From the Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26077, São Paulo 05513-970, São Paulo, Brazil and ** Universidade Federal de São Paulo, São Paulo 04023-900, São Paulo, Brazil
Received for publication, December 20, 2000, and in revised form, March 1, 2001
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ABSTRACT |
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The infective trypomastigote stage of
Trypanosoma 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-MK2
are 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 by
T. 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.
Chagas' disease is a chronic and incapacitating illness, caused
by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells (1). 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 (2-7). Our laboratory was the first to describe
trypomastigote-specific 85-kDa surface glycoproteins, suggesting their
role in host cell invasion by the parasite (6-10). These proteins,
collectively denominated Tc85, form a population of heterogeneous
glycosylphosphatidylinositol-anchored surface glycoproteins with
similar molecular masses but different electric charges (7-8, 11).
Tc85 proteins belong to the gp85/trans-sialidase gene superfamily (12)
and share common motifs with bacterial neuraminidases (1, 12-13).
Interestingly, all members of the superfamily contain a conserved
sequence (VTVXNVFLYNR) (12) 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% (6, 7). 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
(10).
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 (14-20).
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 (10), 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.
Parasite Strain and Culture--
T. cruzi strain Y
was used throughout. Culture conditions for parasites and mammalian
cells are described elsewhere (21).
Peptide Synthesis--
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 (22).
Binding of Cells to Synthetic Peptides--
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
(6) 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 (23).
Binding of 125I-Peptide J to Mammalian
Cells--
Peptide J was radiolabeled with 125I (Amersham
Pharmacia Biotech) using the chloramine-T method (24) and purified by
reverse-phase HPLC, resulting in a specific activity of 2 × 107 cpm µg Isolation and Biotinylation of Cell Surface
Proteins--
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 (25) and solubilized
in 100 mM Affinity Chromatography--
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
Western Blot--
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).
Cross-linking of 125I-Peptide J with Cell
Surfaces--
Membrane fractions from LLC-MK2 and K562
cells were prepared as described (25) 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 mM
glycine, pH 6.8. Samples were then washed twice with PBS and
solubilized for 2 h at 4 °C in PBS containing 100 mM Binding of 125I-CK18 to T. cruzi--
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
(24) 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 Infection of Mammalian Cells by Trypomastigotes--
T.
cruzi trypomastigotes were cultured as described (6).
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 µM
alanine-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 t test.
Immunofluorescence Microscopy--
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.
Characterization of CK18--
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).
Definition of the Tc85-1 Cell-binding Site--
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. 1A,
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. 1B).
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 (27) 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 a
KD of 175 ± 56 nM. The number of binding sites is comparable with that determined for plasminogen binding to CK8 (15). Interestingly, cytokeratin 8 associates with CK18
to form an intermediate filament heteropolymer in several cell
types.
Mapping of the Amino Acids Required for the Binding of Peptide J to
the Host Cell by Alanine Scanning--
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. 1B), 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.
Identification of the Host Cell Receptor for the Truncated Common
Sequence of Tc85--
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 by T. 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.
Cytokeratin 18 Is a Host Cell-binding Site for the Most Conserved
Domain of the Tc85 Family--
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 1 M 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.
6A, 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. 6B).
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.
CK18 Is Present on the Surface of Intact LLC-MK2 Cells
and Binds to Trypomastigotes--
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-MK2
cells 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).
CK18 and Anti-CK18 Antibody Inhibit Binding of
LLC-MK2Cells to Peptide J--
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 µM
peptide J-Ala had no effect.
Effects of Peptide J and CK18 on Host Cell Infection by T. cruzi in
Vitro--
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 (28) by a mechanism different from
phagocytosis. Invasion is preceded by an adhesion step involving
surface molecules from both the parasite and the host cell. Members of
the Tc85 glycoprotein family, expressed on the surface of the
infectious form of T. cruzi, were first suggested to be
involved in the adhesion step necessary for parasite invasion of host
cells (8, 11, 29) because monoclonal antibodies against Tc85 molecules
were able to partially inhibit host cell invasion (6), and a cloned
member of the Tc85 family (Tc85-11) was shown to bind to laminin (10). Other laboratories have also implicated the involvement of several ~85-kDa proteins in parasite-host cell interaction (30-32),
including an 85-kDa protein, probably from the same superfamily but
different from Tc85-11, that was described as a fibronectin receptor
(33). The Tc-85 family of glycoproteins belong to the
gp85/trans-sialidase supergene family, which comprises ~1,000 genes
(34). The whole superfamily represents 1-2% of the T. cruzi genome, with highly redundant and simultaneously expressed
members, a stumbling block that eliminates the possibility of employing
genetic approaches to their functional analysis. It is our working
hypothesis that the gp85/trans-sialidase gene family, in addition to
members coding for trans-sialidase activity, comprises a family coding
for adhesion proteins, with several of its members interacting with
specific ligands. It is worth noting that the 3.5-h half-life of the
Tc85 family is considerably short (35). This fast turnover could facilitate the progression of the parasite from blood vessels to the
cells if different continuously expressed subsets of the family bound
to different ligands on cell surfaces, extracellular matrices, and
basal laminae. It would seem likely that cruzipain (36, 37) and other
proteases (38) could be operative in breaking the successive
protein-protein interactions, thus facilitating parasite progression.
An additional, but not exclusive, possibility is that individual
members of the Tc85 family may interact with two or more ligands on the
cell surface.
The latter hypothesis is favored by our data, because Tc85-11 has two
different binding sites. One of these sites for laminin binding is
under investigation and is known to be located somewhere in the 100 amino acids upstream from the peptide J conserved region of the Tc85-1
clone. The other binding site to CK18 comprises the most conserved
region of all members of the gp85/trans-sialidase family. This is the
first time that multiple adhesion sites on a T. cruzi
molecule, a common feature in other systems (39, 40), have been defined.
The discovery that the most conserved sequence of the
gp85/trans-sialidase family, the trypomastigote adhesion peptide
TXVFLYXR, is a CK18-binding domain for T. cruzi adhesion on vertebrate host cells expands our understanding
of the function of this protein family in the molecular mechanisms of
cell invasion. Our findings may also contribute to the unraveling of
the yet unknown function of trans-sialidases in invasion, because
antibodies against the catalytic site of this enzyme, which is distant
from the conserved peptide J sequence, did not inhibit parasite
internalization into host cells (41). The search for similar motifs in
other pathogen-host cell interactions unrelated to T. cruzi
infection may elucidate our general understanding of cell infection.
Primary infection of human beings by T. cruzi naturally
occurs through skin lesions from the insect bite, by direct contact of
contaminated feces with the dermal layer, or by contact with epithelial
and endothelial tissue. Our work with epithelial cells suggests that
the interaction of T. cruzi with cytokeratin may be
important for the parasite to cross the eye epithelial mucosa (as an
early event leading to the typical Romaña's sign) or the trophoblast epithelium (explaining the congenital transmission of
Chagas' disease).
CK18 associates with CK8 to form a component of intermediate filaments
in simple epithelia and many epithelial cell-derived neoplasms. CK18
can be aberrantly expressed in many non-epithelial cancers, including
lymphomas, melanomas, gliomas, and sarcomas, and in many cases, CK18
has been correlated with increased tissue invasion in vitro
and in vivo (14). Additionally, recent data indicate the
presence of CK18 and CK7 in endothelia of normal veins, venules,
lymphatics, and capillaries in the skin; subcutaneous soft tissues;
mucosal sites; skeletal muscle and smooth muscle cells in the placenta
(42); and the synovial microvasculature (43). This is
consistent with the hypothesis that trypanosomes may bind to CK18 to
traverse the endothelial barrier.
The view that cytokeratins and other intracellular proteins are
confined solely to the cytosol has recently undergone revision. The
fact that various intracellular proteins are also expressed on the cell
surface and there exercise specific functions is now well established
(14-20, 44). CK1 was described as a putative kininogen-binding protein on the surface of endothelial cells (18),
CK13 binds to cable-piliated Burkholderia cepacia (19), and
CK8 is a plasminogen and tissue plasminogen-activator receptor on the
surface of hepatocytes and breast cancer cells (14-16) as well as a
receptor for group B streptococci and other Gram-positive cocci (20).
In particular, CK18 was identified as the binding site for
thrombin-antithrombin III complexes on the plasma membrane of rabbit
hepatocytes (17).
In summary, the results herein presented favor the hypothesis that the
conserved common sequence of the gp85/transialidase family is an
important docking domain to the host cell surface, although other sites
should not be ruled out (10, 27). Along with growing evidence for
surface expression of intracellular proteins and the expression of
cytokeratins also in epithelial and other tissues, our data indicate
CK18 as a putative mammalian cell receptor for T. cruzi
and/or a binding protein that is necessary for further receptor
activation. Other studies have shown that transforming growth factor
Previous incubation of LLC-MK2 cells with peptide J or
Tc85-11 increases cell invasion by T. cruzi, suggesting a
role for the gp85/trans-sialidase family as signaling molecules that
enhance receptiveness of the host cell for the parasite by a yet
unknown mechanism. Members of this family possessing a relatively short half-life (35) are constitutively shed into culture medium (49), suggesting that contact between the surface of the parasite or shed
Tc85 proteins and CK18 on the mammalian cell may promote signaling
events in the host cell, thus facilitating T. cruzi infection.
T. cruzi internalization requires host cell lysosome
recruitment, inducing localized clustering and fusion of host cell
lysosomes with the plasma membrane at the site of trypomastigote
attachment. This process requires host cell
[Ca2+]i transients and transient rearrangement of
actin microfilaments, which might facilitate lysosome
access to the plasma membrane during parasite invasion (48). Because
other filaments are connected to actin microfilaments in the cytosol,
it is possible that parasite binding to CK18 may also influence the
lysosome migration process. It is worth noting that
thrombin-anti-thrombin complexes, which are internalized via the CK18
receptor on the surface of hepatocytes, are degraded by lysosomes
(17).
T. cruzi infection is a complex process involving several
host and parasite molecules in the recognition process as well as the
involvement of enzymatic reactions and bivalent ions. The present study
indicates a new and physiologically relevant role for the most
conserved sequence of the gp85/trans-sialidase super gene family. We
have shown that this sequence is involved in host cell binding during
the infection process and that CK18 is a putative trypomastigote
receptor on epithelial cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
-D-n-octyl glucoside for 2 h at 4 °C.
-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
-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 8 M urea as above. The collected fractions were dialyzed,
concentrated, and analyzed by SDS-PAGE (26) in 9% gels.
-D-n-octyl glucoside, 1 mM EDTA, 2 µg/ml aprotinin, 1 mM
N-
-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.
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The most conserved sequence of the
gp85/trans-sialidase superfamily binds to LLC-MK2
cells. A, each well of a 24-well plate was coated with
40 µg of peptides A-K corresponding to the 131 amino
acids of the carboxyl terminus of Tc85-11 in the presence of BSA to
diminish unspecific binding and then incubated with 1 × 105 LLC-MK2 cells in 0.5 ml of DME medium for
1 h at 37 °C. The wells were washed three times with DME
medium, and analysis by inverted microscopy revealed that cells adhered
significantly only to the well coated with peptide J but not to wells
coated with BSA or other peptides. The figure shows representative results of 15 independent
experiments. Peptide sequences are shown on the top of each
panel. B, the peptide J region is highly conserved in a
number of T. cruzi surface molecules. Dots
indicate identical amino acids, and the numbers in the left column
correspond to data base accession numbers; the GenBankTM
accession number of Tc85-11 is AF085686.
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Fig. 2.
Saturation analysis of
125I-peptide J binding to the LLC-MK2 cell
surface. Increasing amounts of 125I-peptide J (20-800
nM) were incubated with LLC-MK2 cells in the
presence and absence of a 100-fold molar excess of unlabeled peptide J. Each data point represents the average ± S.D. of triplicate
determinations of two independent experiments. The binding of
125I-peptide J to LLC-MK2 cells in the presence
(continuous line) and absence (dotted line) of
unlabeled peptide is shown in the inset.
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Fig. 3.
Determination of the minimal sequence of
peptide J required for its biological effect. LLC-MK2
cells were incubated for 1 h at 37 °C in wells coated with
peptide J in the absence (control) and presence of competing peptides:
the peptide J itself and 11 peptides where each individual position of
the sequence VTNVFLYNRPL was substituted by alanine. The figure shows
the percentage of binding compared with the control. The wells were
washed three times with DME medium, and the cells were stained with
crystal violet (23). Each data point represents the average of
triplicate determinations of three independent experiments. The
asterisk indicates one of the alanine-substituted peptides
that does not inhibit cell binding to peptide J. This peptide,
denominated J-Ala, was used in further experiments as a negative
control to test the binding specificity of peptide J.
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Fig. 4.
Identification of a 45-kDa molecule as a
receptor for peptide J in mammalian cells. The LLC-MK2
cell surface was biotinylated as described under "Experimental
Procedures" and lysed. The plasma membrane fraction was incubated
with the peptide J affinity gel. The column was eluted with 1 M NaCl and 8 M urea. The collected fractions
were analyzed by SDS-PAGE, and the proteins were transferred to a
supported nitrocellulose membrane and tested by Western blot with a
streptavidin-peroxidase conjugate. The biotinylated 45-kDa molecule was
eluted from the column with 8 M urea. The arrow
marks the 45-kDa region. This is a representative figure of at least 15 independent experiments.
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Fig. 5.
A 45-kDa host cell surface molecule is
specifically labeled by 125I-peptide J but not by
125I-peptide J-Ala. Membrane fractions of
LLC-MK2 and K562 cells were prepared and incubated for
2 h on ice with 125I-peptide J (Pep J) or
125I-peptide J-Ala (Pep J-Ala) in the presence
(+) and absence ( ) of a 100-fold excess of
unlabeled peptide. The receptor-ligand complexes were separated from
free ligands by centrifugation, and chemical cross-linking was
performed with ethyl-3-(3-dimethylaminopropyl)carbodiimide as described
under "Experimental Procedures." After washing and solubilization,
proteins were separated by SDS-PAGE, and 125I-peptide
J-protein complexes were detected by autoradiography. The
arrow marks the 45-kDa region. Identical results were
obtained in six independent experiments.
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Fig. 6.
CK18 binds to peptide J. A,
detergent extracts of plasma membrane fractions from K562 and
LLC-MK2 cells were incubated with the peptide J affinity
gel. The columns were eluted with 1 M NaCl and 8 M urea. The collected fractions were analyzed by SDS-PAGE,
and the proteins were transferred to a supported nitrocellulose
membrane and tested by Western blot with anti-PAN-CK
antibodies. This experiment was repeated twice. B,
125I-CK18 was incubated with the peptide J affinity gel.
The column was washed with PBS and eluted with 1 M NaCl and
8 M urea. The collected fractions were separated by
SDS-PAGE, and 125I-CK18 was detected by autoradiography.
The arrow marks the 45-kDa region. No 45-kDa molecule was
eluted from columns that were saturated with BSA instead of peptide
J.
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Fig. 7.
CK18 is expressed on the surface of
LLC-MK2 but not on K562 cells. Viable, impermeabilized
LLC-MK2 cells adhered to peptide J- or FCS-coated
wells, and K562 cells were fixed with 4% paraformaldehyde and
incubated with a specific anti-CK18 antibody followed by fluorescein
isothiocyanate anti-mouse antibody IgG. LLC-MK2 cells
showed a patchy fluorescent pattern of cytokeratin, whereas no
fluorescence was observed in viable, impermeabilized K562 cells. When
the cells were permeabilized with 0.05% saponin, a diffuse cytoplasmic
fluorescence pattern of cytokeratin was observed in LLC-MK2
and K562 cells. The light colored panels show phase
contrast, and the darker colored panels show
immunofluorescence images. Similar results were observed in three
independent experiments.
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Fig. 8.
125I-CK18 binds to T. cruzi trypomastigote cells. 9 × 106
purified trypomastigotes were incubated with 1 × 106
cpm 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.
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Fig. 9.
CK18 is the LLC-MK2 cell surface
receptor to peptide J. Each well of a 24-well plate was coated
with 40 µg of peptide J or BSA, washed with PBS, and incubated for
2 h with 1% BSA/PBS. LLC-MK2 (1 × 105) cells in 0.5 ml of DME medium were incubated with BSA
(control), 100 µM peptide J, 100 µM peptide
J-Ala, 0.1 mg/ml CK18, and anti-CK18 antibody (diluted 1:20) for 15 min, added to the peptide-coated wells, and incubated for 1 h at
37 °C. The wells were washed with DME medium, and the cells were
stained with crystal violet (23). The figure shows representative
results of four independent experiments.
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Fig. 10.
Importance of peptide J and CK18 on the
infection of mammalian cells by T. cruzi
trypomastigotes. LLC-MK2 cells previously
incubated with 200 µM peptide J-Ala, 100 or 200 µM peptide J (1xJ and 2xJ,
respectively), 10 µM Tc85-11, mouse IgG, anti-CK18
antibody (1:20), or peptide J plus anti-CK18 antibody (1:20) were
assayed for invasion by T. cruzi trypomastigotes
(LLC-MK2 cells:parasites, 1:100). All experiments were done
in triplicate, and 12 photos of each replicate were made, enabling the
counting of infected and non-infected cells in samples each containing
200 cells. The data were compared for statistical significance using
the unpaired Student's t test. The plot represents the
average of triplicate determinations ± S.D. of five independent
experiments. Except for peptide J-Ala and mouse IgG, the other
experimental points differed significantly from the control
(p < 0.05).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors are required for the infection of mammalian cells by
T. cruzi (45), but the parasite ligand is unknown.
Interestingly, cells treated with epidermal growth factor plus
transforming growth factor
express higher levels of CK18 (46). The
fact that CK18 can be phosphorylated (47) suggests an involvement of
cytokeratin in the intracellular signaling induced by T. cruzi (48).
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ACKNOWLEDGEMENTS |
---|
Cytokeratin 18 was sequenced at HHMI Biopolymer Laboratory and W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University, New Haven, CT. We acknowledge Dr. Chuck S. Farah for critically reading the manuscript.
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FOOTNOTES |
---|
* This work was supported by Grants 95/4562-3 and 99/12459-9 from the Fundação de Amparo à Pesquisa do Estado de São Paulo (to M. J. M. A. and W. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Submitted as part of a doctoral thesis at the Universidade
de São Paulo, São Paulo, Brazil.
§ A post-doctoral fellow from the Fundação de Amparo à Pesquisa do Estado de São Paulo.
¶ Present address: M. D. Anderson Cancer Center, Houston, Texas 77030.
A visiting professor with a Fundação de Amparo
à Pesquisa do Estado de São Paulo-Deutscher Akademischer
Austauschdienst joint fellowship.
To whom correspondence should be addressed: Departamento de
Bioquímica, Instituto de Química, Universidade de
São Paulo, Caixa Postal 26077, São Paulo 05599-970,
São Paulo, Brazil. Tel.: 55-11-3818-3810 ext. 233; Fax:
55-11-3815-5579; E-mail: mjmalves@iq.usp.br.
Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M011474200
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ABBREVIATIONS |
---|
The abbreviations used are: CK, cytokeratin; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DME, Dulbecco's modified Eagle's; FCS, fetal calf serum; PAGE, polyacrylamide gel electrophoresis; kb, kilobase (pairs); RT, room temperature; WGA, wheat germ agglutinin..
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