Two Trans-sialidase Forms with Different Sialic Acid Transfer and Sialidase Activities from Trypanosoma congolense
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m212909200
ISSN1083-351X
AutoresEvelin Tiralongo, Silke Schrader, Hans Lange, Hilmar Lemke, Joe Tiralongo, Roland Schauer,
Tópico(s)Research on Leishmaniasis Studies
ResumoTrypanosomes express an enzyme called trans-sialidase (TS), which enables the parasites to transfer sialic acids from the environment onto trypanosomal surface molecules. Here we describe the purification and characterization of two TS forms from the African trypanosome Trypanosoma congolense. The purification of the two TS forms using a combination of anion exchange chromatography, isoelectric focusing, gel filtration, and subsequently, antibody affinity chromatography resulted, in both cases, in the isolation of a 90-kDa monomer on SDS-PAGE, which was identified as trans-sialidase using micro-sequencing. Monoclonal antibody 7/23, which bound and partially inhibited TS activity, was found in both cases to bind to a 90-kDa protein. Both TS forms possessed sialidase and transfer activity, but markedly differed in their activity ratios. The TS form with a high transfer-to-sialidase activity ratio, referred to as TS-form 1, possessed a pI of pH 4–5 and a molecular mass of 350–600 kDa. In contrast, the form with a low transfer-to-sialidase activity ratio, referred to as TS-form 2, exhibited a pI of pH 5–6.5 and a molecular mass of 130–180 kDa. Both TS forms were not significantly inhibited by known sialidase inhibitors and revealed no significant differences in donor and acceptor substrate specificities; however, TS-form 1 utilized various acceptor substrates with a higher catalytic efficiency. Interestingly, glutamic acid-alanine-rich protein, the surface glycoprotein, was co-purified with TS-form 1 suggesting an association between both proteins. Trypanosomes express an enzyme called trans-sialidase (TS), which enables the parasites to transfer sialic acids from the environment onto trypanosomal surface molecules. Here we describe the purification and characterization of two TS forms from the African trypanosome Trypanosoma congolense. The purification of the two TS forms using a combination of anion exchange chromatography, isoelectric focusing, gel filtration, and subsequently, antibody affinity chromatography resulted, in both cases, in the isolation of a 90-kDa monomer on SDS-PAGE, which was identified as trans-sialidase using micro-sequencing. Monoclonal antibody 7/23, which bound and partially inhibited TS activity, was found in both cases to bind to a 90-kDa protein. Both TS forms possessed sialidase and transfer activity, but markedly differed in their activity ratios. The TS form with a high transfer-to-sialidase activity ratio, referred to as TS-form 1, possessed a pI of pH 4–5 and a molecular mass of 350–600 kDa. In contrast, the form with a low transfer-to-sialidase activity ratio, referred to as TS-form 2, exhibited a pI of pH 5–6.5 and a molecular mass of 130–180 kDa. Both TS forms were not significantly inhibited by known sialidase inhibitors and revealed no significant differences in donor and acceptor substrate specificities; however, TS-form 1 utilized various acceptor substrates with a higher catalytic efficiency. Interestingly, glutamic acid-alanine-rich protein, the surface glycoprotein, was co-purified with TS-form 1 suggesting an association between both proteins. The flagellated protozoa, trypanosomes, the agents of several diseases, express a unique type of glycosyltransferase, called trans-sialidase (TS), 1The abbreviations used are: TS, trans-sialidase; FCS, fetal calf serum; GARP, glutamic acid-alanine-rich protein; IEF, isoelectric focusing; mAb, monoclonal antibody; MU, 4-methylumbelliferone; MUGal, 2′-(4-methylumbelliferyl)galactoside; MULac, 2′-(4-methylumbelliferyl)lactoside; MUNeu5Ac, 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid; Neu5Ac, N-acetylneuraminic acid; Neu2en5Ac, 5-N-acetyl-2-deoxy-2,3-didehydroneuraminic acid; SA, sialidase; α2,3-SL, sialyllactose (Neu5Acα2,3-lactose); ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; GPI, glycosylphosphatidylinositol. which is believed to play an important role in maintaining pathogenicity of the parasites (1Cross G.A. Takle G.B. Annu. Rev. Microbiol. 1993; 47: 385-411Google Scholar, 2Schenkman S. Eichinger D. Pereira M.E. Nussenzweig V. Annu. Rev. Microbiol. 1994; 48: 499-523Google Scholar). Unlike typical sialyltransferases, which require CMP-activated sialic acid as the monosaccharide donor (3Paulson J.C. Colley K.J. J. Biol. Chem. 1989; 264: 17615-17618Google Scholar), TS catalyzes the transfer of, preferably, α2,3-carbohydrate-linked sialic acids to another carbohydrate forming a new α2,3-glycosidic linkage to galactose or N-acetylgalactosamine. In the absence of an appropriate acceptor TS acts like a sialidase (SA), similar to viral, bacterial, mammalian, and trypanosomal SA, hydrolyzing glycosidically linked sialic acids (1Cross G.A. Takle G.B. Annu. Rev. Microbiol. 1993; 47: 385-411Google Scholar, 2Schenkman S. Eichinger D. Pereira M.E. Nussenzweig V. Annu. Rev. Microbiol. 1994; 48: 499-523Google Scholar). TS was first described in the bloodstream form of the American trypanosome Trypanosoma cruzi (4Schenkman S. Jiang M.S. Hart G.W. Nussenzweig V. Cell. 1991; 65: 1117-1125Google Scholar), the pathogen of Chagas disease, afflicting millions of people in Latin America. TS has also been reported to occur in the procyclic insect forms of the African trypanosomes Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense (5Engstler M. Schauer R. Brun R. Acta Trop. 1995; 59: 117-129Google Scholar), which are the cause of human sleeping sickness. Furthermore, TS has been found in procyclic forms of other African trypanosomes, such as Trypanosoma brucei brucei (6Pontes de Carvalho L.C. Tomlinson S. Vandekerckhove F. Bienen E.J. Clarkson A.B. Jiang M.S. Hart G.W. Nussenzweig V. J. Exp. Med. 1993; 177: 465-474Google Scholar, 7Engstler M. Reuter G. Schauer R. Mol. Biochem. Parasitol. 1993; 61: 1-13Google Scholar) and Trypanosoma congolense (5Engstler M. Schauer R. Brun R. Acta Trop. 1995; 59: 117-129Google Scholar). These parasites are the agents of Nagana, the trypanosomiasis in African ruminants. Trypanosomes are unable to synthesize sialic acids; instead they utilize TS to transfer sialic acid from the environment onto trypanosomal surface molecules. In the case of T. cruzi,TS is employed to acquire sialic acid from mammalian host glycoconjugates to sialylate mucin-like acceptor molecules in the parasite plasma membrane (8Schenkman S. Ferguson M.A. Heise N. de Almeida M.L. Mortara R.A. Yoshida N. Mol. Biochem. Parasitol. 1993; 59: 293-303Google Scholar). Furthermore, TS sialylates host cell glycoconjugates to generate receptors, which are used for parasite adherence and subsequent entry into host cells (2Schenkman S. Eichinger D. Pereira M.E. Nussenzweig V. Annu. Rev. Microbiol. 1994; 48: 499-523Google Scholar). In the African species T. brucei brucei and T. congolense, where TS is only expressed in the procyclic insect stage, the enzyme is used to sialylate the major cell surface glycoprotein of the parasites (e.g. procyclic acidic repetitive protein and GARP) in the vector (tsetse fly). Thus, a negatively charged glycocalyx is formed, which is believed to protect the parasites from digestive conditions in the fly gut, or from immunocompetent substances present in the fly's blood meal and may enable them to interact with epithelial cells (6Pontes de Carvalho L.C. Tomlinson S. Vandekerckhove F. Bienen E.J. Clarkson A.B. Jiang M.S. Hart G.W. Nussenzweig V. J. Exp. Med. 1993; 177: 465-474Google Scholar, 9Engstler M. Schauer R. Parasitol. Today. 1994; 10: 180Google Scholar). Additionally, it has recently been reported that T. cruzi TS itself directs neuronal differentiation in PC12 cells (10Chuenkova M.V. Pereira M.A. Neuroreport. 2001; 12: 3715-3718Google Scholar), stimulates interleukin-6 secretion from normal human endothelial cells (11Saavedra E. Herrera M. Gao W. Uemura H. Pereira M.A. J. Exp. Med. 1999; 190: 1825-1836Google Scholar), and potentiates T cell activation through antigen-presenting cells (12Gao W. Pereira M.A. Eur. J. Immunol. 2001; 31: 1503-1512Google Scholar). Investigating TS has become increasingly attractive over the last years, not only because of its involvement in trypanosomal pathogenicity but also because of its biotechnological importance. That is, TS is a unique enzyme that, because of its ability to transfer Neu5Ac in a stereo- and regio-specific manner, can be utilized to synthesize a variety of biologically relevant structures of the type Neu5Acα2,3Galβ1-R (13Takahashi N. Lee K.B. Nakagawa H. Tsukamoto Y. Kawamura Y. Li Y.T. Lee Y.C. Anal. Biochem. 1995; 230: 333-342Google Scholar, 14Vetere A. Ferro S. Bosco M. Cescutti P. Paoletti S. Eur. J. Biochem. 1997; 247: 1083-1090Google Scholar). To date, only two trypanosomal TS have been studied in detail, the American T. cruzi TS (15Schenkman S. Pontes D.C. Nussenzweig V. J. Exp. Med. 1992; 175: 567-575Google Scholar, 16Pereira M.E. Mejia J.S. Ortega-Barria E. Matzilevich D. Prioli R.P. J. Exp. Med. 1991; 174: 179-191Google Scholar, 17Scudder P. Doom J.P. Chuenkova M. Manger I.D. Pereira M.E. J. Biol. Chem. 1993; 268: 9886-9891Google Scholar) and the African T. brucei brucei TS (6Pontes de Carvalho L.C. Tomlinson S. Vandekerckhove F. Bienen E.J. Clarkson A.B. Jiang M.S. Hart G.W. Nussenzweig V. J. Exp. Med. 1993; 177: 465-474Google Scholar, 7Engstler M. Reuter G. Schauer R. Mol. Biochem. Parasitol. 1993; 61: 1-13Google Scholar, 18Engstler M. Reuter G. Schauer R. Mol. Biochem. Parasitol. 1992; 54: 21-30Google Scholar), with different genes encoding T. cruzi TS (19Uemura H. Schenkman S. Nussenzweig V. Eichinger D. EMBO J. 1992; 11: 3837-3844Google Scholar, 20Parodi A.J. Pollevick G.D. Mautner M. Buschiazzo A. Sanchez D.O. Frasch A.C. EMBO J. 1992; 11: 1705-1710Google Scholar, 21Buschiazzo A. Frasch A.C.C. Campetella O. Cell Mol. Biol. 1996; 42: 703-710Google Scholar) and T. brucei brucei TS (22Montagna G. Cremona M.L. Paris G. Amaya M.F. Buschiazzo A. Alzari P.M. Frasch A.C. Eur. J. Biochem. 2002; 269: 2941-2950Google Scholar) being identified and analyzed. Furthermore, the SA expressed by Trypanosoma rangeli, a non-pathogenic relative of T. cruzi, has been isolated and characterized biochemically (23Reuter G. Schauer R. Prioli R.P. Pereira M. Glycoconj. J. 1987; 4: 339-348Google Scholar) and genetically (24Buschiazzo A. Campetella O. Frasch A.C. Glycobiology. 1997; 7: 1167-1173Google Scholar). Although the crystal structure of T. cruzi trans-sialidase has recently been published (25Buschiazzo A. Amaya M.F. Cremona M.L. Frasch A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Google Scholar), a number of questions concerning the exact TS transfer mechanism remain un-answered. Because native and recombinant enzymes can differ in their glycosylation, antibody specificity, and biochemical properties, it is important that the native enzyme be purified and characterized, with the subsequent aim of obtaining sequence information. This is especially important, because several genes encoding TS/SA enzymes, or even silent genes may exist in trypanosomes, as has been shown for T. cruzi (26Cremona M.L. Campetella O. Sanchez D.O. Frasch A.C.C. Glycobiology. 1999; 9: 581-587Google Scholar). Here we describe the purification and characterization of two TS forms from the African trypanosome T. congolense and their identification using micro-sequencing. Moreover, we report on the production of monoclonal antibodies raised against both enzyme forms and their subsequent use in purification. Additionally, we present characterization studies that reveal significant differences between both TS forms concerning their transfer to SA ratios and catalytic efficiencies using various acceptor substrates. Materials—Unless otherwise stated analytical grade reagents from Sigma (Deisenhofen, Germany), Merck (Darmstadt, Germany), ICN (Eschwege, Germany), and Roche Diagnostics GmbH (Mannheim, Germany) were used throughout this study. Galβ1,4-[14C]GlcNAc was purchased from Hartmann Analytic GmbH (Braunschweig, Germany). Materials for chromatography, including Q-Sepharose FF and Sephadex G150 SF were obtained from Amersham Biosciences (Freiburg, Germany). Substances—2′-(4-Methylumbelliferyl)lactoside (MULac) was provided by Dr. T. Yoshino (Tokyo, Japan), 4-amino-Neu2en5Ac and 4-guanidino-Neu2en5Ac by Dr. M. von Itzstein (Gold Coast, Australia), suramin was a gift from Dr. P. Nickel (Bonn, Germany), and recombinant T. cruzi TS and T. brucei brucei TS were a gift from Dr. A. C. C. Frasch (Buenos Aires, Argentina). Neu5Acα2,3-lactose (α2,3-SL) and Neu5Acα2,6-lactose were isolated from cow colostrum according to Veh et al. (27Veh R.W. Michalski J.C. Corfield A.P. Sander-Wewer M. Gies D. Schauer R. J. Chromatogr. 1981; 212: 313-322Google Scholar). Neu5Acα2,3-N-acetyllactosamine was purchased from Dextra Laboratories (Reading, UK) and fetuin from ICN. Sialyloligosaccharides from bovine and human milk, as well as glycomacropeptide and apolactoferrin were provided by Numico Research (Friedrichsdorf, Germany). Sialyl-Lewisx, N-acetyllactosamine, lacto-N-biose I, lacto-N-neotetraose, lacto-N-tetraose, lactose, lactitol, mannose, galactose, glucose, maltose, galactose-α1,4-galactose, and Neu5Ac were obtained from Calbiochem-Novabiochem GmbH (Bad Soden, Germany). Chondroitin sulfate A, heparan sulfate, dextran sulfate, heparin (high and low molecular weight), Neu5Ac, Neu2en5Ac, 2′-(4-methylumbelliferyl)galactoside (MUGal) and N-(4-nitrophenyl)oxamic acid were purchased from Sigma. Antibodies—Antiserum to T. cruzi TS was generously provided by Dr. I. Marchal (Lille, France). Anti-T. congolense procyclin (GARP) mAb was purchased from Cedarlane (Toronto, Canada). Horseradish peroxidase-conjugated affinity-pure donkey anti-rabbit IgG antibody was from Dianova (Hamburg, Germany). Peroxidase-conjugated anti-mouse IgG antibody and biotin-conjugated anti-mouse IgG3 antibody from Southern Biotechnology Associates Inc. was purchased from Dunn Labortechnik GmbH (Asbach, Germany). Peroxidase-conjugated streptavidin was purchased from Roche Applied Science (Mannheim, Germany). Cultivation—Procyclic culture forms of T. congolense (STIB 249, kindly provided by Dr. Retro Brun from the Swiss Tropical Institute, Basel, Switzerland) were cultivated axenically in SM/SDM 79 medium (28Brun R. Schoenberger M. Acta Trop. 1979; 36: 289-292Google Scholar), containing 10% fetal calf serum (FCS, PAA Laboratories, Austria) and 0.001% hemin. After 3–4 days of cultivation, the trypanosomes were transferred into new SM/SDM 79 medium without FCS and hemin. Following a further 3 days, the culture supernatant was harvested via centrifugation. Assays—For all enzyme assays the formation of product was linear with respect to time and protein amount. In all activity tests, controls were performed in the absence of enzyme sample or using heat-inactivated enzyme. For fluorescence detection a 96-well-plate fluorometer (Fluorolite 1000, Dynatech Laboratories) was used. SA activity was routinely tested in the presence of 1 mm MUNeu5Ac in 20 mm Bis/Tris buffer, pH 7.0 (29Warner T.G. O'Brien J.S. Biochemistry. 1979; 18: 2783-2787Google Scholar). The reaction mixture was incubated for 120 min at 37 °C in black 96-well-plates (Microfluor, Dynex). By the addition of 0.08 m glycine/NaOH buffer, pH 10, the reaction was terminated, and the fluorescence of MU released measured immediately at an excitation and emission wavelength of 365 and 450 nm, respectively. The instrument was calibrated with MU standard solutions. One unit of SA activity equals 1 μmol of MU released per minute, which is equivalent to 1 μmol of sialic acid released per minute. TS activity was routinely tested using the non-radioactive assay described by Schrader et al. 2S. Schrader, E. Tiralongo, G. Paris, T. Yoshino, and R. Schauer, submitted for publication. Briefly, TS activity was monitored by incubating 25 μl of enzyme solution in 50 mm Bis/Tris buffer, pH 7.0, containing 1 mm α2,3-SL as the donor and 0.5 mm MUGal as the acceptor in a final volume of 50 μl at 37 °C for 2 h. The reaction was terminated by the addition of ice-cold water and, subsequently, applied to mini-columns containing Q-Sepharose FF. After washing, the sialylated product was eluted with 1 m HCl, hydrolyzed at 95 °C for 45 min, and cooled on ice. The samples were neutralized, adjusted to pH 10, and MU released was measured as stated above. One unit of TS activity equals 1 μmol of MU released per minute, which is equivalent to 1 μmol of sialic acid transferred per minute. During the course of this study the TS test described above was modified by applying the assay principle to a 96-well plate format. Because of its enhanced throughput, all TS tests for mAb screening, as well as kinetic experiments were performed using the 96-well-plate assay (Schrader et al.). 2S. Schrader, E. Tiralongo, G. Paris, T. Yoshino, and R. Schauer, submitted for publication. Protein concentration was determined using either the BCA protein assay kit from Pierce (Cologne, Germany) or the Bio-Rad protein assay (30Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar) from Bio-Rad (Munich, Germany), as described by each manufacturer. All assays were performed in 96-well plates employing bovine serum albumin as the standard, and photometric determinations were performed using a 96-well plate photometer (Tecan Sunrise, Tecan Deutschland GmbH). Total amounts of bound sialic acid were measured by the micro-adaption of the orcinol/Fe3+/HCl reaction (31Schauer R. Methods Enzymol. 1987; 138: 132-161Google Scholar). Separation and Purification of the Two TS Forms—The crude culture supernatant was filtered (1.2-μm membrane, Millipore GmbH, Schwalbach, Germany) and concentrated in an Amicon ultrafiltration device (molecular mass cut off 20 kDa, Sartorius, Göttingen, Germany) prior to undergoing chromatography. Following all purification steps, fractions were concentrated with the aid of the following devices depending on the volume: Centrex UF-2 (molecular mass cut off 30 kDa, Schleicher & Schuell, Dassel, Germany), Centricon Plus-20 (molecular mass cut off 30 kDa, Millipore, Eschborn, Germany), or an Amicon ultrafiltration device (molecular mass cut off 20 kDa). Unless otherwise stated all purification experiments were performed at 4 °C. The separation of two major TS activity peaks was provided by chromatography on Q-Sepharose FF. The concentrated culture supernatant was applied to a column (2 × 20 cm) of Q-Sepharose FF, equilibrated with 20 mm Bis/Tris buffer, pH 7.0, at a flow rate of 0.6 ml/min. Following extensive washing bound TS activities were eluted using a 600-ml continuous NaCl gradient (0–0.8 m) in 20 mm Bis/Tris buffer, pH 7.0. Fractions of 6 ml were collected and analyzed for transfer and SA activity. A larger Q-Sepharose column could not be employed due to poor separation of the two TS forms. Therefore, several Q-Sepharose runs were performed using the column size stated above, with separated TS-form 1 and TS-form 2 following each run being combined and further purified individually by isoelectric focusing (IEF). Isoelectric focusing was carried out in a 16-ml Rotor cell (Rotofor Preparative Isoelectric Focusing Cell, Bio-Rad) using ampholytes that provided a pH range between pH 4 and 6 (Biolyte pH 4–6, Bio-Rad). The buffer contained in the collected fractions was immediately exchanged, and fractions were concentrated and activity determined. Active fractions were pooled and further purified by gel filtration chromatography. Each individual TS form was applied to a column (1 × 90 cm) of Sephadex-G150 SF equilibrated with 20 mm Bis/Tris buffer, pH 7.0, containing 100 mm NaCl at a flow rate of 0.125 ml/min, which had been calibrated using the high molecular weight calibration kit (Amersham Biosciences, Freiburg, Germany) as described by the manufacturer. Fractions of 500 μl were collected and analyzed for activity. Active fractions were pooled, concentrated, and analyzed by SDS-PAGE. T. congolense TS Antibody Production, Detection, Isolation, and Iso-typing—BALB/c (H-2d) mice, obtained from Harlan/Winkelmann (Borchen, Germany) and reared under conventional conditions, were used for the production of monoclonal antibodies (mAb). Female BALB/c mice 6 weeks of age were injected three times intraperitoneally with 25 μg of the partially purified TS forms adsorbed to 2 mg of Al(OH)3 (Imject® Alum, Pierce, Rockford, IL). Three days after the last injection spleen cells of one mouse was fused with non-secretor Ag8.653 myeloma cells (32Kearney J.F. Radbruch A. Liesegang B. Rajewsky K. J. Immunol. 1979; 123: 1548-1550Google Scholar) by the conventional polyethylene glycol-mediated fusion technique (33Peters J.H. Baumgarten H. Monoclonal Antibodies. Springer Verlag, Stuttgart1992Google Scholar). After fusion, cells were plated in 288 wells of 24-well hybridoma plates (Greiner, Nürtingen, Germany) in RPMI 1640 supplemented with 10% FCS, 100 units/ml penicillin, 100 μg/ml streptomycin, 100 μm hypoxanthine, 0.4 μm aminopterin, and 16 μm thymidine. The medium was further supplemented with 10% conditioned medium from the J774 cell line. Wells containing antigen-specific IgG-secreting hybridomas were identified via ELISA using mouse-IgG-specific antiserum and an enzyme immunoassay (TS activity binding assay). Clones in positive wells were subcloned and reanalyzed. The TS activity-binding assay was performed using Dynabeads M-450 goat anti-mouse IgG (Dynal, Hamburg, Germany) and a magnetic particle concentrator for microcentrifuge tubes (Dynal MPC-S, Dynal, Hamburg, Germany). Briefly, 200 μl of beads were washed twice with phosphate-buffered saline (PBS) as described by the manufacturer. Following incubation with putative anti-T. congolense TS mAb at room temperature for 1 h the beads were washed again five times with 900 μl of PBS buffer and further incubated with 200 μl of TS-containing solution at 4 °C for 1 h. The incubation was terminated by transferring the supernatant to a new cap, and the beads were subsequently washed five times with 900 μl of PBS buffer. In the supernatant, as well as on the beads, TS activity was determined and compared with a control provided by binding non-TS-specific IgG2b antibodies to the Dynabeads. The reduction of TS activity in the TS-containing solution in comparison to the control, as well as the activity detected on the beads, enabled the determination of a clone producing anti-T. congolense TS-specific mAb. Purification of the anti-T. congolense TS mAb from hybridoma supernatant was performed by affinity chromatography using rProtein A-Sepharose FF (Amersham Biosciences, Freiburg, Germany) according to the manufacturer. The antibody concentration of the eluted preparation was determined with an enzyme immunoassay for the quantitative determination of mouse IgG (Roche Diagnostics GmbH, Mannheim, Germany). Immunoglobulin subclass determination was performed with the Hybridoma Subtyping kit (Roche Diagnostics GmbH). Immunoaffinity Chromatography—Purified anti-T. congolense TS mAb (7/23, 24 mg) were incubated for 2 h with rProtein A-Sepharose FF (5 ml) and equilibrated with binding buffer (20 mm Na2HPO4, NaH2PO4, pH 7.0). Following washing with 70 ml of binding buffer, the matrix was further washed with cross-linker buffer (0.2 m triethylamine, pH 8.5) and, subsequently, incubated with 10 ml of cross-linker reagent (20 mm dimethyl pimelimidate in cross-linker buffer) at room temperature for 1 h. The cross-linking reaction was terminated by washing the column with 70 ml of 0.2 m ethanolamine, pH 9, followed by 70 ml of binding buffer and 70 ml of sodium citrate buffer, pH 3.0. A flow rate of 1–1.25 ml/min was used at all stages of matrix preparation. The column was washed with binding buffer prior to immunoaffinity chromatography. The immunoaffinity matrix equilibrated in binding buffer was incubated with the partially purified TS forms overnight at 4 °C. Unbound protein was removed by washing with binding buffer. TS activity was eluted stepwise with 70 ml of 100 mm Bis/Tris, pH 7.0; 100 mm Bis/Tris, pH 7.0, containing 1 m NaCl; 20 mm sodium citrate, pH 4.5; and 20 mm sodium citrate, pH 3.0, at a flow rate of 1–1.25 ml/min. The last fraction was immediately neutralized prior to TS activity determination. Micro-sequencing—In-gel trypsin digestion and mass spectrometric analysis of peptides were performed by WITA GmbH (Berlin, Germany). Kinetic Studies—Kinetic data for both enzyme forms were obtained by making suitable modifications to the standard SA and TS assay. To measure transfer activity various concentrations of the acceptor substrates MUGal and MULac (0–2 mm) were used at a constant donor substrate (α2,3-SL) concentration of 1 mm. The kinetic parameters for α2,3-SL were obtained by varying α2,3-SL concentrations (0–3 mm) at a constant concentration of MUGal (0.5 mm). Various concentrations of MUNeu5Ac (0–0.2 mm) were used to obtain kinetic data for SA activity. Apparent Vmax and apparent Km values were determined by non-linear regression using the computer program Enzfitter from Elsevier Biosoft. Additionally, the temperature and pH optima of both forms were investigated in the range of 5–55 °C and pH 4.5–10.5, respectively. Donor and Acceptor Substrate Specificities and Inhibitor Studies—A number of glycoconjugates, as well as mono- and oligosaccharides (Table IV) were assayed as potential donors using the TS assay described. Known viral and bacterial SA inhibitors, as well as salts (NaCl and KCl), cations (20 mm Ca2+, Mg2+, Mn2+; 5 mm Cu2+, Zn2+, Fe2+, Co2+), and other compounds, including anti-T. congolense TS mAb (0–20 μg/ml), were assayed for their ability to inhibit TS activity using essentially the standard TS assay described, except additives were preincubated in the assay mixture for 30 min at room temperature prior to starting the reaction. Potential TS acceptors (Table IV) were assayed in a similar manner to that described for potential inhibitors.Table IVSubstrate specificity of both T. congolense TS formsSubstratesConcentrationaThe concentration of potential donors is stated as the concentration of bound sialic acid.Relative transfer activitybThe same total transfer activity was used for both TS forms in all assays.TS-form 1TS-form 2mM%DonorSialyl-α2,3-N-acetyllactosamine; Neu5Acα2,3Galβ1,4GlcNAc1111115Sialyl-α2,3-lactose; Neu5Acα2,3Galβ1,4Glc11001000.590900.257070Sialyl-α2,6-lactose; Neu5Acα2,6Galβ1,4Glc13126Sialyl-Lewisx; Neu5Acα2,3Galβ1,4[Fucα1,3]GlcNacβ1,3Galβ1,4Glc0.251111Fetuin0.57878Sialyloligosaccharides, bovine milk0.57970Sialyloligosaccharides, human milk0.57066Glycomacropeptide0.57771Apolactoferrin0.54520Relative transfer activity (decrease)mM%AcceptorN-Acetylactosamine; Galβ1,4GlcNAc14924Lacto-N-biose I; Galβ1,3GlcNAc14013Lacto-N-neotetraose; Galβ1,4GlcNAcβ1,3Galβ1,4Glc15639Lacto-N-tetraose; Galβ1,3GlcNAcβ1,3Galβ1,4 Glc13112Lactose; Galβ1,4Glc14524Lactitol16135Galactose-β1,4-galactose; Galβ1,4Gal13715Galactose5147Glucose500Mannose500Maltose500a The concentration of potential donors is stated as the concentration of bound sialic acid.b The same total transfer activity was used for both TS forms in all assays. Open table in a new tab SDS-PAGE and Immunoblot Analyses—SDS-PAGE was performed according to Laemmli (34Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) in a Mini Protean II Cell (Bio-Rad, Munich, Germany) in the presence of a reducing agent (dithiothreitol). Polyacrylamide gels usually consisted of 8% resolving and 4% stacking gel, with the exception of gels used for immunoblot analyses using anti-T. congolense GARP mAb, where the resolving gel was 12%. As molecular weight markers, pre-stained SDS-PAGE standards from Bio-Rad (for immunoblotting) or SDS-PAGE Marker High Range from Sigma were applied (for staining). Gels were subsequently stained with either silver (35Ansorge W. J. Biochem. Biophys. Methods. 1985; 11: 13-20Google Scholar) or Coomassie Brilliant Blue R-250 (36Meyer T.S. Lamberts B.L. Biochim. Biophys. Acta. 1965; 107: 144-145Google Scholar). For immunoblot analyses, after SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) using a Mini-V 8–10 Blot Module (Life Technologies, Eggenstein, Germany) as described by the manufacturer. For immunodetection, blots were blocked overnight at 4 °C in TBS (Tris-buffered saline) buffer containing 0.05% Tween 20 (TBST) and 5% skim milk (blocking buffer), washed six times with TBST for 5 min, and then incubated for either 24 h at 4 °C (antiserum to T. cruzi TS, dilution 1:5000) or1hat room temperature (anti-T. congolense TS mAb, dilution 1:3000 or anti-T. congolense procyclin (GARP) mAb, dilution 1:1000) in blocking buffer solution containing the appropriate primary antibody. Following incubation, the blots were washed again six times with TBST buffer and then incubated for1hat room temperature with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:10000). After washing six times for 10 min with TBST buffer, bands were visualized using the ECL immunoblotting detection reagent kit (Amersham Biosciences, Braunschweig, Germany) as described by the manufacturer. Immunoprecipitation—Immunoprecipitation was performed in a similar manner to that described for the TS activity-binding assay. Briefly, 30 μl of Dynabeads M-450 goat anti-mouse IgG were washed twice with PBS buffer and incubated with either 30 μl of PBS buffer (control) or 30 μl of anti-T. congolense TS mAb (mAb7/23) at room temperature for 1 h. Following incubation, the beads were washed again five times with 900 μl of PBS buffer and further incubated at 4 °C for 1 h with 200 μl of TS containing concentrated culture supernatant. The beads were washed five times with 900 μl of PBS buffer, subsequently boiled in SDS-PAGE sample buffer (containing SDS and dithiothreitol) at 95 °C for 5 min, analyzed by S
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