From the Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri 63110
Received for publication, September 25, 2002, and in revised form, December 3, 2002
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ABSTRACT |
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Host cell invasion by apicomplexan parasites is
accompanied by the rapid, polarized secretion of parasite proteins that
are involved in cell attachment. The Toxoplasma
gondii micronemal protein MIC2 contains several extracellular
adhesive domains, a transmembrane domain, and a short cytoplasmic tail.
Following apical secretion, MIC2 is transiently present on the parasite surface before being translocated backward and released by proteolytic cleavage. Mutations in the extracellular domain of MIC2, directly upstream of the transmembrane domain, prevented processing and release
of the soluble protein into the supernatant. A conserved basic residue
in MIC2 was essential for cleavage, and basic residues are similarly
positioned in other microneme proteins. Following the induction of
secretion, MIC2 processing mutants were stably expressed on
the surface of the parasite. Surface MIC2-expressing mutants
showed increased adhesion to host cells, yet were impaired in their
capacity to invade. These data demonstrate that proteolysis is
essential for releasing cell surface adhesins prior to cell entry by
apicomplexan parasites.
Toxoplasma gondii is member of the phylum Apicomplexa,
a group of medically and economically important obligate intracellular parasites that includes Plasmodium, the causative agent of
malaria. One of the first events in host cell recognition by T. gondii is the polarized release of parasite adhesins from
secretory vesicles called micronemes (1). Micronemal proteins contain a
variety of conserved adhesive domains, and several have been
demonstrated to bind to host cells (2). Despite their important role in cell attachment, microneme proteins only transiently occupy the parasite cell surface during the brief interval of cell invasion before
being released into the extracellular medium. MIC2 is composed of a
short cytoplasmic tail, a transmembrane
(TM)1 domain, and an
extracellular region composed of an integrin A-domain and six type 1 thrombospondin (TSP-1)-like repeats (3). The T. gondii microneme protein MIC2 belongs to the family of
thrombospondin-related anonymous proteins (TRAPs) previously shown to
be essential for host cell invasion by Plasmodium
sporozoites and ookinetes (4-6). MIC2 is normally stored in the
micronemes and is rapidly discharged at the apical tip of the parasite
when it contacts the host cell (7). Following secretion, MIC2 is
translocated backward as the parasite enters the cell, occupying the
junction that forms between the host and parasite cell membranes. MIC2
is released into the medium by C-terminal processing that is carried
out by a parasite protease called MPP1 (8). The cleavage specificity of
this protease is unknown, and it is insensitive to a variety of
commonly used protease inhibitors. Throughout the processes of
secretion, translocation, and release, MIC2 is complexed with another
protein called MIC2-associated protein (M2AP), which is comprised of
two extracellular domains but is not predicted to be anchored in
the membrane (9).
The cleavage of micronemal proteins upon secretion is a common feature
of this protein family that occurs in MIC2, MIC6, MIC8, MIC12 (10), and
AMA-1 (11). It was recently reported that extensive mutations within
the conserved TM domains of MIC2, MIC6, and MIC12 prevented their
release into the medium, suggesting that processing of micronemal
proteins may occur within the membrane (10). Intramembrane processing
has been reported for proteins produced by a broad range of organisms
from bacteria to humans (12). However, in other examples of
intramembrane processing, cleavage within the membrane-spanning domain
is always preceded by a separate proteolytic processing step that
releases the extracellular domain (12).
We show here that mutations in the exodomain of MIC2, directly upstream
of the TM domain, prevent the cleavage and release of the protein from
the cell surface, revealing that microneme proteins are likely
processed in multiple steps. Parasites expressing uncleaved MIC2 on
their surface showed increased adherence to host cells but impaired
invasion, demonstrating an important role for the processing of MIC2
during cell entry.
Antibodies--
MIC2 was detected with monoclonal antibody 6D10
as described previously (3). The c-Myc epitope (EQKLISEEDL) was
detected with mouse monoclonal antibody 9E10 (Zymed
Laboratories Inc.), and the HA9 epitope was detected using rabbit
antisera (Zymed Laboratories Inc.). Rabbit anti-SAG1
was provided by Lloyd Kasper (Dartmouth Medical School). M2AP and MIC5
were detected using rabbit antisera provided by Vern Carruthers (Johns
Hopkins University). Chemicals were obtained from Sigma.
Growth of Host Cells and Toxoplasma Strains--
T.
gondii tachyzoites of the RH hxgprt Construction of an Expression Cassette for the MIC2
Gene--
Constructs for expression of MIC2 in T. gondii
were based on the HXGPRT selection plasmids described previously
(13). The full-length MIC2 cDNA encoding the complete
open reading frame and ~ 1 kb of 5'- and 3'-flanking genomic
regions was inserted into this vector to create MIC2 expression
plasmids for T. gondii. PCR-based cloning was used to add an
HA9 tag (consisting of the residues YPYDVPDYAL) after amino acid 769, followed by a stop codon. The c-Myc epitope was inserted in-frame,
replacing blocks of 10 amino acids at a time upstream of the TM domain
of MIC2 from Ser-659 to Gly-698. PCR amplification was used to create point mutations in a plasmid bearing the MIC2 gene harboring
the c-Myc tag from position Glu-679 to Gly-688 and the HA9 tag at position 770. Gene-specific primers were designed to change the residues 692SKKE695 to SAAE, SAKE, SKAE, SRAE,
or SKER. The correct sequence of constructs was confirmed by cycle
sequencing, and expression of the epitope-tagged forms of MIC2 was
verified by transient expression in T. gondii as described
previously (13).
Microneme Secretion Assay--
Freshly harvested parasites were
purified and resuspended in 100 µl of PBS-Ca2+ containing
1% ethanol and incubated for 15 min at 37 °C. After centrifugation
(400 × g, 10 min), the supernatant containing secreted microneme proteins was used for Western blot experiments and the parasite pellet was used for Western blot and immunofluorescence experiments. Samples were separated by 10% SDS-PAGE, and proteins were
transferred semi-dry to nitrocellulose membranes. Western blots were
performed as described previously using ECL (Pierce) (8).
Immunoprecipitation--
Polyclonal anti-M2AP or pre-immune
rabbit sera were incubated separately with 100 µl of rec-protein
A-coated Sepharose beads (Zymed Laboratories Inc.) in
300 µl of PBS overnight at 4 °C. Antibody coated beads were washed
three times in 500 µl of RIPA buffer (50 mM Tris-HCl, pH
7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 100 mM NaCl2, 5 mM EDTA). Purified
parasites (5 × 107) were resuspended in 100 µl of
RIPA buffer and incubated for 15 min at room temperature. Insoluble
material was removed by centrifugation at 14,000 rpm for 10 min at room
temperature. The lysates were added to the beads and incubated at
4 °C for 4 h. Beads were washed five times with RIPA buffer,
and proteins bound to the beads were dissolved in 40 µl of SDS-PAGE
sample buffer. The amount of proteins loaded onto SDS-PAGE gels was
normalized to the percentage of parasites expressing each
epitope-tagged MIC2 protein. Proteins were transferred to
nitrocellulose membranes for immunoblotting with the mouse monoclonal
antibody anti-c-Myc.
Indirect Immunofluorescence (IF) Microscopy--
To detect
microneme proteins within the parasite, HFF cell monolayers were
infected with parasites and incubated overnight prior to being fixed
and processed for IF (14). Permeabilized cells were incubated with
anti-c-Myc, anti-MIC2, or anti-MIC5 antibodies and then washed and
incubated with secondary antibodies conjugated to Alexa 494 or Alexa
488 (Molecular Probes, Eugene, OR). Coverslips were washed and mounted
in Vectashield containing DAPI (Vector Laboratories, Burlingame, CA).
To detect microneme proteins at the cell surface, parasites were
stimulated to secrete (see above) and then fixed and processed for IF
as described previously (15). Non-permeabilized parasites were
incubated with anti-c-Myc, rabbit anti-SAG1, or rabbit anti-M2AP
primary antibodies followed by the appropriate secondary antibodies,
and the cells were mounted in Vectashield. Slides were examined using a
Zeiss Axioplan (Carl Zeiss, Inc.) microscope equipped with phase
contrast and epifluorescence optics. Images were obtained using a Zeiss
AxioCam cooled charge-coupled device camera directed by Zeiss
Axiovision software and processed using Photoshop 4.0 (Adobe Systems,
San Jose, CA).
Parasite Adherence and Invasion Assays--
HFF monolayers were
infected with parasites during a 5-min pulse, washed, fixed gently in
3.4% paraformaldehyde, and incubated with anti-SAG1 antibodies to
reveal extracellular parasites. Following detergent permeabilization,
cells were incubated with an anti c-Myc antibody to reveal transfected
parasites expressing epitope-tagged forms of MIC2. Monolayers were
washed and incubated with appropriate secondary antibodies and mounted
in Vectashield containing DAPI.
To determine the percentage of transfected parasites that were
adherent, the proportion of c-Myc-positive parasites in the starting
inoculum (c-Myc positive) was compared with the proportion of parasites
that were attached to host cells following a brief invasion pulse and
staining for the surface protein SAG1 (c-Myc positive and SAG
positive). Data are presented as fold increase that was calculated as
the percentage of c-Myc-positive parasites bound to host cells per the
percentage of c-Myc-positive parasites in the inoculum. Values
represent the mean ± S.E. from three experiments.
To determine the percentage of transformed parasites that successfully
entered the host cells, the number of c-Myc positive parasites that was
attached to host cells (SAG1 positive and DAPI positive) was compared
with the number that was intracellular (SAG1 negative but DAPI
positive). Values are presented as the percentage of invasion based on
three similar experiments (mean ± S.E.).
Mutations in the Extracellular Domain of MIC2 Block
Processing--
The c-Myc epitope was inserted to generate in-frame
mutations upstream of the predicted TM domain of MIC2 (Fig.
1A). To detect processing, the
proteins were also tagged with the HA9 epitope at the C terminus (Fig.
1A). Immunofluorescence staining with antibodies to c-Myc
and another microneme protein, MIC5, revealed that all the
epitope-tagged proteins were faithfully targeted to micronemes in the
parasite (Fig. 1B). Epitope-tagged genes were transiently
transfected into T. gondii, and secretion of the expressed
proteins was evaluated by Western blot analysis of proteins released
into the supernatant. Insertion of the c-Myc epitope at residues
689-698 in the construct referred to as mut4 completely prevented the
release of the protein into the supernatant following stimulation of
secretion (Fig. 2A). The
region of MIC2 that was removed in creating mut4 contains two lysines
located at positions 692 and 693, and this site resembles the
substrates for trypsin-like enzymes, including subtilisins. To test the
involvement of these residues, the two lysines were replaced by
alanines to create mutAA (Fig. 1A). The mutAA form of MIC2
was not released into the supernatant after induction of secretion
(Fig. 2A). This lack of processing was not a consequence of
the c-Myc epitope, as placement of the tag at residues 679-688 in the
construct referred to as MIC2-TAG, or at other sites upstream of this,
did not prevent secretion (Fig. 2A, and data not shown).
Transient expression of epitope-tagged forms of MIC2 did not
discernibly affect secretion of the endogenous wild type MIC2 protein
(Fig. 2A, and data not shown). The HA9 tag was only detected
in the full-length form of the protein found in the cell pellet (Fig.
2A), indicating that, when secreted, the C terminus was
removed by proteolytic processing as described previously (8).
MIC2 Processing Mutants Remain Associated with the Parasite Surface
after Secretion--
MPP1 is a parasite protease that acts on MIC2
only once it has been released onto the parasite surface (8). Thus, the
lack of secretion of mut4 and mutAA could be due to their failure to be
released onto the cell surface following stimulation of secretion. The
localization of epitope-tagged forms of MIC2 was examined after
stimulation of microneme discharge. Parasites were stained with
antibodies in the absence of permeabilization to reveal proteins on the
cell surface (Fig. 2B). Epitope-tagged MIC2 (MIC2-TAG) was
not detected on the parasite cell surface (Fig. 2B),
consistent with the fact that this protein is rapidly secreted,
processed, and released into the supernatant as described previously
(8) (see also Fig. 2A). In contrast, parasites expressing
mut4 or mutAA were strongly labeled with anti c-Myc antibodies on their surface as shown by colocalization with staining for the cell surface
protein SAG1 (Fig. 2B). Moreover, mut4 and mutAA were often
concentrated at the posterior end of the parasites, whereas the
extruded conoid, located at the apical end, was weakly labeled (Fig.
2B). Absence of staining with antibodies to the HA9 epitope, which is attached to the cytoplasmic domain (8), was used to verify
that the parasites that stained positively for epitope-tagged MIC2 were
not permeabilized during the IF procedure (data not shown).
During secretion and surface translocation, MIC2 remains associated
with the protein M2AP (9). Epitope-tagged MIC2 proteins co-immunoprecipitated with M2AP (Fig.
3A), showing that the
mutations and/or the insertion of the c-Myc tag do not alter the
interaction with M2AP. In parasites expressing the non-processed forms
of MIC2, M2AP was retained on the cell surface (Fig. 3B). In
contrast, in parasites expressing wild type epitope-tagged MIC2
(MIC2-TAG), M2AP was not retained on the surface of the cell (Fig.
3B). To examine whether the surface localization of other
MIC proteins was affected by the expression of non-processed forms of
MIC2, we performed surface IF assay staining as described above. No accumulation of MIC4, MIC5, or MIC6 was observed in parasites expressing non-processed forms of MIC2 (data not shown). Collectively, these data show that, upon secretion, mutAA and mut4 are
transferred to the surface of the parasite where they are
complexed with M2AP; however, they are not released into the
supernatant because of the failure of MIC2 to be proteolytically
processed.
MIC2 Processing Mutants Show Enhanced Adherence to Host Cells but
Are Impaired in Invasion--
We analyzed whether the enhanced
retention of MIC2 on the surface of parasites expressing processing
mutants would result in alterations in binding to host cells. Because
the percentage of cells expressing the epitope-tagged MIC2 proteins
(c-Myc positive parasites) was different between constructs and in
separate experiments (a natural feature of transient expression
assays), binding was expressed as a fold change in the proportion of
positively expressing cells that were attached to host cells
versus the proportion that was present in the challenge
inoculum (varied from 5-35%). Parasites expressing the processing
mutant mut4 or mutAA strains showed enhanced binding to HFF cells by
3.8- and 2.8-fold, respectively (Fig.
4A). In contrast, the ratio
observed for parasites expressing MIC2-TAG was 0.9, indicating that
their capacity to adhere to the cells was not significantly different
to that observed in the untransfected population (Fig. 4A).
These data indicate that parasites expressing non-processed forms of
MIC2 on their cell surface have an enhanced capacity to adhere to host
cells.
To determine whether the enhanced attachment to host cells by surface
MIC2-expressing parasites also affected invasion, the efficiency of
cell entry was evaluated. When parasites transfected with the MIC2-TAG
form were used to challenge host cell monolayers, between 50-70% of
the parasites that were associated with the monolayer were also able to
enter the host cell (Fig. 4B). Although the efficiency of
invasion was reduced in parasites expressing MIC2-TAG, it was not
significantly different from that of non-transfected parasites (Fig.
4B). In contrast, only 10% of adherent parasites expressing
mut4 or mutAA were able to enter the cells, a result that was
significantly different from that observed with MIC2-TAG (p < 0.05, Student's t test). These data
indicate that the unusual persistence of MIC2 at the surface of the
parasite enhances binding but does not lead to productive cell entry.
A Conserved, Basic Residue Is Required for the Cleavage of
MIC2--
To analyze the contribution of Lys-692 and Lys-693 to the
cleavage of MIC2, we substituted each amino acid independently with an
alanine leading to the construction of mutAK and mutKA constructs (Fig.
5A). Additionally, we
generated a mutant, mutRA, in which Lys-692 was substituted by arginine
(Fig. 5A). These constructs were transiently expressed in
T. gondii, and the capacity of the parasite to secrete the
epitope-tagged proteins was analyzed by Western blotting as described
above. The supernatants from parasites expressing MIC2-TAG, mutKA, and
mutRA contained the secreted form of MIC2, indicating that these forms
were properly cleaved by MPP1 (Fig. 5B). In contrast,
epitope-tagged MIC2 was not found in the supernatants of mutAA- and
mutAK-expressing parasites (Fig. 5B). These data demonstrate
that Lys-693 is not essential for the cleavage of MIC2, whereas
Lys-692, which can be replaced by arginine, is required for processing.
The sequence KER, which is found upstream of the TM domain of MIC6, was
also inserted in place of the 691SKK693 of MIC2
to create mutKER. When transfected into T. gondii, the mutKER form of MIC2 was properly secreted, processed, and released into
the supernatant (Fig. 5B). The ability of this sequence to be recognized in the context of MIC2 suggests that it may fulfill a
similar role in MIC6. Furthermore, analysis of other microneme proteins
from T. gondii (TgMIC6, TgMIC12, and TgMIC8),
Plasmodium falciparum (PfTRAP) or Neospora caninum
(NcMIC2) revealed that a conserved lysine is located within residues
Proteolytic processing plays an important role in the maturation
and activation of microneme protein apicomplexans (2). Processing of
the micronemal protein MIC3 has been shown to be essential to activate
its adhesive properties (16). Similarly, processing of the malarial
protein MSP1 on the surface of merozoites is important for exposing an
epidermal growth factor (EGF)-like domain that is involved in cell
recognition (17). Following the release of full-length MIC2 from the
anterior end of the parasite, it is translocated to the posterior end
of the cell before being released into the medium (8, 14). MIC2 occurs
as a complex with M2AP that is released from the cell surface following
cleavage of MIC2 by a surface or secreted protease called MPP1, which
is insensitive to a variety of inhibitors (8).
We demonstrate here that mutations in a sequence just upstream of the
predicted transmembrane domain of MIC2 disrupt the processing and
prevent release of the protein complex into the medium. Importantly, these mutant forms of MIC2 are still normally packaged in the micronemes, secreted upon stimulation, and efficiently delivered to the
cell surface. Processing of cell surface MIC2 was critically dependent
on a basic amino acid positioned Our results lead to a different model from that recently proposed for
the release of microneme proteins by intramembrane processing (10).
Mutations in the conserved TM domains of MIC2 and MIC6 blocked release
of chimeric, heterologous proteins into the supernatant (10). This
result is somewhat unexpected, because in other well studied examples
of intramembrane processing there is little sequence specificity to the
cleavage site (18). Rather, the major determinant of which TM domains
are susceptible to intramembrane proteolysis is the lack of a
significantly sized lumenal or extracellular domain (18). Furthermore,
in other examples of intramembrane protein processing, cleavage within
the membrane is secondary and is always preceded by a primary cleavage
that removes the extracellular or lumenal domain of the protein (12,
19). Our findings clearly establish that the extracellular domain of
MIC2 is essential for proteolytic processing that releases MIC2 into the supernatant. Interestingly, Opitz et al., (10) also
reported that insertion of c-Myc outside the TM domain disrupted
processing of MIC2; however, they did not follow up on the precise
residues involved in this effect. Our studies implicate a basic residue just outside the TM domain as being critical for processing of MIC2.
Whether this basic residue is involved in the cleavage site per se or is simply part of an extended
recognition domain will require further studies to determine.
MIC2 is a homologue of the malaria protein called TRAP, which is
essential for gliding motility and cell invasion by malaria sporozoites
(5, 20). TRAP mediates entry into mammalian and insect cells,
indicating that it contains recognition motifs for highly conserved
receptor molecules (21, 22). Recognition of host cell receptors by TRAP
is likely mediated by an integrin A domain and a single type 1 thrombospondin domain that have been shown to bind to sulfated glycans
in vitro (23-25). MIC2 contains similar adhesive domains
for the recognition of glycosaminoglycans and binds to host cells
in vitro, suggesting that it may participate in the
recognition of host cell glycosaminoglycans by the parasite (14,
26, 27).
Parasites expressing mutant forms of MIC2 on the cell surface exhibited
enhanced binding to host cells, suggesting that MIC2 plays a role in
cell attachment. Recently, it was shown that disruption of the M2AP
gene results in reduced targeting of MIC2 to the micronemes and
decreased attachment and entry of host
cells.2 Collectively, these
findings indicate that the MIC2/M2AP complex plays an important role in
cell recognition. Although the stable expression of MIC2 on the
parasite surface led to enhance binding to host cells, it had the
opposite effect on entry. These findings indicate that the expression
of MIC2/M2AP on the cell surface must be carefully regulated and that
turnover of the complex is essential for productive entry. Precedent
for such a dual role in binding and regulated release has been reported
for the vertebrate cell surface hyaluronic acid receptor CD44, which
participates in cell-cell and cell-matrix interactions. Proteolytic
cleavage of CD44 from the surface of vertebrate cells decreases
attachment with the substratum, thus facilitating cell migration, a
process important for metastasis of some tumor cells (28).
Typically, adhesins are stably expressed on the surface of
microorganisms, where their ability to bind host cell receptors often
facilitates entry by activating host cell endocytosis (29). Apicomplexan parasites accomplish this process in a fundamentally different manner. Adhesins are stored in apical secretory organelles and discharged at the time of cell contact. The directional
translocation of adhesin-receptor complexes from the anterior to the
posterior end of the parasite accompanies host cell penetration. TRAP
family adhesins participate in this directional attachment and likely facilitate cell entry by interaction with the parasite's actin-myosin cytoskeleton, which provides the essential propulsive force for cell
entry (30-32). Importantly, uncoupling of this process by the
prevention of proteolytic processing of MIC2 enhanced binding to host
cells but did not lead to productive entry. Our findings underscore the
fact that polarized secretion, translocation, and proteolytic
processing must be tightly coupled for efficient invasion.
Proteolysis is important in controlling a variety of complex processes
in parasites, including cell entry and egress by Plasmodium (33), tissue migration by Schistosoma (34), and development in Giardia (35). Proteolysis is known to activate parasite
proteins, enabling them to recognize host cell receptors in a
variety of adhesins including MIC3 (16), MSP1 (36), and AMA-1
(37). We demonstrate here that proteolysis is also critically
important for releasing parasite adhesins from the cell surface,
thus breaking the attachment to host cell receptors prior to the
completion of cell invasion by apicomplexan parasites.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strain (obtained form David Roos, University of Pennsylvania) were
maintained by growth in monolayers of human foreskin fibroblast (HFFs),
propagated in Dulbecco's modified Eagle medium containing 10% fetal
bovine serum, 2 mM glutamine, 20 mM HEPES, pH
7.5, and 20 µg ml
1 gentamicin (referred to as D10).
Transformed strains were cultivated in D10 supplemented with 25 µg
ml
1 of mycophenolic acid and 50 µg ml
1 of xanthine.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Epitope-tagged and mutant constructs of MIC2
are normally packaged in the micronemes. A, schematic
representation of MIC2 constructs. The wild type amino acid sequence
from position 689 to 696 is shown beneath the wild type protein
(top) and in the epitope-tagged form MIC-TAG
(second from top). The location of the
c-Myc tag (gray box) is indicated in parentheses where the
amino acids (single letter code) and position within the protein are
given in superscript. Point mutations introduced in the mutant mutAA
are shown beneath the lower construct. The HA9 epitope is located
between residues 770 and 780 in each construct (white
box). Black bars represent the
transmembrane domain. B, MIC2-TAG and mutant forms of MIC2
were correctly localized to micronemes. Intracellular parasites were
permeabilized, incubated with anti-c-Myc and anti-MIC5 antibodies, and
the proteins were revealed using secondary antibodies coupled to
Alexa 594 (red) and Alexa 488 (green),
respectively. Scale bar = 2 µm.
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Fig. 2.
MIC2 processing mutants are stably retained
on the cell surface following secretion. A, Western
blot analysis of secreted microneme proteins by transiently transfected
parasites. Although epitope-tagged MIC2 (MIC2-TAG) was secreted
normally and appeared in the supernatant (S), insertion of
the c-Myc tag within mut4 or the point mutations in mutAA disrupted
secretion and resulted in retention in the pellet (P). The
tagged proteins were detected using antibodies against c-Myc
(middle panel) or HA9 (bottom panel). As an
internal control for secretion of the endogenous form of MIC2, the
monoclonal antibody against MIC2 (6D10) was used in the top
panel. All three panels contain proteins from the MIC2-TAG,
mut4, and mutAA parasites, respectively. B,
immunolocalization of epitope-tagged MIC2 proteins at the surface of
the parasites following secretion. Although the wild type
(MIC2-TAG) protein was turned over rapidly, both mut4 and
mutAA forms accumulated on the surface, primarily at the posterior end
of the cell. Following stimulation of secretion, cells were fixed and
then incubated with anti-c-Myc and anti-SAG1 antibodies in the absence
of permeabilization. Epitope-tagged MIC2 proteins and SAG1 were
detected using secondary antibodies coupled to Alexa 494 (red) and Alexa 488 (green), respectively. The
arrows represent the extruded conoid at the apical end of
the parasites. Scale bar = 2 µm.
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Fig. 3.
M2AP interacts with epitope-tagged MIC2
proteins. A, immunoprecipitation of epitope-tagged MIC2
using M2AP-specific antibodies. Lysates of strains producing MIC2-TAG,
mut4, and mutAA were incubated with beads coated with rabbit anti-M2AP
(M2AP) or prebleed sera (prebleed). Proteins
bound to the beads were resolved on 10% SDS-PAGE gels and Western
blotted using anti-c-Myc antibodies. B, immunolocalization
of endogenous M2AP and epitope-tagged MIC2 at the surface of parasites
following secretion. Although wild type parasites (MIC2-TAG)
rapidly shed MIC2/M2AP from the surface, this complex accumulated on
the cell surface in parasites expressing mut4 and mutAA mutants.
Following stimulation of secretion, parasites were fixed and then
incubated with anti c-Myc and anti M2AP antibodies in the absence of
permeabilization. The presence of the proteins at the surface of the
parasites was detected using a secondary antibody coupled to Alexa 494 (red) and Alexa 488 (green), respectively.
Unlabeled parasites correspond to untransfected parasites.
Scale bar = 2 µm.
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Fig. 4.
MIC2 processing mutants show enhanced cell
binding but decreased entry. Analysis of cell attachment and
invasion by parasites expressing epitope-tagged MIC2. MIC2 processing
mutants showed enhanced attachment but are inhibited in cell entry.
A, capacity of the mutants to adhere to the surface of the
cells. The proportion of adherent parasites was compared with the
frequency of positively expressing cells in the inoculum
(i.e. percentage of positively expressing cells bound per
the percentage of positively expressing cells in the inoculum) to
determine a fold difference in attachment. An asterisk (*) indicates a
statistically significant difference versus MIC2-TAG,
p < 0.05 (Student's t test). B,
capacity of the mutants to invade host cells. The percentage of the
parasites able to enter host cells is shown for each construct.
CTL denotes control or non-transfected parasites. An
asterisk (*) indicates a statistically significant difference
versus CTL or MIC2-TAG-expressing parasites,
p < 0.05 (Student's t test).
11 and
13 from the putative beginning of their transmembrane
domains (Fig. 5C).
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Fig. 5.
A similar basic residue is found upstream of
the TM domain in a variety of MIC proteins. A, schematic
representation of MIC2 containing point mutations between residues 691 and 693. The position of the c-Myc tag is indicated in parentheses. The
HA9 is located between positions 770 and 780 in each mutant.
Black bars represent the transmembrane domain,
white bars the HA9 tag, and gray
bars the c-Myc tag. B, Western blot analysis of
microneme proteins secreted by parasites transiently expressing mutant
proteins. Following stimulation of secretion, proteins associated with
the parasites (pellet, P) or released into the medium
(supernatant, S) were electrophoresed on a 10%
polyacrylamide gel and transferred to a nitrocellulose membrane. The
mutant proteins were detected using antibodies against c-Myc.
C, comparison of the region upstream of the transmembrane
domains of microneme proteins from apicomplexans. The beginning regions
of the predicted transmembrane domains are underlined.
Boldfaced lysines represent amino acids thought to be
important for the cleavage of the proteins. Their localization upstream
the transmembrane domain is numbered. Accession numbers are as follows:
TgMIC2, AAB63303; TgMIC6, AAD28185; TgMIC12, AAK58479; TgMIC8,
AF353165; NcMIC2, AF061273; and PfTRAP, AAC18657.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
11 residues upstream of the
predicted TM domain, and a mutation of this residue abolished processing. A similar basic residue is found 11-13 residues upstream of the TM domains of a variety of microneme proteins, suggesting a
similar requirement in proteolytic processing. Collectively, these
results suggest that MPP1 is responsible for cleavage of a variety of
MIC proteins from the cell surface and that it may recognize a sequence
that includes a basic residue. A more complete characterization of the
processing site of MPP1 awaits additional mutational analysis and
determination of the specific cleavage site by peptide sequencing or
mass spectrometry.
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ACKNOWLEDGEMENTS |
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We thank Antonio Barragan and Dan Goldberg for fruitful discussions and Vern Carruthers for critical review of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant AI34036.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.
Partially supported by National Institutes of Health Institutional
Training Grant AI010172 (to Washington University).
§ Recipient of a Scholar Award in Molecular Parasitology from the Burroughs Wellcome Fund and to whom correspondence should be addressed. Tel.: 314-362-8873; Fax: 314-362-3203; E-mail: sibley@borcim.wustl.edu.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc. M209837200
2 V. B. Carruthers, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: TM, transmembrane; TRAP, thrombospondin-related anonymous protein; MPP1, microneme protein protease 1; M2AP, MIC2-associated protein; HFF, human foreskin fibroblast; HA, hemagglutinin; RIPA, radioimmune precipitation assay buffer; IF, immunofluorescence; DAPI, 4',6-diamidino-2-phenylindole.
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