclic cells (60). In accordance with our hypothesis, we suggest that TbMSP-B also mediates the endoproteolytic release of VSG during differentiation, being up-regulated during this process and remaining on the surface of fully differentiated procyclic cells. Developmental expression of TbMSP-B is consistent with this theory: Northern analysis indicates that, although TbMSP-A and TbMSP-C mRNAs are expressed predominantly in the bloodstream stage of the parasite, TbMSP-B mRNA is about 2-fold more abundant in procyclic trypanosomes than in bloodstream stage cells (60). An RNAi strategy to assess the role of TbMSP-B in VSG release during differentiation is currently under development. In the meantime, we cannot dismiss the possibility that the TbMSP-A and -C classes also function in VSG release. In any case, our findings raise the question of why VSG is not released by endoproteolysis from dividing long slender bloodstream cells. Answers to this question must await development of specific immunoreagents to look at TbMSP expression at the protein level. Presumably, however, if these enzymes are expressed, they must be tightly regulated or topologically sequestered to prevent inappropriate release of VSG.

GPI-PLC is not essential for differentiation (23) and proteolysis presumably compensates in its absence to ensure that surface coat exchange occurs. The converse, however, cannot be stated because the effects of blocking proteolysis during differentiation are unknown. We have attempted in vitro differentiation assays with the inclusion of the peptidomimetic inhibitors over an extended period of time, but the compounds were found to be nonspecifically toxic to procyclic cells in long-term culture at the concentration used to block VSG release.2 Hopefully, the RNAi strategy mentioned above will provide some insight into the need for endoproteolysis during differentiation. In any case, it is likely that both GPI-PLC and the protease provide redundant means to release the old VSG coat.

One explanation for redundancy would be that GPI hydrolysis provides a back-up function for proteolysis. Our data indicate that LouTat 1.0 VSG is more susceptible to proteolytic cleavage than AnTat 1.1 VSG. Furthermore, cleavage site selection varies as evidenced by differently sized VSG fragments released from the two differentiating cell lines. Ziegelbauer et al. (22) mapped the proteolytic cleavage site of MITat 1.4 VSG to a region within the C-terminal domain that forms a loop between two subdomains. Little is known about the structure of the C-terminal domains of different VSGs, but they can be divided into several classes based on the number and spacing of conserved cysteine residues (61). Variations in the structure of the C-terminal domains could influence cleavage site selection and accessibility to the protease active site, making certain VSGs inherently more cleavable than others. In cases where a VSG is less susceptible to proteolysis, GPI-PLC would be the primary mediator of release, e.g. AnTat 1.1.

If these functions are redundant and GPI hydrolysis is capable of being the sole mediator of VSG release, the question remains as to why proteolysis occurs at all. Proteolysis may not be as efficient as GPI hydrolysis in mediating release of certain VSGs, and in addition, if the protease cleaves VSG first the C terminus must still be removed, presumably by GPI-PLC. Perhaps proteolysis serves an additional function in the procyclic insect stage of the parasite, the absence of which would go undetected during in vitro differentiation. However, if the TbMSP-B family is responsible for the zinc metalloprotease activity, it has been shown that RNA interference against TbMSP-B mRNA does not affect the growth rate of procyclic T. brucei in culture (60). This does not rule out the possibility that TbMSP-B is playing an essential role in vivo, within the midgut of the tsetse fly.

In summary, VSG release during differentiation is mediated in part by proteolysis and in part by GPI hydrolysis. We have identified the proteolytic activity as a zinc metalloprotease and propose that it is a member of the TbMSP-B family. Work is currently underway to test this hypothesis. The protease would release VSG during differentiation and then remain on the surface of fully differentiated procyclic cells, perhaps serving an essential function within the midgut of the tsetse fly. In addition, our results demonstrate that the other major mediator of VSG release, GPI-PLC, is localized to the extracellular side of the plasma membrane in intact short stumpy cells. Thus, GPI-PLC does gain access to its VSG substrate and plays a more prominent role in VSG release than previously thought. Finally, it cannot be coincidental that VSG is susceptible to these modes of release while procyclins are resistant. Procyclins contain C-terminal repeat motifs that are inherently protease resistant. Furthermore, as procyclins are expressed during differentiation, the core GPI structure changes from the diacylglycerol anchor of VSG to a lysoacylglycerol anchor with a direct inositol fatty acylation (8, 62, 63) that confers resistance to GPI-PLC. Thus, there is a "molecular checklist" for differentiation ensuring that only the unwanted VSG coat is shed as the new procyclin coat is simultaneously displayed.


    FOOTNOTES
 
* This work supported in part by National Institutes of Health (NIH) Grant AI35739 (to J. D. B.) and in part by NATO Collaborative Research Grant 972491 (to J. D. B. and N. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by NIH Cellular and Molecular Parasitology Training Grant AI07414. Back

** A Burroughs Welcome Fund New Investigator in Molecular Parasitology. To whom correspondence should be addressed: Dept. of Medical Microbiology and Immunology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-3110; Fax: 608-262-8418; E-mail: jdbangs{at}facstaff.wisc.edu.

1 The abbreviations used are: VSG, variant surface glycoprotein; sVSG, soluble VSG; GPI, glycosylphosphatidylinositol; GPI-PLC, glycosylphosphatidylinositol-specific phospholipase C; P27, morpholinourea-phenylalanine homophenylalanine-benz-{alpha}-pyrone; RT, reverse transcriptase; CRD, cross-reacting determinant; HSP70, 70-kDa heat shock protein; GP63, 63-kDa glycoprotein; RNAi, RNA interference; MSP, major surface protease; PBS, phosphate-buffered saline; PIC, protease inhibitor cocktail; RIPA, radioimmune precipitation assay; PAS, Protein A-Sepharose; HRP, horseradish peroxidase; Bicine, N,N-bis(2-hydroxyethyl)glycine. Back

2 A. E. Gruszynski and J. D. Bangs, unpublished observation. Back


    ACKNOWLEDGMENTS
 
We are indebted to Drs. Doug LaCount, John Donelson, and Anant Menon for providing experimental results prior to publication and/or thoughtful discussions and critical reading of the manuscript.



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