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• W2094509346 abstract "The infective trypomastigote stage ofTrypanosoma cruzi expresses a set of surface glycoproteins that are known collectively as Tc85 and belong to the gp85/trans-sialidase supergene family. A member of this family, Tc85–11, with adhesive properties to laminin and cell surfaces was recently cloned. In this report, the Tc85–11 domain for cell binding and its corresponding receptor on epithelial cell LLC-MK2are described. Using synthetic peptides corresponding to the Tc85–11 carboxyl-terminal segment, we show that the mammalian cell-binding domain colocalizes to the most conserved motif of the trypanosome gp85/trans-sialidase supergene family (VTVXNVFLYNR). Even though Tc85–11 binds to laminin, the 19-residue cell-binding peptide (peptide J) does not contain the laminin-binding site, because it does not bind to laminin or inhibit cell binding to this glycoprotein. The host cell receptor for the peptide was characterized as cytokeratin 18. Addition of anti-cytokeratin antibodies to the culture medium significantly inhibited the infection of epithelial cells byT. cruzi. Tc85–11 is a multiadhesive glycoprotein, encoding at least two different binding sites, one for laminin and one for cytokeratin 18, that allow the parasite to overcome the barriers imposed by cell membranes, extracellular matrices, and basal laminae to reach the definitive host cell. This is the first description of a direct interaction between cytokeratin and a protozoan parasite. The infective trypomastigote stage ofTrypanosoma cruzi expresses a set of surface glycoproteins that are known collectively as Tc85 and belong to the gp85/trans-sialidase supergene family. A member of this family, Tc85–11, with adhesive properties to laminin and cell surfaces was recently cloned. In this report, the Tc85–11 domain for cell binding and its corresponding receptor on epithelial cell LLC-MK2are described. Using synthetic peptides corresponding to the Tc85–11 carboxyl-terminal segment, we show that the mammalian cell-binding domain colocalizes to the most conserved motif of the trypanosome gp85/trans-sialidase supergene family (VTVXNVFLYNR). Even though Tc85–11 binds to laminin, the 19-residue cell-binding peptide (peptide J) does not contain the laminin-binding site, because it does not bind to laminin or inhibit cell binding to this glycoprotein. The host cell receptor for the peptide was characterized as cytokeratin 18. Addition of anti-cytokeratin antibodies to the culture medium significantly inhibited the infection of epithelial cells byT. cruzi. Tc85–11 is a multiadhesive glycoprotein, encoding at least two different binding sites, one for laminin and one for cytokeratin 18, that allow the parasite to overcome the barriers imposed by cell membranes, extracellular matrices, and basal laminae to reach the definitive host cell. This is the first description of a direct interaction between cytokeratin and a protozoan parasite. cytokeratin high pressure liquid chromatography phosphate-buffered saline bovine serum albumin Dulbecco's modified Eagle's fetal calf serum polyacrylamide gel electrophoresis kilobase (pairs) room temperature wheat germ agglutinin. Chagas' disease is a chronic and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells (1Colli W. FASEB J. 1993; 7: 1257-1264Crossref PubMed Scopus (122) Google Scholar). The protozoan is transmitted to humans by wound contamination with insect feces during blood sucking. A particularly important portal of entry is the ocular conjunctiva that is put in contact with contaminated insect feces by involuntary scratching from nearby bites on a sleeping person's face, leading to a periorbital swelling known as Romaña's sign. Other forms of transmission such as blood transfusion, congenital transmission, and breast feeding are also important, particularly in northern hemisphere regions that received intense migratory currents from Ibero-American countries. In recent years, 85–90-kDa parasite surface proteins have been implicated in host cell invasion by different investigators (2Burleigh B.A. Andrews N.W. Annu. Rev. Microbiol. 1993; 49: 175-200Crossref Scopus (192) Google Scholar, 3Ortega-Barria E. Pereira M.E.A. Infect. Agents Dis. 1992; 1: 136-145PubMed Google Scholar, 4Ouaissi M.A. Cornette J. Afchain D. Capron A. Gras-Masse H. Tartar A. Science. 1986; 234: 603-607Crossref PubMed Scopus (79) Google Scholar, 5Ramirez M.I. Ruiz R. Araya J.E. Silveira J.F. Yoshida N. Infect. Immun. 1993; 61: 3636-3641Crossref PubMed Google Scholar, 6Alves M.J.M. Abuin G. Kuwajima V.J. Colli W. Mol. Biochem. Parasitol. 1986; 21: 75-82Crossref PubMed Scopus (109) Google Scholar, 7Abuin G. Colli W. Souza W. Alves M.J.M. Mol. Biochem. Parasitol. 1989; 35: 229-238Crossref PubMed Scopus (58) Google Scholar). Our laboratory was the first to describe trypomastigote-specific 85-kDa surface glycoproteins, suggesting their role in host cell invasion by the parasite (6Alves M.J.M. Abuin G. Kuwajima V.J. Colli W. Mol. Biochem. Parasitol. 1986; 21: 75-82Crossref PubMed Scopus (109) Google Scholar, 7Abuin G. Colli W. Souza W. Alves M.J.M. Mol. Biochem. Parasitol. 1989; 35: 229-238Crossref PubMed Scopus (58) Google Scholar, 8Katzin A.M. Colli W. Biochim. Biophys. Acta. 1983; 727: 403-411Crossref PubMed Scopus (63) Google Scholar, 9Giordano R. Chammas R. Veiga S.S. Colli W. Alves M.J.M. Mol. Biochem. Parasitol. 1994; 65: 85-94Crossref PubMed Scopus (65) Google Scholar, 10Giordano R. Fouts D.L. Tewari D. Colli W. Manning J.E. Alves M.J.M. J. Biol. Chem. 1999; 274: 3461-3468Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). These proteins, collectively denominated Tc85, form a population of heterogeneous glycosylphosphatidylinositol-anchored surface glycoproteins with similar molecular masses but different electric charges (7Abuin G. Colli W. Souza W. Alves M.J.M. Mol. Biochem. Parasitol. 1989; 35: 229-238Crossref PubMed Scopus (58) Google Scholar, 8Katzin A.M. Colli W. Biochim. Biophys. Acta. 1983; 727: 403-411Crossref PubMed Scopus (63) Google Scholar, 11Andrews N.W. Katzin A.M. Colli W. Eur. J. Biochem. 1984; 140: 599-604Crossref PubMed Scopus (73) Google Scholar). Tc85 proteins belong to the gp85/trans-sialidase gene superfamily (12Cross G.A.M. Takle G.B. Annu. Rev. Microbiol. 1993; 47: 385-411Crossref PubMed Scopus (144) Google Scholar) and share common motifs with bacterial neuraminidases (1Colli W. FASEB J. 1993; 7: 1257-1264Crossref PubMed Scopus (122) Google Scholar, 12Cross G.A.M. Takle G.B. Annu. Rev. Microbiol. 1993; 47: 385-411Crossref PubMed Scopus (144) Google Scholar, 13Schenkman S. Eichinger D. Parasitol. Today. 1993; 9: 218-222Abstract Full Text PDF PubMed Scopus (116) Google Scholar). Interestingly, all members of the superfamily contain a conserved sequence (VTVXNVFLYNR) (12Cross G.A.M. Takle G.B. Annu. Rev. Microbiol. 1993; 47: 385-411Crossref PubMed Scopus (144) Google Scholar) upstream from the carboxyl terminus and absent in bacterial neuraminidases. The involvement of at least one member of the Tc85 family in parasite-host cell interactions is indicated by the observation that the monoclonal antibody H1A10, which specifically recognizes Tc85 glycoproteins, inhibits host cell invasion by the parasite in vitro by 50–90% (6Alves M.J.M. Abuin G. Kuwajima V.J. Colli W. Mol. Biochem. Parasitol. 1986; 21: 75-82Crossref PubMed Scopus (109) Google Scholar, 7Abuin G. Colli W. Souza W. Alves M.J.M. Mol. Biochem. Parasitol. 1989; 35: 229-238Crossref PubMed Scopus (58) Google Scholar). An acidic 786-amino acid member of the Tc85 family (Tc85–11) and a recombinant fusion protein of the monoclonal antibody H1A10 epitope-containing carboxyl-terminal segment of Tc85–11 (Tc85–1) both showed adhesive properties to isolated laminin and to entire cells (10Giordano R. Fouts D.L. Tewari D. Colli W. Manning J.E. Alves M.J.M. J. Biol. Chem. 1999; 274: 3461-3468Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The high plasticity of the cytoskeleton is often exploited by pathogens to enter non-phagocytic cells. Increasing evidence has been provided for the expression of cytoskeletal proteins on cell surfaces that serve as receptors for different ligands. For example, intermediate filament proteins belonging to the cytokeratin family are expressed on the cell surface and act as receptors for bacteria as well as for plasminogen and tissue plasminogen activator, high molecular weight kininogen, and thrombin-antithrombin complexes (14Hembrough T.A. Vasudevan J. Allietta M.M. Glass II W.F. Gonias S.L. J. Cell Sci. 1995; 108: 1071-1082PubMed Google Scholar, 15Hembrough T.A. Li L. Gonias S.L. J. Biol. Chem. 1996; 271: 25684-25691Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 16Hembrough T.A. Kralovich K.R. Li L. Gonias S.L. Biochem. J. 1996; 317: 763-769Crossref PubMed Scopus (53) Google Scholar, 17Wells M.J. Hatton M.W. Hewlett B. Podor T.J. Sheffield W.P. Blajchman M.A. J. Biol. Chem. 1997; 272: 28574-28581Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Hasan A.A.K. Zisman T. Schmaier A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3615-3620Crossref PubMed Scopus (162) Google Scholar, 19Sajian U.S. Sylvester F.A. Forstner J.F. Infect. Immun. 2000; 68: 1787-1795Crossref PubMed Scopus (59) Google Scholar, 20Tamura G.S. Nittayajarn A. Infect. Immun. 2000; 68: 2129-2134Crossref PubMed Scopus (38) Google Scholar). The present work demonstrates that the conserved common sequence VTVXNVFLYNR of the gp85 glycoprotein/trans-sialidase supergene family is a mammalian cell-binding domain. Its host cell receptor for this motif was purified and characterized as cytokeratin 18 (CK18)1 present on the surface of LLC-MK2 cells (monkey kidney epithelial cells). Because Tc85 also binds to laminin (10Giordano R. Fouts D.L. Tewari D. Colli W. Manning J.E. Alves M.J.M. J. Biol. Chem. 1999; 274: 3461-3468Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), the results presented herein suggest that the Tc85 family is composed of multiadhesive glycoproteins that bind to different receptor molecules either located on the cell surface or belonging to components of the extracellular matrix. T. cruzi strain Y was used throughout. Culture conditions for parasites and mammalian cells are described elsewhere (21Andrews N.W. Colli W. J. Protozool. 1982; 14: 447-451Google Scholar). Peptides were synthesized in an automated bench top simultaneous multiple solid phase peptide synthesizer (PSSM 8 system from Shimadzu) using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) procedure. The synthesized peptides were deprotected and purified by semipreparative HPLC using an Econosil C-18 column (10 μm, 22.5 × 250 mm) and a two-solvent system: (A) trifluoroacetic acid/H2O (1:1000) and (B) trifluoroacetic acid/MeCN/H2O (1:900:100). The peptides were separated at a flow rate of 5 ml/min and a gradient from 10 (or 30) to 50 (or 60)% of solvent B. Analytical HPLC was performed using a binary HPLC system (Shimadzu) with an SPD-10AV Shimadzu UV-visible detector and a Shimadzu RF-535 fluorescence detector, coupled to an Ultrasphere C-18 column (5 μm, 4.6 × 150 mm), which was eluted with solvent systems A1 (H3PO4/H2O, 1:1000) and B1 (MeCN/H2O/H3PO4, 900:100:1) at a flow rate of 1.7 ml/min and a 10–80% gradient of B1 over 15 min. The HPLC column eluates were monitored by their absorbance at 220 nm and by fluorescence emission at 420 nm following excitation at 320 nm. The purity of obtained peptides was checked by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) spectroscopy in the reflectron mode (TofSpec-E from Micromass, Manchester, UK) and by amino acid sequencing, performed with a Shimadzu sequencer, model PPSQ-23 (22Kates S.A. Alberecio F. Solidphase Synthesis: A Practical Guide. Marcel Dekker, Inc., New York2000Google Scholar). In a 24-well plate, 40 μg of each peptide in 200 μl of 10% Me2SO were dried overnight at 37 °C with agitation, washed with PBS (140 mm NaCl, 2.7 mm KCl, 10 mm phosphate buffer, pH 7.3), and incubated for 2 h with 1% BSA/PBS. LLC-MK2 cells were cultured as described (6Alves M.J.M. Abuin G. Kuwajima V.J. Colli W. Mol. Biochem. Parasitol. 1986; 21: 75-82Crossref PubMed Scopus (109) Google Scholar) in a 75-cm2 bottle, removed by trypsin, and resuspended in 5 ml of DME medium supplemented with 10% FCS. The cells were incubated for 1 h at 37 °C in 50-ml polyethylene tubes (Corning) and washed twice with DME medium to remove FCS. Then, 1 × 105 cells in 0.5 ml of DME medium were added to the peptide-coated wells and incubated for 1 h at 37 °C. The wells were washed three times with DME medium and analyzed using an inverted microscope. In binding competition assays, LLC-MK2 cells were preincubated for 15 min with the peptide acting as a competitor and added to peptide-coated wells. After incubation and washing as described, the number of bound cells was quantified following staining with crystal violet (23Morla A. Zhang Z. Ruoslahti E. Nature. 1994; 367: 193-196Crossref PubMed Scopus (266) Google Scholar). Peptide J was radiolabeled with 125I (Amersham Pharmacia Biotech) using the chloramine-T method (24Roth J. Methods Enzymol. 1975; 37: 223-233Crossref PubMed Scopus (167) Google Scholar) and purified by reverse-phase HPLC, resulting in a specific activity of 2 × 107 cpm μg−1. LLC-MK2 cells were collected as described above, and 6 × 105 cells were incubated for 2 h on ice in the presence of increasing concentrations of the radiolabeled peptide. Nonspecific binding was determined in the presence of a 100-fold excess of unlabeled peptide J. The reaction mixtures were washed three times to remove unbound radiolabeled ligand, and cells were lysed in 1% SDS for 10 min and directly assayed for radioactivity by scintillation counting. Each experiment was performed in triplicate. LLC-MK2 and K562 cells were collected as described, washed three times with PBS, and biotinylated with the EZ-Link-Sulfo-NHS-biotinylation kit (Pierce) as recommended by the manufacturer. Plasma membranes were prepared (25Borrow P. Oldstone M.B.A. J. Virol. 1992; 66: 7270-7281Crossref PubMed Google Scholar) and solubilized in 100 mm β-d-n-octyl glucoside for 2 h at 4 °C. Peptide J was synthesized with an additional cysteine at the amino terminus. One mg of peptide J was coupled to a solid matrix (UltraLinkTM iodoacetyl, Pierce) and used for affinity chromatography experiments. The supernatants from solubilizations in β-d-n-octyl glucoside were incubated overnight with the peptide J affinity gel at 4 °C with agitation. The gel was loaded into a column, and the column was washed with 40 volumes of 25 mm β-d-n-octyl glucoside in incubation buffer. The gel was then washed with 1 m NaCl, followed by agitation for 1 h at room temperature. The column was washed again with 40 column volumes of PBS and then incubated with 8m urea as above. The collected fractions were dialyzed, concentrated, and analyzed by SDS-PAGE (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) in 9% gels. Following analysis by SDS-PAGE, proteins were transferred to a supported nitrocellulose membrane using 25 mm Tris, 150 mm glycine, and 20% methanol (pH 8.3) as transfer buffer. The blots were blocked with 3% BSA in TBSTT (Tris-buffered saline (TBS; 10 mm Tris, pH 7.5, 150 mm NaCl) containing 0.05% Tween 20 and 0.03% Triton X-100) and incubated for 2 h at room temperature with ExtrAvidin-peroxidase (Sigma) or anti-PAN-cytokeratin antibody (Sigma), as recommended by the manufacturer. The latter recognizes cytokeratins 4, 5, 6, 8, 10, 13, and 18. The membrane was washed with TBSTT and, when necessary, incubated with the secondary antibody conjugated to peroxidase. The reaction was developed with the ECL kit (Amersham Pharmacia Biotech). Membrane fractions from LLC-MK2 and K562 cells were prepared as described (25Borrow P. Oldstone M.B.A. J. Virol. 1992; 66: 7270-7281Crossref PubMed Google Scholar) and incubated for 2 h on ice with 125I-peptide J in the presence and absence of a 100-fold excess of unlabeled peptide. The receptor-ligand complexes were separated from unbound ligands by centrifugation and washed once with PBS, 0.01% Triton X-100. Chemical cross-linking was initiated by addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 100 mm final concentration (Sigma). After 30 min of incubation on ice the reaction was stopped with 150 mmglycine, pH 6.8. Samples were then washed twice with PBS and solubilized for 2 h at 4 °C in PBS containing 100 mm β-d-n-octyl glucoside, 1 mm EDTA, 2 μg/ml aprotinin, 1 mm N-α-p-tosyl-l-lysine chloromethyl ketone, and 1 mm phenylmethylsulfonyl fluoride. The proteins were separated by SDS-PAGE, and 125I-peptide J-protein complexes were detected by autoradiography. Trypomastigotes were washed twice with DME medium and purified by centrifugation over a Lymphoprep gradient (Nycomed Pharma AS) to eliminate contaminating host cells and cell debris. CK18 (Research Diagnostics) was radioiodinated using the chloramine-T method (24Roth J. Methods Enzymol. 1975; 37: 223-233Crossref PubMed Scopus (167) Google Scholar) and purified on a G-25 Sepharose column. Purified trypomastigotes (9 × 106) in a total volume of 200 μl were incubated with 1 × 106 cpm of 125I-CK18 (8.6 × 105 cpm μg−1) in the presence and absence of a 20-fold excess of unlabeled CK18 for 2 h on ice. The incubation mixtures were separated by filtration over nitrocellulose filters previously saturated with 0.1% BSA, and the filter-bound radioactivity was quantified using scintillation counting. T. cruzi trypomastigotes were cultured as described (6Alves M.J.M. Abuin G. Kuwajima V.J. Colli W. Mol. Biochem. Parasitol. 1986; 21: 75-82Crossref PubMed Scopus (109) Google Scholar). LLC-MK2 cells grown in an 8-well dish were washed three times with DME medium, and each well was incubated for 15 min in DME medium supplemented with 2% FCS containing 200 μmalanine-substituted peptide (peptide J-Ala), 100 or 200 μm peptide J, 10 μm Tc85–11, mouse IgG (Sigma), or a 1:20 dilution of an anti-CK18 antibody (Research Diagnostics). These cells were infected with trypomastigotes in a 1:100 ratio for 2 h at 37 °C and washed twice with PBS. The nonadherent parasites were removed by addition of Lymphoprep to the cell layers, followed by two washes with PBS. The cells were incubated with DME medium supplemented with 2% FCS for 48 h at 37 °C, fixed with 100% methanol, and stained with chromomycin A (Molecular Probes). All experiments were performed in triplicate, and 12 photos of each replicate were made using a digital video-imaging fluorescence microscope, enabling the counting of infected and non-infected cells in samples containing 200 cells each. The data were compared for statistical significance using the unpaired Student's ttest. LLC-MK2 cells were adhered to peptide J or FCS-coated 30-mm glass coverslips as described above and then fixed in 4% paraformaldehyde. Cells were washed with PBS and incubated for 30 min at 37 °C with either anti-PAN-cytokeratin antibody (Sigma) or specific anti-CK18 antibody (Research Diagnostics), diluted as recommended by the manufacturers, washed five times with PBS, and then incubated under the same conditions with fluorescein isothiocyanate-labeled goat anti-mouse IgG (1:30 dilution) and rinsed five times. In experiments where K562 cells, which do not bind to peptide J, were used, cells were washed by centrifugation at 1,000 × g. The same protocol was used for saponin-treated cells, except that 0.05% saponin was added during incubation with the antibodies. Fractions of the affinity column-purified and biotinylated 45 kDa-protein from LLC-MK2 cells were determined to be cytokeratin 18 by mass spectrometry of tryptic peptides at the HHMI Biopolymer Laboratory and W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University (New Haven, CT). To define the Tc85–1 cell-binding site, we synthesized 11 peptides with five amino acid overlaps that spanned the carboxyl-terminal segment of the recombinant Tc85–11 protein. These peptides were used to coat the surface of 24-well plates and mediate LLC-MK2 cell adhesion. As shown in Fig. 1 A, the cells only adhered significantly to the well coated with peptide J (19 amino acids) that contained the VTVTNVFLYNR motif. This motif is highly conserved and present in all members of the gp85/trans-sialidase superfamily. This observation implicates the importance of that common sequence in the binding of members of the gp85/trans-sialidase supergene family to their host cell receptors (Fig. 1 B). Other cell lines that are invaded by T. cruzi were tested for their affinity for peptide J. In addition to LLC-MK2 cells, tumor cells (B16F10), human umbilical cord endothelial cells (ECV), macrophage-like cells (J774), mouse fibroblast cells (3T3), and mouse pheochromocytoma cells (PC-12) bound to peptide J (data not shown). Consistent with a crucial function of peptide J in cell invasion, mouse erythrocytes and K562 erythroleukemia cells (27Ruiz R.C. Favoreto Jr., S. Dorta M.L. Oshiro M.E. Ferreira A.T. Manque P.M. Yoshida N. Biochem. J. 1998; 330: 505-511Crossref PubMed Scopus (140) Google Scholar) that are not invaded by T. cruzi did not bind to peptide J. Additional evidence for the physiological relevance of peptide J binding to mammalian cells is that a 10 μm concentration of this peptide inhibited the binding of the recombinant Tc85–11 protein to the host cell. As opposed to Tc85–11, peptide J does not bind to laminin; nor does it inhibit cell binding to laminin. Furthermore, it was established that peptide J does not bind to cells at the same receptor used by laminin or to laminin on its cell-binding domain, because different concentrations of this glycoprotein did not affect LLC-MK2 adhesion to peptide J. The combined data strongly suggest that the Tc85–11 recombinant protein is a molecule with multiple adhesion sites, specific for different ligands of the vertebrate host cell. Radioiodinated peptide J binds to LLC-MK2 cells in a specific, saturable manner, as shown by nonlinear saturation analysis (Fig. 2). The data suggest 1.66 ± 0.16 × 106 binding sites with aK D of 175 ± 56 nm. The number of binding sites is comparable with that determined for plasminogen binding to CK8 (15Hembrough T.A. Li L. Gonias S.L. J. Biol. Chem. 1996; 271: 25684-25691Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Interestingly, cytokeratin 8 associates with CK18 to form an intermediate filament heteropolymer in several cell types. To identify the minimal sequence that is relevant for the binding of peptide J to the host cell, truncated peptides were constructed spanning the whole sequence of peptide J. Cells were layered on peptide J-coated plates, and truncated peptides were checked for their competing ability for cell adhesion. The minimal inhibitory sequence was VTNVFLYNRPL (data not shown). To identify the residues responsible for this binding, each amino acid of the minimal inhibitory sequence was consecutively substituted by alanine, and the modified sequences were tested for their inhibitory effects in adhesion assays of peptide J to cells. It was observed that substitution in some positions resulted in the loss of the inhibitory effect on the binding of cells to peptide J (Fig.3). These experiments strongly indicate that the amino acid sequence VTXVFLYXR, conserved in most members of the 85-kDa trypomastigote surface glycoprotein family, is essential for parasite-cell interaction. In 40 analyzed sequences (Fig. 1 B), LYXR was present in all members of the family, whereas the first Val residue that is found in 80% of the sequences was substituted in the remaining sequences by Leu or Ala, which are also apolar amino acids. Threonine, at position 2, showed a smaller degree of conservation (38%), most often being replaced by other polar residues: Ser (15%), Asn (20%), and Lys (15%). The valine at position 4 is again highly conserved (95%), and Leu substitutes Phe at position 5 in 38% of the molecules. To characterize the host cell receptor for peptide J, the peptide was coupled to an affinity matrix and used for purification of the receptor, employing chromatographic methods. The affinity matrix was incubated with solubilized membranes from biotinylated LLC-MK2 cells. The 8 m urea eluates of the LLC-MK2 cell extracts revealed a biotinylated 45-kDa molecule that was detected by Western blot (Fig.4). To obtain further evidence that the 45-kDa molecule is the host cell receptor, radioiodinated peptide J was chemically cross-linked with LLC-MK2 cells. For a negative control, we also performed the experiment using K562 cells, which were neither infected byT. cruzi nor adhered to surfaces coated with peptide J. As an additional control, an alanine-substituted peptide, peptide J-Ala (VTNVFAYNRPL), that does not inhibit cell binding to peptide J was radiolabeled and cross-linked to LLC-MK2 cells. Following solubilization and separation of plasma membrane proteins by SDS-PAGE, a protein migrating with a molecular mass of 45 KDa was detected only in the LLC-MK2 cell extract (Fig.5). The labeling of the 45-kDa protein was specific, because it could be inhibited by a 100-fold molar excess of unlabeled peptide. As expected, no specific labeling of K562 cells by 125I-peptide J was observed, and the peptide J-Ala did not bind to LLC-MK2 cells. These results strongly suggest that a 45-kDa molecule present on LLC-MK2 cells is involved in adhesion of the parasite to these cells. Purified 45-kDa biotinylated protein fractions, as described in Fig. 4, were digested with trypsin, and the peptides were analyzed by mass spectrometry. The identified peptides from three independent experiments indicated that the 45-kDa molecule was biotinylated CK18. In agreement with the mass spectrometry analysis, the isolated protein comigrated with authentic cytokeratin 18 with an apparent molecular mass of 45 kDa and a pI of 5.4 in a two-dimensional SDS-PAGE (data not shown). To further confirm these results, LLC-MK2 and K562 plasma membrane extracts were incubated with the peptide J affinity column, and after elution with 1m NaCl and 8 m urea, the eluates were analyzed by SDS-PAGE and tested by Western blot with anti-PAN-cytokeratin antibody. As shown in Fig.6 A, a cytokeratin molecule of 45 kDa is present only in 8 m eluates of LLC-MK2 cells. As expected, no K562 cytokeratin could be eluted from the peptide J column. Furthermore, 125I-CK18 bound to the peptide J affinity column and showed the same elution pattern as CK18 from LLC-MK2 cells (Fig. 6 B). Control BSA columns did not bind CK18. The fact that the anti-PAN-cytokeratin antibody, which recognized many proteins in the cell extract, was able to recognize only CK18 in the column eluate suggests a highly specific binding. Intact LLC-MK2 and K562 cells were tested for the presence of cytokeratin by immunofluorescence microscopy with fluorescent anti-CK18-specific antibody (Fig. 7). Whereas CK18 is present in the cytoplasm of both cell lines, only LLC-MK2cells express cytokeratin on the surface. Moreover,125I-labeled CK18 binds in a specific manner to trypomastigotes (Fig. 8) but not to epimastigotes, the non-invasive developmental form of T. cruzi (data not shown).Figure 8125 I-CK18 binds to T. cruzi trypomastigote cells. 9 × 106purified trypomastigotes were incubated with 1 × 106cpm of 125I-CK18 in the presence (black bar) and absence (white bar) of a 20-fold excess of unlabeled CK18. The incubation mixtures were separated by filtration over nitrocellulose filters that were previously saturated with 0.1% BSA, and the filter-bound radioactivity was quantified using scintillation counting. The data show the average of triplicate determinations ± S.D. of two experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As shown in Fig. 9, previous incubation of peptide J-coated wells with CK18 (100 μg/ml) completely inhibited cell adhesion to the wells. Furthermore, previous incubation of LLC-MK2 cells with anti-CK18 antibody inhibited cell adhesion to peptide J by 75%. Addition of 100 μm peptide J completely inhibited the binding, whereas 100 μmpeptide J-Ala had no effect. The effects of peptide J, Tc85–11, and CK18 on the invasion of LLC-MK2 cells by trypomastigote forms were analyzed by invasion assays in the presence of these molecules. The data show statistically significant (p < 0.05) differences among the number of infected cells in the absence and presence of peptide J, recombinant Tc85–11, and anti-CK18 antibody and when both peptide J and anti-CK18 antibodies were added simultaneously (Fig. 10). Thus previous incubation of LLC-MK2 cells with peptide J and Tc85–11 increases cell invasion by T. cruzi, whereas the anti-CK antibody inhibits invasion by more than 60%. The effect of peptide J on cell invasion depends on the concentration used, suggesting a role for the conserved sequence in Tc-85 as a signaling molecule that will prepare the epithelial host cell for parasite invasion. When host cells were previously incubated with anti-CK18 antibody, the increase in infection promoted by peptide J could not be observed (Fig. 10). As controls, invasion assays were performed in the presence of mouse IgG and peptide J-Ala, both of which had no effect on invasion. T. cruzi invades non-phagocytic cells in an energy-dependent manner (28Schenkman S. Robbins E.S. Nussenzweig V. Infect. Immun. 1991; 59: 645-654Crossref PubMed Google Scholar) by a mechanism different from phagocytosis. Invasion is preceded by an adhesion step involving surface molecules from both the parasite and the host c" @default.
• W2094509346 created "2016-06-24" @default.
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• W2094509346 date "2001-06-01" @default.
• W2094509346 modified "2023-10-18" @default.
• W2094509346 title "Infection by Trypanosoma cruzi" @default.
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