From the Departamento de
Bioquímica, Instituto de Química, Universidade de
São Paulo, Caixa Postal 26077, São Paulo 05599-970, S. P.,
Brazil and the ¶ Department of Molecular Biology and Biochemistry,
University of California, Irvine, California 92717
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ABSTRACT |
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Trypomastigotes of Trypanosoma cruzi express a set of surface glycoproteins known, collectively, as Tc-85. A monoclonal antibody to these proteins, named H1A10, inhibits (50-90%) in vitro parasite interiorization into host cells, thus implicating these glycoproteins in the infection process. Two DNA inserts, a genomic DNA fragment and a full-length cDNA encoding the H1A10 epitope, have now been cloned and characterized. Results show that both have high sequence identity with all reported members of the gp85/trans-sialidase gene family, although the H1A10 epitope exists only in the Tc-85 subset of the family. The epitope has been mapped by competition of antibody binding to a Tc-85 recombinant protein with peptides having sequences predicted by the Tc-85 DNA sequence, which contains also putative N-glycosylation sites and COOH-terminal glycosylphosphatidylinositol anchor insertion sites, as expected, since an N-glycan chain and a glycosylphosphatidylinositol anchor have been characterized previously in the Tc-85 subset. The protein encoded by the full-length cDNA insert binds to cells and in vitro to laminin, but not to gelatin or fibronectin, in a saturable manner.
For the first time it was possible to assign a defined ligand to a
sequenced glycoprotein belonging to the gp85 family. This fact,
together with the reported binding of family members to cell surfaces,
reinforces the hypothesis that this family encodes glycoproteins with
similar sequences but differing enough as to bind to different ligands
and thus forming a family of adhesion glycoproteins enabling the
parasite to overcome the barriers interposed by cell membranes,
extracellular matrices, and basal laminae.
The trypomastigote form of Trypanosoma cruzi, which is
the invasive form of the parasite for vertebrates and elicits in humans an ailment known as Chagas' disease, has to overcome a formidable barrier in order to migrate from blood vessels, where they circulate, to the interior of the cells cytoplasm where they differentiate to the
replicative amastigote forms (1-4). In addition to cell membranes and
the extracellular matrix the parasite has to surpass the basal laminae,
barriers found throughout the body surrounding organs and blood vessels
(5-7). The most important macromolecules of the basal laminae are
laminin, entactin, collagen-IV, and heparan sulfate. Laminins belong to
a family of glycoproteins, heterotrimers of Our laboratory has implicated glycoproteins of 85,000 Da molecular mass
as involved in host cell invasion by the parasite. They exist only in
the invasive trypomastigote form and are absent from epimastigotes, the
insect form of the parasite that is unable to invade vertebrate cells.
These glycoproteins, collectively called Tc-85, form a population of
heterogeneous GPI1-anchored
surface glycoproteins with similar molecular masses but different
electric charges (13-15). Involvement of one or more of these family
members with adhesion and/or internalization of the parasite to host
cells is indicated by the observation that monoclonal antibody H1A10
(mAbH1A10), which recognizes and defines the Tc-85 glycoproteins,
partially inhibits invasion of the host cell by the parasite in
vitro (15, 16).
A trypomastigote specific surface 85 kDa glycoprotein, which binds to
laminin-Sepharose (LBG, laminin-binding glycoprotein) and belongs to
the most acidic components of the Tc-85 glycoprotein family, was
reported as the putative laminin receptor (17). Anti-laminin antibodies
also partially inhibit host cell invasion by trypomastigotes and
laminin binds to the surface of the parasite in a specific and
saturable manner, suggesting that it may be involved in the infection
process (17). Interaction of pathogens with laminin, fibronectin, and
extracellular matrix proteins have been described by others (18-22).
The present work describes the cloning of a full-length cDNA
encoding a polypeptide recognized by mAbH1A10 with the expected properties of the LBG, thus showing that at least one of the Tc-85 family members is an adhesion protein to a defined ligand. The corresponding cDNA insert has significant sequence identity with members of the gp85/trans-sialidase gene family of T. cruzi
(23-25), raising the interesting hypothesis that several members of
that superfamily may possess adhesive capacity to different receptor molecules either located on the cell surface or belonging to components of the extracellular matrix.
Parasite Strain and Culture--
Clone 3, isolated from the
T. cruzi Peru strain by micromanipulation, was utilized for
these studies. Growth and maintenance are described elsewhere (26).
Properties and cloning conditions for the mAbH1A10 are as reported (15,
16).
Nucleic Acid Isolation, Radiolabeling, Southern and Northern
Transfers, and Restriction Enzymes--
Parasite nuclear DNA,
bacterial plasmid DNA, and phage DNA were isolated as described
previously (27). Agarose gel electrophoresis of DNA, Southern and
Northern transfers, hybridization, and filter washing were performed as
described previously (28). DNA restriction fragments were radiolabeled
with [ Screening of DNA Subcloning and Fusion Protein Isolation--
The insert from
the PCR--
PCR was carried out using Taq DNA polymerase
(Boehringer Mannheim) in a DNA Thermal Cycler, according to a standard
protocol (28). Test reactions were conducted to optimize template DNA, MgCl2, and dNTP amounts. PCR radiolabeling was performed by
substituting the non-radioactive dCTP by 30 µCi of
[ Polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
Protein samples were subjected to one-dimensional PAGE
and either stained or transferred to nitrocellulose membranes as
described (16). Western blot reactions were carried out by blocking the membranes with TBS-Tween (50 mM Tris, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) supplemented with 3% non-fat
dry milk. Incubations with the first antibody were always overnight at
4 °C in 50 mM Tris, pH 8.6, 150 mM NaCl,
0.03% Tween 20, and 0.05% Triton X-100. After washing three times for
5 min with TBS-Tween the membranes were incubated with the secondary
antibody (anti-mouse 125I-labeled, Amersham Pharmacia
Biotech) overnight at 4 °C in TBS-Tween supplemented with 3%
non-fat dry milk, washed again as before, and exposed to x-ray film.
DNA Sequencing and Sequence Analysis--
DNA sequencing was
performed by dideoxy chain termination (31). Sequencing was performed
on DNA fragments subcloned into Bluescript using
[ Peptide Synthesis--
Peptides were synthesized with an Applied
Biosystems 430A Synthesizer using ABI-fastmocTM chemistry
cycles. Peptides were cleaved from the resin using 90% trifluoroacetic
acid and a scavenger. Cleaved peptides were filtered through a
0.45-µm filter, precipitated in cold ether, and the precipitate
collected by centrifugation. Peptides were dried under nitrogen and
purified by reverse phase high performance liquid chromatography over a
Vydac-C18 column (1.0 × 25 cm) in 0.1% trifluoroacetic
acid/H2O and 0.1% trifluoroacetic acid/acetonitrile. Peptides were eluted from the column with a gradient of acetonitrile (0-100%) over 100 min at a flow rate of 4 ml/min. Absorbance was monitored at 210 nm.
Inhibition of Binding of mAbH1A10 with Synthetic
Peptides--
Microtiter wells were coated with Tc85-1 fusion protein
(50 µl of a 10 µg/ml solution in PBS (PBS = 140 mM
NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH
7.3)) for 1 h at room temperature, washed three times with PBS,
and quenched with blocking buffer (0.25% BSA, 0.05% Tween 20 in PBS)
for 2 h at room temperature. All peptides were prepared as 20 mM stock solutions in Me2SO. Coated microtiter
wells were incubated overnight at 4 °C with 50 µl of a 2 mM solution of individual peptides in PBS + 10%
Me2SO containing mAbH1A10 (10 ng/ml), washed with PBS,
incubated with peroxidase-conjugated anti-mouse sera for 1 h at
room temperature, again PBS-washed, and developed with ABTS (Boehringer
Mannhein). For direct binding of mAbH1A10, microtiter wells were coated
overnight at room temperature with 50 µl of a 100 µg/ml solution of
each of the individual peptides in either 50 mM carbonate
buffer, pH 9.6, or 50 mM acetate buffer, pH 4.0. The wells
were washed, quenched with blocking buffer for 2 h at room
temperature, and incubated with mAbH1A10 in blocking buffer (1 µg/ml)
overnight at 4 °C. Wells were washed with PBS as before and
incubated with peroxidase-conjugated anti-mouse sera for 1 h at
room temperature, washed with PBS, and developed with ABTS.
Cell Binding Assay--
E. coli BL21 DE3 pPlysS cells
were transformed with the pET21-Tc85-11 construct and the cells grown
on LBCC (Luria broth medium supplemented with 200 µg/ml carbenicillin
and 35 µg/ml chloramphenicol) agar overnight at 37 °C/200 rpm.
Several colonies were grown on 40 ml of LBCC medium until
A595 nm ~0.5. The cells were harvested by
centrifugation (5,000 × g/10 min at 4 °C), washed once with minimal M9 medium, resuspended in 40 ml of the same medium
supplemented with a mixture of 18 amino acids, except methionine and
cysteine (25-50 µg/ml each), and further cultured for 1 h at
37 °C/200 rpm. Induction was made with 1 mM
isopropyl-1-thio- Laminin Binding Assay to the Tc85-11 Fusion Protein--
Wells
of microtiter plates were coated with 50 µl of purified Tc85-11
protein (10 µg/ml in PBS supplemented with 8 M urea) for
2 h at room temperature, washed with PBS, blocked with PBS-1% BSA
(2 h at room temperature), and incubated with increasing amounts of
laminin in TCMM (TCMM = 25 mM Tris, pH 7.5, 50 mM NaCl, 5 mM CaCl2, 5 mM MgCl2, 1 mM
MnCl2)-1% BSA (overnight at 4 °C). The wells were
washed, incubated for 2 h at room temperature with anti-laminin
rabbit serum, washed again, incubated with peroxidase-conjugated anti-rabbit sera for 1 h at room temperature, and developed with ortho-phenylenediamine (Sigma). Laminin was purified from
Engelbreath-Holm-Swarm tumor, as described (17).
Isolation of a Genomic DNA (Tc85-1) Insert That Encodes the
mAbH1A10 Epitope and Shares Identity with Members of the
gp85/Trans-sialidase Gene Family--
A genomic DNA library
constructed in
To determine whether Tc85-1 encodes a polypeptide that could represent
a portion of an 85-kDa trypomastigote-specific protein, the insert was
subcloned into the pTrcHis vector, expressed in E. coli, and
the fusion protein was enriched by affinity chromatography. Sera from
immunized mice with the fusion protein were employed to develop Western
blots containing epimastigote and trypomastigote lysates, as well as
the fusion protein. As shown in Fig. 2,
only the fusion protein and an 85-kDa trypomastigote-specific protein were recognized. This result is identical to that observed with mAbH1A10 and suggests that the Tc85-1 insert encodes a portion of an
85-kDa protein recognized by mAbH1A10.
A sequence identity search of the GenBankTM data base with
the Tc85-1 sequence revealed a high percentual identity with all reported members of the gp85/trans-sialidase gene family (23, 24).
Those with the highest percent identity were genes encoding trypomastigote-specific surface glycoproteins of
Mr 85,000-110,000 (Fig.
3). Consistent with the
GenBankTM search and the Western blot results, the Tc85-1
insert hybridized with trypomastigote, but not epimastigote,
poly(A)+ RNA of length 3.7 kb as well as with a large
number of different size restriction fragments of a genomic Southern
blot containing EcoRI- and BamHI-digested genomic
DNA (data not shown).
Cloning and Sequencing the cDNA Insert Tc85-11--
A
trypomastigote cDNA library constructed in
The open reading frame of 2,361 base pairs codes for a peptide of 786 amino acids with a molecular mass of 84,496 daltons, a theoretical pI
of 5.00, and contains two ASP boxes
(S-X-D-X-G-X-T-W) present
in bacterial neuraminidases, as well as the
V-T-V-X-N-V-F-L-Y-D-R sequence, both
common to all members of the trans-sialidase supergene family of
T. cruzi (23, 24). The presence of
N-glycosylation and GPI anchor insertion sites is in
accordance with previous observations showing that Tc-85 is a
GPI-anchored membrane glycoprotein (33, 34) with N-linked
carbohydrates (35, 36). The Tc85-11 polypeptide has 12 putative
N-glycosylation sites as well as two signal peptides, one at
the amino-terminal portion of the molecule, which directs the protein
to the endoplasmic reticulum, and the other at the carboxyl-terminal
portion for anchor insertion (shown in Fig. 4).
Mapping the H1A10 Epitope in Tc85-1--
Peptides of length 15 amino acids, which overlap in sequence by 5 amino acids and span the
Tc85-1 polypeptide, were synthesized and used to map the mAbH1A10
binding site by a competitive inhibition assay (Fig.
6A). Microtiter wells coated
with the Tc85-1 fusion protein were incubated with mAbH1A10 in the
presence of individual peptides, and inhibition of binding of mAbH1A10
to Tc85-1 was measured by ELISA. Only peptide G significantly
inhibited the ability of the monoclonal antibody to react with Tc85-1,
indicating that the epitope is contained within this 15-amino acid
sequence (Fig. 6B). The same result was achieved when the
peptides were immobilized and the direct binding of mAbH1A10
determined by ELISA. Only peptide G promoted the binding of mAbH1A10 in
a reproducible and specific manner (data not shown). As shown in Fig.
6C, inhibition of the binding of mAbH1A10 to Tc85-1 by
peptide G is clearly concentration-dependent, while peptide F
shows no inhibition of binding of mAbH1A10 within the concentration
range of 0.1 to 1.5 mM. Comparison of both Tc85-1 and
Tc85-11 encoded peptides show that only peptides A, F, and G are 100%
identical in both sequences. Peptides E and H have only one amino acid
change at their ends when both sequences are compared (Fig.
6D). The peptide G sequence is contained in the most
conserved region of both proteins, and as shown in Fig. 6C, only peptide G, but not peptide F, inhibits mAbH1A10 binding. Our
result suggests that the peptide G-containing region is involved with
mAbH1A10 binding to Tc-85, and its position in the whole protein is
indicated in Fig. 4.
The Tc85-11 Protein Binds to the Host Cell--
Tc-85 is possibly
involved in the process of invasion of host cells by the parasite. To
verify whether the protein encoded by the Tc85-11 gene binds to the
host cell, the coding sequence of the gene was subcloned (minus the
first 26 amino acids comprising the amino-terminal signal peptide) into
the pET21 vector, and the recombinant protein was expressed in E. coli as described under "Experimental Procedures." Using this
protocol, labeled Tc85-11 recombinant protein was produced by bacterial
cells under the control of the rifampicin-insensitive T7 RNA
polymerase, while expression of the other E. coli genes was
inhibited by rifampicin, since they remained under the control of the
bacterial polymerase. As judged by the total extract autoradiography
(Fig. 7B, total extract), the
labeling was restricted to the Tc85-11 protein, since the major product
is clearly the 85-kDa polypeptide. Some bands, which appear below this
band, are probably degradation products of the main protein. The 85-kDa
polypeptide is also specifically immunopreciptated by the mAbH1A10
(data not shown). Upon incubation of this extract with
glutaraldehyde-fixed LLC-MK2, the bound radioactivity was
eluted with SDS, and an aliquot was precipitated with trichloroacetic acid. Fig. 7A shows that binding to the cells was
concentration dependent. The remainder of the eluted radioactivity was
analyzed by SDS-PAGE and fluorography revealing an 85-kDa, as well as a 37-kDa, polypeptide (Fig. 7B). The 37-kDa polypeptide might
be a proteolytic fragment of the 85-kDa polypeptide, which maintained a
cell binding domain.
The Tc85-11 Gene Codes for the LBG--
It has been shown
previously that acidic components of Tc-85 family (pI values of
5.5-6.5) bind to laminin (17). Considering that the Tc85-11 protein
binds to the host cell, and the predicted pI for the polypeptide is
5.00, an investigation was undertaken in order to show that laminin may
mediate Tc85-11 attachment to the host cell.
[35S]Methionine metabolically labeled Tc85-11 protein was
incubated with laminin and other extracellular proteins. As shown in
Fig. 8A, Tc85-11 binds
specifically to laminin, but not to gelatin or fibronectin previously
immobilized on plastic wells. This result suggests that the Tc85-11
cDNA insert encodes the LBG gene. Interestingly, although the
37-kDa polypeptide bound to cells, it did not bind to laminin in
vitro. This result suggests the possible existence of two
different domains on Tc85-11, one for laminin and the other for another
receptor located on the host cell membrane. To further confirm that the
Tc85-11 gene actually codes for a polypeptide with adhesive properties
to laminin, the recombinant protein was purified from E. coli inclusion bodies and used to coat microtiter wells, which
were then incubated with increasing concentrations of laminin. Laminin
bound to Tc85-11 protein in a saturable manner (Fig.
8B).
After the pioneer reports on the existence of a specific
glycoprotein(s) of 85 kDa, called Tc-85, on the surface of the
infectious form of T. cruzi (13, 14, 37), and the finding
that monoclonal antibodies against Tc-85 were able to inhibit partially
the entrance of the parasite into host cells (16), several proteins of
equivalent molecular weight were implicated in parasite-host cell
interaction (38-40). Antibodies developed against proteins of similar
size have been found to be inhibitors of parasite penetration (41), and
an 85-kDa protein was described as a fibronectin receptor (19). No
sequence data are available for most of the glycoproteins of that size
(19, 38, 39), but it is likely that all may belong to the
gp85/trans-sialidase family (cf. Refs. 23 and 24), since all
gp85 genes cloned to date are members of this gene family (4).
This work shows that the Tc-85 glycoproteins, which bind to wheat germ
agglutinin (13) and are recognized by mAbH1A10 (16), also belong to the
gp85/trans-sialidase supergene family. As genetic analysis of members
of this family has previously shown putative sites for
N-glycosylation as well as for GPI anchor insertion (cf. Refs. 23 and 24), it is safe to generalize that all
members of the family are indeed membrane GPI-anchored glycoproteins, since an N-linked oligosaccharide anthena (35, 36) and a
lysoalkylphosphatidylinositol anchor (33, 34) have been chemically
demonstrated as part of the Tc-85 glycoprotein molecules. Another
GPI-anchored glycoprotein from metacyclic trypomastigotes with adhesive
properties to cells also belongs to the gp85/trans-sialidase family
(40). The present work shows that at least one of the members of the
Tc-85 subfamily binds to the host cell through laminin. This is the
first time a chemically defined ligand is demonstrated for a member of
the 85-kDa glycoprotein superfamily with adhesive properties.
Laminins are found in basal laminae, which are present as a continuous
sheet surrounding each muscle fiber and pass through the synaptic cleft
at the neuromuscular junction. Basal laminae are also found surrounding
vessels and organs, beneath the epithelium and endothelium (5).
Interestingly, muscle and the neuromuscular junction are the preferred
sites of T. cruzi-induced lesions. During the beginning of
infection, the parasite has to leave the site of infection (usually the
insect bite wound) and reach the bloodstream. The interaction of
T. cruzi with the extracellular matrix is thus a crucial
step for the success of infection, as the parasite must leave the blood
probably by adhering to endothelial cells and crossing the basal
laminae as well as interacting with the extracellular connective
milieu. Thus, the presence of a laminin receptor on the surface of the
infective parasite can be very important during this stage.
Antibodies directed against integrins partially block parasite cell
invasion, and the authors suggested that VLA-4 and VLA-5, both
fibronectin receptors are involved, although other It is our working hypothesis that the gp85/trans-sialidase gene family
comprises a family coding for adhesion proteins, with several of its
members interacting with specific ligands. Glycoproteins of about the
same molecular mass have been described as a fibronectin ligand (19)
and a cell adhesion molecule (39, 40), although a 60-kDa glycoprotein
(penetrin), which promotes adhesion of trypomastigotes to heparin,
heparan sulfate, and collagen, has also been described (2). The
Tc85/LBG binds to laminin but not to fibronectin, showing the
specificity of the interaction. Kahn et al. (43) have
reported for the whole family the existence of at least 100 genes of
about 2-2.5 kb representing at least 1% of the T. cruzi genome. It is worth noting that the 3.5 h half-life of the Tc85 family is considerably short (44). This fast turnover could facilitate
the progression of the parasite from blood vessels to the cells if
different continuously expressed subsets of the family would bind to
different ligands on the cell surfaces, extracellular matrices, and
basal laminae. Another possibility is that one single member may
interact with two or more ligands on the surface of the cell. In
multicellular organisms, it is common to find situations in which the
same adhesion molecules interact with several proteins or cell surface
receptors (45, 46). For instance, laminin binds to heparin, laminin
itself, collagen-IV, entactin, integrins, and other putative
non-integrin receptors (10, 11). Thus, during the migration through the
basal lamina, the parasite can be covered with laminin, fibronectin,
collagen, and/or heparin, allowing contact between the nearest cell and
the invasive trypomastigote through integrin receptors. Detachment from
binding could be provided by shedding of the 85,000-Da glycoprotein
adhesion molecule, a phenomenon that is constantly occurring (34, 47)
and/or by proteases, which could cleave the matrix elements. At least
two proteases that might play a role in T. cruzi infection
have been described, cruzipain (48, 49) and an 80-kDa collagenase (50). Shedding and protease digestion could help the parasite to be freed of
the successive haptotactic interactions, allowing its migration through
the basal lamina and the extracellular milieu.
Pathogen surface proteins belonging to multigenic families are
frequently related with protection and escape from the immune system
(51). For T. cruzi, the gp85/trans-sialidase family members have also been proposed to play a role in evasion of the immune system
by the parasite (52, 53), which obviously does not exclude their
proposed function as adhesion molecules.
The high amount of nuclear information that the parasite has kept
through evolution in order to maintain highly similar genomic sequences
is suggestive of the importance of that family for species survival.
Thus, considering that T. cruzi, as opposed to African trypanosomes, needs to enter cells in order to survive, it is conceivable that this parasite may have found a mechanism by which, making subtle sequence modifications in otherwise very similar genes
coding for different gp85 proteins, it could adhere to different molecules of the cell membrane, the basal lamina, and the cell matrix.
The molecular evolution of this family may have been driven on one side
by the pressure of the host immune system and on the other by the
ability of the parasite to enter host cells. Thus, parasites were
selected not only by their capacity to survive in the bloodstream but
also to invade cells more efficiently.
INTRODUCTION
Top
Abstract
Introduction
References
,
, and
chains,
involved in the assembly and shape of basal laminae (8, 9). Their
involvement with adhesion, spread, differentiation, and several other
cell functions is well known and discussed elsewhere (10-12).
EXPERIMENTAL PROCEDURES
-32P]dCTP by nick translation. All restriction
enzymes were purchased from Boehringer Mannheim and used as recommended.
Libraries--
A genomic DNA expression library
constructed in phage
gt11 using DNA isolated from trypomastigotes
was screened with the mAbH1A10 as described below for Western blot
analysis. A cDNA library constructed in
ZAP II using
trypomastigote poly(A)+ RNA was screened with Tc85-1
following PCR radiolabeling of the insert in Bluescript (Stratagene,
Inc.) using the KS and SK primers as described below.
gt11 clone identified by reaction with mAbH1A10 (Tc85-1) was
isolated by digestion with EcoRI and gel electrophoresis.
The insert was excised from the gel, purified, and subcloned into the
plasmid Bluescript for sequencing and pGEX-1 for expression in
Escherichia coli cells (29). The recombinant Tc85-1 protein
described in the text was obtained from a construct of the Tc85-1
insert subcloned as AvaI/EcoRI fragments into the polyhistidine fusion protein expression vector pTrcHis-B (Invitrogen, Inc.) and expressed and purified as described by the manufacturer. The
fragment was generated by PCR with the original subclone in Bluescript
using the KS primer of the vector as the 3' primer and a clone-specific
primer, which introduced and AvaI site at the 5' end of the
insert (5'-GAATTCCCCTCGAGCGAGAAAAC). The Tc85-11 insert was subcloned
into the plasmids Bluescript and pUC19 for sequencing and as a
BamHI/XhoI fragment into the polyhistidine fusion
vector pET21 (Novagen, Inc.) for expression and purification of
protein. The BamHI/XhoI insert was obtained by
PCR with two synthetic oligonucleotides to introduce the restriction
enzyme sites at the 5' and 3' ends, respectively
(5'-CTTGGATCCACGACCGCTCAGG and 5'-GACACCTCGAGCGCAGTAGCAAGGGCCCAC). The
Tc85-11 fusion protein was purified from inclusion bodies prepared as
described (30). The fusion protein was solubilized in 10 mM
phosphate buffer, pH 7.8, 500 mM NaCl, and 6 M
guanidine (overnight at room temperature). The extract was clarified by
centrifugation (30,000 × g for 30 min), applied to a
Ni2+/nitrilotriacetic acid column (Qiagen), and purified as
described by the manufacturer. About 500 µg of purified protein were
obtained from 3 liters of E. coli culture. The presence of
the purified protein was checked by SDS-PAGE and Western blot with
mAbH1A10 and T7.Tag (Invitrogen, Inc.). The latter reacts with the 10 amino acids coded for by the pET21 vector, which are fused with the amino-terminal portion of the fusion protein.
-32P]dCTP in a 30-cycle reaction after optimization
of the template and MgCl2 concentrations. PCR products were
purified using Spin Bind units (FMC BioProducts) as recommended.
-35S]dATP for incorporation and either the KS and SK
primers of Bluescript or with oligonucleotides synthesized from
sequence data; on the 5' end of Tc85-1 subcloned in the expression
vector pGEX-1 using a primer synthesized from sequences upstream of the
glutathione S-transferase cloning site; and on both 5' and
3' ends of subclones in the pTrcHis expression vector with primers
based upon sequences upstream and downstream of the multiple cloning
site of the vector. Nucleotide sequence data were compiled and analyzed
using the IBI Pustell Sequence Analysis programs for the IBM computer.
-D-galactopyranoside for 10 min,
followed by the addition of rifampicin (200 µg/ml) and incubation at
42 °C in a water bath for 20 min. At this point 500 µCi of
[35S]methionine were added, and the cells were grown for
another 10 min at 37 °C/200 rpm, harvested by centrifugation
(5,000 × g/10 min at 4 °C), lysed by the addition
of 1 ml of lysis buffer (PBS containing 0.2% Nonidet P-40, 1 µg/ml
lysozyme, and 1 mM each phenylmethylsulfonyl fluoride and
N
-p-tosyl-L-lysine
chloromethyl ketone), and further incubated for 30 min at room
temperature. The bacterial extract was clarified by centrifugation
(16,000 × g for 15 min), diluted with PBS-1.5% BSA
(final concentration), and incubated with cells or extracellular matrix
proteins. Proteins were incubated on 24-well plates (100 µl of 50 µg/ml in PBS) for 2 h at room temperature, washed with PBS, and
blocked with PBS-3% BSA for 2 h, followed by the addition of the
bacterial extract, as described below. LLC-MK2 cells were grown overnight in 24-well plate (105 cells/well), washed
with PBS, fixed with 2% glutaraldehyde in PBS for 1 h at room
temperature, washed with PBS, and blocked with 10 mM Tris,
pH 7,5, 150 mM NaCl (TBS) for 1 h at room temperature. The wells were blocked with PBS-3% BSA for 2 h at room
temperature, incubated overnight at 4 °C with the clarified cell
extract containing 1.5% BSA in PBS, washed with PBS, and the
radioactivity bound to the cell or to extracellular matrix proteins was
eluted with 100 µl of 1% SDS. The radioactivity was determined in a
10% trichloroacetic acid precipitate of a 20-µl aliquot, and the
remaining eluate (80 µl) was concentrated on Speed-Vac and analyzed
by SDS-PAGE, followed by exposure to x-ray film.
RESULTS
gt11 was screened with the mAbH1A10 as described
under "Experimental Procedures" and one positive clone, named
H3.3, was selected. Restriction enzyme mapping analysis of the
genomic DNA insert, from now on designated as Tc85-1, revealed a 0.4-kb
insert whose complementary strands have been sequenced. Translation of
either complementary strand of Tc85-1 resulted in a complete open
reading frame. To identify which strand encoded the epitope recognized
by mAbH1A10, the Tc85-1 was subcloned into pGEX-1 plasmid, and two
bacterial cell lines containing the insert in opposite orientations
were selected, grown, and induced. Western blot analysis showed that
only one of the two orientations produced a 40-kDa protein recognized
by mAbH1A10 (Fig. 1B). The
nucleotide sequence of Tc85-1, with the conceptual amino acid
translation, is shown in Fig. 1A.
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Fig. 1.
Isolation of a genomic DNA fragment from
T. cruzi containing the epitope for the mAbH1A10.
A, nucleotide sequence of the 395-base pair insert Tc85-1.
The 5' end of the sequence, deduced from direct sequence analysis of
the clones in pGEX, is indicated. The sequence includes the artificial
EcoRI linker on the 5' end, which was added during the
library construction. The amino acid translation is shown above the
nucleotide sequence. B, Western blot of fusion proteins of
Tc85-1 in pGEX-1 reacted with H1A10 monoclonal antibody. Two clones are
shown, one with the insert in the correct 5' to 3' orientation
(lanes 1 and 2) and the other with the insert in
the opposite orientation (lanes 3 and 4).
Lanes 1 and 3 are lysates from uninduced
bacteria, and lanes 2 and 4 are lysates following
3 h of induction with 1 mM
isopropyl-1-thio- -D-galactopyranoside.
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Fig. 2.
Western blots of epimastigote lysate
(lanes 1, 4, and 7), trypomastigote
lysate (lanes 2, 5, and 8), and
pTrcHis/Tc85-1 fusion protein (lanes 3, 6, and
9) were reacted with: normal mouse serum (A),
mouse anti-pTrcHis/Tc85-1 fusion protein (B), and mAbH1A10
(C). Anti-pTrcHis/Tc85-1 fusion protein reacts with a
trypomastigote-specific 85-kDa protein (B, lane
5) as well as with the pTrcHis/Tc85-1 fusion protein
(B, lane 6) but did not react with epimastigote
protein (B, lane 4).
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Fig. 3.
Comparison of the predicted amino acid
sequence of Tc85-1 with members of the gp85/trans-sialidase gene
family. Amino acid differences are shown. Dashed lines
represent deletions in the individual sequences.
ZAP II was screened
with the Tc85-1 insert, and the clones that contained a full-length
cDNA of size >2 kb encoding the mAbH1A10 epitope were selected,
restricted, subcloned into the pTrcHis vector, and expressed in
E. coli. One clone, named Tc85-11, encoded an expression
product that reacted positive with mAbH1A10 by Western blot and was
selected for this work. The whole coding region of the Tc85-11 gene was sequenced in both of the complementary strands by ExoIII/S1
nuclease partial digestion (Fig. 4). As
expected, the DNA sequence has significant identity with Tc85-1 (89%;
118 identical amino acids out of 132) and with several previously
described members of the gp85/trans-sialidase supergene family.
Northern blot analysis shows that the Tc85-11 insert hybridizes to a
3.7-kb trypomastigote stage mRNA but not to epimastigote or
LLC-MK2 mRNA, as expected (Fig.
5A). Chromosome mapping
resulted in hybridization of the Tc85-11 insert with 15 out of 20 chromosome bands of the CL Brener clone genome (Fig. 5B)
(32).
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Fig. 4.
DNA sequence and amino acid sequence of the
open reading frame of the Tc85-11 cDNA insert
(GenBankTM accession number AF085686). The
asterisk indicates putative N-glycosylation
sites. The two ASP boxes, the common sequence to all members of the
gp85/trans-sialidase family, and the peptide G region are indicated by
boxes. At the amino- and carboxyl-terminal two putative
signal peptides are underlined with the arrows
indicating the possible sites of cleavage. Sites for GPI anchor
insertion can be seen at the carboxyl-terminal upstream of the
hydrophobic sequence.
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Fig. 5.
Northern blot analysis and chromosome
location of the Tc85-11 insert. A, Northern blot
analysis shows that the Tc85-11 cDNA insert hybridizes to
trypomastigote mRNA (lane 3) but not to epimastigote
(lane 2) or LLC-MK2 mRNA (lane 1).
B, chromosome mapping of the Tc85-11 gene. A pulse-field gel
of epimastigote of the clone CL Brener was stained with ethidium
bromide (lane 1) and blotted to the nylon membrane. The blot
was hybridized with a telomeric probe (lane 2), with the
Tc85-11 cDNA insert (lane 3) and with a
-tubulin probe (lane 4). The numbers at the
left side indicate the defined chromosome bands, and the
arrows point to bands that hybridized with the Tc85-11
probe.
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Fig. 6.
Epitope mapping. A, peptides
used in the competition experiment for epitope mapping. The figure
shows their sequences and how they overlap. B, results of
the competition experiment with the individual peptides and mAbH1A10.
The amount of mAbH1A10 bound to the wells in the presence of each
peptide was determined by ELISA. The values represent the intensity of
reaction in relation to the control (mAbH1A10 in PBS + 10%
Me2SO equals 100% with a S.D. for this control always
below 5%). The bars show the average of three distinct
experiments. C, results showing the effect of concentration
of peptide on the binding of mAbH1A10 to Tc85-1. mAbH1A10 was incubated
with increasing concentrations of either peptide F or G in PBS + 10%
Me2SO and binding was measured by ELISA. D,
comparison between Tc85-1- and Tc85-11-encoded peptides. Amino acid
differences are shown. Dashed lines represent deletions in
the individual sequences. The peptide G region is indicated.
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Fig. 7.
Binding of Tc85-11 to LLC-MK2
cells. Metabolically labeled Tc85-11 protein was incubated with
glutaraldehyde-fixed LLC-MK2 cells. An aliquot of the
lysate was electrophoresed in SDS-PAGE (lane labeled "Total
Extract"). The cells were thoroughly washed by flushing buffer
on the wells, after which the bound material was eluted with 1% SDS.
A shows cell bound radioactivity with increasing
concentrations of the bacterial lysate, and B shows an
autoradiography of SDS-PAGE performed with the corresponding bound
fractions eluted from the cells.
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Fig. 8.
Binding of Tc85-11 to laminin.
Metabolically labeled Tc85-11 protein was incubated on plastic dishes
previously coated with gelatin (Gel), fibronectin
(Fn), or laminin (Lam). The wells were thoroughly
washed by flushing buffer and eluted with 1% SDS. A shows
the autoradiography of an aliquot of the bound fraction. In
B, wells were coated with purified Tc85-11 protein and
incubated with laminin in TCMM buffer supplemented with 1% BSA. The
wells were washed, incubated with rabbit anti-laminin serum, washed,
and the bound laminin evaluated by ELISA.
DISCUSSION
1,
but not
2, integrins also participate in the invasion
process (42). It has been proposed that fibronectin could work as a
bridge between the parasite and the cell (19), and it is likely that
the same mechanism would be operative in the case of laminin reported herein.
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ACKNOWLEDGEMENTS |
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The assistance of Barbara Granger (University of California at Irvine) and Roberto Zangrandi (Universidade de São Paulo) in maintenance and growth of parasite cultures in part of the experiments is acknowledged. Dr. José Franco da Silveira Filho (Universidade Federal de São Paulo) helped us with the chromosome mapping experiment as part of the T. cruzi genome project.
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FOOTNOTES |
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* This work was supported by a thematic grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (95/04562-3 (to W. C., and M. J. M. A.) and National Institutes of Health Grant AI18873 (to J. E. M.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U11500 and AF085686.
§ Supported by a postdoctoral fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo. Also supported (for 1 year in the laboratory of J. E. M.) by fellowships from Conselho Nacional de Desenvolvimento Científico e Tenológico (CNPq) and Campanha de Aperfeiçoamento do Pessoal do Ensino Superior (CAPES).
To whom correspondence should be addressed: Dept. de
Bioquímica, Instituto de Química, Universidade de
São Paulo, Caixa Postal 26077, São Paulo 05599-970, S.P.
Brazil. Tel.: 55-11-818-3812; Fax: 55-11-815-5579; E-mail:
mjmalves{at}quim.iq.usp.br.
The abbreviations used are: GPI, glycosylphosphatidylinositol; ABTS, 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate(6)] diammonium salt crystals; PBS, phosphate-buffered saline; BSA, bovine serum albumin; kb, kilobase pair(s); LBG, laminin-binding glycoprotein; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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