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• W1997852904 abstract "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 Leishmaniamajor 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. 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 Leishmaniamajor 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. 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: VSG 1The abbreviations used are: VSG, variant surface glycoprotein; sVSG, soluble VSG; GPI, glycosylphosphatidylinositol; GPI-PLC, glycosylphosphatidylinositol-specific phospholipase C; P27, morpholinourea-phenylalanine homophenylalanine-benz-α-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. in bloodstream trypanosomes (1Cross G.A.M. Parasitology. 1975; 71: 393-417Google Scholar) and procyclin in the procyclic insect stage (2Mowatt M.R. Clayton C.E. Mol. Cell. Biol. 1987; 7: 2833-2844Google Scholar, 3Roditi I. Carrington M. Turner M. Nature. 1987; 325: 272-274Google Scholar, 4Richardson J.P. Beecroft R.P. Tolson D.L. Liu M.K. Pearson T.W. Mol. Biochem. Parasitol. 1988; 31: 203-216Google Scholar). 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 (5Cross G.A.M. Bioessays. 1996; 18: 283-291Google Scholar). Unlike VSG, procyclin is a monomeric coat protein that can be divided into two classes based on distinct C-terminal repeat motifs (6Roditi I. Clayton C. Mol. Biochem. Parasitol. 1999; 103: 99-100Google Scholar). 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 (7Field M.C. Menon A.K. Cross G.A.M. EMBO J. 1991; 10: 2731-2739Google Scholar, 8Ferguson M.A.J. Homans S.W. Dwek R.A. Rademacher T.W. Science. 1988; 239: 753-759Google Scholar). 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 (9Rolin S. Hanocq-Quertier J. Paturiaux-Hanocq F. Nolan D. Salmon D. Webb H. Carrington M. Voorheis P. Pays E. J. Biol. Chem. 1996; 271: 10844-10852Google Scholar, 10Voorheis H. Bowles D. Smith G. J. Biol. Chem. 1982; 257: 2300-2304Google Scholar). 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 (11Czichos J. Nonnengaesser C. Overath P. Exp. Parasitol. 1986; 62: 283-291Google Scholar, 12Matthews K. Gull K. J. Cell Sci. 1997; 110: 2609-2618Google Scholar). When initiated with enriched short stumpy populations, differentiation is essentially synchronous, allowing for the precise description of temporally regulated events (13Ziegelbauer K. Quinten M. Schwarz H. Pearson T. Overath P. Eur. J. Biochem. 1990; 192: 373-378Google Scholar). 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 (13Ziegelbauer K. Quinten M. Schwarz H. Pearson T. Overath P. Eur. J. Biochem. 1990; 192: 373-378Google Scholar, 14Overath P. Czichos J. Stock U. Nonnengaesser C. EMBO. 1983; 2: 1721-1728Google Scholar, 15Roditi I. Schwarz H. Pearson T.W. Beecroft R.P. Liu M.K. Richardson J.T. Buhring H.-J. Pleiss J. Bulow R. Williams R.O. Overath P. J. Cell Biol. 1989; 108: 737-746Google Scholar). After VSG shedding has begun, the kinetoplast is repositioned with respect to the nucleus (16Brown R.C. Evans D.A. Vickerman K. Int. J. Parasitol. 1973; 3: 691-704Google Scholar). Differentiating trypanosomes enter into their first procyclic cell cycle following these morphological changes (17Matthews K. Sherwin T. Gull K. J. Cell Sci. 1995; 108: 2231-2239Google Scholar). 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 (7Field M.C. Menon A.K. Cross G.A.M. EMBO J. 1991; 10: 2731-2739Google Scholar, 18Ferguson M.A.J. Haldar K. Cross G.A.M. J. Biol. Chem. 1985; 260: 4963-4968Google Scholar). GPI hydrolysis releases a soluble form of VSG (sVSG), which retains the GPI core glycan. This type of VSG release occurs upon cell lysis (19Cardoso de Almeida M.L. Turner M.J. Nature. 1983; 302: 349-353Google Scholar) 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 (20Seyfang A. Mecke D. Duszenko M. J. Protozool. 1990; 37: 546-552Google Scholar, 21Bulow R. Nonnengasser C. Overath P. Mol. Biochem. Parasitol. 1989; 32: 85-92Google Scholar). GPI-PLC-mediated release of VSG also occurs during differentiation of bloodstream stage trypanosomes (22Ziegelbauer K. Stahl B. Karas M. Stierhof Y.-D. Overath P. Biochemistry. 1993; 32: 3737-3742Google Scholar). The second mode of VSG release, endoproteolysis, only occurs during differentiation as evidenced by the shedding of soluble VSG fragments (21Bulow R. Nonnengasser C. Overath P. Mol. Biochem. Parasitol. 1989; 32: 85-92Google Scholar, 22Ziegelbauer K. Stahl B. Karas M. Stierhof Y.-D. Overath P. Biochemistry. 1993; 32: 3737-3742Google Scholar). 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 (23Webb H. Carnall N. Vanhamme L. Rolin S. Van Den Abbeele J. Welburn S. Pays E. Carrington M. J. Cell Biol. 1997; 139: 103-114Google Scholar). Moreover, GPI-PLC has been reported to localize to the cytoplasmic face of intracellular vesicles, sequestering the enzyme away from extracellular VSG (24Bulow R. Griffiths G. Webster P. Stierhof Y.-D. Opperdoes F.R. Overath P. J. Cell Sci. 1989; 93: 233-240Google Scholar). 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 (25Bangs J.D. Ransom D.M. McDowell M.A. Brouch E.M. EMBO J. 1997; 16: 4285-4294Google Scholar). Release is inhibited by the zinc chelator bathophenanthroline and by peptidomimetic inhibitors of mammalian collagenase-type zinc metalloproteases (26Bangs J. Ransom D. Nimick M. Christie G. Hooper N. Mol. Biochem. Parasitol. 2001; 114: 111-117Google Scholar). 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. Compounds—Bathophenanthroline was obtained from Sigma. SB227961 and SB227962 were kindly supplied by SmithKline Beecham Pharmaceuticals (SB, Harlow, UK) and have been described previously (26Bangs J. Ransom D. Nimick M. Christie G. Hooper N. Mol. Biochem. Parasitol. 2001; 114: 111-117Google Scholar). P27 (morpholinourea-phenylalanine homophenylalanine-benz-α-pyrone) was a generous gift from Dr. James McKerrow (University of California-San Francisco). All compounds were dissolved as 50–100× 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 (27Cunningham I. J. Protozool. 1977; 24: 325-329Google Scholar). 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 (28Bangs J.D. Brouch E.M. Ransom D.M. Roggy J.L. J. Biol. Chem. 1996; 271: 18387-18393Google Scholar) and inserts were sequenced to confirm identity. Stable transformation of cultured Lister 427 strain procyclic trypanosomes has been described (28Bangs J.D. Brouch E.M. Ransom D.M. Roggy J.L. J. Biol. Chem. 1996; 271: 18387-18393Google Scholar). Antibodies—Rabbit antiserum against Tbhsp70 (gene 4) has been described (29McDowell M.A. Ransom D.A. Bangs J.D. Biochem. J. 1998; 335: 681-689Google Scholar). 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 (30Cross G.A.M. J. Cell. Biochem. 1984; 24: 79-90Google Scholar). Anti-cross-reacting determinant (anti-CRD) (31Barbet A.F. McQuire T.C. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 1989-1993Google Scholar) 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 α(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 (25Bangs J.D. Ransom D.M. McDowell M.A. Brouch E.M. EMBO J. 1997; 16: 4285-4294Google Scholar, 26Bangs J. Ransom D. Nimick M. Christie G. Hooper N. Mol. Biochem. Parasitol. 2001; 114: 111-117Google Scholar). 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 (32Overath P. Czichos J. Hass C. Eur. J. Biochem. 1986; 160: 175-182Google Scholar)). 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 (33Lanham S.M. Godfrey D.G. Exp. Parasitol. 1970; 28: 521-534Google Scholar) 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 × g, 37 °C). The cells in suspension were collected and washed once with 37 °C BBS. Cells were diluted to ∼5 × 106 cells/ml in HMI9 media (34Hirumi H. Hirumi K. Parasitol. Today. 1994; 10: 81-84Google Scholar) 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 × 106 cells/ml in 27 °C Cunningham's media supplemented with 3 mm each of citrate and cis-aconitate (11Czichos J. Nonnengaesser C. Overath P. Exp. Parasitol. 1986; 62: 283-291Google Scholar). Cells were cultured at 27 °C, and time points were taken over a 48-h period. At each time point, 1.5 × 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 × 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 × 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 (33Lanham S.M. Godfrey D.G. Exp. Parasitol. 1970; 28: 521-534Google Scholar). 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 10× PBS and centrifuged (5 min, 12,000 × 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 × 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 (33Lanham S.M. Godfrey D.G. Exp. Parasitol. 1970; 28: 521-534Google Scholar). 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 × 108 cells/ml in RIPA buffer supplemented with PIC. Cell lysates were pre-cleared by centrifugation (5 min, 12,000 × g). To remove contaminating mouse IgG that copurifies with short stumpy trypanosomes, cell lysates (2 × 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). 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 (25Bangs J.D. Ransom D.M. McDowell M.A. Brouch E.M. EMBO J. 1997; 16: 4285-4294Google Scholar). 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 (26Bangs J. Ransom D. Nimick M. Christie G. Hooper N. Mol. Biochem. Parasitol. 2001; 114: 111-117Google Scholar). 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 (22Ziegelbauer K. Stahl B. Karas M. Stierhof Y.-D. Overath P. Biochemistry. 1993; 32: 3737-3742Google Scholar). 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, (25Bangs J.D. Ransom D.M. McDowell M.A. Brouch E.M. EMBO J. 1997; 16: 4285-4294Google Scholar)), 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 (26Bangs J. Ransom D. Nimick M. Christie G. Hooper N. Mol. Biochem. Parasitol. 2001; 114: 111-117Google Scholar). Collectively, these results confirm that LouTat 1.0 VSG is subject to endoproteolytic release by the endogenous procyclic zinc metalloprotease activity. 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 (11Czichos J. Nonnengaesser C. Overath P. Exp. Parasitol. 1986; 62: 283-291Google Scholar). 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 (35Matthews K. Gull K. Mol. Biochem. Parasitol. 1998; 95: 81-95Google Scholar, 36Vassella E. Abbeele J.V. Butikofer P. Renggli C.K. Furger A. Brun R. Roditi I. Genes Dev. 2000; 14: 615-626Google Scholar): 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. VSG Release during Differentiation—Both endoproteolysis and GPI hydrolysis by endogenous GPI-PLC have been implicated in VSG release during differentiation (22Ziegelbauer K. Stahl B. Karas M. Stierhof Y.-D. Overath P. Biochemistry. 1993; 32: 3737-3742Google Scholar). GPI hydrolysis removes dimyristoyl glycerol, releasing the intact sVSG protein that retains the glycan portion of the GPI anchor (19Cardoso de Almeida M.L. Turner M.J. Nature. 1983; 302: 349-353Google Scholar, 37Ferguson M.A.J. Low M.G. Cross G.A.M. J. Biol. Chem. 1985; 260: 14547-14555Google Scholar). 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 (38Zamze S.E. Ferguson M.A.J. Collins R.A. Dwek R.A. Rademacher T.W. Eur. J. Biochem. 1988; 176: 527-534Google Scholar). 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 differenti" @default.
• W1997852904 created "2016-06-24" @default.
• W1997852904 creator A5021063040 @default.
• W1997852904 creator A5050214094 @default.
• W1997852904 creator A5081365611 @default.
• W1997852904 creator A5090809624 @default.
• W1997852904 date "2003-07-01" @default.
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• W1997852904 title "Surface Coat Remodeling during Differentiation of Trypanosoma brucei" @default.
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