From the Zentrum für Molekulare Biologie
Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany,
§ Department of Molecular Microbiology, Washington
University School of Medicine, St. Louis, Missouri 63110, and
Unit Nuffield Department of Pathology, University of Oxford,
John Radcliff Hospital, Oxford OX3 9DU, United Kingdom
Received for publication, September 11, 2000
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The initial stage of invasion by apicomplexan
parasites involves the exocytosis of the micronemes-containing
molecules that contribute to host cell attachment and penetration. MIC4
was previously described as a protein secreted by Toxoplasma
gondii tachyzoites upon stimulation of micronemes exocytosis. We
have microsequenced the mature protein, purified after discharge from
micronemes and cloned the corresponding gene. The deduced amino acid
sequence of MIC4 predicts a 61-kDa protein that contains 6 conserved
apple domains. Apple domains are composed of six spacely
conserved cysteine residues which form disulfide bridges and are also
present in micronemal proteins from two closely related apicomplexan
parasites, Sarcocystis muris and Eimeria
species, and several mammalian serum proteins, including kallikrein.
Here we show that MIC4 localizes in the micronemes of all the invasive
forms of T. gondii, tachyzoites, bradyzoites, sporozoites,
and merozoites. The protein is proteolytically processed both at the N
and the C terminus only upon release from the organelle. MIC4 binds
efficiently to host cells, and the adhesive motif maps in the most
C-terminal apple domain.
Toxoplasma gondii is a ubiquitous protozoan pathogen
infecting human and animals. Like other members of the phylum of
Apicomplexa, this parasite possesses an elaborate apical apparatus
dedicated to host cell invasion. The successive exocytosis of secretory compartments, including rhoptries and micronemes, plays a key role in
the invasion process. Micronemal proteins are apparently used for host
cell recognition, binding, and motility, whereas the content of the
rhoptries likely contributes to the formation of a functional
parasitophorous vacuole. T. gondii is remarkable for its
ability to invade almost any nucleated cell within its mammalian hosts.
This broad host cell specificity suggests that adhesion involves the
recognition of ubiquitous surface-exposed host molecules or,
alternatively, the presence of various parasite attachment molecules
able to recognize multiple host cell receptors. Micronemal proteins
identified in several Apicomplexa share common structural features
(1) and, in select cases, can even sustain functional complementation
across species (2). For example, a family of adhesive proteins
containing thrombospondin (thrombospondin-like) and integrin A domains
have been described in the micronemes of Plasmodium,
Eimeria, Toxoplasma, and
Cryptosporidium (3, 4). This family is named from the
original member described in Plasmodium as
thrombospondin-related anonymous protein or TRAP (1). In T. gondii, three major micronemal proteins have been characterized so
far. 1) MIC1 contains only two degenerate thrombospondin-1-like domains
(6), MIC2 is the homologue of TRAP (5), and MIC3 contains epidermal
growth factor-like domains and forms dimers (7). MIC4 was previously
identified as a component of the micronemes (8). In this study, we
present the complete characterization of MIC4, a novel type of
micronemal adhesin in T. gondii for which homologues exist
in two other Apicomplexa; MIC5 in Eimeria tenella (9) and
the lectin (SML) in Sarcocystis muris (10).
Reagents and Antibodies--
Chemical reagents were obtained
from Sigma unless otherwise specified. Mouse hybridomas were obtained
by immunization with excretory-secretory antigens
(ESA)1 as previously
described (11), and mAbs were screened by immunofluorescence assay
(IFA) and Western blotting. Polyclonal rabbit serum reacting to
Toxoplasma actin (anti-ACT1) was described previously (12). The mAb Tg17-43, which reacts to the dense granule protein GRA1, was
provided by Dr. Marie-France Cesbron-Delauw (Lille, France). The
monoclonal anti-MIC2 (T34A11) was provided by Dr. Jean-François Dubremetz, and the hybridome BB2, producing mAbs against Ty1 tag, was
provided by Dr. Keith Gull (University of Manchester,
Manchester, UK).
Host Cells and Parasite Cultures--
Tachyzoites of the
RH strain T. gondii were propagated in human foreskin
fibroblast (HFF) monolayers grown in Dulbecco's modified Eagle's
medium containing 3.7 g/liter sodium bicarbonate, 10 mM HEPES, 1 mM L-glutamine, 10% fetal calf serum,
and 10 µg/ml gentamicin (referred to as D-10). Parasites were
harvested from freshly lysed cultures in Hanks' balanced salt
solution containing 10 mM HEPES and 0.1 mM EGTA (HHE) as previously described (8). Bradyzoites were
obtained from mouse brains chronically infected with the ME49 strain of
T. gondii (kindly provided by Dr. Steve Parmley, Palo Alto
Medical Research Foundation). Oocysts, including nonsporulated, partially sporulated, and fully sporulated preparations, were obtained from cats infected with the VEG strain of T. gondii
(kindly provided by Dr. Michael White, Montana State University). A
clonal isolate of the RH hxgprt Cloning of MIC4 Genomic Locus and DNA Sequencing and
Analysis--
The cosmid library used the SuperCos vector modified
with SAG1/ble Toxoplasma selection cassette inserted into
its HindIII site. The library was prepared from a
Sau3AI partial digestion of RH genomic DNA ligated into the
BamHI cloning site (kindly provided by D. Howe).
Inserts from TgEST phage clones were amplified using T3 and T7
primers and cloned into pCR2.1 (Invitrogen). DNA sequencing was
conducted by cycle-sequencing using ABI Prism Big Dye terminator cycle
sequencing reaction kits (ABI, Foster City, CA) and resolved on ABI 377 DNA sequencers. Sequence analysis was conducted with the Genetics
Computer Group programs (13), programs available through the National
Center for Biotechnology, and programs at the ExPasy site.
Construction of Expression Plasmids--
The vector pGEXMIC4A1
was constructed by cloning a PCR product corresponding to the
N-terminal region of MIC4 and encompassing the first apple domain A1
into pGEX-4T vector (Amersham Pharmacia Biotech). The sense and
antisense primers used for PCR amplification are
5'-cgcggatcctggtttggagtggctaaagccc-3' and
5'-ggttaattaagtggatcccaacacccctcgttccttaa-3', respectively. In
parallel, the same A1 fragment was cloned into a pET vector, and the
nonfusion protein could also be produced in native soluble form in
Escherichia coli BL21.
The expression vector for T. gondii pTMIC4mycHXGPRT
was obtained by cloning a PCR product corresponding to the complete
coding sequence of MIC4 between the EcoRI and
PacI sites of the pTmycHXGPRT vector previously described
(14). The primers for PCR were
5'-cggaattccctttttcgacaaaatgagagcgtcgctccc-3' and
5'-ccttaattaaaatgcatcttctgtgtctttcgcttc-3'. An additional epitope tag
was introduced at the N terminus of MIC4 to generate pTty1MIC4mycHXGPRT. The 11 amino acid Ty-1 tag coding sequence (LEVHTNQDPLD) was inserted as double-stranded oligonulcleotides into
the unique PstI site at amino acid 47 of MIC4:
5'-gaggtccacacgaaccaggacccgctcgaccatgca-3' and
5'-tggtcgagcgggtcctggttcgtgtggacctctgca-3' (15). A vector expressing MIC4 with a stretch of eight histidine residues at the C
terminus was obtained by inserting double-strand oligonucleotides 5'-gggcaccaccatcaccaccatcaccattaat-3' and
5'-taatggtgatggtggtgatggtggtgccctgca-3' into the unique PstI
and PacI sites of the expression vector pT-HXGPRT. The
vector pTMIC4 Production of Polyclonal Antibodies--
The preparation of the
glutathione S-transferase (GST) fusion protein was obtained
by cloning a fragment encompassing the first apple domain of the
predicted MIC4 protein. The DNA sequence coding for the amino acids
19-205 was cloned into the pGEX-4T vector (Amersham Pharmacia Biotech)
for production as a fusion protein in E. coli. Expression of
the recombinant MIC4 fragment fused to GST was achieved in the E. coli strain BL21 after a 4-h induction with
isopropyl- SDS-Polyacrylamide Gel Electrophoresis and Western
Blotting--
SDS-PAGE was performed according to Laemmli (16).
Freshly released tachyzoites were harvested and washed in PBS. Samples were boiled in SDS sample buffer with (reduced) or without (nonreduced) 144 mM Selection of Stable Transformants Using HXGPRT as a Selectable
Marker--
To generate stable transformants, 5 × 107 extracellular RHhxgprt IFA--
All manipulations were carried out at room temperature.
Tachyzoite-infected HFF cells on glass coverslips were fixed with 3%
paraformaldehyde, 0.05% glutaraldehyde or 4% paraformaldehyde only
for 20 min followed by a 3-min incubation with 0.1 M
glycine in PBS. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 20 min and blocked in 2% fetal calf serum or BSA in PBS for
20 min. The cells were then stained with the primary antibodies followed by Cy2- and Cy3-conjugated goat anti-mouse antibodies (Bio-Rad). Confocal images were collected with a Leica laser-scanning confocal microscope (TCS-NT DM/IRB) using a 100 × Plan-Apo
objective with a numerical aperture of 1.30. Single optical sections
were recorded with an optimal pinhole of 1.0 (according to Leica
instructions) and 16× averaging. All other micrographs were obtained
with a Zeiss Axiophot equiped with a camera (Photometrics Type CH-250). Adobe Photoshop (Adobe Systems, Mountain View, CA) was used for image processing.
Electron Microscopy--
Thin sections of
paraformaldehyde-fixed, LR White-embedded materials were mounted
on nickel grids. The grids with sections of enteric forms, tachyzoites,
or tissue cysts were floated on drops of 1% BSA in Tris/HCl buffer, pH
7.2, to reduce nonspecific staining followed by the rabbit anti-MIC4
antibodies appropriately diluted in Tris buffer. After washing, the
grids were floated on secondary antibody conjugated to either 5- or
10-nm colloidal gold particles. Sections were stained with uranyl
acetate before examination in the electron microscope.
Preparation of Micronemal Proteins--
For large scale
preparation of excretory-secretory antigens (ESA), ~5 × 109 tachyzoites were resuspended in 1 ml of HHE and
stimulated to discharge micronemes by the addition of ethanol to a
final concentration of 1.0% and warming to 37 °C for 30 min (8).
Cells were removed by centrifugation at 2,000 × g, and
the supernatant was kept for binding experiments. To purify the
contents of micronemes, ~5 × 109 tachyzoites were
harvested in HHE as above and subjected to sonication and cell
fractionation as described previously (8). Briefly, parasites were
resuspended in cold HHE at ~109/ml and sonicated while on
ice (3 × 30-s pulses at setting 35 on a BioSonik III microprobe
sonicator (Bronwill Scientific, Rochester, NY). After sonication, large
cellular debris was removed by centrifugation at 2,000 × g, 10 min, 4 °C, and the supernatant was further
clarified by spinning at 8,000 × g for 20 min,
4 °C. The micronemes were recovered from the supernatant by
centrifugation at 30,000 × g for 30 min at 4 °C.
The 30,000 × g pellet was resuspended in PBS, pH 6.0, containing a mixture of protease inhibitors (1 µg/ml E64, 10 µg/ml
(4-amidinophenyl)methanesulfonyl fluoride (APMSF), 10 µg/ml
TLCK, 1 µg/ml leupeptin) and subjected to three rapid freeze/thaw cycles. The suspension was then sonicated 3 × 15 s using the
maximum setting for the microprobe sonicator (550 Sonic Dismembrator, Fisher). The suspension was centrifuged at 100,000 × g
for 1 h to remove unbroken micronemes and membranes, and the
supernatant containing soluble micronemal proteins was kept for cell
binding experiments.
Cell Binding Assays--
Confluent monolayers of HFF cells grown
in 6-well plates were rinsed in PBS and blocked for 30 min at 12 °C
with 1% BSA in PBS containing 1 mM CaCl2 and
0.5 mM MgCl2 (CM-PBS). Excess BSA was removed
by rinsing in CM-PBS, and micronemal proteins (20 µg/ml total) were
added in a volume of 1 ml of CM-PBS and incubated at 12 °C for
1 h. The unbound fraction (referred to as supernatant) was
removed, and the monolayers were rinsed four times in cold CM-PBS
(referred to as W1, W2, W3, and W4). The cell-bound fraction (CBF) was
collected by lysing the monolayer in 1 ml of radioimmune precipitation
buffer (50 mM Tris, pH 7.5, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.2% SDS, 100 mM NaCl, 5 mM
EDTA). Fractions were acetone-precipitated and resuspended in SDS
sample buffer containing 2% Characterization of Cellular and Secreted Forms of MIC4 and
N-terminal Microsequencing of the Secreted Form--
To characterize
secretory proteins of Toxoplasma, we generated mAbs to the
ESA fraction released by extracellular tachyzoites. When the mAb 5B1
was used to probe Western blots, it recognized a sharp band that
migrated at 72 kDa in tachyzoite cell lysates and 70 kDa in ESA
resolved under reducing conditions. Both bands migrated more rapidly
and diffusely in the absence of reduction, suggesting the presence of
internal disulfide bonds (Fig.
1A). To identify the gene
corresponding to the protein recognized by mAb 5B1, we isolated ESA on
a large scale and resolved the proteins by SDS-PAGE. Parallel strips
were transferred to nitrocellulose for Western blotting to identify the
band recognized by mAb 5B1 (Fig. 1B) and to polyvinylidene
difluoride membranes for microsequencing. N-terminal sequencing of the
band corresponding to the 72-kDa form yielded a partially degenerate
sequence of 12 residues
(X(G/S)E(P/N)(D/A)(K/P)LDLA(P/L)V).
Identification and Sequencing of the MIC4 Gene--
Comparison of
the N-terminal sequence against the Toxoplasma dbEST data
base using BLAST identified one hit that matched at 9 of 12 residues to
the clone TgESTzy06c08.r1. The sequence of this clone was used to
identify other overlapping ESTs. A radioactive probe derived from the
TgESTzy26b09.r1 clone was used to screen a cosmid library, and an
extended genomic sequence of the locus was determined by primer walking
across 5000 base pairs on a positive clone
(GenBankTM accession number AF143487). The sequence
of the gene codes for a protein of 580 amino acids with a predicted
mass of 61 kDa. The short hydrophobic stretch that follows the
start codon has the hallmark of a putative signal peptide (Fig.
1C). Hydropathy analysis indicates no other hydrophobic
stretch on the protein.
Comparison of the complete coding sequence against the nonredundant
GenBankTM data base using BLAST revealed that the gene was
homologous to several micronemal antigens previously described from
S. muris (18, 19) and to the recently reported E. tenella micronemal protein EtMIC5 (9). These proteins share the
feature of containing conserved, cysteine-rich domains known as apple
motifs, which were detected using Prosite (ExPasy). Such a domain
contains six half-cystine residues at highly conserved positions that
form a structure resembling an apple (20, 21). The consensus for the
internal four cysteine residues of this sequence is
CX3CX5CX11C. The six apple domains of MIC4 are arranged as follows A1 (amino acids
67-139), A2 (amino acids 140-230), A3 (amino acids 231-303), A4
(amino acids 304-417), A5 (amino acids 418-490), and A6 (amino acids
491-580) (for alignment of the six apple domains, see Ref. 9). In the
case of human plasma prekallikrein, it has been shown that three highly
conserved disulfide bonds are linking the first and sixth, second and
fifth, and third and fourth half-cystine residues in each domain (14).
Since these cysteine residues are conserved in MIC4, it is likely that
disulfide bond formation in MIC4 is similar to prekallikrein. The
sequence analysis of MIC4 predicts a signal peptide
cleavage site between residues Ala25 and His26
(ExPasy). Although we have not been able to verify the N-terminal sequence of the 72-kDa form of the protein found in cells, it likely
corresponds to the mature N terminus generated within the secretory
pathway, with removal of an additional 32 residues occurring at the
time of secretion into the medium.
Features of MIC4 Gene--
The 1743-base pair open reading frame
of MIC4 contains no introns. The putative transcription
start site of MIC4 was determined by sequencing several
clones obtained by 5' rapid amplification of cDNA ends PCR. A
sequence analysis of the promoter region revealed the lack of TATA box
and no element resembling an initiator element (Inr) (22). However, a
consensus sequence (heptamer motif) found multiple times in the
5'-flanking sequences of several T. gondii genes (23) is
present in the promoter region of MIC4 (Fig. 1C). Two heptamer motifs are positioned at Subcellular Localization and Pattern of Expression of MIC4--
We
previously reported that the antigen recognized by mAb 5B1 is secreted
from Toxoplasma in a manner consistent with it originating from micronemes (8, 26). To confirm that the gene described here
corresponds to a micronemal protein, we produced a bacterial recombinant GST-A1 fusion of the N-terminal 186 amino acids of MIC4
encompassing the first apple domain (A1) and raised polyclonal antibodies against the purified protein. The rabbit antisera obtained were tested on immunoblots loaded with the recombinant nonfusion A1 and
GST-A1 fusion expressed in E. coli, T. gondii
tachyzoites, and Vero cell lysates (Fig.
2A). The sera recognized
specifically a 72-kDa protein in tachyzoites. Neither anti-GST
antibodies nor the preimmune rabbit sera reacted with T. gondii proteins on Western blots (data not shown).
To determine the pattern of expression of MIC4 in the different life
stages of the parasite, cell lysates of tachyzoites, bradyzoites, and
oocysts were resolved by SDS-PAGE and probed by Western blotting (Fig.
2B). MIC4 was not detected in unsporulated or partially
sporulated oocysts but was present at approximately equal levels in
fully sporulated oocysts (sporozoites), bradyzoites, and tachyzoites.
As anticipated, the mAb 5B1 specifically recognized micronemes by
indirect immunofluorescence and immunoelectron microscopy (data not
shown). IFA studies with rabbit polyclonal antibodies raised against
GST-A1 confirmed a typical staining pattern for proteins located in the
apical microneme organelles of the parasites (punctate fluorescence
pattern at the apical pole). MIC4 colocalized perfectly with
the other micronemal protein MIC2 (27) (Fig. 2B). IFA
performed on extracellular parasites only stained parasites permeabilized with Triton X-100 before incubation with the first antibody (data not shown). This observation suggests that MIC4 is
predominantly localized in the micronemes and absent from the cell
surface. Ultrastructural examination confirmed that the polyclonal antibodies recognized a protein located within the micronemes of
tachyzoites (Fig. 2C, a) and bradyzoites (Fig.
2C, b). The few micronemes in the merozoite of
mature schizonts were also labeled (Fig. 2C, c).
In keeping with the nomenclature of previously established
Toxoplasma proteins (27), we named this antigen MIC4.
MIC4 Is Proteolytically Cleaved Only after Release by the
Micronemes--
The T. gondii micronemal proteins
characterized so far are subjected to extensive proteolytic remodeling
during their transport and/or secretion (27). Comparison of the amino
acids sequence deduced from the MIC4 gene with the
information obtained from N-terminal sequencing of the mature
protein is indicative of proteolytic cleavage. To determine
whether MIC4 is proteolytically cleaved during its transport to the
micronemes, we generated recombinant parasites expressing MIC4 tagged
at both ends with epitopes. The construct pTMIC4mycHXGPRT produced MIC4
with epitope tags at the N terminus and/or the C terminus. An
additional Ty-1 epitope tag was introduced 10 amino acids upstream of
the cleavage site mapped previously by the N-terminal sequencing of
secreted form to generate pTty1MIC4mycHXGPRT. Both constructs were
stably integrated into T. gondii tachyzoites, and expression
of MIC4myc or Ty-1MIC4myc was examined by Western blot and by IFA. The
mAbs anti-Myc and anti-Ty-1 recognized the 72-kDa form of MIC4 on
Western blot (Fig. 3A) and
gave a typical microneme staining on IFA (Fig. 3B). These results demonstrate that the form of MIC4 stored in the micronemes is
not proteolytically cleaved beside the cotranslational removal of the
signal peptide and, as for MIC2, the processing on MIC4 occurred
uniquely post-exocytosis.
MIC4 Is Processed at Both Ends after Release by the Micronemes, and
MPP2 Is Likely to Be Responsible for the C-terminal
Cleavage--
Immunoblot analysis of tachyzoite lysates and ESA
material using mAb 5B1 and the rabbit polyclonal antisera revealed the
existence of an additional proteolytic processing at the C terminus
that generates the two products of ~50 and 15 kDa (Fig.
3C, lanes 1 and 2, upper
and lower panels). The 72-kDa precursor form of MIC4 is
present in the micronemes, whereas the processed forms are uniquely
detectable in ESA. The polyclonal antibodies were raised against the
apple domains A1 and A2 and recognized the 50-kDa form only, whereas
the 15-kDa product was detected exclusively by the mAb 5B1. Together,
these results document that processing occurs at the surface of the
parasite only after release by the micronemes and allowed the mapping
of the epitope recognized by the mAb 5B1 within the A6 at the C
terminus of MIC4. Recently, distinct protease activities for MIC2 have
been described using a variety of protease inhibitors (28). Upon
release from the micronemes, MIC2 is proteolytically modified at
multiple sites by two distinct enzymes, microneme protein protease 1 (MPP1) and microneme protein protease 2 (MPP2), which probably operate
on the parasite surface (28). A subset of serine and cysteine protease inhibitors was shown to block MPP2 activity. Similarly, examination of
MIC4 in ESAs from parasites pretreated with various protease inhibitors
revealed that the processing of MIC4 into 50- and 15-kDa species was
blocked by chymostatin,
N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal, and
N-acetyl-L-leucinyl-L-leucinyl-methioninal
but not any of the other protease inhibitors tested (Fig.
3C). The profile of sensitivity to protease inhibitors
strongly suggests that MPP2 processes both MIC2 and MIC4.
MIC4 Binds to Host Cells--
Several previous reports indicate
that micronemal proteins bind to host cells and may participate in
parasite/cell attachment (6, 7, 28, 29). To determine whether MIC4
binds to host cells, we incubated HFF monolayers with mixtures of
micronemal proteins isolated from intact cells or from ESAs. Binding
assays were conducted at 12 °C to prevent internalization by
endocytosis as previously described (28). After incubation, monolayers
were washed, and the cell-bound fraction (CBF) was obtained by
detergent lysis. MIC4 present in ESA (70 kDa) or in parasite lysates
(72 kDa) bound tightly to HFF cells and was recovered in the CBF (Fig. 4A). Comparison of the input
fraction with the recovered material by phosphorimaging
analysis revealed that 11% of MIC4 in the microneme preparation and
26.7% of MIC4 in ESA preparations remained bound to the cell surface
(Fig. 4B). Collectively, these data indicate that
MIC4 binds substantially to the surface of human fibroblasts. As
control, GRA1, which is abundantly present in the ESA, does not bind
detectably to host cells. The processed forms of MIC4 were examined
separately for their ability to bind to host cells using the mAb 5B1
after resolution on 15% SDS-PAGE or the rabbit antiserum (Fig.
4A, right panel). Interestingly, the 15-kDa form but not the 50-kDa form of MIC4 bound to host cells. The mAb 5B1 recognizing the 15-kDa product inhibits about 50% of cell binding by
the 70-kDa form.2 From these
results, we concluded that the 15-kDa C-terminal product, which
corresponds approximately to the A6 domain, carries the adhesive
properties of MIC4.
As mentioned above, the apple structure is maintained by the formation
of three disulfide bridges. To test whether these disulfide bridges are
necessary for MIC4 binding to host cells we pretreated ESA with the
strong reducing agent MESNA. This treated completely abolished MIC4
binding to host cells, providing additional evidence that the adhesive
properties of MIC4 are dependent on the presence of intact cystine
residues (Fig. 4C). MESNA is not acting on a host cell
receptor since preincubation of the ESA with MESNA at room temperature
or the addition of the reducing agent to host cells during the binding
assay did not impair MIC4 adhesiveness (data not shown). The generation
of recombinant parasites expressing a truncated form of MIC4 confirmed
these observations. We compared the binding activity of MIC4 with
Ty-1MIC4Myc, MIC4his carrying a stretch of eight histidine residues at
the C terminus, and a deletion mutant of MIC4 lacking the last 12 amino
acids (MIC4 The Nature of the Interaction between MIC4 and the Host
Cells--
The MIC4-related major micronemal protein of S. muris contains two apple domains and has been shown previously to
function as a dimeric lectin with high affinity for galactose (10). To test if the A5-6 domains of MIC4 exhibit similar properties, we have
undertaken host cell binding assays in the presence of increasing concentrations of galactose, N-acetylgalactosamine, or
N-acetylglucosamine. These competition experiments revealed
that the interaction of MIC4 with host cells could be enhanced in the
presence of a low concentration of carbohydrates (1 mg/ml), whereas
high concentrations (50 mg/ml) are inhibitory (Fig.
5). The carbohydrates tested here showed
similar competitive effect, with galactose slightly more potent
compared with the others. Our findings on MIC4 structure, processing,
and adhesive activities are summarized in the Fig. 6.
Micronemal proteins are thought to be critical ligands determining
host cell specificity at the time of invasion. Recent studies provide
strong evidence that the transmembrane micronemal proteins of the TRAP
family contribute not only to attachment but also to gliding motility
and, thus, actively participate in the invasion process (2, 31). To
ensure delivery of ligands at the right time and optimal place,
micronemes exocytose adhesins and other factors in a regulated fashion
onto the parasite surface during an early phase of invasion (8). This
apical secretion is sensitive to the kinase inhibitor staurosporine and
can be stimulated by calcium ionophore or ethanol treatment (8, 26).
These characteristics were used to explore the content of micronemes
and to develop a strategy for the identification of novel micronemal
proteins and cloning of their corresponding gene (32).
We present here the identification and characterization of a novel
micronemal protein identified by this approach. The gene corresponding
to MIC4 revealed the existence of a distinct type of adhesive motif
called an "apple domain." MIC4 contains six apple domains and shows
a high degree of homology with a small major micronemal protein of
S. muris containing two apple domains (18, 19) and the much
larger micronemal protein from E. tenella EtMIC5 (9) with 11 apple domains. The S. muris protein is proteolytically processed and released at the apical tip of invading merozoites (33).
This protein, called SML, was shown to form noncovalent homodimers and
to recognize N-acetylgalactosamine as the dominant sugar
(10). Apple domains have been described previously on plasma proteins
such as factor XI and prekallikrein (20, 34) and is composed of six
half-cystine residues at highly conserved positions. Several studies
show that apple domains are implicated in specific interactions between
factors of the blood coagulation cascade (35, 36). A single apple
domain can exhibit a very specific affinity, as illustrated by the
interaction of the third domain A3 of activated factor XI with factor
IX (34).
MIC4 has a calculated molecular mass of 61 kDa, and the deduced amino
acid sequence from the gene predicts the presence of a signal peptide
and six apple domains. We showed that the protein is localized to the
micronemes of all infective stages of the parasite. One surprising
incidental finding was that the polyclonal anti-MIC4 stained a
sub-population of dense granules (wall-forming bodies, type 1) in the
macrogametocyte and the outer veil of the early oocyst in the cat
intestine. However, it was not possible to identify the molecule
recognized as MIC4 or a closely related MIC4-like protein (37).
MIC4 is synthesized and stored in the parasites as a full-length 72-kDa
form. Upon discharge from the micronemes, MIC4 is rapidly cleaved at
the N terminus to produce a 70-kDa form and less efficiently at the C
terminus. The C-terminal cleavage of the 70-kDa species into 50- and
15-kDa products causes a gap in size, as the processed forms do not add
up to form 70 kDa. We can not exclude additional cleavage events, but
the most likely explanation is inaccuracies in the size estimates or a
change in conformation that effects migration. The C-terminal cleavage probably results from the protease activity of MPP2, which mediates the
N-terminal processing of MIC2 at the surface of the parasite (28). In
the case of MIC2, processing at the C terminus by another protease
(MPP1) released the protein from the surface of the parasites and
alters drastically the adhesive properties of MIC2. MIC4 binds efficiently to host cells, and the analysis of the diverse processed forms revealed that the adhesive properties of the molecule are confined within the apple domain at the C terminus. In contrast to
MIC2, cleavage of MIC4 does not appear to influence the binding properties of MIC4. The 72-kDa precursor as well as 70 and 15 kDa
processed forms of MIC4 bind to host cells, and therefore, the
biological significance of MIC4 processing is not clear yet. The fact
that the 50-kDa form of MIC4 failed to bind to host cells suggested
that the adhesive properties of MIC4 are confined strictly to the last
15-kDa form of the protein, which corresponds to the domain A6.
A deletion of 12 amino acids at the C terminus of MIC4 confirmed the
importance of this region of the molecule for binding. In addition, a
pretreatment of ESA with the reducing agent MESNA at 37 °C (but not
at room temperature) abbrogates completely MIC4 binding, suggesting
that an intact apple structure hold by disulfide bridges is
prerequisite for adhesion.
In T. gondii, several studies point to a crucial role of
sugar-binding proteins in host cell recognition. The glycoprotein, BSA-glucosamide, competitively blocks infection of human fibroblasts by
tachyzoites and depends on the presence of the major surface antigen
SAG1 (38). Incubation of tachyzoites in the presence of gold-labeled
albumin-N-acetyl-D-glucosamine or
albumin-galactose but not in the presence of albumin-mannose led to
labeling of the rhoptries in a pattern similar to that observed with
the lectins (39). More recent studies suggest that host recognition by
T. gondii is mediated by parasite lectins (40) and that
sulfated proteoglycans are one determinant used for substrate and cell recognition by MIC2 (30). In competition experiments, MIC4 binding to
host cells in the presence of increasing concentrations of carbohydrates showed a diphasic effect. Host cell binding was enhanced
at lower concentrations of competitors, but minimal binding was
observed at higher doses. The relatively high doses imposed to induce
competition suggest that the specificity of the lectin has not yet been
identified, and possibly multivalent or more complex carbohydrate
structures are involved. A previous study reported the identification
of 45, 65, and 71 kDa lectins in T. gondii tachyzoites (40).
The association of the 65- and 71-kDa proteins with host cells was
abolished in presence of fucoidan, and a biphasic effect was reported
in the competition experiments, similar to our observations. MIC4
binding to host cells in the presence of increasing amounts of fucoidan
showed no competition, which likely rules out that MIC4 corresponds to
the 71-kDa protein described by Ortega-Barria and Boothroyd
(40). Alternatively, like the apple domain studied in the
coagulation factors, a yet unknown very specific protein-protein
interaction might be responsible for binding.
Since MIC4 lacks a transmembrane or lipid anchor, it likely contributes
to parasite adhesion by acting as a bridge between a receptor on the
parasite and a receptor on the host cell. Indeed, a recent study has
revealed that MIC4 forms a complex with two other micronemal proteins,
MIC1 and MIC6.3 MIC6 is a transmembrane protein that
functions as a cargo receptor and ensures proper sorting of MIC1 and
MIC4 to the micronemes. MIC6 also likely retains these soluble adhesins
at the surface of the parasite during invasion. In this complex, MIC1
is directly and stably associated to MIC4, since the two proteins
coimmunoprecipitate even in absence of MIC6. The region of MIC4
interacting with MIC1 is currently being investigated. MIC1, like MIC4,
has previously been shown to bind to host cells (6) and does it also in
the absence of MIC4 in mutant
mic4ko.4 Therefore MIC1
association with MIC4 represents an interfering parameter in our
competition studies that might explain the high doses of galactose
necessary to abolish completely host cell binding. For further studies,
the nature of the interaction between MIC4 and host cells will have to
be examined in the absence of MIC1.
As also observed for other types of micronemal proteins, structural
homologues of MIC4 exist in other Apicomplexa (1). The major micronemal
antigen of S. muris contains two domains, whereas EtMIC5
exhibits 11 apple domains (GenBankTM accession number
AJ245536). A search through the current status of the genome sequencing
project of Plasmodium falciparum and the ESTs available for
Plasmodium vivax and Plasmodium berghei failed to
reveal the presence of a homologue in these members of Apicomplexa.
Intriguingly S. muris, T. gondii, and
Eimeria infect their hosts via the digestive tract. In
contrast, Plasmodium species that are transmitted by an
insect vector enter their mammalian host directly by injection into the
blood. The existence of several distinct types of adhesins in T. gondii, including MIC1, MIC2, MIC3, and now MIC4, illustrates the
diversity of strategies used by the parasite to establish interactions
with the host. This diversity confers either a functional redundancy or
might accommodate the broad range of host cell type specificity. It
will be interesting to examine the possible role of MIC4 in the context
of tissue specificity and to determine the nature of the receptor on
host cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
of T. gondii
was used as the recipient strain for all the transfection experiments.
C12 corresponds to a deletion mutant at the C terminus
of MIC4 lacking the last 12 amino acids, which was generated by PCR
using the antisense primer
5'-ccttaattaatcaggatccattgtcacagaaagtatagggtc-3'.
-D-thiogalactopyranoside. The protein was
purified under native conditions according to the manufacturer. Rabbit
polyclonal sera were made against the purified GST-A1-2 fusion. The
initial immunization was performed with 500 µg of protein with
complete Freund's adjuvant, whereas 300 µg of protein in Gerbu
adjuvant (LQ) were used for the subsequent boosts.
-mercaptoethanol and separated on 8.5 or 10%
polyacrylamide gels. Gels were stained with Coomassie or transferred to
nitrocellulose membranes for Western blotting and to polyvinylidene
difluoride nylon membranes for N-terminal sequencing. Western blots
were probed with antibodies to Toxoplasma proteins followed
by goat anti-mouse or goat anti-rabbit IgG peroxidase and developed by chemiluminescence using the ECL system (Roche Molecular Biochemicals) or SuperSignal (Pierce). Western blots were quantified by exposure of
blots to GS-250 imaging screen CH using a model 363 Molecular Imaging
System (Bio-Rad) and analyzed using the Molecular Analyst software.
parasites were transfected and selected as previously described (17),
with the following modifications. Parasites were transfected with
80-100 µg of linearized plasmid. Twenty-four hours later, parasites
were subjected to mycophenolic acid/xanthine exposure and cloned 3 to 5 days later by limiting dilution in 96-well microtiter plates containing
HFF cells in the presence of mycophenolic acid/xanthine. Stable
transformants were analyzed for the presence of the recombinant protein
by IFA.
-mercaptoethanol. Dilution standards of
the micronemal protein preparations were loaded in parallel to simulate
the contents of 1, 5, and 10% of the total input material. ESA treated
with 2-mercaptoethanesulfonic acid (MESNA, Sigma) was preincubated at
37 °C for 30 min in the presence of 50 mM MESNA and then
diluted 50 times in CM-PBS for the binding assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (64K):
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Fig. 1.
Cloning and characterization of the gene
coding for MIC4. A, Western blot analysis of MIC4 in
lysates of tachyzoites (Cells) and excretory secretory
(ESA) fractions. The cellular form of the protein migrates
slightly slower in both cases. The broad smear in the absence of
reduction suggests the presence of internal disulfide bonds. Proteins
were resolved by SDS-PAGE in the presence of 2% -mercaptoethanol
(Red.) or without reduction (Non-Red.) and
Western-blotted with the mAb 5B11. Molecular standards are given in
kDa. B, ESA proteins were resolved by SDS-PAGE and stained
with Coomassie (left) or transferred to nitrocellulose and
Western-blotted with mAb 5B1 (right). The corresponding band
was cut-out from a polyvinylidene difluoride membrane and subjected to
N-terminal sequencing. C, nucleotide sequence of the
MIC4 gene and predicted amino acid sequence
(GenBankTM accession number AF143487). The initiation of
transcription mapped by 5' rapid amplification of cDNA ends PCR is
indicated by an arrow. Two conserved heptamer motifs present
in the promoter region of MIC4 are boxed. The
predicted signal sequence is underlined. An arrow
indicates the N-terminal proteolytic cleavage site. The amino acids
obtained from the N-terminal sequencing of the purified protein are in
bold and boxed.
716 (AGAGACG) and
496 (TGAGACG) from the transcription start site. These elements have been
previously mapped and shown to be critical for transcription of the
family of GRA genes (23) and are also included in the 27-base pair repeat element of SAG1 gene (24). A single
in-frame ATG lies 58 residues upstream of the N-terminal sequence that was obtained from the purified protein. This ATG probably serves as the
translational initiation codon based on the facts that 1) it is the
first in-frame ATG, 2) the six nucleotides preceding the ATG (CACAAA)
are consistent with the consensus sequence for translational initiation
in T. gondii (GNCAAA) (25), and 3) the ATG immediately
precedes a sequence predicted to encode a hydrophobic signal peptide.
Northern blot and Southern blot analyses confirmed that MIC4
is present as a single copy gene in T. gondii genome that
produces a single transcript of the expected size in tachyzoites (data
not shown).
View larger version (64K):
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Fig. 2.
MIC4 is a micronemal protein expressed in all
invasive stages of T. gondii. A, left
panel shows a Western blot analysis of bacterial lysates
expressing GST-A1, nonfusion A1, and whole cell lysates from T. gondii tachyzoites and Vero cells. Right
panel, stage-specific expression of MIC4 analyzed by Western
blotting using mAb 5B1. MIC4 was expressed in approximately equal
amounts by bradyzoites (B) , tachyzoites (T), and
fully sporulated oocysts (FS) but was not detectable in
nonsporulated (NS) or partially sporulated (PS)
oocysts. Blotting in parallel with rabbit anti-Toxoplasma
actin provided loading control. B, coimmunofluorescence.
Rabbit polyclonal anti-MIC4 and mAb anti-MIC2. C,
immuno-localization by electron microscopy of MIC4 using the rabbit
anti-serum. MIC4 was found in micronemes (arrows) clustered
at the apical end of the tachyzoite (a), bradyzoite
(b), and merozoite (c). Sections were incubated
with rabbit anti-MIC4 followed by goat anti-rabbit IgG conjugated to
10-nm gold. DG, dense granule; R, rhoptry;
CW, cell wall; C, conoid; PG,
polysaccharide granules.
View larger version (49K):
[in a new window]
Fig. 3.
MIC4 is proteolytically processed at
the N and C terminus after release from the micronemes.
A, Western blot analysis of wild type (wt) RH and
parasites expressing MIC4myc or Ty-MIC4Myc using rabbit anti-MIC4,
anti-Ty-1, or anti-myc antibodies. Both epitopes are present on the
72-kDa form of MIC4. B, IFA analysis of
HFF-infected-transformed parasites expressing MIC4Myc or Ty-1MIC4myc
using rabbit anti-MIC4 and colocalization with anti-Myc and anti Ty-1
antibodies. The N- and C-terminal-tagged MIC4s accumulate in the
micronemes. C, effect of protease inhibitors on proteolytic
processing of MIC4. Western blots of tachyzoites lysate (lane
1) or ESA derived from tachyzoites treated with a solvent control
(Me2SO, lane 2) or protease inhibitors
(lane 3, pepstatin; lane 4, EDTA; lane
5, 1,10-phenanthroline; lane 6, MMP-1; lane
7, 2,3-dichloroisocoumarin; lane 8,
4-(2-aminoethyl)benzenesulfonyl) fluoride hydrochloride;
lane 9, (4-amidinophenyl)methanesulfonyl fluoride (APMSF);
lane 10, chymostatin; lane 11, TLCK; lane
12, leupeptin; lane 13, E64; lane 14,
N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal;
lane 15,
N-acetyl-L-leucinyl-L-leucinyl-methioninal;
lane 16, EGTA). The 50-kDa cleavage product (closed
arrowhead) was detected with rabbit anti-MIC4 (upper
panel), and the 15-kDa cleavage product (open
arrowhead) was recognized by mAb 5B1 (lower panel).
Chymostatin,
N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal,
and
N-acetyl-L-leucinyl-L-leucinyl-methioninal
blocked production of the 50- and 15-kDa cleavage products, whereas
little or no effect was observed with the other protease inhibitors
tested.
View larger version (70K):
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Fig. 4.
MIC4 binds to host cells, and the adhesive
motif is restricted to the C terminus encompassing the A6 domain.
A, Western blot analysis of cell binding. The micronemal
protein MIC4 bound specifically to host cells, whereas binding of the
dense granule protein GRA1 was not detected. After incubation with
total micronemal proteins (Microneme prep.) or ESA, the
unbound fraction was removed (Sup), and cells were washed
(W1 and W4 correspond to the first and fourth
wash, respectively). The CBF was recovered by detergent extraction, and
samples were resolved by SDS-PAGE and Western-blotted for MIC4 (mAb
5B1) and GRA1 (Tg17-43). Standards correspond to 10, 5, and 1% of the
input material from the microneme prep. The loading of the SUP
represents 5%, whereas all other fractions represent 20% of the total
material recovered. To determine the host cell binding activity of the
processed forms of MIC4, the CBF was analyzed on a 15% SDS-PAGE using
the rabbit anti-MIC4 or the mAb-5B1. The 70- and 15-kDa forms bound to
host cells, whereas no binding was detected with 50-kDa form.
B, quantification of the binding of MIC4 to host cells.
Approximately 11% of MIC4 in the microneme preparation and 26% of
MIC4 in ESA was bound to the host cell (*). In contrast, GRA1 was not
detected in the CBF. Values are plotted as relative intensity as
determined by phosphorimage analysis and compared with loading
standards for 1, 5, and 10% of the starting material. C,
ESA prepared from wild type RH and TyMIC4Myc were analyzed in the cell
binding assay. The ESA from RH was preincubated for 30 min at 37 °C
in absence or presence of 50 mM strong reducing agent
MESNA. The treated ESA was then diluted 50 times in CM-PBS before
incubation with host cells. D, ESAs prepared from RH and
parasites expressing MIC4His, TyMIC4Myc, or two independent clones of
MIC4 C12 were tested and compared in host cell binding assays. The
wild type and mutated forms of MIC4 are indicated by an
arrow. The rabbit serum anti-MIC4 used in this experiment
showed a cross-reaction with host cells, indicated by an
asterisk. This signal was also detectable in the sample of
HFF cells, which was not incubated in presence of ESA
C12). MIC4his and MIC4
C12 were expressed in a clone of
mic4ko mutant parasites lacking MIC4 gene that had
been deleted by double homologous recombination.3 Western blot
and IFA analysis of the transformed parasites confirmed that MIC4
mutants were of the expected size and appropriately targeted to the
micronemes (data not shown). ESAs prepared from these parasites were
tested in host cell binding assays. As for the endogenous MIC4, the
TyMIC4myc and MIC4his proteins bound substantially to host cells. In
contrast, ESA corresponding to MIC4
C12 failed to bind to host cells
(Fig. 4D). Two additional deletion mutants of MIC4 with
deletion of apple domains A5-6 or A3-6 were generated and failed to
bind to host cells (data not shown), consistent with the 50 kDa
protein lacking adhesive properties. Truncation of the last 12 C-terminal amino acids abrogated the adhesiveness of MIC4, possibly by
compromising the proper folding of the domain A6.
View larger version (28K):
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Fig. 5.
Binding of MIC4 in presence of carbohydrates
as competitors. ESA from RH was tested in the presence of
increasing concentrations of galactose,
N-acetylgalactosamine, or
N-acetylglucosamine.
View larger version (20K):
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Fig. 6.
Schematic representation of the structural
and functional domains of MIC4. The sites of proteolytic cleavages
on MIC4 are indicated by arrows. The processed forms of MIC4
are depicted, and their corresponding adhesiveness to host cells is
indicated with a plus (+) or minus ( ). ER, reticulum
endoplasmic; VT, valine threonine; SS, serine
serine; rAb, rabbit antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are grateful to James Ajioka for the screening of the cosmid library. We thank Steve Parmley, Michael White, Keith Gull, and Marie-France Cesbron-Delauw for the generous donation of materials used here and Amy Crawford and Maren Lingnau for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by Deutsche Forschungsgemeinschaft Grants 366/1-1 and SO366/1-2) (to D. S.), National Institutes of Health Grant AI34046 (to L. D. S.), and by a grant from the Wellcome Trust.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.
¶ Present address: The W. Harry Feinstone Dept. of Molecular Microbiology and Immunology, Johns Hopkins University School of Hygiene and Public Health, 615 N. Wolfe St., Baltimore, MD, 21205.
** To whom correspondence should be addressed. Tel.: 49 6221 54 6870; Fax: 49 6221 54 5892; E-mail: soldati@zmbh.uni-heidelberg.de.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M008294200
2 V. B. Carruthers, unpublished information.
3 M. Reiss, N. Viebig, S. Brednt, M.-N. Fourmaux, M. Soete, M. DiCristina, J. F. Dubremetz, and D. Soldati, submitted for publication.
4 U. Jäkle, and D. Soldati unpublished information.
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ABBREVIATIONS |
---|
The abbreviations used are:
ESA, excretory-secretory antigen;
mAb, monoclonal antibody;
PCR, polymerase
chain reaction;
PBS, phosphate-buffered saline;
BSA, bovine serum
albumin;
CBF, cell-bound fraction;
PAGE, polyacrylamide gel
electrophoresis;
MIC4, micronemal protein 4;
HXGPRT, hypoxanthine-xanthine-guanine-phosphoribosyltransferase;
HFF, human foreskin fibroblast;
IFA, immunofluorescence assay;
MPP1, microneme protein protease 1;
MESNA, 2-mercaptoethanesulfonic acid;
TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone or
N-p-tosyl-L-lysine
chloromethyl ketone.
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---|
1. | Tomley, F. M., and Soldati, D. (2001) Parasitol. Today, in press |
2. |
Kappe, S.,
Bruderer, T.,
Gantt, S.,
Fujioka, H.,
Nussenzweig, V.,
and Menard, R.
(1999)
J. Cell Biol.
147,
937-944 |
3. | Coppel, R. L., Brown, G. V., and Nussenzweig, V. (1998) Curr. Opin. Microbiol. 1, 472-481[CrossRef][Medline] [Order article via Infotrieve] |
4. | Naitza, S., Spano, F., Robson, K. J. H., and Crisanti, A. (1998) Parasitol. Today 14, 479-484[CrossRef] |
5. | Wan, K. L., Carruthers, V. B., Sibley, L. D., and Ajioka, J. W. (1997) Mol. Biochem. Parasitol. 84, 203-214[CrossRef][Medline] [Order article via Infotrieve] |
6. | Fourmaux, M. N., Achbarou, A., Mercereau-Puijalon, O., Biderre, C., Briche, I., Loyens, A., Odberg-Ferragut, C., Camus, D., and Dubremetz, J. F. (1996) Mol. Biochem. Parasitol. 83, 201-210[CrossRef][Medline] [Order article via Infotrieve] |
7. | Garcia-Réguet, N., Lebrun, M., Fourmaux, M-N., Mercereau-Puijalon, O., Mann, T., Beckers, C. J. M., Samyn, B., Van Beeumen, J., Bout, D., and Dubremetz, J. F. (2001) Mol. Microbiol., in press |
8. | Carruthers, V. B., and Sibley, L. D. (1999) Mol. Microbiol. 31, 421-89[CrossRef][Medline] [Order article via Infotrieve] |
9. | Brown, P. J., Billington, K. J., Bumstead, J. M., Clark, J. D., and Tomley, F. M. (2000) Mol. Biochem. Parasitol. 107, 91-102[CrossRef][Medline] [Order article via Infotrieve] |
10. | Klein, H., Loschner, B., Zyto, N., Portner, M., and Montag, T. (1998) Glycoconj J. 15, 147-153[CrossRef][Medline] [Order article via Infotrieve] |
11. | Wan, K. L., Blackwell, J. M., and Ajioka, J. W. (1996) Mol. Biochem. Parasitol. 75, 179-186[CrossRef][Medline] [Order article via Infotrieve] |
12. | Dobrowolski, J. M., Niesman, I. R., and Sibley, L. D. (1997) Cell. Motil. Cytoskeleton 37, 253-262[CrossRef][Medline] [Order article via Infotrieve] |
13. | Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395[Abstract] |
14. |
Hettmann, C.,
Herm, A.,
Geiter, A.,
Frank, B.,
Schwarz, E.,
Soldati, T.,
and Soldati, D.
(2000)
Mol. Biol. Cell
11,
1385-1400 |
15. | Bastin, P., Bagherzadeh, Z., Matthews, K. R., and Gull, K. (1996) Mol. Biochem. Parasitol. 77, 235-239[CrossRef][Medline] [Order article via Infotrieve] |
16. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
17. |
Donald, R.,
Carter, D.,
Ullman, B.,
and Roos, D. S.
(1996)
J. Biol. Chem.
271,
14010-14019 |
18. | Eschenbacher, K. H., Klein, H., Sommer, I., Meyer, H. E., Entzeroth, R., Mehlhorn, H., and Ruger, W. (1993) Mol. Biochem. Parasitol. 62, 27-36[Medline] [Order article via Infotrieve] |
19. | Klein, H., Mehlhorn, H., and Ruger, W. (1996) Parasitol. Res. 82, 468-474[CrossRef][Medline] [Order article via Infotrieve] |
20. | McMullen, B. A., Fujikawa, K., and Davie, E. W. (1991) Biochemistry 30, 2050-2056[Medline] [Order article via Infotrieve] |
21. | McMullen, B. A., Fujikawa, K., and Davie, E. W. (1991) Biochemistry 30, 2056-2060[Medline] [Order article via Infotrieve] |
22. | Nakaar, V., Bermudes, D., Peck, K. R., and Joiner, K. A. (1998) Mol. Biochem. Parasitol. 92, 229-239[CrossRef][Medline] [Order article via Infotrieve] |
23. | Mercier, C., Lefebvre-van Hende, S., Graber, G., Lecordier, L., Beauchamps, P., Capron, A., and Cesbron-Delauw, M.-F. (1996) Mol. Microbiol. 21, 421-428[Medline] [Order article via Infotrieve] |
24. | Soldati, D., and Boothroyd, J. C. (1995) Mol. Cell. Biol. 15, 87-93[Abstract] |
25. | Seeber, F., and Boothroyd, J. C. (1996) Gene 169, 39-45[CrossRef][Medline] [Order article via Infotrieve] |
26. | Carruthers, V. B., Moreno, S. N., and Sibley, L. D. (1999) Biochem. J. 342, 379-386[CrossRef][Medline] [Order article via Infotrieve] |
27. | Achbarou, A., Mercereau-Puijalon, O., Autheman, J. M., Fortier, B., Camus, D., and Dubremetz, J. F. (1991) Mol Biochem. Parasitol. 47, 223-233[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Carruthers, V. B.,
Sherman, G. D.,
and Sibley, L. D.
(2000)
J. Biol. Chem.
275,
14346-14353 |
29. | Entzeroth, R., Kerckhoff, H., and Konig, A. (1992) Eur. J. Cell Biol. 59, 405-413[Medline] [Order article via Infotrieve] |
30. |
Carruthers, V. B.,
Hakansson, S.,
Giddings, O. K.,
and Sibley, L. D.
(2000)
Infect. Immun.
68,
4005-4011 |
31. | Sultan, A. A., Thathy, V., Frevert, U., Robson, K. J., Crisanti, A., Nussenzweig, V., Nussenzweig, R. S., and Menard, R. (1997) Cell 90, 511-522[Medline] [Order article via Infotrieve] |
32. | Brydges, S. D., Sherman, G. D., Nockemann, S., Loyens, A., Däubener, W., Dubremetz, J.-F., and Carruthers, V. B. (2000) Mol. Biochem. Parasitol. 111, 51-66[CrossRef][Medline] [Order article via Infotrieve] |
33. | Pohl, U., Dubremetz, J. F., and Entzeroth, R. (1989) Parasitol. Res. 75, 199-205[Medline] [Order article via Infotrieve] |
34. |
Sun, Y.,
and Gailani, D.
(1996)
J. Biol. Chem.
271,
29023-29028 |
35. |
Baglia, F. A.,
Jameson, B. A.,
and Walsh, P. N.
(1995)
J. Biol. Chem.
270,
6734-6740 |
36. |
Baglia, F. A.,
and Walsh, P. N.
(1996)
J. Biol. Chem.
271,
3652-3658 |
37. | Ferguson, D. J. P., Brecht, S., and Soldati, D. (2000) Int. J. Parasitol. 30, 1203-1209[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Mineo, J. R.,
McLeod, R.,
Mack, D.,
Smith, J.,
Khan, I. A.,
Ely, K. H.,
and Kasper, L. H.
(1993)
J. Immunol.
150,
3951-3956 |
39. | de Carvalho, L., Souto-Padron, T., and de Souza, W. (1991) J. Parasitol. 77, 156-161[Medline] [Order article via Infotrieve] |
40. |
Ortega-Barria, E.,
and Boothroyd, J. C.
(1999)
J. Biol. Chem.
274,
1267-1276 |