Produção Nacional
Revisado por pares
trans-Sialidase from Trypanosoma cruziBinds Host T-lymphocytes in a Lectin Manner
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m203185200
ISSN1083-351X
AutoresAdriane R. Todeschini, Murielle Girard, Jean‐Michel Wieruszeski, Marise P. Nunes, George A. DosReis, Lúcia Mendonça‐Previato, José O. Previato,
Tópico(s)Galectins and Cancer Biology
ResumoTrypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, expresses on its surface an uncommon membrane-bound sialidase, known astrans-sialidase. trans-Sialidase is the product of a multigene family encoding both active and inactive proteins. We report here that an inactive mutant of trans-sialidase physically interacts with CD4+ T cells. Using a combination of flow cytometry and immunoprecipitation techniques, we identified the sialomucin CD43 as a counterreceptor for trans-sialidase on CD4+ T cells. Using biochemical, immunological, and spectroscopic approaches, we demonstrated that the inactivetrans-sialidase is a sialic acid-binding protein displaying the same specificity required by active trans-sialidase. Taken together, these results suggest that inactive members of thetrans-sialidase family can physically interact with sialic acid-containing molecules on host cells and could play a role in host cell/T. cruzi interaction. Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, expresses on its surface an uncommon membrane-bound sialidase, known astrans-sialidase. trans-Sialidase is the product of a multigene family encoding both active and inactive proteins. We report here that an inactive mutant of trans-sialidase physically interacts with CD4+ T cells. Using a combination of flow cytometry and immunoprecipitation techniques, we identified the sialomucin CD43 as a counterreceptor for trans-sialidase on CD4+ T cells. Using biochemical, immunological, and spectroscopic approaches, we demonstrated that the inactivetrans-sialidase is a sialic acid-binding protein displaying the same specificity required by active trans-sialidase. Taken together, these results suggest that inactive members of thetrans-sialidase family can physically interact with sialic acid-containing molecules on host cells and could play a role in host cell/T. cruzi interaction. The surface of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas' disease (American Trypanosomiasis), displays a unique enzyme known astrans-sialidase (TS) 1The abbreviations used for: TS, trans-sialidase; iTS, inactive trans-sialidase; irTS, recombinant inactive trans-sialidase; rTS, recombinant active trans-sialidase; α2–3-SL, α2–3-sialyllactose; α2–6-SL, α2–6-sialyllactose; βGalp , β-galactopyranose; Fuc, fucose; SLeX, NeuAcα2–3Galβ1–3(Fucα1–4)-GlcNAc; PAA, polyacrylamide; PE, phycoerythrin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; FCM, flow cytometry; STD, saturation transfer difference; TOCSY, total correlation spectroscopy; mAb, monoclonal antibody. 1The abbreviations used for: TS, trans-sialidase; iTS, inactive trans-sialidase; irTS, recombinant inactive trans-sialidase; rTS, recombinant active trans-sialidase; α2–3-SL, α2–3-sialyllactose; α2–6-SL, α2–6-sialyllactose; βGalp , β-galactopyranose; Fuc, fucose; SLeX, NeuAcα2–3Galβ1–3(Fucα1–4)-GlcNAc; PAA, polyacrylamide; PE, phycoerythrin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; FCM, flow cytometry; STD, saturation transfer difference; TOCSY, total correlation spectroscopy; mAb, monoclonal antibody. (1Previato J.O. Andrade A.F.B. Pessolani M.C.V. Mendonça-Previato L. Mol. Biochem. Parasitol. 1985; 16: 85-96Crossref PubMed Scopus (173) Google Scholar, 2Schenkman S. Jiang M.S. Hart G.W. Nussenzweig V. Cell. 1991; 65: 1117-1125Abstract Full Text PDF PubMed Scopus (374) Google Scholar). TS is a modified sialidase (3Roggentin P. Rothe B. Kaper J.B. Galen J. Lawrisuk L. Vimr E.R. Schauer R. Glycoconj. J. 1989; 6: 349-353Crossref PubMed Scopus (157) Google Scholar) sharing the catalytic mechanism (4Todeschini A.R. Mendonça-Previato L. Previato J.O. Varki A. van Halbeek H. Glycobiology. 2000; 10: 213-221Crossref PubMed Scopus (45) Google Scholar) and the active site architecture (5Buschiazzo A. Tavares G.A. Campetella O. Spinelli S. Cremona M.L. Paris G. Amaya M.F. Frasch A.C. Alzari P.M. EMBO J. 2000; 19: 16-24Crossref PubMed Scopus (121) Google Scholar) with other known sialidases (6Crennell S.J. Garman E.F. Laver W.G. Vimr E.R. Taylor G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9852-9856Crossref PubMed Scopus (234) Google Scholar, 7Crennell S. Garman E. Laver G. Vimr E. Taylor G. Structure. 1994; 2: 535-544Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 8Garskell A. Crennell S.J. Taylor G. Structure. 1995; 15: 1197-1205Abstract Full Text Full Text PDF Scopus (191) Google Scholar). However, instead of releasing sialic acid, TS preferentially transfers sialic acid from β-galactopyranosyl (βGalp)-containing exogenous donor molecules to terminal βGalp-containing acceptors, attaching it in an α2–3 linkage configuration (9Vandekerckhove F. Schenkman S. Pontes de Carvalho L. Tomlinson S. Kiso M. Yoshida M. Hasegawa A. Nussenzweig V. Glycobiology. 1992; 2: 541-548Crossref PubMed Scopus (124) Google Scholar). T. cruzi TS belongs to a large family of proteins (10Uemura H. Schenkman S. Nussenzweig V. Eichinger D. EMBO J. 1992; 11: 3837-3844Crossref PubMed Scopus (76) Google Scholar, 11Cremona M.L. Sanchez D.O. Frasch A.C.C. Campetella O. Gene (Amst.). 1995; 160: 123-128Crossref PubMed Scopus (88) Google Scholar), and several other members of this family, which lack enzymatic activity (11Cremona M.L. Sanchez D.O. Frasch A.C.C. Campetella O. Gene (Amst.). 1995; 160: 123-128Crossref PubMed Scopus (88) Google Scholar), are expressed. Comparison of the deduced amino acid sequences shows that enzymatic activity requires a Tyr at position 342, whereas inactive members contain a His at the same position (11Cremona M.L. Sanchez D.O. Frasch A.C.C. Campetella O. Gene (Amst.). 1995; 160: 123-128Crossref PubMed Scopus (88) Google Scholar). The Tyr342 residue is involved in the stabilization of the transition-state sialyl carbocation formed during the hydrolysis reaction (4Todeschini A.R. Mendonça-Previato L. Previato J.O. Varki A. van Halbeek H. Glycobiology. 2000; 10: 213-221Crossref PubMed Scopus (45) Google Scholar, 5Buschiazzo A. Tavares G.A. Campetella O. Spinelli S. Cremona M.L. Paris G. Amaya M.F. Frasch A.C. Alzari P.M. EMBO J. 2000; 19: 16-24Crossref PubMed Scopus (121) Google Scholar). Indirect evidence suggests that an enzymatically inactive recombinant TS acts as a lectin, agglutinating desialylated erythrocytes (12Cremona M.L. Campetella O. Sanchez D.O. Frasch A.C.C. Glycobiology. 1999; 9: 581-588Crossref PubMed Scopus (62) Google Scholar). However, no direct evidence for a βGalp binding site or for its role in the parasite host interaction has been established. Trypomastigote-derived TS is anchored to the membrane through a glycosylphosphatidylinositol anchor and is released to the extracellular medium during acute T. cruzi infection in humans (13Frasch A.C. Parasitol. Today. 2000; 16: 282-286Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), thus acting distant from the parasite. Besides a role in mammalian cell invasion (14Burleigh B.A. Andrews N.W. Annu. Rev. Microbiol. 1995; 49: 175-200Crossref PubMed Scopus (187) Google Scholar), the soluble TS functions as a virulence determinant molecule. Chuenkova and Pereira (15Chuenkova M. Pereira M.E. J. Exp. Med. 1995; 181: 1693-1703Crossref PubMed Scopus (78) Google Scholar) demonstrated thatin vivo injection of minute amounts of purified native TS increases subsequent parasitemia and mortality in T. cruzi-infected mice. As TS injection into severe combined immunodeficiency mice did not affect parasitemia or mortality, it was suggested that TS acts on the host adaptive immune response (15Chuenkova M. Pereira M.E. J. Exp. Med. 1995; 181: 1693-1703Crossref PubMed Scopus (78) Google Scholar). It is well known that T lymphocytes bearing conventional αβ T cell receptors are required for control of parasitemia and mortality in murine infection by T. cruzi (16DosReis G.A. Parasitol. Today. 1997; 13: 335-342Abstract Full Text PDF PubMed Scopus (86) Google Scholar). Host CD4+ T cells are also involved in the immunopathology ofT. cruzi infection, and their exacerbated function can lead to mortality in susceptible hosts (17Hunter C.A. Ellis-Neys L.A. Slifer T. Kanaly S. Gruning G. Fort M. Rennick D. Araújo F.G. J. Immunol. 1997; 158: 3311-3316PubMed Google Scholar). Recently, we demonstrated that both enzymatically active and inactive TS costimulated CD4+T cell activation in vitro and in vivo and blocked activation-induced cell death in CD4+ T cells fromT. cruzi-infected mice through CD43 engagement (18Todeschini A.R. Nunes M.P. Pires R.S. Lopes M.F. Previato J.O. Mendonça-Previato L. DosReis G.A. J. Immunol. 2002; 168: 5192-5198Crossref PubMed Scopus (58) Google Scholar). In the present work, we extended our studies and, using CD43−/− mice, we show that the major lymphocyte mucin CD43 is the counterreceptor for the inactive TS on host CD4+ T cells. We also employed NMR spectroscopy and immunochemical approaches to investigate the nature of the CD43 epitope that functions as ligand for TS, and we demonstrated that inactive TS binds to sialic acid-containing molecules with the same specificity exhibited by active TS. Most of the chemical products used were from Sigma or Fisher. The following materials were obtained from other sources: microtiter plates were from Nunc; protein G-Sepharose, prepacked Ni2+-chelating HP HiTrap, Mono Q HR 10/10 and Mono S HR 5/5 columns, and enhanced chemiluminescence (ECL) hyperfilm were from Amersham Biosciences; biotin-conjugated polyacrylamide (PAA) probes substituted with sialylated glycans (α2–3-sialyllactose-PAA (α2–3-SL-PAA), α2–6-sialyllactose-PAA, (α2–6-SL-PAA), NeuAcα2–3Galβ1–3(Fucα1–4)-GlcNAc-PAA (SLeX-PAA)) were from Glycotech; anti-rat Ig κ chain mAb MAR 18.5 was from Cedarlane Laboratories; the sialic acid-dependent anti-CD43 mAb S7 (19Jones A.T. Federsppiel B. Ellies L.G. Williams M.J. Burgener R. Duronio V. Smith C.A. Takei F. Ziltener H.J. J. Immunol. 1994; 153: 3426-3439PubMed Google Scholar), fluorescein isothiocyanate-labeled anti-CD43 mAb S7, anti-CD16/CD32 mAb 2.4G2 (Fc block), phycoerythrin (PE)-conjugated streptavidin, horseradish peroxidase-conjugated anti-mouse IgG, and peroxidase-conjugate streptavidin were from Pharmingen; serum-free Dulbecco's modified Eagle's medium was from Invitrogen. Recombinant active TS (rTS) and inactive TS (irTS), containing the C-terminal repeats, were obtained fromEscherichia coli MC1061 electro-transformed with plasmids containing either the wild-type TS insert, TSREP (11Cremona M.L. Sanchez D.O. Frasch A.C.C. Campetella O. Gene (Amst.). 1995; 160: 123-128Crossref PubMed Scopus (88) Google Scholar), or the inactive mutant TS insert bearing a Tyr342 → His substitution, pTrcHisA (11Cremona M.L. Sanchez D.O. Frasch A.C.C. Campetella O. Gene (Amst.). 1995; 160: 123-128Crossref PubMed Scopus (88) Google Scholar). Bacteria were grown in supplemented Terrific broth in the presence of 100 μg/ml ampicillin. When the culture reached an A 600 nm of 1.0, 30 mg/liter isopropyl-1-thio-β-d-galactopyranoside was added, and incubation continued overnight. Bacteria were lysed at 4 °C in 20 mm Tris-HCl containing 2.0 mg/ml lysozyme, 2% Triton X-100, 0.1 μm phenylmethylsulfonyl fluoride, 5.0 μg/ml leupeptin, 1.0 μg/ml trypsin inhibitor, and 0.1 μmiodoacetamide. Both rTS and irTS containing a poly-His tag were purified as described by Buschiazzo et al. (20Buschiazzo A. Frasch A.C. Campetella O. Cell. Mol. Biol. (Oxf.). 1996; 42: 703-710PubMed Google Scholar) and modified by Todeschini et al. (4Todeschini A.R. Mendonça-Previato L. Previato J.O. Varki A. van Halbeek H. Glycobiology. 2000; 10: 213-221Crossref PubMed Scopus (45) Google Scholar) using Ni2+-chelating chromatography on a HiTrap column and eluted with an imidazole gradient (0–1 m). The eluates were dialyzed against 20 mm Tris-HCl, pH 7.6, further purified by ion exchange chromatography on Mono Q and Mono S columns, applying a linear NaCl gradient (0–1 m), and stored in 20 mm Tris-HCl buffer, pH 7.6, at 4 °C until used. The homogeneity of proteins was evaluated on 10% SDS-PAGE. For flow cytometry (FCM) and Western blotting analyses, irTS was biotin-conjugated as described previously (21Harlow E. Lane D. Antibodies: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 340-354Google Scholar). Enzyme activity was assayed by incubating rTS preparations in 5 mm cacodylate buffer, pH 7.0, in the presence of 0.25 μmol of α2–3-sialyllactose (α2–3-SL) and 0.25 μmol of [d-glucose-1-14C]lactose (400,000 cpm) (4Todeschini A.R. Mendonça-Previato L. Previato J.O. Varki A. van Halbeek H. Glycobiology. 2000; 10: 213-221Crossref PubMed Scopus (45) Google Scholar). After incubation for 30 min at 37 °C, the reaction mixture was diluted with 1 ml of water and applied to a column containing 1 ml of Dowex 2X8 (acetate form) equilibrated with water. To remove the excess of [d-glucose-1-14C]lactose, the column was washed with 10 ml of water, and sialylated [d-glucose-1-14C]lactose was eluted with 3 ml of 0.8 m ammonium acetate and quantitated by scintillation counting (Beckman LS 6500). One unit was defined as the amount of rTS required to catalyze the incorporation of 1 μmol of sialic acid into lactose per minute. BALB/c mice (male, aging 4–5 weeks) were obtained from Fundação Oswaldo Cruz, Rio de Janeiro, Brasil; CD43−/− and wild-type control mice (22Manjunath N. Correa M. Ardman M. Ardman B. Nature. 1995; 377: 535-538Crossref PubMed Scopus (186) Google Scholar) were from Universidade Federal de São Paulo, São Paulo, Brasil, animal facilities. All experiments were conducted according to protocols approved by the Committee on Ethics and Regulations of Animal Use of Instituto de Biofı́sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil. Primary T cell-enriched suspensions were obtained by nylon-wool filtration of fractionated splenocytes depleted of red cells by treatment with Tris-buffered ammonium chloride (23Lopes M.F. Veiga V.F. Santos A.R. Fonseca M.E.F. Dos Reis G.A. J. Immunol. 1995; 154: 744-752PubMed Google Scholar). Purified CD4+ T cells were nylon-nonadherent cells treated with anti-CD8 mAb for 30 min at 4 °C followed by anti-rat Ig κ chain mAb MAR 18.5 plus 10% rabbit complement for 45 min at 37 °C. 1 × 107 CD4+ T cells were lysed in PBS containing 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 0.1 μm iodoacetamide, 1 μg/ml trypsin inhibitor, and 0.1% Triton X-100 for 1 h at 4 °C under vigorous shaking. After pelleting (10,000 × g for 30 min), the supernatant was precleared overnight at 4 °C with 100 μl of protein G-Sepharose beads and centrifuged, and the supernatant was incubated with 3 μg of anti-CD43 mAb S7 for 2 h at 4 °C, under shaking. Protein G-Sepharose (50 μl) was added and incubated for 3 h. The beads were separated by centrifugation, washed three times, and incubated with SDS sample buffer for 5 min at 100 °C. The supernatant was run on 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked overnight with 2% bovine serum albumin in Tris-buffered saline containing 0.2% Tween 20, incubated with anti-CD43 mAb S7 for 2 h followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG. The reaction was detected using enhanced chemiluminescence (ECL) in Hyperfilm-ECL according to the manufacturer's instructions. For irTS immunoprecipitation, streptavidin beads and biotin-conjugated irTS were used. 1 × 107 T cells were resuspended in serum-free Dulbecco's modified Eagle's medium and incubated for 30 min at 37 °C with 0.05 units of Clostridium perfringens (Type X) sialidase in a total volume of 500 μl. Sialidase was removed by washing three times with serum-free Dulbecco's modified Eagle's medium. Desialylated T cells (5 × 106) were resialylated by incubation with 0.05 units of recombinant rTS in the presence of 1 mm α2–3-SL for 30 min at room temperature and washed as described above. Desialylated and resialylated T lymphocytes were resuspended in sorting buffer (10 mm PBS, pH 7.4, containing 2% bovine serum albumin, 0.02% NaN3, and 0.1 m lactose) incubated with biotin-conjugated irTS for 30 min at 4 °C, washed, stained with PE-conjugated streptavidin, and analyzed by FCM as described below. T cell-enriched suspension was incubated with 10 μg/ml Fc block for 5 min at 4 °C followed by addition of 10 μg/ml fluorescein isothiocyanate-labeled anti-CD43 mAb S7 or 10 μg/ml biotin-conjugated irTS for 30 min at 4 °C. The T cells were then washed in sorting buffer, incubated for 30 min with PE-conjugated streptavidin at 4 °C, washed again, and resuspended in 0.4 ml of sorting buffer plus 2% paraformaldehyde. T lymphocytes were gated by forward scatter and side scatter parameters, and 10,000 cells were analyzed on a fluorescence-activated cell sorter Xcalibur system using Cell Quest software. For irTS inhibition assay, biotin-conjugated irTS was preincubated with α2–3- or α2–6-SL in a range of 0–1 mm for 30 min at 4 °C. Analysis of irTS binding to sialic acid-containing molecules was done by ELISA (24Brinkman-van der Linden E.C.M. Varki A. J. Biol. Chem. 2000; 275: 8625-8632Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Wells in microtiter plates were coated overnight at 4 °C with a monoclonal antibody against the TS repeats (25Costa F. Franchin G. Pereira-Chioccola V.L. Ribeirão M. Schenkman S. Rodrigues M.M. Vaccine. 1998; 16: 768-774Crossref PubMed Scopus (101) Google Scholar) (500 ng/well) in 50 mm carbonate/bicarbonate buffer, pH 9.5. The plates were washed with ELISA buffer (3% bovine serum albumin in PBS, pH 7.4) and incubated overnight at 4 °C with irTS (500 ng/well) in the same buffer. Plates were blocked with ELISA buffer containing Triton X-100 1% for 1 h at room temperature and subsequently washed. Wells were then incubated with biotin-conjugated PAA probes substituted with sialylated glycans at a final concentration of 5.0 μg/well or a range between 0.25 and 10.0 μg/well for 2 h at room temperature. Following washing, wells were incubated for 1 h at room temperature with peroxidase-conjugated streptavidin, diluted (1:500) in blocking buffer, and developed with the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diamonium substrate system (100 μl/well). The plates were read at 405 nm in an automatic microplate reader (Bio-Tek Instruments). Control wells received a polyclonal antibody against TS catalytic domain (anti-TS) (25Costa F. Franchin G. Pereira-Chioccola V.L. Ribeirão M. Schenkman S. Rodrigues M.M. Vaccine. 1998; 16: 768-774Crossref PubMed Scopus (101) Google Scholar) (500 ng/well) for 1 h. Both mono- and polyclonal antibodies were generously supplied by Dr. Maurı́cio M. Rodrigues from the Universidade Federal de São Paulo, São Paulo, Brasil. The sialylated PAA probes (40 μg) were oxidized with 200 μl of 2 mm NaIO4 in PBS for 30 min at 4 °C in the dark and reduced with 200 μl of 10 mm NaBH4in PBS saline for 1 h at 4 °C (24Brinkman-van der Linden E.C.M. Varki A. J. Biol. Chem. 2000; 275: 8625-8632Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The products were diluted 5× with ELISA buffer and used in the assay. For sham treatment, 2 mm NaIO4 and 10 mmNaBH4 were mixed for 1 h at 4 °C and diluted with ELISA buffer, and sialylated PAA probes were added to this mixture just before use in the assay. Sialylated PAA probes were carboxyl-reduced with sodium borohydride treatment after esterification with iodoethane (24Brinkman-van der Linden E.C.M. Varki A. J. Biol. Chem. 2000; 275: 8625-8632Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Briefly, 100 μg of lyophilized sialylated PAA probes was solubilized in 350 μl of dimethyl sulfoxide, esterified with 35 μl of CH3CH2I for 1 h at room temperature, and then reduced by addition of 1.115 ml of PBS buffer containing 10 mm NaBH4. The reaction products were diluted 4× with ELISA buffer and directly used in the assay. For sham treatment, the same procedures were performed without adding CH3CH2I. Microtiter plates were coated with biotinylated PAA probes (200 ng/well) overnight at 4 °C in 50 mmcarbonate/bicarbonate buffer, pH 9.5. After blocking with ELISA buffer and subsequent washing, the wells were incubated for 1 h at room temperature with peroxidase-conjugated streptavidin diluted 1:500 times and developed with 100 μl/well 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamonium substrate system. The plates were read at 405 nm in an automated microplate reader. α2–3-SL, α2–6-SL, SLeX, or methyl α-mannoside was dissolved in deuterated PBS, pH 7.6 (not corrected for isotope effects). irTS solution in 20 mm Tris-HCl was exchanged with deuterated PBS by gel filtration on a G25 column. 20 μl of a stock solution containing 10 mg/ml of irTS was added to a solution of sialyl glycoside (2 mm final concentration), and the total volume was adjusted to 500 μl. NMR spectra were obtained at a probe temperature of 20 °C on a Bruker DMX 600 equipped with a 5-mm self-shielded gradient triple resonance probe or on a Bruker DRX 600 with a 5-mm triple resonance probe. One-dimensional STD experiments were performed by low power presaturation of the methyl region of the protein during the 2-s relaxation delay. The pulse scheme was as follows: relaxation delay with or without presaturation of the protein resonances, 90 degrees pulse, and acquisition of 256 scans (16,000 points for 10 ppm of spectral width). The data were obtained with interleaved acquisition of on-resonanse and control spectra to minimize the effects of temperature and magnet instability. Total Correlation Spectroscopy (STD-TOCSY) spectra were recorded with a mixing time of 66 ms, 32 scans per t 1 increment. 200t 1 increments were collected in an interlaced mode with presaturation on or off for 2 s. Prior to subtraction, both spectra were processed and phased identically. The acquisition time for the two-dimensional experiments were typically 16 h. The spectra were multiplied with a square cosine bell function in both dimensions and zero-filled two times. All spectra were referenced relative to trimethyl-silyl-2,2′,3,3′-d 4-propionic acid-sodium salt (δ = 0.0 ppm). We recently demonstrated that irTS from T. cruzi binds CD4+ T cells and that this binding is abrogated by prior treatment with anti-CD43 S7 mAb (18Todeschini A.R. Nunes M.P. Pires R.S. Lopes M.F. Previato J.O. Mendonça-Previato L. DosReis G.A. J. Immunol. 2002; 168: 5192-5198Crossref PubMed Scopus (58) Google Scholar). To prove that CD43 is the counterreceptor for TS on CD4+ T cells, we investigated the interaction between irTS and the leukosialin (CD43), using splenic CD4+ T cells from CD43-deficient mice (CD43−/−). As compared with wild type, CD4+ T cells from CD43−/− mice failed to bind either anti-CD43 mAb S7 (Fig. 1 A) or biotin-conjugated irTS (Fig. 1 B). To confirm that CD43 is the counterreceptor for irTS, CD4+ T cell extracts from wild-type and CD43−/− mice were immunoprecipitated with biotin-conjugated irTS or with anti-CD43 mAb S7. Precipitates were immunoblotted and revealed with anti-CD43 mAb S7 (Fig. 1 C). Remarkably, both irTS and anti-CD43 mAb S7 immunoprecipitated the same 115-kDa protein band expected for CD43 (19Jones A.T. Federsppiel B. Ellies L.G. Williams M.J. Burgener R. Duronio V. Smith C.A. Takei F. Ziltener H.J. J. Immunol. 1994; 153: 3426-3439PubMed Google Scholar). However, this protein was absent when CD4+ T cells from CD43−/− mice were submitted to the same treatment (Fig. 1 C). These results indicate that CD43 expression is required for irTS binding on T cells, showing that soluble irTS physically interacts with CD43. CD43 is the most abundant glycoprotein bearing α2–3- and α2–6-linked sialic acids expressed on the surface of T cells (26Ostberg J.R. Barth R.K. Frelinger J.G. Immunol. Today. 1998; 19: 546-550Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). To investigate whether sialic acid is the epitope for irTS, T cells were treated with C. perfringens sialidase, and the binding of biotinylated irTS was tested. Fig.2 A (a) shows that irTS binds to T cells, and this binding is abrogated by sialidase treatment (Fig. 2 A (b)). irTS binding was reconstituted with high intensity by resialylation of T cells with rTS in the presence of α2–3-SL as donor substrate (Fig. 2 A(c)). Since TS catalyzes the transfer of sialic acid residues from NeuAcα2–3Galpβ1-x-containing donors and attaches them in an α2–3 linkage to terminal βGalp-containing molecules (9Vandekerckhove F. Schenkman S. Pontes de Carvalho L. Tomlinson S. Kiso M. Yoshida M. Hasegawa A. Nussenzweig V. Glycobiology. 1992; 2: 541-548Crossref PubMed Scopus (124) Google Scholar), our results clearly show that irTS binds α2–3-linked sialic acid. The specificity of the interaction of irTS with α2–3-linked sialic acid was confirmed by inhibition studies using α2–3- (Fig. 2 A (d)) or α2–6-SL (Fig. 2 A (e)). As shown in Fig.2 A (d), binding of irTS to T cells was abrogated by the previous incubation of irTS with α2–3-SL but not with α2–6-SL (Fig. 2 A (e)). These results are consistent with the irTS bright pattern of staining observed after resialylation of T cells by rTS (Fig. 2 A (c)) since these cells now must bear almost exclusively α2–3-linked sialic acid. In addition, previous treatment of irTS with α2–3-SL completely disrupt irTS binding to resialylated T cells (Fig.2 A (f)). This inhibition was dose-dependent and had an IC50 of 0.49 mm (Fig. 2 B). The irTS preference for α2–3-linked sialic acid was further investigated using sialylated PAA probes. Fig.3 shows that irTS strongly binds to α2–3-linked sialic acid. Binding was dependent on the concentration of α2–3-SL-PAA, being maximal at 10 μg/ml, and relies on the carboxylate group of sialic acid since irTS binding is abrogated after carboxyl reduction (Fig. 3). Furthermore, when α2–6-SL- or SLeX-PAA probes were used as ligands, low level binding or no binding to irTS was observed, respectively (Fig. 4).Figure 4Binding of irTS to sialylated PAA probes. Sialylated PAA probes conjugated to α2–3-SL, α2–6-SL, or SleX were either untreated or treated with periodate and added to immobilized irTS in the presence or absence of anti-TS (antibodies against the TS catalytic site). Binding was tested by ELISA as described under "Experimental Procedures." Data show the mean ± S.D. of duplicates. Black column, PAA sham-treated; White column, PAA mild periodate-treated;Gray column, PAA + anti-TS.View Large Image Figure ViewerDownload (PPT) Periodate treatment of sialylated PAA probes did not reduce irTS binding (Fig. 4), demonstrating that the sialic acid side chain is not required for irTS recognition. Taken together, these data suggest that irTS displays a binding site that recognizes α2–3-linked sialic acid and its 7-carbon analog and that this binding can be abolished by either fucosylation or carboxyl reduction. Interactions of irTS with all sialylated PAA probes were inhibited in the presence of a polyclonal antibody directed against epitopes present in the catalytic domain of the active TS (Fig. 4) (25Costa F. Franchin G. Pereira-Chioccola V.L. Ribeirão M. Schenkman S. Rodrigues M.M. Vaccine. 1998; 16: 768-774Crossref PubMed Scopus (101) Google Scholar). To better understand the binding specificity observed for irTS, NMR spectroscopy studies were employed. Saturation transfer difference (STD) experiments were used to verify soluble α2–3-SL interaction with irTS. The binding assay was done in the presence of the methyl α-mannoside, used as negative control. Fig.5 shows the one-dimensional STD experiment of irTS in the presence of α2–3-SL and methyl α-mannoside in D2O PBS, pH 7.6, at 20 °C. Binding of α2–3-SL can be followed by measuring the signal at 2.050 ppm assigned to the 5NAc protons of N-acetylneuraminic acid (NeuAc) (27Reuter G. Schauer R. Methods Enzymol. 1994; 230: 168-199Crossref PubMed Scopus (147) Google Scholar) and by the NeuAc H3 equatorial (eq) and axial (ax) resonances at 2.727 and 1.812 ppm, respectively. Binding of methy α-mannoside would be detected by observation of the H1 and methyl protons at 4.775 and 3.405 ppm, respectively, in the STD spectrum. The subtraction of the spectrum in which protein resonance was presaturated (0.5 ppm) (Fig. 5 B) from the reference spectrum without protein saturation (Fig. 5 A) reveals the resonances at 2.727, 2.050, and 1.812 ppm (Fig. 5 C) of NeuAc from α2–3-SL ligand involved in the binding to the protein. No signals arising from methyl α-mannoside were observed, showing that α2–3-SL binds to irTS in a specific interaction. irTS interaction with α2–3-SL was further verified by two-dimensional NMR experiments, which show in detail the key structural elements of α2–3-SL involved in binding to irTS. A reference TOCSY spectrum of irTS in the presence of α2–3-SL and methyl α-mannoside was recorded without protein presaturation and with protein presaturation (0.5 ppm) (Fig.6 A). In the STD-TOCSY obtained (Fig. 6 B), the on-diagonal cross-peaks identify hydrogens in close proximity with the protein in the complex. It is evident that α2–3-SL binds to irTS. As the relative signal intensities on the STD spectrum correlate with the proximity to the protein, we can conclude that NAc and the H3ax protons from the NeuAc are in close contact with irTS. In the STD-TOCSY spectrum, off-diagonal cross-peaks help identify key hydrogens involved in binding. It is clear that the H3eq, H4, and H5 from the NeuAc residue and the H1, H3 and H4 from the βGalp ring also interact with the irTS as they receive saturation from the protein (Fig. 6 B). The H1 and H2 peaks from the α-anomer of Glcp are low in intensity, indicating a loose contact with the irTS binding site. Cross-peaks arising from α-Glcp or from methyl α-mannoside we
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